In order to reveal the diversity characteristics of endophytes in different species and different organs,the correlation between endophyte community structure and host species and organ types was preliminarily clarified.The stems,leaves and leaf sheaths,which were respectively collected from the plants of the foxtail millet blast-susceptible varieties Shawan millet and Jigu 22 and the foxtail millet blast resistant varieties Xiaoqinggu and Shiliuzi,were used to carry out the endophytic diversity by high-throughput sequencing based on the 16S rDNA V3—V4 region.There were certain differences in endophyte species composition between susceptible and resistant varieties.In all tested samples,the dominant groups at the phylum level were Proteobacteria and Actinobacteriota.Bacteroidota,Chloroflexi,Myxococcota,Firmicutes followed.Alpha diversity analysis showed that the susceptible varieties(Shawan millet,Jigu 22)had higher abundance of endophyte in leaves.PCoA analysis revealed that the organ type had a greater impact on the endophyte community structure than the variety.Species composition analysis showed that the susceptible varieties Shawan millet and Jigu 22 contained endophytic flora that were significantly different from those of Xiaoqinggu and pomegranate(resistant to foxtail millet blast).The susceptible varieties(Shawan millet,Jigu 22)contained Entotheonellaeota phylum in leaves,while resistant varieties had Hydrogenedentes phylum in leaf sheaths.It clarified that the diversity and community structure of endophytes in different organs and millet varieties susceptible and resistant to foxtail millet blast were different,and organ types had a greater impact on the community structure of endophytes than varieties.

The SiPRR73 gene was cloned from Yangu 11 using RT-PCR technology,and through analyzing tissue-specific expression,responsive features of SiPRR73 to different photoperiods,photo-thermal combinational treatments and five abiotic stress treatments,the regulation mode of photoperiod and temperature on SiPRR73,and the responsive pattern of SiPRR73 to abiotic stresses in foxtail millet were explored. The results showed that totally 2 928 bp cDNA sequence of SiPRR73 was obtained from Yangu 11,which included 2 283 bp CDS region,encoding 760 amino acids. The SiPRR73 proteins of C4 crops including Panicum miliaceum,Panicum hallii,Sorghum bicolor and Zea mays showed relatively close relationship with SiPRR73. The second parietal leaf was the highest expression tissue of SiPRR73,but the expression level at root,stem and panicle tissues was relatively lower. The expression level of SiPRR73 was higher at light period than that at dark period under both short-day and long-day conditions,and during the whole vegetative growth phase,SiPRR73 showed higher expression level under long-day compared to short-day,which indicated that the expression of SiPRR73 was induced by light and controlled by photoperiod. The temperature determined expression peak number of SiPRR73 and the photoperiod determined occurrence time of expression peaks,so temperature and photoperiod participated in regulating of SiPRR73 expression mutually. PEG and low temperature stresses induced SiPRR73 expression totally,NaCl induced SiPRR73 expression at early stress stage,but inhibited it at later stress stage. Fe stress inhibited SiPRR73 expression at early stage,but induced it at later stage. ABA stress caused the close responsive feature of SiPRR73 to NaCl. This study indicated that SiPRR73 showed light-dependent expression feature,and photoperiod and temperature regulated SiPRR73 by interaction pattern,suggesting that SiPRR73 participated in adaptability regulation process to different photo-thermal conditions and might play a certain role in coping with drought,low temperature,ABA,NaCl and Fe stresses in foxtail millet.

In order to understand the function of SibHLH19 in foxtail millet,the CDS sequence and promoter sequence of SibHLH19 gene were separately cloned with the leaf cDNA and genomic DNA from resistance material Shilixiang as template by PCR.Promoter cis-acting elements and biological characteristics were analyzed using bioinformatics online tools.Then the expression patterns of SibHLH19 in different tissues and during the process to rust resistance were surveyed by qRT-PCR,respectively.Lastly the prokaryotic expression characteristics for the gene were detected by SDS-PAGE,laying a theoretical foundation for further research on SibHLH19 gene function and disease resistance mechanism.The results showed that the CDS sequence of the SibHLH19 transcription factor was 843 bp in length,encoding a total of 280 amino acids,the predicted protein molecular weight was 29.97 ku.The theoretical isoelectric point was 5.85,and the encoded protein chemical formula was C₁₂₉₆H₂₀₇₁N₃₉₇O₄₀₀S₁₁,containing a bHLH conserved domain,belonging unstable hydrophilic protein.The largest element of the protein's secondary structure was random coils,and the smallest element was a β-turn.Evolutionary analysis showed that SibHLH19 had the higher homology to the amino acid sequences of Panicum miliaceum (RLM85279.1),Panicum hallii (PUZ71581.1)and Panicum virgatum (XP_039835205.1),and had the lowest homology with Triticum aestivum(KAF7059972.1)and Aegilops tauschii subsp.strangulata (XP_040244423.1).The analysis of the promoter cis-acting elements showed that there were multiple response elements such as hormones and stresses in the promoter region of the SibHLH19 gene.Tissue expression analysis showed that the gene was mainly expressed at the seedling stage with the highest expression in the aboveground part,and was almost no expression at the booting stage.Within 24 hours of the response to the biotic stress of rust disease in foxtail millet,the SibHLH19 gene expression was up-regulated at 8 and 16 h in the disease resistance response,while its expression was only slightly up-regulated at 16 h and down-regulated at the rest of the time points in the susceptible response.It was speculated that SibHLH19 played a positive regulatory role in the resistance response to rust disease in foxtail millet.The constructed prokaryotic expression vector pET30a-SibHLH19 could express the SibHLH19 fusion protein with an apparent molecular weight of about 44 ku after being induced by 0.1 mmol/L IPTG.

In order to alleviate the continuous cropping obstacle of millet,provide a reference for optimizing millet planting mode,millet continuous cropping(Si)was named as control(CK),millet-corn(Si-Zm),millet-potato-corn(Si-St-Zm),millet-corn-soybean(Si-Zm-Gm)and millet-soybean-potato(Si-Gm-St)were used to analyze the effects of different rotation patterns on the physiological indicators,photosynthetic characteristics,agronomic characters,yield and downy mildew incidence rate during the critical millet growth periods.The results showed that compared with CK,in the Si-St-Zm,Si-Zm-Gm and Si-Gm-St rotation patterns,the activeness of superoxide dismutase,peroxidase and polyphenol oxidase in millet flag leaves were significantly increased,with the largest increases of 45.55%,41.55% and 109.09%,respectively.In the Si-Zm-Gm and Si-Gm-St rotation patterns,millet plant height,stem thickness,root length and root branch number were significantly increased,with the largest increases of 30.48%,30.50%,31.76% and 13.79%, respectively.In addition,compared with CK,under the Si-Gm-St rotation system,the H₂O₂ and MDA content in the millet flag leaves were significantly decreased,with the maximum reductions of 18.78% and 47.29%, respectively;and the stomatal conductance,net photosynthetic rate,transpiration rate and relative chlorophyll content were significantly improved by 31.94%-101.43%,35.74%-234.00%,16.44%-46.97% and 24.15%-66.16%,respectively;with millet ear length,1000-grain weight and yield increased by 14.90%,17.09% and 10.58%,respectively;and millet downy mildew incidence rate significantly reduced by 12.33%.In short,compared with CK,the Si-Gm-St rotation system significantly increased the activeness of SOD,POD and PPO,and improved photosynthetic efficiency in millet flag leaves,meanwhile,the millet yield and disease resistance enhanced.Therefore,compared with Si-Zm,Si-St-Zm and Si-Zm-Gm rotation patterns,Si-Gm-St rotation system has the best effect on alleviating continuous cropping obstacles,which can provide a reference for optimizing millet planting system.

In order to explore the effective methods for the identification of drought tolerance of foxtail millet during the entire growth period,select the identification indices of drought tolerance of foxtail millet,and speed up the process of drought tolerance breeding of foxtail millet,a field experiment was conducted using a randomized incomplete block design(alpha-lattice design)and repeated thrice in 2019—2020. Each experiment consisted of 30 foxtail millet genotypes treated under drought stress(DS)and normal water supply(CK). The agronomic traits and drought tolerance coefficient(DC)of the genotypes were assessed during the entire growth period. Combined analysis of variance showed that soil water environment had significant effects on 1000-grain weight,and extremely significant effects on the other indices. Genotype had significant effects on spike weight and grain weight per plant,and extremely significant effects on the other indices. Besides,the interaction between genotype and soil water environment extremely significantly affected the growth traits of foxtail millet,but not yield traits(except 1000-grain weight). Under drought stress,the characteristic value of each index decreased in varying degrees compared with CK,and the sensitivity of each index to drought stress was different. The results of t test showed that the effect of drought stress was significant(except 1000-grain weight). GGE biplot explained 71.15% of the total variation of data.The drought tolerance coefficient of each index was correlated in different degrees,in which the drought tolerance coefficients of plant height,spike length,stem and leaf dry weight and top second leaf area were significant positive correlation,and that of spike weight per plant,grain weight per plant,kernels per spike and yield were also significant positive correlation. The drought tolerance of foxtail millet could be reflected by different agronomic traits. According to the distance from ideal drought-tolerant varieties and ideal drought-tolerant evaluation indices,the drought tolerance and evaluation indices of foxtail millet materials were ranked. Taixuan 26,Chaogu 62,Chaogu 13 and other materials had strong drought resistance,plant height and spike weight per plant could be used as indices for drought tolerance identification in foxtail millet. GGE biplot provided an objective and effective new visual identification method for the breeding of drought resistant foxtail millet varieties.

In order to clarify the drought resistance and drought resistance mechanism of Allium polyrhizum in Inner Mongolia grassland,potted water control method was adopted.Two gradients were set,75% of the maximum field water capacity as the control(CK),and 25% of the soil relative water content under 30 days of drought stress.The root morphology,physiological characteristics and leaf photosynthetic characteristics of Allium polyrhizum from different sources were analyzed under drought stress. The result showed that Allium polyrhizum in Siziwang Banner compared with the Chifeng and Ordos was remaining relatively high root surface area,root volume and root length 0-0.5 mm in diameter. Allium polyrhizum root growth was relatively less affected by drought stress,chloroplasts were relatively stable and strong drought resistance in Siziwang Banner. Drought stress significantly decreased the above ground biomass,below ground biomass and total biomass of Allium polyrhizum in Chifeng and Ordos,but had no significant effects on Siziwang Banner(P>0.05). The Pn,Tr,Gs,Ci and leaves relative water content of Allium polyrhizum seedlings were significantly decreased and the relative permeability of cytoplasmic membrane was markedly increased under the whole drought stress treatment. Drought stress induced stomatal closure of Allium polyrhizum leaves and reduced transpiration to decrease water loss.The root characteristics,relative water content,chlorophyll and photosynthetic characteristics of Allium polyrhizum in different sources were certain differences under the same treatment. In general,the effect of drought stress on Allium polyrhizum in Siziwang Banner was relatively weak,it was indicating stronger drought tolerance.

To provide a solid foundation for studying the biological function of WRKY family members in kenaf in response to salt stress,all members of WRKY family were identified and their expression patterns were analyzed.Physical and chemical properties,phylogeny and conserved functional domains of WRKY gene family members were analyzed by bioinformatics method.The expression characteristics of WRKY gene family members under salt stress were analyzed by RT-PCR.The results showed that a total of 33 WRKY family members were identified,which were unevenly distributed on 12 chromosomes.There were certain differences in the physical and chemical properties of each member,such as amino acid number,molecular weight and theoretical isoelectric point.The conserved sequence WRKYGQK of each member did not change.Phylogenetic tree analysis showed that 33 WRKY family members were divided into 3 groups,GroupⅠ,GroupⅡ,Group Ⅲ,of which Group Ⅲ contained 5 subgroups.The real-time fluorescence quantitative RT-PCR results showed that there were 26 WRKY family members induced by salt stress,of which 23 had positive regulation and 3 had negative regulation.A total of 33 WRKY family members of kenaf were identified,of which 26 WRKY family members were involved in the salt stress response of kenaf.

To increase crop yields,reduce the application of chemical fertilizers,and improve nutrient utilization efficiency,N-efficient maize cultivars were screened and popularized. An understanding of nitrogen uptake,utilization,and field balance in maize cultivars with contrasting nitrogen efficiency response to N management is essential for efficient breeding and cultivation of maize to produce fodder and bio-energy. To determine the effects of N management on these factors during maize cultivation,a two-year field experiment was conducted in 2015 and 2016 in a subtropical semi-humid climate zone. The results showed that the proportion of N in the stem plus sheath and leaves in ZH311 during VT and R6 was significantly higher than that of XY508. In addition,the N accumulation into grain post-silking(NAG)and contribution of NAG to grain(CNAG)of ZH311 were significantly higher than those of XY508,while the N redistribution rate(NRR)and contribution of NRA to grain yield(CNRA)of ZH311 were significantly lower than those of XY508. The higher proportion of N in the vegetative organs of a N-efficient cultivar,ZH311,led to a significantly higher N accumulation in each stage than that observed for the N-inefficient cultivar XY508. The N accumulation advantage of ZH311 was higher after silking than before silking. The high post-silking N accumulation of ZH311 inhibited the pre-silking N transport that determines the N transport rate and contribution rate to grain of pre-silking N accumulation,which were significantly lower than those of XY508. Meanwhile,the N uptake efficiency,N recovery efficiency,and N partial productivity of ZH311 were significantly higher than those of XY508. Compared with that of XY508,the root system of ZH311 could more effectively absorb and utilize inorganic N in the 40-80 cm soil layer,reduce N deposition,and significantly decrease apparent N losses. The differences in apparent N losses between the two cultivars were mainly elicited post-topdressing. In summary,ZH311 has not only a higher yield per unit area than XY508,but also lower N losses,consequently reducing environmental risks.

In order to explore the effects of saline-alkali stress on the physiological and molecular mechanism of Brassica napus L.,using Huayouza 62 as experimental material.The seedlings of Brassica napus L.were treated with different concentrations of compound salt,compound alkali and compound saline-alkali solution.Physiological indexes such as seed germination rate,chlorophyll content,proline content,soluble sugar content and antioxidant enzyme activity in Brassica napus L.leaves were determined.The accumulation of betaine in rape leaves was determined by high performance liquid chromatography(HPLC).The key enzyme gene choline monooxygenase gene(CMO)in betaine synthesis pathway was analyzed by qRT-PCR technique.The results showed that the degree of damage to seed germination in artificially simulated saline-alkali solution of different concentrations was compound saline-alkali>alkali>salt.Low concentration saline-alkali solution promoted chlorophyll formation in rape leaves,while high concentration saline-alkali solution inhibited chlorophyll formation,saline-alkali stress significantly increased the contents of proline and soluble sugar,and the contents of proline and soluble sugar in high saline-alkali solution(YJ75,saline-alkali 75 mmol/L)for 21 d were 65.99 and 5.21 times higher than those in the control group,respectively,and the content of malondialdehyde was increased by saline-alkali stress.Saline-alkali stress significantly increased the activity of peroxidase(POD).Compared with the control group,the content of POD in high saline-alkali solution(YJ75)increased by 2.26 times after 21 d,and the content of POD reached the highest value on the 14th day after treatment with compound salt and compound alkali,however, the activity changes of superoxide dismutase (SOD) and catalase (CAT) were not obvious, and the role of species in the process of saline-alkali stress was low.Saline-alkali stress significantly increased the expression of key enzyme gene CMO,thus regulating the accumulation of betaine.In summary,the damage degree of saline-alkali stress to Brassica napus L. was compound saline-alkali > alkali > salt.Under high saline-alkali stress,Brassica napus would accumulate a large amount of betaine to reduce the damage.

Yield and nitrogen accumulation of maize will decline under soil acidification,but the physiological mechanism is not clear.Field experiment was conducted with four different soil acidity gradients:nautral acid(pH=7,CK),weak acid(pH=6,T1),medium acid(pH=5,T2)and strong acid(pH=4,T3),comparing yield,nitrogen accumulation,grain protein content,nitrogen metabolism-related enzyme activities,gene expression,nitrate nitrogen,ammonium nitrogen,soluble protein,free amino acid content in leaf and stem of maize.The results showed that compared with CK,the yield of T1,T2 and T3 treatments declined by 4.2%,30.7% and 52.3%,respectively.Grain number per spike decreased by 1.8%,28.1% and 42.8%;grain protein content showed a downward trend with T3 treatment significantly reduced by 14.5%.At the big flare stage,with the increase of soil acidity,nitrogen accumulation in leaves showed a downward trend,it was significantly decreased in T2 and T3 treatment by 28.1% and 56.2%,respectively.In stem,the nitrogen accumulation increased firstly and then decreased.Compared with CK,T1 treatment was significantly increased by 33.1%,and T3 treatment significantly decreased by 65.4%.At the big flare stage, the activities of nitrate reductase, glutamate dehydrogenase and glutamate synthase in leaf and stem under T3 treatment were significantly higher than those in CK, while the activities of glutamine synthase in leaf were significantly decreased. The amino acids in stem decreased first and then increased.With the increase of soil acidity,the expressions of ZmGln2 and ZmFd-GOGAT were up-regulated,which promoted the assimilation of {{\mathrm{NH}}_4}^{+} released by photorespiration and {{\mathrm{NH}}_4}^{+} produced by {{\mathrm{NO}}_3}^{-} reduction;the down-regulated expression of ZmGln1.2-ZmGln1.4, ZmNADH-GOGAT2 in leaf and ZmNADH-GOGAT1 in stem decreased the assimilation of {{\mathrm{NH}}_4}^{+} released by catabolism.By up-regulating or down-regulating the expression of relevant genes,maize could promote the production of more free amino acids and soluble proteins during nitrogen metabolism to resist acidification stress,but also reduced the nitrogen accumulation,resulting in lower yield and grain protein content.

NRL(NPH3/RPT2-Like)is a type of light-responsive protein unique to plants and plays a vital role in the phototropic signal pathway. To reveal the NRL gene maize genome's characteristics and expression,we analyzed them using bioinformatics methods combined with qRT-PCR technology. The property,structure,evolution of their encoded proteins,and growth period tissue expression and stress expression were analyzed. 31 ZmNRL genes identified were located in nine maize chromosomes,encoding protein amino acids 464-749 aa,which predicted to have chloroplast,nuclear and cytoplasmic locations. According to protein conservation,ZmNRL family was divided into four categories. Their gene structure also presented certain conservation,the most contained four exons. Analysis of the cis-elements of gene promoters revealed a large number of abscisic acids,jasmonic acid,light response,and anti-oxidation elements,among which G-box and Sp1 were two types of light-related elements. The expression of ZmNRL family genes in tissues during the growth period showed a temporal and spatial specificity,and the majority expression level was not high. Only ZmNRL2,ZmNRL4,ZmNRL24,and ZmNRL29 highly expressed. Furthermore,the characteristic modules were produced based on the data of the tissue co-expression genes. And the GO enrichment analysis of a particular leaf growth module containing six ZmNRL genes,mainly associated with the plastid organization biological processes and rRNA binding molecular functions. The expression of ZmNRL5,ZmNRL7,ZmNRL12,and ZmNRL19 genes were analyzed by qRT-PCR under salt,drought,high temperature,and Rhizoctonia solani inoculation treatments. The results showed that ZmNRL12 was significantly up-regulated in maize seedlings treated with high temperature,while ZmNRL5,ZmNRL7 and ZmNRL19 genes were down-regulated in drought,salt and pathogen treatments. In summary,31 ZmNRL genes were identified in the maize genome. They not only had apparent specific tissue expression but also participated in biotic and abiotic stress responses.

In order to investigate the effect of fertilizer reduction combined with organic fertilizer on the yield and quality of silage corn and soil nutrient,in 2019 and 2020,the effects of fertilizer reduction and organic fertilizer on agronomic traits,yield, quality,soil nutrient content and fertilizer utilization rate of silage corn were studied.As a result,the yield of silage corn was significantly affected by different fertilization treatments,and the yield of mixed application of organic fertilizer and slow control fertilizer reached 55 084.75 kg/ha,dry matter production reached 24 192.11 kg/ha.There was no significant difference in yield between slow controlled fertilizer constant and slow controlled fertilizer reduction of 20%,that was,excessive fertilization had no significant effect on yield.The accumulation of N,P and K under T2 treatment was 234.83,173.75,35.72 kg/ha,and significantly higher than other treatments.The maximum nitrogen fertilizer productivity of silage corn under T4 treatment was 166.46 kg/kg,and the maximum nitrogen fertilizer utilization efficiency under T1 treatment was 0.80 kg/kg,which indicated that the mixed application of organic fertilizer and inorganic fertilizer could improve the productivity of silage corn.After T4 treatment,the crude fat content of silage corn was the highest,and the application of organic fertilizer could increase the crude fat content of silage corn,and also increase the content of acid washing fiber.The yield of silage maize was correlated with urease,catalase and available phosphorus content,the correlation coefficients were 0.845,0.798,0.784.The results showed that fertilizer reduction and organic fertilizer application in Southwest China could significantly improve the yield and quality of silage corn,which was beneficial to the protection of farmland ecological environment and the sustainable utilization of soil fertility.

To investigate the function of soybean GmPP2C89 gene in plant abiotic stress response and adaptation. The expression patterns of GmPP2C89 under NaCl,PEG and mannitol treatments were detected by transcriptome data and Real-time quantitative PCR. Then,the cis-acting elements on the promoter of GmPP2C89 in response to abiotic stress were analyzed,and promoters of different lengths were cloned according to the distribution of cis-elements to construct fusion GUS vectors to obtain the corresponding transgenic Arabidopsis. The response of the promoters to NaCl,PEG and mannitol was analyzed by GUS staining. Transgenic Arabidopsis overexpressing the GmPP2C89 was constructed,and the root length,leaf MDA content and electrolytic leakage,and the expression of salt stress-related genes(SOD,POD,CAT,RD26,RD29A,and RD29B)were measured under normal and NaCl treatment conditions. The results showed that NaCl,PEG and mannitol treatments all led to a significant increase in the expression level of soybean GmPP2C89;the promoter region contained many cis-acting elements such as ABRE,DRE,G-box,MBS,MYB,MYC and TC-rich repeats which were involved in abiotic stress response,and this promoter was more responsive to NaCl treatment. In addition,under the salt treatment,the root length of transgenic Arabidopsis GmPP2C89-OX was significantly greater than that of WT,while the MDA content and electrolytic leakage were significantly lower than those of WT,and the salt tolerance was significantly enhanced;the expression of antioxidant enzyme genes(SOD and POD)and ABA pathway key gene RD29B in GmPP2C89-OX was significantly higher than that in WT. These results indicated that soybean GmPP2C89 was induced by NaCl,PEG and mannitol,and GmPP2C89 overexpression could enhance the salt tolerance of transgenic Arabidopsis by activating antioxidant and ABA pathways.

This study investigated the dynamics of soil phosphorus,yield responses to soil phosphorus and phosphate fertilizer in winter wheat-summer maize rotation system in the last decades,which was important for the scientific application of phosphate fertilizer in continuous high-yielding cropping.A combined method that included in situ phosphate fertilizer experiment under different soil fertility conditions,the analysis of changes of soil available P since 1978,the response of yield to phosphate fertilizer was used.Average content of soil available P of winter wheat-summer maize rotation area was 22.43 mg/kg,and the soil available P of the piedmont plain of Taihang mountain was greater than the alluvial plain.During 1996-1999,the soil available P content of the the piedmont plain and the alluvial plain was 15.09,11.90 mg/kg in cultivated land,respectively,the application rate of P₂O₅ in the winter wheat season of the rotation system was 180 kg/ha;the soil P supply capacity for winter wheat in these two regions piedmont were 83.9%,75.8%,respectively,and for summer maize they were 83.3%,89.7%,respectively.Under the condition of winter wheat straw returning,soil P surplus of these two regions was estimated to be 52.8%,55.4%,respectively.During 2010-2012,the soil available P of the piedmont plain of Taihang mountain was 27.22 mg/kg,the application rate in winter wheat and summer maize season were 108,60 kg/ha,respectively.The soil P supply capacity in winter wheat season was 84.6%,90.1% in summer maize season.Soil P surplus was estimated to be 6.7% in winter wheat season,and soil P of deficit was estimated to be 47.1% in summer maize season without straw returning.The application rate of P₂O₅ for the maximal yield production of winter wheat and summer maize were calculated according to the yield responses of winter wheat and summer maize to the P fertilization rates at multiple sites during 2002-2006 and 2012-2016.For winter wheat they were 107.3,125.1 kg/ha,respectively,and for summer maize they were 52.0,58.9 kg/ha,respectively.The accumulated P increaded the yield of winter wheat and summer maize for excess application 3 time of P fertilizer in 3 years 6 crop.The recommendation of P₂O₅ rate for winter wheat and summer maize with wheat straw returning were 90-100 kg/ha and 30 kg/ha,respectively,and they were 100-120 kg/ha and 45 kg/ha without wheat straw returning.

In order to preliminarily explore the key regulatory networks and genes involved in maize cold resistance,identify the key regulatory pathways and genes in response to low temperature stress,which laying a foundation for further research on the molecular mechanism of cold stress resistance.Here,a cold tolerance variety Mintian 6855 was employed to determine the gene expression pattern at 24,48 and 72 hours post low temperature of 5 ℃ stress by using transcriptome technique.The PCA analysis revealed that the repeated samples were well clustered together and significantly separated from CK samples.The results of difference analysis showed that about 4 000—7 000 difference genes expressed after cold stress treatments,while,only about 100—2 000 showed difference expressing among low temperature treated samples,indicating that low temperature was the main factor results in genes difference expressing,and the difference expression genes were mainly responded in the early stage.Meanwhile,KEGG annotation analysis results revealed that the differentially expressed genes were enriched in pathways of plant hormone signal transduction and MAPK,suggesting that these two signaling pathways actively respond to cold stress.In addition,different express genes were also enriched in plant-pathogen interaction as well as circadian rhythm plant,strongly implied that there were overlapping or common regulatory pathways in biological and abiotic stress pathways,while,genes that regulate circadian rhythms also playing a key role in plant adaptation to low temperatures.

To investigate the potential functions of DUF760s in rice growth and development,the genome-wide identification,classification,promoter sequence and expression profile analysis of the DUF760 gene family were performed.This research identified 6 and 8 members of DUF760 gene family in rice and Arabidopsis respectively by bioinformatics analysis.Phylogenetic tree analysis divided these members into two subfamilies,there were also some characteristic differences between the two subfamilies in protein conserved motif and gene structure.Multiple cis-acting elements responding to stresses and phytohormones existed in the promoter regions of rice DUF760 family genes.ABRE(abscisic acid response)element was present in the promoter sequence of all DUF760 family members,the promoter region of OsDUF760-1 possessed 9 abscisic acid(ABA)related response elements.The transcriptional expression level of OsDUF760-1 in rice was significantly down-regulated after ABA treatment,while OsDUF760-3 was significantly up-regulated.The expression change patterns of these two genes in rice after drought stress treatment were consistent with that after ABA treatment,which indicated that these two genes may participate in rice drought stress response through ABA signaling pathway,and play different roles.In addition to strong responses to ABA and drought stress treatments,members of rice DUF760 family also showed relatively strong expression changes in response to JA(Jasmonic acid),low temperature and M.oryzae treatments.

Tillering stage and jointing—booting stage are important for the formation of panicle number and spikelets per panicle of rice,respectively.Nitrogen application affects the formation of panicle number and spikelet per panicle.Bacteria involve in soil nitrogen cycle.Two high-yield rice varieties,Y Liangyou 900 and Zaofengyou 69,were used to study the difference of soil bacterial number and community structure between tillering stage and jointing—booting stage,and the relationship between them and soil nitrogen,rice yield under two application ratios of nitrogen fertilizer(7:3 and 6:4 application ratios of basic tiller fertilizer to panicle-spikelet fertilizer).The results showed that there were significant differences in the structures of dominant bacterial groups in paddy soil between the tillering stage and jointing—booting stage,which were mainly Proteobacteria,Bacteroidetes,Chloroflexi and Acidobacteria.The relative abundance of Chloroflexi at the tillering stage was 6.16 percent points higher than that at the jointing—booting stage.The relative abundance of Acidobacteria at the tillering stage was 2.65 percent points higher than that at the jointing—booting stage.The relative abundance of Proteobacteria at the jointing—booting stage was 0.69 percent points higher than that at the tilling stage.The relative abundance of Bacteroidetes at the jointing—booting stage was 1.09 percent points higher than that at the tillering stage.The results of correlation analysis showed that rice yield was significantly negatively correlated with the number of soil bacteria at the tillering stage,but significantly positively correlated with the number of bacteria and total nitrogen content in the soil at the jointing—booting stage.There was a significant positive correlation between the number of effective panicles and the number of soil bacteria at the tillering stage.The number of spikelets per panicle was significantly positively correlated with the ammonia oxidation potential at the tillering stage and the jointing—booting stage.Redundancy analysis(RDA)showed that the bacterial community structures were affected by many factors.The contents of soil ammonium nitrogen and the ammonia oxidation potential were the main factors affecting the bacterial community structures at the tillering stage and the jointing—booting stage,respectively.The function prediction of FAPROTAX further indicated that the denitrification in the 7:3 treatment at the tillering stage was enhanced,especially in the soil of Y Liangyou 900.Therefore,increasing the number of soil bacteria at the tillering stage could enhance the number of effective panicles.Ammonia oxidation involved by soil bacteria at the jointing—booting stage can promote the increase of spikelets per panicle in rice.For the high-yielding rice varieties,Y Liangyou 900 and Zaofengyou 69 under different nitrogen application ratios,the formation of yield,effective panicle number and spikelets per panicle is closely related to soil bacterial number,community composition and ecological function.

Gibberellin pathway is an important pathway in plant flowering regulation.In order to understand the role of gibberellin pathway related genes in the regulation of radish flowering. The structure,physicochemical properties,chromosome distribution,promoter cis-elements and tissue-specific expression of gibberellin biosynthesis and signal transduction related genes in radish were analyzed by bioinformatics.The expression levels of these genes in radish varieties with different florescence were detected by Real-time fluorescence quantitative PCR(qPCR).The results showed there were 46 genes related to gibberellin biosynthesis and signal transduction in radish genome,among them,the gene numbers of CPS,KS,KO,KAO,GA20OX,GA3OX,GA2OX,GAI,RGA,RGL,GID1 and SKP2 were 2,1,2,2,9,5,12,1,1,4,3 and 4,respectively.They were unevenly distributed on 9 chromosomes,molecular weight of their coding proteins were 21.32—127.80 ku,and the isoelectric points of the proteins were from 4.72 to 9.04.The analysis of gene structure and conserved domain showed that the number of exons of these 46 genes ranged from 1 to 21,and some conserved motifs were shared by most genes.Promoter cis-elements analysis showed that the promoters of these 46 genes contained cis-elements related to light,gibberellin,auxin,ABA,SA,low temperature,drought,etc.Using radish gene expression database analysis,it was found that the expression levels of these 46 genes were different in different tissues and at different developmental stages;qPCR detection showed that there were significant differences in the expression of these genes between early flowering material Xinlimei and late flowering material wild radish,suggesting that they may be closely related to the reproductive growth of radish.

Zinc finger proteins are transcription factors widely studied in eukaryotes,and play important roles in plant growth and development,and responses to stresses.In order to deeply understand the gene function of zinc finger protein in soybean,the full-length CDS sequence of GmZAT12 gene was cloned from Shangdou 1201,and the characteristics of coded protein by this gene was predicted by bioinformatics analysis.The subcellular localization of GmZAT12 protein was detected by the tobacco epidermal injection system, and the expression pattern of GmZAT12 gene in different tissues of soybean and abiotic stress was analyzed by Real-time PCR (qRT-PCR) technology. The results showed that GmZAT12 CDS contained 516 bp,encoding 171 amino acids and the molecular weight of the protein was 19.264 28 ku with a theoretical isoelectric point(pI)of 9.02.Its main components were random coils and α-helices and it contained 20 phosphorylation sites, mainly serine phosphorylation sites.Sequence analysis indicated that GmZAT12 possessed two conserved C2H2 zinc finger domains.The result of subcellular location indicated that GmZAT12 protein was localized in the nucleus.The results of qRT-PCR showed that CmZAT12 gene expressed mainly in roots,leaves and seeds of soybean,while low expression in flower and stem, and was induced by high temperature,low temperature,NaCl and ABA.The fact implied that this gene might be involved in abiotic stress signaling pathways.

The objectives of this study were to investigate the effects of nitrogen application rate on the grain yield,nitrogen accumulation and use efficiency of spring maize under different film mulching methods,and to provide a theoretical basis for efficient nitrogen application management of spring maize film mulching in high altitude area of Guizhou.Field trials were carried out from 2018 to 2019.The plots were arranged in a split-plot design.The main plots were assigned to the film mulching methods(wide film and narrow film) to be studied and divided into subplots according to the five nitrogen application levels(0,80,160,240 and 320 kg/ha).To study the effects of different film mulching methods and nitrogen application rates on yield,nitrogen accumulation,transport characteristics and utilization efficiency in different organs of spring maize.Film mulching,nitrogen application rate and their interaction significantly increased the yield of spring maize.The results showed that compared to narrow film mulching,wide film mulching not only increased the yield of spring maize by 17.8%,but also significantly enhanced the nitrogen accumulation and the retransfer of nitrogen accumulated before silking,then significantly increased the grain nitrogen accumulation.Moreover,compared to narrow film mulching,wide film mulching achieved significantly higher NUTE,NUPE,AEN,NPFP and NUE values showing increases of 4.9%,21.4%,23.5%,12.2% and 4.23 percentage points,respectively.Nitrogen application realized the synergistic growth of grain yield and plant nitrogen accumulation of spring maize,and could significantly affect nitrogen absorption,accumulation and transport.As the N application rate increased,the N translocation and the contribution rate of nitrogen transfer from vegetative parts to grain after silking could be effectively promoted,as well as enhanced.However,the nitrogen use efficiency,including the NDGPE,NHI,NUTE,NUPE,NUE,AEN,NPFP of spring maize,significantly decreased by increasing of N application rate.The regression analysis had shown that the optimal yield and nitrogen application rate for wide film mulching were reduced nitrogen application by 55 kg/ha and increased yield by 12.3% compared with narrow film mulching.The combination of wide film mulching and suitable nitrogen application rate was beneficial to the plant accumulation and absorption of nitrogen,so as to achieve high yield and nitrogen productivity,and then achieve the purpose of saving fertilizer and increasing yield.Considering the grain yield,N accumulation,transport and N use efficiency of spring maize,the reasonable N application rate of wide film mulching for spring maize in high altitude and other similar ecological zones in Guizhou was 160 kg/ha,which yield could reach 11 404.3 kg/ha.

In order to study the reasons for the differences of different types of rice under cadmium stress,it used the cadmium sensitive indica rice restorer line Changhui 891(CH891)and the cadmium tolerant japonica rice variety 02428 as materials,and selected the buds after seed germination,which were continuously treated with cadmium for 3 days and not treated with cadmium for RNA-Seq analysis using high-throughput Illumina HiSeq 4000 sequencing technology.A total of 539 524 490 valid read were obtained,the comparison rate with the reference genome was between 94.81% and 96.82%,and the GC content was above 49%.Through comparative analysis,a total of 7 204 differentially expressed genes(DEGs)were detected,among which 849 genes were specifically expressed in CH891(SCR3),676 genes were specifically expressed in 02428(RCR3),and 770 genes were expressed in both varieties(CCR3),but the expression level was different.The analysis of enrichment pathways of KEGG and GO showed that isoflavone biosynthesis,inositol phosphate metabolism,flavonoid biosynthesis and anthocyanin biosynthesis were significantly enriched in SCR3.Ribosome,regulation of autophagy and insulin resistance pathways were significantly enriched in RCR3.Alpha-linolenic acid metabolism,phagosome,limonene and pinene degradation and fatty acid degradation pathways were significantly enriched in CCR3.The results showed that after Cd treatment,the secondary metabolic process was mainly enriched in CH891,and the protein metabolism was mainly enriched in 02428.In conclusion,the metabolic pathways of CH891 and 02428 were different after Cd treatment,and the molecular mechanism controlling these metabolic pathways might be the main reason for the differences of cadmium stress in different rice varieties.

Protein kinases are important factors in plant defense system,protein kinase SnRK2 is a serine/threonine protein kinase,can play an important role in the plant stress signal transduction pathway through phosphorylation.We analyzed the expression pattern of SnRK2.4 under abiotic stresses,aiming to reveal its role in the regulation of adversity.The SnRK2.4 gene of high-quality cold-season turfgrass Poa pratensis L. was cloned using RT-PCR,the SnRK2.4 gene contained an ORF of 1 092 bp encoding a 363-amino acid protein,and its molecular characteristics were analyzed by bioinformatics.In addition,the expression pattern of this gene in different tissue parts under different abiotic stresses was observed using qPCR.The results showed that the Poa pratensis L.SnRK2.4 gene belonged to the SRK/SAPK superfamily,contained typical STKc_SnRK2 domain,tyrosine kinase phosphorylation site,casein kinase Ⅱ phosphorylation site and serine/threonine-protein kinase activity site,which had the highest homology with Brachypodium distachyon.The qPCR results demonstrated that the Poa pratensis L.SnRK2.4 gene was tissue-specific,highly expressed in panicles,and had no significant differential expression in roots,stems and leaves.Moreover,the Poa pratensis L.SnRK2.4 gene could respond positively to abiotic stresses such as drought,salt,low nitrogen,low phosphorus,ABA and BR.

This study intended to investigate the dynamic shoot characteristics and differences of several super rice cultivars and provide theoretical guidance for super rice breeding and cultivation in the South China rice area.Four super hybrid rice combinations,two super conventional rice varieties,and two high-quality super-high-yielding traditional rice were selected as test materials and applied.We investigated the plant leaf morphology,dry matter weight per unit area,yield at the seedling stage,active tillering stage,secondary panicle branch differentiation stage,initial heading stage and mature stage.The results showed that five dynamic shoot indexes,such as plant height,average number of tillers per plant,leaf morphology,dry matter weight per unit area and yield components,had significant seasonal ecological characteristics.Moreover,except the leave tillering angles from the early and middle stages of growth and development,other key indicators displayed that the super hybrid rice combination was significantly higher than the super conventional rice varieties.In details,the number of effective spikes per unit area,total grains per spike,thousand grains weight,economic coefficient and yield per unit area were significantly higher in the hybrid rice combinations than in the conventional rice varieties;while the spike length and seed setting rate of the conventional rice varieties were extremely significantly higher than those of the hybrid rice combinations.So,the construction of wide adaptation super rice breeding demands early and fast seedling development,high biological yield at mid and late growth stage,and high harvest index at maturity.Meanwhile,conventional rice varieties need to cultivate early and fast development characteristics,improve biological yield,and increase thousand grains weight and total number of grains per spike appropriately as well as maintaining high seed setting rate.Hybrid rice combinations requires to improve seed setting rate.

In order to study the effects of OsBAK1P,a precursor of Brassinosteroid insensitivity 1-associated receptor kinase 1,on the agronomic characteristics of rice.A target sequence fragment with a CDS fragment size of 651 bp was amplified from the rice panicle cDNA using the Zhonghua 11 japonica rice variety as the material according to the specific primers designed by the gene;the PTCK303-OsBAK1P overexpression vector and the PTCK303-OsBAK1P RNA interference vector were successfully constructed by restriction enzyme digestion and ligation methods;the Agrobacterium tumefaciens EHA105 strain was transformed into the correct expression vector plasmid,and the positive Agrobacterium tumefaciens clones were screened out by using CDS amplification primers;the callus of japonica rice cultivar Zhonghua 11 was infected by Agrobacterium tumefaciens genetic transformation to obtain transgenic plants;finally,compared to Zhonghua 11,two overexpressed and RNA-interfered T₁ transgenic plants with similar phenotypes were selected to measure and analyze the agronomic characteristics such as plant height,panicle length,and leaf angle,changes in root length and coleoptile length at the early stage of germination,and response to brassinolide(BL).The results showed that the plant height of OE-OsBAK1P transgenic rice was dwarfed,the panicle length was shorter,and the leaf angle decreased,meanwhile,the root length increased and the seedling length shortened after seed germination,and the leaves were not sensitive to BL.However,the plant height,panicle length and leaf angle of RNAi-OsBAK1P transgenic rice increased,meanwhile,root length shortened and seedling length increased after seed germination,and the leaves were sensitive to BL.To sum up,these results provide theoretical support for changing rice plant structure to increase grain yield and may provide a reference for further studies on other functions of OsBAK1 and its precursor OsBAK1P.

Sowing date and Planting density are two key factors that affect maize yield.In order to clarify the response characteristics of different summer maize cultivars to sowing date and planting density in the Huanghuaihai region.Two maize cultivars Zhongnongda 788 and Kehe 699 were used as test materials.Three sowing dates,June 10,17 and 24,as well as three densities,67 500(A),75 000(B),and 82 500 plants/ha(C)were set and later investigated the growth stages,morphological indexes,yield and yield components.The results showed that the growth process before silking was accelerated and the filling period was prolonged as the sowing dates postponed,maize planted in the third sowing date could not reach physiological maturity.Late sowing date(the third sowing date June 24)compared to early sowing date(the first sowing date June 10),the ear height of Zhongnongda 788 and the plant height,ear height of Kehe 699 increased significantly.The stem diameters of the two cultivars were significantly reduced;the empty stalk and lodging rate of the two cultivars increased with the delayed sowing dates;the yield of Zhongnongda 788 decreased by 21.8% mainly due to the decrease of 1000-kernel weight,the yield of Kehe 699 decreased by 41.3% due to significant reduction of ear number,kernel number per ear and 1000-kernel weight.Compared to density A,the plant height,ear height,empty stalk rate and lodging rate of the two cultivars increased significantly,the stem diameter decreased in density C significantly.Zhongnongda 788 obtained the maximum yield at density B and was significantly higher than density A,which were 12 450,11 097 kg/ha,respectively.With the increase of density,ear number of Kehe 699 didn't increased due to increased empty stalk rate,kernel number per ear decreased,thus yield of density C was significantly lower than density A,which were 7 548,9 464 kg/ha respectively.Interaction between sowing date and density only had extremely significant effect on lodging rate,but had no significant effects on morphological indexes,empty stalk rate,yield and yield components.On the whole,the average yield of Zhongnongda 788 was higher than Kehe 699,the former had lower empty stalk rate,lodging rate and more stable yield under the conditions of late sowing date and high density.In practical production,summer maize should be sown as early as possible,the yield loss caused by late sowing can be reduced by selecting suitable cultivars.To improve yield by increasing density,density resistance cultivar breeding is necessary.

In order to select rotation crops suitable for tomato in southeastern of Shanxi Province,we planted six cropsrotation patterns,namely maize(LVZm),zucchini(LVCp),peanut(LVAh),green onion(LVAf),okra(LVAe),and cucumber(LVCs),taking treatment with continuous tomato cultivation(LLLe,CK)as control. The changes in the microbial community structure and diversity in the ITS1 region of soil fungi were analyzed by Illumina Miseq high-throughput sequencing technology. At the same time,the quality indicators(soluble total sugar,organic acid content,sugar-acid ratio,Vc content,soluble protein,soluble solids,lycopene content,nitrate)and yield indicators(single fruit weight,yield)of the following tomatoes were measured. The results showed that Ascomycote was the dominant phyla in the 7 treatments,and the species and abundance were quite different,LVCs,LVZm,LVAh and LVAe increased the diversity index of fungal,while LVCp decreased the index. LVZm had better taste;LVAe and LLLe had the highest Vc content;no significant difference in soluble protein content;the content of soluble solids were the highest in LLLe and LVCp;the content of lycopene was highest in LVCp;LVAe had the highest nitrate content. The yields significantly increased in LVAe and LVCp patterns,and the quality of single fruit significantly increased in LVCp rotation. Principal component analysis(PCA)showed that the scores of quality and yield among different patterns from high to low were LVCp>LVAe>LLLe> LVAh>LVAf> LVZm>LVCs. In summary,crop rotation changes the soil fungal community structure,affects the index of the fungal,the quality and yield of tomatoes in subsequent crops. Based on various factors,zucchini and okra are the dominant rotation crops suitable for the growth of local tomatoes.

Small GTPases Rabs is a kind of monomer GTP binding protein,which can act as a molecular switch involve in variety of physiological and biochemical reactions in plant cells. In order to explore the relationship between potato small G protein StRab5b and anthocyanin synthesis,the StRab5b gene was overexpressed in potato by Agrobacterium mediated genetic transformation,try to study the effects of StRab5b gene on anthocyanin content and PAL activity. The result showed that overexpression of StRab5b gene significantly increased the anthocyanin content in potato leaves,stems and tubers. Compared with the control,the anthocyanin content in leaves of transgenic potatoes StRab5b-L2,StRab5b-L3 and StRab5b-L5 increased by 42%,24% and 54%,respectively;in stems was increased by 59%,37% and 32%,respectively;in tubers was increased by 118%,82% and 105%,respectively. At the same time,StRab5b gene also promoted the PAL activity,a key enzyme of plant anthocyanin synthesis. Compared with the control,the PAL activity in leaves of transgenic potatoes StRab5b-L2,StRab5b-L3 and StRab5b-L5 increased by 88%,63% and 66%,respectively;in stems increased by 48%,38% and 31%,respectively;in tubers increased by 98%,78% and 64%,respectively. In summary,small GTPases StRab5b increased the content of anthocyanin in potato by regulating PAL activity.

In order to establish a rapid DNA molecule identification system of lettuce varieties,8 lettuce varieties of different types with significant morphological differences were chosen and used for preliminary primer-screening,and 81 pairs of primary primers were selected from 245 pairs of primers by polyacrylamide gel electrophoresis. Labeled with fluorescence at 5' end of upstream,the 81 pairs of primary primers were re-screened with 90 lettuce varieties by capillary electrophoresis,and 22 pairs of core primers were finally screened out. The corresponding reference samples for each of the core primers were also selected according to the size of amplified fragments and a SSR fingerprint database of 233 varieties was constructed based on these core primers. The results showed that 22 pairs of core primers detected a total of 269 alleles in 233 varieties. For each pair of primers,the detected alleles ranged from 4 to 22,with an average of 12.23,and the PIC value ranged from 0.37 to 0.89,with an average of 0.73. Cluster analysis showed that 233 varieties were divided into two major groups of stem lettuce and leaf lettuce. Among the leaf groups,crisphead lettuce cluster into a group,half-hitch lettuce tended to cluster into a group,and looseleaf lettuce was difficult to cluster into a group. 15 varieties in 6 groups with no difference in SSR markers were compared in a field trail. 3 pairs of varieties with no significant morphological differences were determined as the same and other varieties from different groups were determined as similar varieties because of a few morphological differences. Thus,the result of DNA clustering was basically in agreement with that of the field morphological classification. In addition,24 varieties in 8 groups with the same name can be distinguished by the molecular markers. In conclusion,a fingerprint database of 233 lettuce varieties was constructed based on the 22 pairs of core primers and a variety identification system for stem lettuce and leaf lettuce was established,which could be used for rapid identification of lettuce varieties and germplasm resources,genetic diversity analysis and similar variety selection in DUS testing.

The aim of this study was to analyze the variation in meat quality and expression of intramuscular genes between Tan sheep,Dorper sheep and Small-tailed han sheep,and to preliminarily reveal the molecular mechanisms underlying the differences in their meat quality traits and also provide a theoretical basis for the selection and improvement of other sheep breeds. The meat quality of 8-month-old Dorper sheep,Tan sheep and Small-tailed han sheep was analyzed and compared,and the Illumina HiSeq^TM 4000 platform was used to sequence and analyze the transcriptome of the longest dorsal muscle tissue to explore the regulatory genes associated with the differences in their meat quality traits. The results showed that a total of 820 differentially expressed genes were screened,including 99 between Dorper sheep and Tan sheep,436 between Dorper sheep and Small-tailed han sheep and 552 between Tan sheep and Small-tailed sheep. The results of GO functional enrichment analysis of the differential genes showed that each comparison group was significantly enriched in 224,517 and 657 GO entries;and the KEGG pathway enrichment analysis showed that DEGs were enriched in cAMP,MAPK,AMPK,purine metabolism,glycerophospholipid metabolism,amino acid and fatty acid metabolism and glycolysis/gluconeogenesis signalling pathways,further screening for candidate genes related to meat quality regulation and flavor substance metabolism such as FOS,PLA2G4E,LPIN1,AMPD1,AMPD3, NT5C1A,GPI,PFKM and PKM. Real-time fluorescence quantitative PCR validation of several randomly selected differential genes showed consistent expression trends with the transcriptome sequencing results,indicating the reliability of the sequencing results. These differential genes obtained in this study can be used as basic information for the breeding of new breeds(lines)of Ningxia high-quality meat sheep.

In order to identify the response of starch components and physicochemical properties of waxy wheat grains to irrigation,a split plot design was adopted under field conditions.The main plot consisted of two waxy wheat varieties(Linnuo 88,soft;Jinmai 99,hard),and the sub-region was treated with three types of irrigation(S₁,irrigation overwintering water;S₂,overwintering water + jointing water;S₃,overwintering water + jointing water + grouting water; CK,no irrigation),the effects of irrigation on grain yield,starch content,starch composition,particle size distribution,flour gelatinization characteristics and flour quality of two types of waxy wheat were analyzed. The results showed that the yield and constituent factors of two waxy wheat varieties increased with irrigation.Compared with S₁ and S₂, the average two-year Linnuo 88 S₃ treatment increased yield by 63.59% and 9.02%,and Jinmai 99 increased yield by 64.15% and 6.95%,respectively.The starch content of the two waxy wheat varieties under S₂ was the highest, which was 1.75 and 5.54 percentage points higher than CK, respectively. The amylopectin content of Linnuo 88 increased firstly and then decreased with the increase of irrigation,and S₂ treatment was significantly higher than the other treatments. The amylopectin content of Jinmai 99 decreased with the increase of irrigation,and that of S₁ treatment was significantly higher than the other treatments. The amylose/amylopectinratio of starch was the opposite. The particle size distribution of the tested grain starch granules ranged from 1.0 to 45.7 μm,and the number proportion distribution showed a single peak curve.Both volume proportion distribution and surface area proportion distribution of starch granules showed a double peak curve.With the increase of irrigation times,the number of B-type starch granules increased first and then decreased,flour gelatinization temperature first decreased and then increased,and the peak time moved forward,but S₂ irrigation treatment was the most significant;protein content,wet gluten content and sedimentation value all decreased with the increase of irrigation. Under this trial conditions,at the same time as stable output of overwintering water + jointing water,it could increase the starch content and B-type starch volume proportion of Linnuo 88,and reduce amylose/amylopectin ratio;while overwintering water + jointing water + grouting water could increase the yield and decrease amylose/amylopectin ratio of Jinmai 99,and improve the gelatinization characteristics of hard waxy wheat.Irrigation can effectively regulate the starch composition and particle size distribution of waxy wheat grains,thereby changing the physicochemical properties of starch.

CYP79B2 is a related gene that regulates auxin synthesis in Arabidopsis.By cloning the homologous gene BrcCYP79B2-1 of CYP79B2 in B.rapa ssp. cninensis,analyzing its expression in different tissues and periods,and studying its regulatory mechanism on vernal flowering in B.rapa ssp. cninensis,to lay a theoretical foundation for the subsequent functional verification of the auxin-encoding gene in B.rapa ssp. cninensis.The homologous gene of CYP79B2 was cloned from B.rapa ssp. cninensis by qRT-PCR and named as BrcCYP79B2-1.The structure,physicochemical properties and relationship of its protein were analyzed by bioinformatics method,and its protein was analyzed by qRT-PCR method.Expression levels in different tissues and growth stages in B.rapa ssp. cninensis.And its expression in different tissues and growth stages of B.rapa ssp. cninensis was analyzed by qRT-PCR method.The results showed that the full-length coding sequence of BrcCYP79B2-1 gene was 1 623 bp,encoding 540 amino acids.The physicochemical analysis of the protein showed that the molecular mass of the protein was 60.849 73 ku,and the theoretical isoelectric point was 8.71.Compared with the amino acid sequences of other species,it was found that BrcCYP79B2-1 was highly conserved in cruciferous plants,and had the highest homology with turnip,up to 99.52%;the expression levels of different organs of B.rapa ssp. cninensis were analyzed by qRT-PCR, and it was found that the expression level of BrcCYP79B2-1 was the highest in roots, followed by leaves, and the lowest in flower buds.The expression of BrcCYP79B2-1 in seedlings after 0,10,15 and 16 days of low temperature treatment showed that the expresion of BrcCYP79B2-1 reached its peak on the 10th day of low temperature treatment, that is, the vegetative growth period, while the expression of BrcCYP79B2-1 decreased during flower bud differentiation, which indicated that the expression of BrcCYP79B2-1 was related to low temperature vernalization and could affect flower bud differentiation.

In order to explore the effect of low temperature during the flowering period in Brassica napus,Brassica napus L.GZhui(strong resistance to winter cold)and 10B(weak resistance to winter cold)were used as test materials,low-temperature stress (14 h, 12 ℃ at daytime; 10 h, 2 ℃ at nighttime) to these materials for 1, 2, 3, 4, 5 d, and normal environment(14 h,22 ℃ at daytime;10 h,18 ℃ at nighttime)as control,determine the changes of physiological indexes in bolting stem leaves and lower leaves under low-temperature stress,as well as pollen viability and stigma receptivity after the opening of flower buds of different length.The research results showed that the leaves of the two materials were slightly wilted after low-temperature treatment,and there was no obvious damage to the shape of the plants in all treatments,the changes of various physiological indexes of leaves after low-temperature treatment were more complicated,and the three antioxidant enzymes were sensitive to peroxidase(POD),and most of the antioxidant was significantly increased,lower leaves more than bolting stem leaves.The content of soluble sugar(SS)in osmotic adjustment substances changed significantly,GZhui increased significantly.Malondialdehyde(MDA)content increased significantly,10B more than GZhui,and the lower leaves and bolting stem leaves showed different performances.For flower buds larger than 6.0 mm,the pollen viability of the two materials was little affected by low-temperature treatment for 4 days.For less than 3.0 mm of flower buds,the development stopped and eventually died for more than 4 days of cold stress,and the pollen viability decreased significantly after 2—3 days of stress.After the flower buds of all levels,less than 6.0 mm were under low-temperature stress,the pollen viability of GZhui was higher than that of 10B,and the difference was obvious with the flower buds of 3.0—6.0 mm,so it was considered that the 3.0—6.0 mm flower buds could be used as an indicator for identifying different varieties of low-temperature tolerance during flowering.The performance of stigma receptivity was consistent with the trend of pollen vigor.The stigma receptivity of flower buds larger than 3.0 mm was not affected within 3 days of low-temperature treatment,and the receptivity of stigma more than 4 days of treatment was reduced;the flower buds smaller than 3.0 mm,the receptivity of stigma decreased to varying degrees within 3 days of low-temperature treatment.These results indicated that flower buds(smaller than 3.0 mm)were more sensitive to low temperature,resulting in reduced pollen vitality and stigma receptivity,and even abortion.

In order to study the effects of straw returning on soil active organic carbon components and carbon cycle related enzyme activities in rice-rape rotation farmland of Chengdu Plain,we carried out 3-year straw returning field experiment(2017—2020),including no straw(CK),chemical fertilizer alone(NPK),50% straw returning and chemical fertilizer(SR1),100% straw returning and chemical fertilizer(SR2),200% straw returning and chemical fertilizer(SR3).We measured soil physical-chemical properties,soil organic carbon content(SOC),soil labile organic C fractions,carbon cycle related enzymes,and their correlations.The results showed that straw returning could effectively improve soil physical-chemical properties,soil available nitrogen,phosphorus and potassium contents.Compared with CK treatment,straw returning treatments significantly increased SOC,ROC,DOC,and MBC contents by 5.05%—8.55%,18.40%—36.80%,35.76%—66.93% and 27.20%—52.10%,respectively.In general,higher returning dosage resulted in higher C content.On the other side,compared with CK and NPK,straw returning treatments significantly increased soil cellulase,β-glucosidase,catalase,polyphenol oxidase.The activities of soil cellulase,β-glucosidase,and polyphenol oxidase under SR2 treatment were the highest,which were significantly higher than SR1 treatment by 16.25%,8.49%,and 14.69%,respectively.The catalase activity of SR3 treatment was the highest,which was significantly higher than that of SR1 treatment by 25.10%(P<0.05).There were significant positive linear correlations among soil SOC,labile SOC fractions,and carbon cycle related enzyme activities.Consequently,full straw returning has been proved of the most efficient way of improving active organic carbon components,carbon cycle related enzyme activities,and promoting the improvement of soil quality in rice-rape rotation farmland of Chengdu Plain.

In order to explore the effects of the combination of nitrogen application rate and nitrogen application period on the photosynthetic characteristics,senescence characteristics,grain filling characteristics and yield of leaves at ear position of summer maize under the integrated condition of drip irrigation,summer maize variety Zhengdan 958 was selected as the test material.Under the condition of 210 kg/ha,top dressing treatment at jointing stage,belling stage and flowering stage(A1),top dressing treatment at jointing stage and belling stage(A2),top dressing treatment at jointing stage and flowering stage(A3);under the condition of 180 kg/ha,top dressing treatment at jointing stage,belling stage and flowering stage(A4),top dressing treatment at jointing stage and belling stage(A5),top dressing treatment at jointing stage and flowering stage(A6);the traditional border irrigation was set as the control CK,and the total nitrogen application was 240 kg/ha,CK1 was topcoated with nitrogen fertilizer at jointing stage at one time,and CK2 was topcoated with nitrogen fertilizer at jointing stage and belling stage respectively,with a total of 8 treatments.The results showed that compared with CK1,A1 and A4 treatments not only maintained LAI and SPAD values in the late growth stage of summer maize,but also significantly increased the activity of antioxidant enzymes(SOD,POD and CAT),and effectively inhibited the content of peroxide MDA,so as to delay the aging process of its leaves and protect the functional structure of leaf cells.Therefore,the summer maize under this treatment maintained efficient photosynthetic characteristics in its later growth stage,promoted the grain filling rate,and then increased the number of grains per ear and 1 000 grains weight,so that the maize yield was significantly improved.The yield difference between A1 and A4 treatments was not significant difference,but the nitrogen application rate of A4 was reduced by 14.3% compared with A1,which reduced the input of nitrogen fertilizer and saved the input cost.It was the recommended treatment in this experiment.

In order to explore the germplasm resources of male sterile line and identify the male fertility genes, which would provide the basic materials for the maize seed production by the male sterile line. Using the male sterile mutant x50 as the experimental material,the male sterile phenotype of x50 was studied,and the F₁ and F₂ populations of x50 and inbred line Mo17 were constructed to determine the genetic pattern of the male sterile trait in x50.With the F₂ population,the male fertility gene X50 was identified by the map-based cloning.Furthermore,the candidate gene was confirmed by the allelism test.The results showed that compared with the wild type,the x50 anthers did not emerge from the glume,and was smaller and wilted,and no mature pollen grains were formed.All F₁ plants were fertile,and F₂ plants displayed 3∶1 segregation ratio between fertile and sterile plants,indicating that the monofactorial recessive inheritance of x50.The gene X50 was mapped to the interval from the molecular marker 2-4901 to 2-4963 on chromosome 2 with a physical range of 237.42 to 241.39 Mb by map-based cloning.Candidate gene analysis found that the male fertility gene ZmMs33 was located in the mapping region.In addition,the test crosses of the ms33 mutants(ms33-6029 and ms33-6052)and heterozygous +/x50 revealed 1∶1 segregation ratio between fertile and sterile plants.These results suggested that x50 was an allele mutant of ZmMs33 gene.The identification of the male sterile mutant x50 provided germplasm resources for maize hybrid seed production and functional study of ZmMs33 gene.

Luyu 13 is an excellent maize hybrid bred by the Millet Research Institute of Shanxi Agricultural University.It has strong drought and stress tolerance,and has achieved remarkable social benefits.In order to compare the drought and stress tolerance characteristics of the parents 1572 and Hai 921 of maize hybrid Luyu 13,we analyzed the related parameters of seed germination and seedling growth under simulated drought stress with 20% PEG solution.Results showed that there was no significant difference in the germination parameters between Luyu 13,1572 and Hai 921 under normal conditions,but the germination rate,germination energy,germination index and sprout index were inhibited under drought conditions,while the inhibition degree of Hai 921 was significantly higher than that of 1572 and Luyu 13.These results showed that the seed vigor of 1572 was higher than that of Hai 921,indicating inbred line 1572 had higher germination rate and germination advantage under drought stress,and possess stronger adaptability to drought.Subsequently,the important stresses response genes,ZmCIPK16 and ZmSAM1,were selected and analyzed their expression in 1572 and Hai 921 inbred lines under different abiotic stresses at seedling stage and under drought stress at different growth stages by Real-time PCR.Results showed that ZmCIPK16 and ZmSAM1 were widely responses to drought,salt,low temperature,high temperature,and ABA at seedling stage of 1572 and Hai 921 and responses to drought at different growth stages.ZmCIPK16 and ZmSAM1 showed more actively participated in the response to drought and other stresses in 1572.These results indicated that the inbred line 1572 was an excellent germplasm with drought and stress tolerance,and its contribution to drought resistance of hybrid Luyu 13 was greater than that of inbred line Hai 921.

To explore the relationship level between CaMADS6 expression and floral organ development of pepper,CaMADS6 was cloned for bioinformatics prediction from the sterile line 9704A and the maintainer line 9704B,and the spatiotemporal expression characteristics of this gene were analyzed.The results showed that the CaMADS6 cloned from 9704A and 9704B had the same coding sequence,with a full length of 744 bp and encoding 247 amino acid residues.CaMADS6 protein had the relative molecular weight of 28.67 ku and theoretical isoelectric point of 8.98.It was a hydrophilic protein with no transmembrane structure.The secondary structure of CaMADS6 consisted of 57.49% α-helices,8.91% extended chains and 28.74% irregular coils.The CaMADS6 protein shared 100% homology with CaFUL2,which possessed a typical MADS-box characteristics.CaMADS6 was expressed in different organs of 9704A and 9704B at different developmental stages and the expression was the highest in flower buds,followed by leaves and stems,and almost no expression in roots.The expression of CaMADS6 in stems of 9704A was significantly lower than that of 9704B at seedling,flower bud and adult stages,and the expression of CaMADS6 in leaves was significantly lower than that of 9704B at the adult stage.The expression level of CaMADS6 in flower buds of 9704A was extremely significantly higher than that of 9704B at flower bud and adult stage,and its expression level was 2.2 and 3.5 times higher than in 9704B,respectively;CaMADS6 gene was expressed in different parts of flower organs of 9704A and 9704B,and the expression of CaMADS6 gene from high to low was calyx,corolla,ovary and anther.The expression level of CaMADS6 in the calyx and corolla of 9704A was extremely significantly or significantly lower than that of 9704B,the expression level of CaMADS6 were 66%,83% of 9704B,respectively.The expression level of CaMADS6 in 9704A anther was 34 times higher than in 9704B,and the difference between the two groups was extremely significant,predicting the abnormal expression of CaMADS6 in anthers was closely related to pepper male sterility.

In order to explore the responses of kernel position effect of summer maize to plant density and its carbon and nitrogen metabolism characteristic,field experiments were conducted during 2020 and 2021 growing reasons at Dishang Experimental Station,Institute of Cereal and Oil Crops,Hebei Academy of Agriculture and Forestry Sciences. Three plant densities(PD1:60 000 plants/ha;PD2:75 000 plants/ha;PD3:90 000 plants/ha)were arranged,with the objectives to study the effect of plant density on grain filling and kernel weight ratio of inferior and superior kernel and its physiological characteristics of carbon and nitrogen metabolism. Compared with the superior kernel,the response of inferior kernel to plant density was more obvious,significant differences in grain-filling rate and kernel weight of inferior kernel were observed since 20—25 d and 30—35 d after pollination,respectively. As the plant density increases,the kernel weight ratio of inferior to superior kernel significantly decreased,PD3 decreased the kernel weight ratio of inferior to superior kernel by 8.45% on average,compared to that of PD1. The single plant dry matter accumulation significantly decreased as the plant density decreased,this was mainly due to the significant decrease of post-silking dry matter accumulation. The analysis of carbon and nitrogen metabolism of kernel showed that the increased plant density exacerbate the difference in starch and protein contents between inferior and superior kernels;and the increased plant density also exacerbate the difference in SPSase,ADPGase and GS activities between inferior and superior kernels,which mainly attributed to the significant decrease in SPSase,ADPGase and GS activities of inferior kernel. In conclusion,the increased plant density exacerbate the kernel position effect of summer maize,this was related to the lower grain-filling rate and kernel weight of inferior kernel since mid-grain filling stage,the lower grain filling rate in inferior kernel under dense planting was not only related to the insufficient post-silking dry matter accumulation,and it was also closely related to the lower activities of SPSase,ADPGase,and GS in inferior kernels.

In order to explore the function of ACS gene in herbaceous peony,a full-length cDNA sequence of PlACS cDNA in Paeonia lactiflora was obtained,RACE technique and bioinformatic methods were used to analyze the protein sequence which it encoded.The CDS of PlACS was subcloned,the prokaryotic expression vector of PlACS was constructed based on pET32a vector,and then the highly efficient prokaryotic expression system was established.The results showed that the total length of PlACS cDNA(GenBank accession JX512359)was 1 752 bp,which encoded 492 amino acids.Seven conserved regions and active sites K₂₇₈ were detected in PlACS protein.Phylogenetic tree analysis showed that PlACS was highest homological with ACS of P.suffruticosa.PlACS protein was determined structurally to be 40.04% α-helix,16.26% β-extended strand,6.91% β-turn and 36.79% random coil.Protein 3D structure homology modeling predicted that PlACS existed as homodimers.The optimal expression condition of PlACS protein was that when the cell density of genetic engineering strain A₆₀₀ reached 0.2,IPTG with a final concentration of 0.1 mmol/L was added,and the recombinant protein was expressed for two hours at 37 ℃.It was of great significance to acquire PlACS recombinant protein with biological activity by denaturation & renaturation and identify its enzymatic activity in vitro.

In the early stage, the dwarf dwarf-12 was successfully selected by distant hybridization of dwarf sorghum and local maize.After previous studies, the dwarf gene may be controlled by br2.Since there were no adverse traits, in order to utilize and discover excellent dwarf inbred lines, and then crossed dwarf-12 with local white maize, and selected the excellent dwarf inbred line d8227. The dwarf maize material d8227 obtained by predecessors was combined with maize of different heights, and it was identified that it has good combining ability. In order to increase the germplasm resources of dwarf maize and improve the yield of maize, in-depth research was carried out.d8227 and dwarf-12 were used as research materials to compare the differences in main agronomic traits between dwarf parents and progeny to observe the differences in stem cytology;d8227 and four inbred lines with different backgrounds were used for genetic mating design to analyze dwarf culm.Inheritance mode of genes; construct a targeting population, perform high-throughput sequencing with BSA method, conduct preliminary mapping of dwarf genes, and perform allelic identification of dwarf materials with known positioning intervals to clarify target genes and known genes relationship. The results showed that the plant height of d8227 increased by 9.35%, the ear position increased by 31.50%, the leaves of d8227 decreased, and the length of stem nodes increased.dwarf-12 increased by 52.21%, 5.26%, 23.76%, 6.93%, 12.02%; using the paraffin section method, observe the characteristics of transverse and longitudinal cells on the upper, ear and lower ear of d8227 and dwarf-12 with a microscope, and d8227 was longitudinally sectioned.The cells were loosely arranged and the cells were obviously elongated;the dwarf-12 cells were arranged regularly and compactly.After measuring the cell area, ear and lower ear cell area of the d8227 were significantly increased than that of the dwarf-12, which was mainly caused by the elongation of the d8227 cells. Through genetic analysis, the dwarf stalk gene was a single recessive gene, and the gene was preliminarily located.The dwarf stalk gene were located at 190-215 Mb of chromosome 1.The dwarf maize that had been located in the interval was selected for allelic identification.The two-year planting results showed that d8227 and 123d, Na360 were not alleles, but may be alleles with 125d and 123d, which needed follow-up fine mapping and in-depth research. On the whole, d8227 is a medium dwarf material with excellent characters and has breeding potential, but further studies such as fine positioning are needed to judge its utilization value.

In order to understand the main flavonoids and the key synthase genes related to flavonoids and clarify the differences of flavonoids among different pepino.Combined metabolomic and transcriptomic analyses were carried out with the mature pepino(LOF and SRF)currently main cultivars in China.The results showed that 9 kinds of secondary metabolites and 41 kinds of tertiary metabolites were identified,and the flavonols content was the highest.There were 21 metabolites with significant differences in flavonoid components,which were distributed in 6 secondary metabolites such as flavanols,flavonoid carbonoside.The total content of dihydroflavonol,flavanols and isoflavones was higher in the Light-oval fruit(LOF)than in the Sweet-round fruit(SRF),while other compounds including flavonols were higher in the SRF.A total of 503 genes related to flavonoid synthesis were identified by RNA-seq analysis,the expression of Phenylalanine ammonia-lyase(PAL),Cinnamyl alcohol dehydrogenase(CAD),and Flavanone-3-Hydroxylase(F3H)which were key genes for upstream stage of the whole process of pepino flavonoid synthesis,were all up-regulated in LOF compared to SRF.Flavonol synthase gene expression was consistently higher in SRF than LOF.According to the result of transcriptome,the expressed trend of 4 flavonoid synthases agreed with the testing by the RT-PCR.The results showed that a large number of flavonoid compounds and their synthase genes were present in different cultivars of pepino.However,the content of different flavonoid components and the activities of enzymes related to its assimilation and is varied among different resources.

In order to find the loci associated with quality traits in wheat,the mapping of quantitative trait loci(QTL)was conducted using the high-density genetic linkage map based on single nucleotide polymorphism(SNP)and the quality parameters surveyed in four environments including grain protein content,wet gluten content,starch content,sedimentation value and extensibility with the recombinant inbred line population derived from Bainong AK58/Bima 4.A total of 60 QTLs were identified by inclusive composite interval mapping,which were distributed on 20 chromosomes except for 6B.21 QTLs were found in two or more environments,of which the positive alleles of 15 QTLs were derived from Bainong AK58 and the positive genes of the remaining 6 QTLs were derived from Bima 4. A QTL cluster was found on chromosome 4A in the interval of 116.4—139.0 cM(629.36—701.53 Mb),and several QTLs such as QGpc.his-4A-2,QWgc.his-4A-2,QSv.his-4A and QEx.his-4A were identified in this region.Furthermore,1BL/1RS translocation caused improvement of protein content,wet gluten content and sedimentation value,but showed a negative effect on starch content.It is supposed that the impact of 1BL/1RS translocation lines on quality may be associated with their genetic background.

Being effective only to the traits controlled by large-effect QTL,conventional marker-assisted selection(MAS)can hardly improve the quantitative traits that are controlled by many small-effect QTL.Later on, the genomic selection (GS) technology proposed in 2001 solves the problem of improving complex traits controlled by the minor polygenic effects due to its high prediction accuracy through estimating the breeding value of individual with high-density molecular markers. At present,it has been successfully applied to the genetic improvement of quantitative traits in animals and plants such as dairy cattle,pigs,sheep,maize,and wheat in the US,Canada,Australia,Germany,France and so on.As a breaking-through breeding technology and a hot spot in research and application now. In this review,we summarized the factors affecting predictive accuracy of GS and the research progress of GS in breeding maize,wheat,rice and rapeseed at home and abroad,and finally discussed the existing problems in its application. This review will provide some reference for GS of the crops in the future.

To select the optimum tillage mode for the fluvo-aquic soil in North Henan,a three-year field experiment was carried out during 2016 to 2019. The experiment was designed with five combinations of three different tillage practices in wheat season as continuous rotary tillage(RT-RT-RT);deep tillage-rotary tillage-rotary tillage(DT-RT-RT);deep tillage-rotary tillage-strip rotary tillage(DT-RT-SRT);deep tillage-strip rotary tillage-strip rotary tillage(DT-SRT-SRT);deep tillage-strip tillage-rotary tillage(DT-SRT-RT). The photosynthetic indexes,soil available nutrients,and wheat yield were measured and analyzed under different treatments. The results showed that compared with RT-RT-RT,photosynthetic characteristics under different rotation tillage treatments improved. Compared with RT-RT-RT,the net photosynthetic rate,stomatal conductance under DT-SRT-RT increased by 10.85%,7.83%,respectively. The chlorophyll content increased gradually with the growth stage under DT-SRT-RT,and it was significantly higher than that under RT-RT-RT at the filling stage,with an increment of 16.52%. The content of available nitrogen,available phosphorus,and available potassium in the 0-50 cm soil layer increased under treatments with rotation tillage. Additionally,the spike number,grains per spike,1000-grain weight,and yield of wheat under DT-SRT-RT were higher than those under RT-RT-RT,therein,the yield under DT-SRT-RT was the highest with an increment of 14.64%. The correlation analysis showed that the net photosynthetic rate was positively correlated with wheat yield,which reached significant level at the flowering period. The contents of nitrate nitrogen,ammonium nitrogen,available phosphorus,and available potassium in surface soil were significantly positively correlated with wheat yield. Generally,for the fluvo-aquic soil area in North Henan,the photosynthetic characteristics of wheat,soil available nutrients content and the components of yield were improved by rotation tillage modes,and therein,the DT-SRT-RT was the optimum one.

In order to explore the molecular mechanism of diploid potato material response to drought stress under different drought stress time treatment,and explore drought resistance related genes.The high drought-resistant diploid resource A90 was used as experimental material,and transcriptome sequencing technology was used to analyze the differentially expressed genes under PEG-6000 stress at different times.Differentially expressed genes involved in drought stress response of potato were predicted by GO enrichment,KEGG pathway analysis and transcription factors,and key genes of drought resistance regulation were preliminarily explored.The expression of three candidate genes was analyzed by RT-qPCR.The results showed that there were 2 519 differentially expressed genes in the A90 treated with 20% PEG-6000 for 3,6,24 h compared with the control,and these genes were mainly enriched in plant hormone signal transduction,glutathione metabolism,phenylpropanoid biosynthesis,pentose and glucuronate interconversions and other drought-related processes.In addition,among 340 codifferentially expressed genes significantly enriched in the pathway,53 genes were annotated to 15 transcription factor families including RLKs,AP2/ERF and Tify.Among which StST/K1,StERF1 and StTify1 had higher expression levels,which might be key genes in drought resistance regulation.StST/K1 and StERF1 were mainly up-regulated in roots and leaves under drought and low temperature stress,StERF1 was up-regulated in roots,stems and leaves under salt stress.StTify1 was up-regulated in stems and leaves of the material under drought,and it was up-regulated in roots,stems and leaves under salt stress.But it was down-regulated only in stems of the material under low temperature stress.These results indicate that the three selected genes can respond to drought,salt and low temperature stress,it can provide a theoretical basis for study on drought resistance candidate genes in potato molecular breeding in the future.

In order to reduce the nitrate content of Brassica rapa L.ssp. chinensis L.,this study carried out nutrient-breaking treatment before the Brassica rapa L.ssp. chinensis L.was harvested.Physiological analysis of Brassica rapa L.ssp. chinensis L.nutrient-breaking treatment showed that Brassica rapa L.ssp.chinensis L.yield did not decrease significantly at 5 d,but the nitrate content of root,petiole and leaf decreased by 49.77%,23.90% and 33.39% respectively.Transcriptome comparison found that 301,2 270,and 2 271 differentially expressed genes(DEGs)were identified in Brassica rapa L.ssp.chinensis L.root,petiole,and leaf at 5 d,respectively.The Gene Ontology(GO)analysis revealed that these DEGs were mainly enriched in nitrogen(N),carbon(C)and reactive oxygen species(ROS)metabolism.In the process of N metabolism,nitrate uptake transporter NPF7.2 genes were down-regulated expression in root,petiole and leaf.The upstream regulator ERF104 of NPF7.2 were down-regulated expression in petiole and leaf.Nitrate retransporter NPF2.13 and NPF1.1 genes were up-regulated expression in leaf.Nitrate assimilation key enzyme glutamine synthetase(GS)genes were up-regulated expression in root and leaf.In the process of C metabolism,the sucrose phosphate synthase(SPS)genes,a key enzyme for sucrose synthesis in petiole and leaf,were up-regulated expression.In the process of ROS metabolism,superoxide dismutase(SOD)and ascorbate peroxidase(APX)genes were up-regulated expression in root.The expression of cytochrome P450,peroxisome and lipoxygenase genes were down-regulated expression in petiole and leaf.Catalase(CAT)and peroxidase(POD)genes were up-regulated expression in leaf.In summary,the nutrient-breaking treatment 5 d before harvesting can not only ensure that the yield of Brassica rapa L.ssp.chinensis L.was not affected,but also improve the quality of Brassica rapa L.ssp.chinensis L..

In order to study the application method of humic acid on grapes and find out the optimal proportion of carbon such as humic acid instead of organic fertilizer,the plot experiment was carried out with simple chemical fertilizer fertilization mode as control(CK treatment)for two consecutive years.Based on farmers' fertilizer practice(FFP treatment),3 ratios of carbon such as humic acid instead of organic fertilizer were set:10% carbon substitution(T1 treatment),20% carbon substitution(T2 treatment)and 30% carbon substitution(T3 treatment).Their effects on grape yield,quality and soil nutrients were studied,and the optimal substitution ratio was screened by principal factor analysis.The results showed that the yield of T2 treatment was the highest for two consecutive years,but there was no significant difference between T2 and T3 treatment.Single cluster weight and single fruit weight in T1,T2,T3 treatments were significantly higher than those in CK treatment,but there was no significant difference between FFP treatment and T1,T2,T3 treatments;on the whole,the contents of soluble solids,soluble sugar,titratable acid and vitamin C in humic acid treatment instead of organic fertilizer were better than those in other treatments.Through correlation analysis,soil available potassium,organic matter and acid phosphatase were positively correlated with grape yield.Available phosphorus,available potassium,organic matter and acid phosphatase in soil were very significantly correlated with soluble solids,soluble sugar and vitamin C content of grape.Regulation of these soil indexes was the way to increase grape yield and quality by replacing organic fertilizer with humic acid;through principal factor analysis,there were 2 principal factors with eigenvalue greater than 1 in 2019 and 2020,the cumulative contribution rate of variance of 2 principal factors reached 88.864%-91.470%,and T2 treatment was the highest comprehensive score for two consecutive years.On the whole,carbon such as humic acid instead of 20% organic fertilizer was the optimal proportion.

In order to clarify the biological function of gibberellin receptor GID1 in Pyrus betulifolia,and provide a good foundation for future development of P.betulifolia dwarf rootstocks using CRISPR/Cas9 gene editing technology.Pyrus betulifolia was used as the test material,and the PbGID1s genes were obtained by homologous cloning method.Bioinformatics analysis software was used to construct the gene structure and design the target sites;construction of sgRNA expression cassettes with targets into CRISPR/Cas9 expression vectors,through the mediation of Agrobacterium,the CRISPR/Cas9 expression vector was transferred into the cotyledons of P.betulifolia.Results showed that four PbGID1s were successfully cloned from P.betulifolia plants and named as PbGID1b-1,PbGID1b-2,PbGID1c-1 and PbGID1c-2. They all consisted of two exons and one intron found by gene structure analysis.Amino acid sequence comparison showed that all PbGID1s had the HGG and GXSXG conserved domains.Five gRNAs that could potentially edit all 4 PbGID1s simultaneously were successfully constructed into a single CRISPR/Cas9 vector,pYLCRISPR/Cas9P_35S-N.The results of the genetic transformation test of P.betulifolia showed that a total of 595 cotyledons of P.betulifolia were infiltrated,176 resistant buds and 33 positive plantlets were obtained,and the transformation efficiency reached 5.55%.A CRISPR/Cas9 vector was successfully constructed that could simultaneously target the PbGID1s family genes of P.betulifolia.Through the mediation of Agrobacterium,the vector was successfully transformed into P.betulifolia cotyledons,and positive plants were obtained.

In order to explore the relationship between the vernalization genes and winter-spring characteristics of wheat varieties in the Northern China Winter Wheat Region,271 wheat cultivars from the Northern China Winter Wheat Region were used as materials to detect the composition and distribution of four vernalization genes Vrn-A1,Vrn-B1,Vrn-D1 and Vrn-B3 by molecular markers.The heading date of these cultivars in the field was observed in Sanya,Hainan Province,and their winter-spring characteristics were investigated with the data recorded.The results showed that in this wheat region:among the four dominant alleles,Vrn-D1 (27.7%)had the highest distribution frequency in the tested cultivars and the distribution frequency decreased gradually from the South to the North;the distribution frequency of dominant Vrn-B1 alleles (3.0%)was very low in wheat materials in this wheat region;none of the test materials contained the dominant alleles Vrn-A1 and Vrn-B3;the most common vernalization gene allele combination was vrn-A1/vrn-B1/vrn-D1/vrn-B3.Further analysis of winterness-springness type of the test cultivars showed that some cultivars containing the dominant allele Vrn-D1 must be vernalized to blossom,while the wheat cultivars containing the dominant allele Vrn-B1 had a weak demand for vernalization.Among the four dominant vernalization genes,only the dominant alleles Vrn-D1 and Vrn-B1 were distributed in the tested cultivars in the Northern China Winter Wheat Region.The effect of Vrn-B1 on the vernalization development characteristics was stronger than that of Vrn-D1.

In order to find out the regularities of formation of panicle grain characteristics of multi-panicle type and large-panicle type varieties to increase rice yield.The conventional field experiments method were conducted to use the multi-panicle type varieties Yueyou 9113(Y9113),the large-panicle type varieties Tianyouhuazhan(TYHZ)and Wufengyou T025(WT025)as test materials.To study the differences in carbon and nitrogen metabolism and yield composition of main stem,functional leaves and young panicles during the young panicle differentiation stages of multi-panicle type and large-panicle type varieties.The results showed that the activities of key enzymes in carbon and nitrogen metabolism(SPS,AMS,NR and GS)and soluble sugar and soluble protein content of the main stem,functional leaves and young panicles of large-panicle type varieties were higher or significantly higher than multi-panicle type varieties in the main young panicle differentiation stages.In the second branch and spikelet primordia differentiation stage,which the number of grains increased rapidly,the soluble protein content of functional leaves and soluble sugar content and GS activity of young panicles of Wufengyou T025 were significantly higher than Yueyou 9113 by 6.77%,35.07% and 20.10%,and the SPS activity of main stem and GS activity of young panicle of Tianyouhuazhan were significantly higher than Yueyou 9113 by 46.72% and 7.81%.In the meiotic stage of the pollen mother cell,which the spikelets are prone to degeneration and the number of grains decreases,the soluble protein content of functional leaves and soluble sugar content and soluble protein content of main stem of Wufengyou T025 were significantly higher than Yueyou 9113 by 9.88%,21.20% and 16.20%,and the soluble sugar content of main stem and SPS activity of functional leaves of Tianyouhuazhan were significantly higher than Yueyou 9113 by 14.67% and 28.55%.Under the conditions of this experiment,compared with the multi-panicle type varieties,the stronger carbon and nitrogen metabolism during the young panicle differentiation stage was one of the mechanisms of the large-panicle formation of the large-panicle type varieties.

To screen out new candidate rapeseed varieties suitable for production in South China region,field trail data of fourteen early-mature rapeseed new varieties in China's regional trial was first analyzed by AMMI models,and then the relationship between G×E interaction and climate factors in eight experimental sites were investigated by GGE biplot method. Then biomass and photosynthetic parameters during wintering were also investigated in a representative variety. This analysis allowed a deeper understanding of the influence of climate factors and physiology on yield formation,by which new varieties with better adaptation can be identified and used in agricultural production. The results showed that the mean yield of 14 varieties varied markedly,with S0013 ranking the highest(2 191.021 kg/ha)and 282081 the lowest (1 328.512 kg/ha, only 60% of the previous one). Combined ANOVA indicated that the majority of variation was from the environment effect(82.27%)and the least from the genotype effect(4.93%). Moreover,variation from genotype×environment interaction(G×E)was also very significant,although it only accounted for 10.17% of total variation,most of which(92.63%)could be further dissected by AMMI model. It seemed that yielding ability was mainly affected by climate factor such as rainfall,while yield stability was largely determined by temperature. For yield stability,the fourteen varieties were ranked,from high to low,as Qianza ZW9001>Qianza J9002>C868>05V11>Qianza J9001>WB203>Chuanza 09NH014>S0013>Yunyouza No.2>Za 1613>08SH60>282081>Ronghua 906>131(CK). Among these,S0013,C868 and WB203 had the highest yield,while 282081 the lowest. The discriminating power for experimental sites were listed,in descending order as Yuxi>Baoshan>Ji'an>Nanchang(Yichun)>Guilin>Changsha>Anshun. Moreover,the biomass of YG131 during wintering was the highest in Guilin. Meanwhile,photosynthetic parameters such as Pn,Tr and Ci were also high and positively correlated with plot yield,suggesting that active photosynthesis promote yield formation. Taken together,the comprehensive evaluation of field performance for new rapeseed varieties in Chinese national field trial through AMMI model and GGE biplot can provide valuable information for the breeding of early maturing rapeseed varieties and the selection of idea experimental sites.

WRKY transcription factors act important regulators in plant response to low phosphorus.Buckwheat performs well in under-fertilized soils with higher phosphorus use efficiency.Taking tartary buckwheat as experimental materials,this study aims to explore the possible regulatory roles of WRKY genes in phosphorus starvation response of buckwheat.The entire coding sequence(CDS)of FtWRKY6 gene was cloned from RNA samples generated from roots treated by low phosphorus using reverse transcription PCR.The obtained CDS of FtWRKY6 was 1 572 bp in length,encoded a polypeptide of 524 amino acid residues which consists of two conserved WRKY domain each with a zinc finger motif of CCHH,and belonged to the WRKY group Ⅰ.FtWRKY6 shared the highest identity(55.5%)at the amino acid level with Camellia sinensis CsWRKY24.Transient expression assay in protoplasts showed that FtWRKY6 protein was localized in nucleus.Yeast one-hybrid assay revealed that FtWRKY6 had transcription-activating activity.Real-time fluorescence quantitative PCR analysis showed that the expression of FtWRKY6 in roots was significantly induced by low phosphorus and three related hormones such as indole acetic acid(IAA),gibberellin(GA)and cytokinin(CTK).Taken together,FtWRKY6 possesses basic structural and biochemical characteristics as a putative transcription factor,and may be involved in low phosphorus response in roots possibly by crosstalk of IAA,GA and CTK signaling pathways.

Clarifying the regulation effect of nitrogen on grain development under heat stress at blister stage of maize is of great significance for rational fertilization,relieving the harm of heat and realizing high and stable yields.The effects of amount of nitrogen application(90,180,270 kg/ha,marked as N90,N180,N270)on the grain development and yield under heat stress during blister stage of maize were investigated by using Xianyu 335(XY335)and Zhengdan 958(ZD958)as materials and setting heat treatment(T)and control(CK).The results showed that heat stress broke the balance of endogenous hormones in maize grains, resulting in the decrease of abscisic acid (ABA) content in the grains of N180 and N270 of two maize varieties and auxin (IAA) content in the grains of N180 and N270 of ZD958; Soluble acid invertase activity (SAI) of upper grains was decreased, grain volume expansion and dry matter accumulation were blocked, abortion rate was increased, grain number per ear was decreased, and yield was significantly decreased. The heat-sensitive variety XY335 was more affected by heat stress than the heat-resistant variety ZD958.With the increase of nitrogen application rate,the negative effect of heat stress on corn grains development was intensified.Under heat stress,the ABA/GA₃ of XY335 and ZD958 decreased,the IAA and ZR contents increased,the grains volume and dry matter were more severely reduced,the abortion rate was significantly increased by 25.55,29.31 percentage points and 15.45,24.49 percentage points,respectively,the grains number per spike was decreased by 42.89%,52.68% and 20.95%,35.25%,respectively,and yield was significantly decreased by 44.29%,52.04% and 26.41%,39.94% respectively,under medium(N180)and high(N270)nitrogen treatments compared with low nitrogen(N90)treatment.Therefore,reasonable nitrogen application rate(N90)could alleviate the adverse effects of heat stress on corn grain development and reduce yield loss.

In order to analyze the molecular mechanism of different watermelon peel firmness,and provided a theoretical basis for discovering key genes related to watermelon peel firmness,the high firmness(901)4-1-1-M and low firmness BSH with similar growth period but significant difference in peel hardness were used as experimental materials.The peel with the maximum difference 30 days after pollination was selected for transcriptome sequencing analysis and the Illumina HiSeq^TM sequencing platform was used to analyze the molecular mechanism of different watermelon peel firmness,Real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the sequencing results.A total of 1 085 differentially expressed genes (DEGs) were screened by transcriptome sequencing,including 555 up-regulated genes and 530 down-regulated genes.Gene Ontology(GO)analysis showed that 1 085 DEGs were significantly enriched in cell components,molecular functions and biological processes,including cell wall,cell periphery,external encapsulation structure,extracellular region,tetrapyrrole skeleton,redox enzyme activity,transferase activity and pectin esterase activity.Kyoto Encyclopedia of Genes and Genomes (KEGG)analysis indicated that 19 DEGs,including Cla97C04G075800,Cla97C02G044950,Cla97C09G165820,Cla97C10G195660,Cla97C01G025380,etc.,were enriched in the most significant enrichment biosynthesis of phenylpropanoid,which eventually lead to the metabolites of Syringyl lignin,5-Hydroxy-guaiacy lifnin,Guaiacy lifnin and P-Hydroxy-phenyl lifnin.The correlation coefficient of DEGs expression levels by qRT-PCR and RNA-seq data was 0.791,which indicated that the transcriptome test data were reliable.This study explained the reason of watermelon peel firmness difference between(901)4-1-1-M and BSH from the transcriptional level.

Carcass muscle growth and development is an important evaluation index that affects the breeding efficiency of goats.The aim of this study was to analyze key candidate genes for muscle growth and development of Guizhou white goats with different genders by RNA-Seq,and provide new reference information for the research on the muscle growth and development of Guizhou white goat.We collected the longissimus dorsi muscle to extract RNA and determined the slaughter performance of 6 Guizhou white goats with different genders,which were two years old and were feeded in same level.Then screened differentially expressed genes,analyzed the signal pathway of related genes.Meanwhile,RT-qPCR was used to verify the screened differentially expressed genes.The results showed that the raw reads which obtained by sequencing were filtered,a total of 78.99 Gb Clean Data were obtained in six samples.Each single sample was obtained Clean reads between 83 030 104 and 95 739 024,and the comparison efficiency with the reference genome was between 93.75%-94.79%,a total of 25 089 transcripts were obtained,of which 1 077 were significant differentially expressed genes,and 194 were new transcripts.Among them,563 were up-regulated and 514 were down-regulated in longissimus doris tissue of male sheep.GO functional enrichment analysis was performed on 1 077 differentially expressed mRNAs,which 587 were significantly enriched(Q-value≥0.05) and concentrated in 35 groups of three major categories.KEGG signaling pathway analysis revealed that annotated differential genes participated in 243 signaling pathways,among which the PI3K/AKT signaling pathway were the most enriched,including 32 genes,among them,17 genes were significantly up-regulated and 1 gene was down-regulated,meanwhile,the coexpression score of COL4A1 and COL4A2 genes were the highest in this pathway,the ITGAV protein interaction with other proteins was the most abundant.6 genes that may be closely related to muscle growth and development in white goat were screened out,among which FHL3,WFIKKN2 and SOX6 genes were up-regulated in male longissimus doris tissue,QSOX2,MYH2 and LAP genes were down-regulated.RT-qPCR showed that the expression trend of the 6 candidate genes were consistent with high-throughput sequencing results,and the expression levels differences of these genes were significant,which showed the sequencing results were reliable.Totally,1 077 differentially expressed mRNAs,which 194 were new transcripts were obtained of longissimus doris tissue in Guizhou white goat with different genders by RNA-Seq technology,the screened differentially expressed genes and PI3K/AKT signaling pathway were related to the growth and development of longissimus dorsi muscle of Guizhou white goats.

RPP13 is a typical R gene that can induce plant immune response by recognizing of the effectors.The transcriptome sequencing analysis of five potato cultivars infected with Potato spindle tuber viroid (PSTVd)showed that RPP13 genes were up-regulated in the five potato cultivars infected with PSTVd.In order to study whether this gene was related to PSTVd resistance in potato,DNAMAN 8.0 software was used to perform sequence alignment analysis on RPP13 upregulated gene sequences obtained by transcriptome sequencing.The common conserved region was selected as the silencing object,and a stem-loop structure inverted repeat with the conserved region as the arm and the intron from somatic embryogenesis receptor-like kinase 1 (SERK1)(Accession number EF175216)as the loop was inserted in plant expression vector pROKⅡ to construct RNAi vector named pROKⅡ-RPP13.The pROKⅡ-RPP13 vector was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method,and the potato cultivar Minfenghong was transformed by Agrobacterium tumefaciens mediated genetic transformation method. Healthy potato leaves of 28 days of seedling age were used as materials. After 2 days of pre-culture, 10 minutes of infection, 2 days of co-culture under dark conditions, roots and buds were induced by the resistance screening medium. The regenerated plants were screened by PCR, and three positive transgenic plants were obtained.Using efla gene of potato as the reference gene,Real-time RT-PCR was used to detect the expression of RPP13 gene in potato transformed plants.The results showed that the expression level of RPP13 gene in the three transgenic plants obtained was significantly different from that in the control without transgenic.In the obtained RNAi transgenic potato plants,the expression level of RPP13 gene was decreased by 35%—60%.

In order to preliminarily identify the function of HvMUTE, a transcription factor gene for stomatal development in barley, and by analyzing its biological characteristics and expression patterns, HvMUTE, a transcription factor gene for stomatal development in barley, was studied by bioinformatics analysis, PCR amplification and qRT-PCR. Because barley G1614 is the sister line of barley MOREX, and the reference genome data of barley MOREX has been published. So HvMUTE gene sequence was obtained by homology Blast of BdMUTE against between the referencre genome of barely G1614. The coding region of the HvMUTE gene with 651 bp in length was isolated from the first leaf emerging from the bud of barley G1614. Bioinformatics analysis indicated that HvMUTE protein was an unstable protein with no obvious hydrophilicity and hydrophobicity, located in the nucleus, and hold a Helix-Loop-Helix (HLH) structure in three demision level. Phylogenetic tree analysis showed that HvMUTE was closely related to MUTEs of wheat and Aegilops. qRT-PCR results showed that there was no significant change in the expression of HvMUTE gene under drought conditions. This result was different from previous studies, and it was speculated that barley was a monocotyledonous plant, and the existence of parathyroid cells in the stomatal structure was different from that of dicotyledonous plants.

The objective of the study was to screen the candidate genes and SNP markers for the total number born(TNB)and number born alive(NBA)in pigs,so as to prepare for the next step of improving the reproduction traits,and breeding new high-proliferation of Canadian line of large white pigs. Illumina porcine 50K chip was used to do a genome-wide SNP scan on 186 Canadian line of large white sows in one pig farm,followed by quality control,beagle fill and SNP genotyping. Combined with the phenotypic traits and population structure analysis results of these experimental pigs,genome-wide association study(GWAS)was carried out. The results of population structure analysis showed that there was no population stratification in the pig samples used in the experiment. A total of 36 867 effective SNP markers were obtained on 18 pairs(36)of autosomes for GWAS analysis of experimental pigs. The results of GWAS showed that NBA in all parities of these Canadian large white pigs was significantly associated with two SNPs:seq-rs323899658 and seq-rs329781338,respectively. The SNP of seq-rs329781338 was annotated with three genes:SSBP1,WEE2 and KIAA1147,respectively. NBA was significantly associated with 7 SNPs,namely:seq-rs81238474,seq-rs321377412,seq-rs340736313,seq-rs80782154,seq-rs81244816,seq-rs81467772 and seq-rs329781338. Five out of seven SNPs had gene annotation,and a total of 22 genes were annotated:EphA1, TAS2R60, FAM131B, CLCN1, CASP2, TMEM139, TRBV21OR9-2, PRSS2, TRBV19, TRBV24-1, U6, SSBP1, WEE2, KIAA1147, NKX2-8, ALKBH3, HSD17B12, U6, HOMER2, WHAMM, FSD2 and SCARNA15. The results of GO function and KEGG pathway analysis showed that the most significant biological process of GO function of these genes was the regulation of multi biological process,the molecular function was non transmembrane/transmembrane protein tyrosine kinase activity,and the KEGG pathway was mismatch repair.Combined with GWAS and gene function annotation results,WEE2 and EphA1 genes could be used as key candidate genes to increase the TNB and NBA of Canadian large white pigs,and provide some reference for the precise cultivation of high-yield pigs in the future.

Polygalacturonase-inhibiting proteins(PGIPs)play an important role during response to pathogen infection.A PGIP gene and its promoter were isolated from Lilium regale Wilson,which has strong resistance to Fusarium oxysporum.The open reading frame of LrPGIP was 1 008 bp in length,which encoded a protein with a length of 335 amino acid residues and contained 7 leucine-rich repeat domains.LrPGIP had high homology with PGIP of Elaeis guineensis,Cocos nucifera and Nicotiana sylvestris,which was 91%,94% and 86%,respectively.The LrPGIP showed closer relation with Phoenix dactylifera,Ensete ventricosum,E.guineensis and C.nucifera.LrPGIP was expressed in roots,stems,leaves,flowers and scales.It had the highest expression level in roots and the lowest expression level in scales.The expression level of LrPGIP was induced by F.oxysporum,and the highest expression level was at 72 h after F.oxysporum infection.The plant signal molecules including salicylic acid(SA),jasmonic acid(JA),ethephon(ETH)and hydrogen peroxide induced the expression of LrPGIP.Among them,the expression level of LrPGIP induced by ETH was the highest,which was followed by JA.The length of LrPGIP promoter fragment was 706 bp and had some cis-acting elements,such as hormone and stress responding elements.The expression cassette of the β-glucuronidase(GUS)driven by the LrPGIP promoter was transferred into tobacco for expression,and the GUS activity was obviously induced by the infection of F.oxysporum and Alternaria alternata as well as the stress of HgCl₂ and NaCl.It also responded to defense-related plant hormones including JA,SA and ETH.The up-regulation of GUS activity by HgCl₂ treatment was the greatest,followed by A.alternata and ETH.These data showed that the LrPGIP promoter activity was induced by plant hormones,biotic and abiotic stresses.The above results indicated that LrPGIP might be an important disease resistance gene in L.regale against F.oxysporum.

In order to clarify the mechanism of pyroligneous acids improving replantation soil,the effect of pyroligneous acids treatment on plant growth was studied by field experiment using apple rootstock-Malus micromalus as experimental material.The main physical and chemical properties and enzyme activities of soil in July and October were analyzed based on Illumina high-throughput sequencing to analyze the changes of microbial diversity in rhizosphere soil.The results showed that compared with the control,the annual increase of plant height,ground diameter and leaf area of Malus angustifolia seedlings were significantly increased after 100-fold pyroligneous acids irrigation.Pyroligneous acids root irrigation treatment significantly increased the contents of main nutrients and enzyme activities in the soil of replanting disease.The contents of soil organic matter,total nitrogen,alkali hydrolyzable nitrogen,available phosphorus and available potassium were 1.31,1.38,1.20,1.60 and 1.65 times of CK1,respectively in July,and 1.12,1.03,1.58,1.40 and 1.25 times of CK2 in October,respectively.Sucrase and urease activities increased significantly in July and October.Pyroligneous acids increased the microbial diversity of rhizosphere soil in summer and autumn.Under CK and pyroligneous acids treatments in July and October,the top five dominant gates of rhizosphere bacteria were Proteobacteria,Acidsobacteria,Gemmatimonadetes,Chloroflexi and Rokubacteria;at the genus level,they were mainly uncultured_bacterium_c_Subgroup_6, uncultured_bacterium_f_Gemmatimonadaceae, uncultured_bacterium_o_Rokubacteriales, RB41 and MND1. The top five dominant gates of rhizosphere fungi were Ascomycota,Basidiomycota, Mortierellomycota,Chytridiomycota and Glomeromycota.At the genus level,they were mainly Cladosporium,Mortierella,Ilyonectria,Guehomyces and Fusarium.According to the correlation network map,the beneficial bacteria RB41 and uncultured_bacterium_f_Gemmatimonadaceae in the rhizosphere and the pathogenic fungi Fusarium and Ilyonectria were negatively correlated with other populations.Pyroligneous acids root irrigation can increase soil nutrients and enzyme activity,increase soil microbial diversity,improve soil microbial community structure,promote plant growth,enhance plant resistance and reduce the harm of replanting disease.

In order to obtain COVID-19 nucleocapsid protein with similar function and activity to natural protein and apply it to practical detection. Firstly,according to Bac-to-bac insect expression system and synthetic COVID-19 nucleocapsid protein(N protein)sequence,BamH Ⅰ and Xba Ⅰ on pFastBac^TMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology,and T-Vector pMD19(simple)vector and pFastBac^TMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBac^TMHTB-COV19-N,and finally construct recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N in DH10Bac cells was expressed in insect cell Sf9. The recombinant protein was obtained and analyzed by SDS-PAGE and WB. The recombinant plasmid pMD19-T(simple)-COV19-N was identified by PCR and double enzyme digestion. The recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N was constructed in DH10Bac cells was identified by PCR and the expected two bands were 2 430,3 690 bp,respectively,which proved that the recombinant bacmid was successfully obtained. The recombinant bacmid was transfected into Sf9 insect cells. At the same time,the recombinant GFP protein control group was established. After 120 h of transfection,the recombinant N protein and recombinant GFP protein were collected and samples were prepared;SDS-PAGE and WB analysis were carried out respectively. HRP-His labeled antibody was used to verify that the transfection was successful,and both recombinant N protein and recombinant GFP protein were successfully expressed in Sf9 cells. The experimental results were consistent with the expectation,and the size of recombinant N protein band was about 46 ku. The eukaryotic expression vector of respiratory coronavirus N gene was successfully constructed and successfully expressed in insect cells,which provides an experimental basis for the establishment of ELISA detection methods and other related research.

Increasing the planting density is still the main way to improve the yield of maize,but the group light will be affected by increasing planting density,which leads to premature leaf senescence. Therefore,it is of great significance to study the effect of the mixture of diethyl aminoethyl hexanoate(DA-6)and chloroquine(CCC)on the photosynthesis intensity and time of maize leaves in the process of improving maize yield by increasing planting density. In 2018 and 2019,a maize variety of Zhengdan 958 was used as experiment material in field. Two planting densities were set(6.75×10⁴,9.00×10⁴ plants/ha)and the mixtures with two concentrations(0 mg/L DA-6+0 g/L CCC and 15 mg/LDA-6+2 g/L CCC)were sprayed on the whole plant at 7-leaf stage. The leaf area index,specific leaf weight,leaf photosynthetic performance,antioxidant capacity and yield differences of maize population of different planting densities were studied under the control of compounding agents,in order to provide a theoretical basis for the application of chemical regulators in maize dense planting. Results showed that when the planting density increased from 6.75×10⁴ plants/ha to 9.00×10⁴ plants/ha,the leaf area index of maize was increased,and the specific leaf weight was also increased at the late silking stage. The leaf area index of the low-density group sprayed with the compounding agent was decreased,while the high-density group had no significant change. The specific leaf weight in the plants sprayed with the compounding agent enhanced,but the difference was not significant compared to the control. After dense planting,the SPAD value and photosynthesis intensity of ear leaves significantly decreased,and the chlorophyll fluorescence parameters negatively affected. In the dense planting group treated with the compounding agent,the SPAD value,the net photosynthetic rate,transpiration rate,stomatal conductance,intercellular carbon dioxide concentration of ear leaves were significantly increased overall as well as the maximum fluorescence,variable fluorescence and Fv/Fm,and initial fluorescence decreased. After dense planting,the relative senescence rate of leaves increased,the activity of antioxidant enzymes decreased,while reactive oxygen species and malondialdehyde accumulated excessively,and the content of soluble protein decreased. In the dense planting group treated with the compounding agent,the relative senescence rate of leaves decreased,the activity of superoxide dismutase,peroxidase,catalase and soluble protein content significantly increased,and the content of ROS and MDA significantly decreased on the whole. A negative effect on the agronomic traits of ears could be observed in dense planting,but the yield was not significantly affected due to the increase in density. After spraying the compounding agent,the photosynthetic capacity and time of maize ears promoted,resulting in improvement of ear traits and significant increase of yield. Compared to the high-density group non-treated by compounding agent,the yields of the group treated by compounding agent increased by 14.61% and 6.64% in 2018 and 2019,respectively. To sum up,the compounding agent increased the material accumulation by improving the photosynthetic capacity and time of maize group,thereby increasing the maize yield.

To provide a theoretical basis for the high-quality production and precise fertilization of Merlot grapes,through the determination of the mineral element content of each growth and different tissue parts,and the nutritional diagnosis and analysis based on the quality index formed by the phenolic substances in the fruit.The comprehensive quality index of fruit phenolics,namely CI value,was calculated by Topsis analysis method.By comparing the correlation between content of various mineral elements in different growth stages/ tissues and CI value,the nutritional diagnostic factors are determined.In order to provide a theoretical basis for the high-quality production and precise fertilization of Merlot grapes,high-CI vineyards were classified by CND method.The results showed that there was a significant synergistic and antagonistic relationship among the mineral elements in the leaves,petioles and fruits of the Merlot grapes.VS_F_N,MS_F_P,VS_F_K,VS_L_Ca,FBS_F_Mg,VS_F_Fe,FBS_L_Mn,MS_F_Zn,FBS_P_Cu,VS_P_B,FBS_L_Mo were selected as plant nutrition diagnostic factors.The quality index inflection point value of high-CI vineyards was 0.735 5,of which 7 orchards met this condition,accounting for 14.58% of the overall sample.According to the mineral element content range of Merlot plant within high-CI vineyards,the appropriate values of nutritional diagnostic factors were:N(8.85—11.81)mg/g,P(1.98—4.26)mg/g,K(14.97—20.70)mg/g,Ca(35.57—68.83)mg/g,Mg(3.69—15.51)mg/g,Fe(70.96—103.26)mg/kg,Mn(166.20—277.67)mg/kg,Zn(10.71—20.27)mg/kg,Cu(9.54—14.90)mg/kg,B(11.44—17.07)mg/kg,Mo(0.69—1.60)mg/kg.The nutritional diagnosis of high-CI vineyards showed that the K,Ca,Mn,and Mg in Merlot plants were on the low side.The obtain the highest fruit quality index as the goal,the recommended fertilizer rates per hectare were N 62.25 kg,P₂O₅ 46.50 kg,K₂O 0.00 kg,CaO 56.25 kg,and MgO 46.50 kg.Ca and Mg fertilizers should be applied in small amounts and multiple times;we should strengthen the management of water and fertilizer in the orchard to increase the absorption of K element by the tree.Through foliar spraying,the lack of Mn elements can be appropriately supplemented.

To investigate the physiological and biochemical responses of maize to gray spot stress.Using the natural inoculation method in the field,two inbred lines K365(resistant)and K169(susceptible)and two hybrids Zhenghong 431(resistant)and Zhenghong 532(susceptible)were used as test materials.To study the effects of gray spot disease on leaf photosynthesis,reactive oxygen species content,antioxidant enzyme activity and endogenous hormone content of different resistant maize inbred lines and hybrids.Gray leaf spot stress led to the decrease of net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci) and transpiration rate (Tr) of maize leaves, except for Ci, the three photosynthetic indexes of resistant materials did not decrease significantly, specifically, Pn, Gs and Tr of K365 decreased by 24.41%,25.00% and 4.20%, respectively. The Pn, Gs and Tr of positive Zhenghong 431 decreased by 11.53%, 21.43% and 0.06%, respectively, while the three photosynthetic indexes of susceptible material decreased significantly, specifically, Pn, Gs and Tr of K169 decreased 59.32%, 95.28% and 80.18%, respectively. The Pn, Gs and Tr of Zhenghong 532 decreased by 85.39%, 69.60% and 56.77%, respectively;at the same time, the content of hydrogen peroxide (H₂O₂), peroxidase (POD) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, salicylic acid (SA) content, and jasmonic acid (JA) in maize leaves were increased under leaf gray spot stress, and the resistant materials could stimulate higher POD,SOD and CAT activities,maintaining a relatively stable H₂O₂ content,more reasonable regulation of SA and JA content,could maintain the balance of endogenous hormones.Resistant material may through rational accumulation of SA,JA signaling molecules to regulate antioxidant system,late in the disease still maintain a high level of defense enzymes in the body to more effectively remove excess H₂O₂,at the same time,adjust the blade endogenous hormone balance to make it adapt to the effects of resistant,thus to enhance maize disease resistance.

In order to deeply understand the genetic variation of yield related traits of flax germplasm,229 flax germplasm resources were used as materials,SSR markers that were significantly associated with yield related traits in flax were explored by association analysis of generalized linear model(GLM)and mixed linear model(MLM). The results showed that the indexes of plant height((58.40±5.67)cm),fruit number per plant(17.50±3.50)and grain weight per plant((0.55±0.15)g)of flax germplasm planted in Hohhot were the largest under four environments. In Jining area,the indexes of branch number(5.00±1.25),fruit grain number(6.52±2.67)and 1000-seed weight((5.58±1.48)g)were the largest. The phenotypic variation coefficients of the seven yield related traits were 11.18%—18.29%. The order of generalized heritability was 1000-seed weight > seeds per capsule>seeds weight per plant>plant height>capsule number per plant>branch number>stem length. When the population structure K=4,229 flax germplasm were divided into 4 groups. A total of 365 bands were amplified by 30 pairs of SSR primers,and 36 SSR markers were detected by GLM correlation analysis with 7 yield related traits of flax. The interpretation rate of phenotypic variation was 1.15%(Lu146)—7.75%(Lub747);MLM association analysis detected 23 SSR markers,and the interpretation rate of phenotypic variation was 2.26%(Lu203a)—7.16%(Lu291). Under GLM and MLM,Lu203a and Lua125a markers were detected on chromosome 12 and 2,respectively. It was concluded that SSR markers in flax could be well correlated with yield-related traits and had a certain explanatory rate.

In order to clarify the regulating effects of different irrigation stages and irrigation times under border irrigation on wheat yield formation and water use efficiency of winter wheat,field experiments were established in 2018-2019 and 2019-2020,and four supplemental irrigation treatments were set up:no-irrigation after emergence(W0),irrigation at jointing stage(W1),irrigation at jointing and anthesis stages(W2),irrigation at rejuvenation,jointing and anthesis stages(W3).The results showed that the spike number and grains per spike of winter wheat increased with the increase of irrigation times under the condition of consistent water management during the sowing period in the two years,and there was a significant difference between treatments. The 1 000-grain weight varied significantly in different years,and it was significantly positively correlated with the amount of pre-anthesis dry matter remobilization.In 2018-2019,the grain yield of W3 treatment reached 10 100.05 kg/ha,with no significant difference compared with W2 treatment,but they were all significantly higher than the other treatments.The grain yield of W3 treatment in 2019-2020 was 9 604.00 kg/ha,significantly higher than other treatments,while the water use efficiency and irrigation benefit were significantly lower than other treatments.There was no significant difference in water use efficiency between W1 and W2,but both were higher than other treatments.Correlation analysis showed that grain yield was significantly positively correlated with post-anthesis dry matter accumulation and contribution of post-anthesis dry matter accumulation to grain.Therefore,irrigation stage and frequency during wheat growing period should be considered in conjunction with the soil moisture during the sowing period and the precipitation in the key growing stages,and supplemental irrigation at jointing and anthesis stages could achieve a synergistic increase of both yield and water use efficiency under the condition of sufficient moisture during the sowing period.

In order to explore the role of melatonin in the interaction between wheat and leaf rust,we studied the incompatible combination between wheat variety Lovrin 10(hereinafter referred to as L10)and physiological race 260 of leaf rust. In this experiment,reactive oxygen species were induced by exogenous injection of Methyl viologen (Methy lviologen(MV)as an oxidant induces the production of superoxide anion,which can effectively increase the content of reactive oxygen species),and the best concentration of melatonin was determined by using the ability of melatonin to scavenge reactive oxygen species;then,melatonin was injected into the leaves of wheat seedlings and inoculated with leaf rust race 260,and the changes of H₂O₂ content were observed by DAB staining. The HR area was detected by Rohringer staining. The effects of exogenous melatonin injection on the antioxidant capacity of wheat were investigated by measuring the peroxidase(POD)and catalase(CAT)activities of wheat;through the above research,the role of melatonin in the interaction between wheat and leaf rust was clarified. The results showed that the reactive oxygen species was increased by exogenous injection of methyl viologen,and the optimum injection concentration of melatonin was 10 μmol/L. The results of DAB staining of incompatible combinations showed that after injection of 10 μmol/L melatonin,the accumulation of H₂O₂ induced by leaf rust infection in wheat leaves was less than that in control group,which indicated that melatonin participated in H₂O₂ scavenging. Rohringer staining showed that the area of wheat HR cells decreased after exogenous melatonin treatment,which effectively enhanced the resistance to leaf rust. In addition,exogenous injection of melatonin increased the activities of antioxidant enzymes POD and CAT,which indicated that the antioxidant capacity of wheat was significantly improved. The results showed that exogenous injection of melatonin was involved in the removal of reactive oxygen species in the process of wheat resistance to leaf rust,and improved the disease resistance of wheat.

Bipolaris sorokiniana is one of the important pathogens of maize root rot.The purpose of the present work was to establish a rapid detection method for B.sorokiniana based on the loop-mediated isothermal amplification(LAMP).Firstly,a set of primers for LAMP assay was designed according to the partial sequence of Brn1 involved in the melanin biosynthetic pathway.Then,the optimal reaction temperature was screened for this primer set,the specificity and sensitivity of LAMP reaction were detected,and the LAMP detection was evaluated by using the maize root rot samples artificially inoculated with B.sorokiniana.The results showed that the primer set designed could amplify the target gene Brn1 at 61—68 ℃,with 66 ℃ was the optimal temperature.In the specific detection,the primer set could specifically detect B.sorokiniana from the genomic DNA of 10 main pathogenic fungi isolated from maize root rot plants.In the sensitivity detection,the minimum detection limit for plasmid DNA carrying Brn1 was 10 copies/μL,and amplification could be achieved in about 25 min at the minimum concentration.And,for the detection of maize root rot samples,B.sorokiniana can be detected in 1 pg/μL of maize root tissue DNA.These results indicated that the LAMP detection method for B.sorokiniana established in this study has robust specificity and high sensitivity.

In order to reveal the molecular mechanism of MiPDCD6 protein suppressing tomato PTI immunity,the MiPDCD6 overexpression seedlings of tomato variety Xinjinfeng 1 were used as experimental materials,and the tissue culture seedlings of tomato variety Xinjinfeng 1 were used as control.Transcriptome sequencing was performed on tomato MiPDCD6 overexpressing seedlings and control seedlings,respectively.With tomato cultivars Heinz 1706,comparing the genome as a reference genome,FPKM method was used to calculate quantity of gene expression,set parameters(|log₂ FC|>1 and P<0.05)in screening the differentially expressed genes(DEGs).Gene ontology(GO)database was used to analyze the GO functional enrichment of DEGs,count the number of DEGs in each GO term,calculate the significance of gene enrichment,and find out the functional term with significant enrichment.KEGG database was used for Pathway enrichment analysis of DEGs,and hypergeometric distribution test was used to calculate the significance of enrichment of DEGs in each Pathway.The enrichment degree of KEGG was measured by FDR and gene number.Combined with gene differential expression analysis and functional enrichment analysis,the effect of MiPDCD6 protein on tomato PTI immune-related pathway genes was studied.The results showed that there were 2 366 DEGs in MiPDCD6 overexpressed tomato plants compared with wild-type tomato,including 1 354 up-regulated genes and 1 012 down-regulated genes.In these DEGs,a large number of differentially expressed genes were concentrated in KEGG pathways such as plant hormone signal transduction(sly4075),plant-pathogen interaction(sly04626),plant MAPK signal pathway(sly04016)and procycloid biosynthesis(sly00940)through GO and KEGG annotation.SA biosynthesis pathways included ICS and PAL.In the MiPDCD6 overexpressed tomato plants,PAL1 and PAL-like genes in SA synthesis pathways and TGA9,TGA10-like and PR1a2 genes in SA signal transduction pathways were significantly down-regulated,suggesting that MiPDCD6 may inhibit SA synthesis thus inhibiting plant PTI immunity.

In order to investigate the effect of 1-methylcyclopropene(1-MCP)on the physiology,storage quality and electronic nose characteristic of Starking delicious apple.Fruits were treated with 1-MCP and then stored at ambient temperature(20±1)℃.Respiration rate,ethylene production rate,fruit intrinsic quality,color difference and the electronic nose response was analyzed during storage.The results showed that the postharvest respiration rate and ethylene release rate of Starking delicious apple increased at first,then decreased after peaking at 10 d and 15 d,respectively.At the same time,fruit firmness and titratable acid(TA)content decreased,soluble solid content(SSC)increased then decreased.Fruit chromaticity varied,a^*,b^*,C^* and ΔE^* values increased.1-MCP treatment inhibited fruit respiration rate and ethylene release rate,delayed the decline of fruit firmness,SSC and TA content,inhibited increasing of a^*,b^*,C^* and ΔE^*.1-MCP treatment significantly reduced the production of sulfides and terpene compounds(W1W),nitrogen oxides(W5S),organic sulfides and aromatic compounds(W2W),methyl aromatic compounds(W1S),alcohols,aldehydes and ketones aromatic compounds(W2S).Linear discriminant analysis(LDA)combined with electronic nose could distinguish control and 1-MCP fruit at different storage periods.Loading analysis showed that W1W,W5S,W2W,W1S and W2S sensors played key roles in differentiating control and 1-MCP treatment fruit during storage.During ambient temperature storage,1-MCP treatment delayed fruit softening and kept a lower solid-acid ratio,effectively maintained fruit flavor and color of Starking delicious apple.However,1-MCP decreased the response value of sensitive sensors in electronic nose,and inhibited the production of fruit volatile substances.According to correlation analysis,electronic nose sensor response values were significantly correlated with the internal quality and color difference,which may provide a basis for the rapid and nondestructive detection of Starking delicious apple.

It conducted to analyze the biological characteristics of Porcine parvovirus type 1 (PPV1) mutant strains.The PPV was isolated and cultured from 67 aborted fetal tissue samples collected from pig farms in different regions of Shandong Province,and identified by electron microscopy and IFA test.DNAMAN V6 was used to demonstrate and compare the secondary structures of 5'UTR and 3'UTR,MEGA V7 was used for genetic evolutionary analysis,and finally the multi-step growth curve was used to compare the proliferation ability of the isolates in susceptible cells.Three strains were isolated and identified as PPV type 1 strains,named SDPV1,SDPV2 and SDPV3,with GenBank accession numbers ON924737,ON924738 and ON924739,respectively;the whole gene sequence lengths of the three isolates were basically the same,among which the 5'-UTR sequence was highly conserved and had a Y-shaped structure,while the 3' UTR had a U-shaped structure with some differences in individual bases;there were five non-synonymous mutation sites in the amino acid sequence of VP2,namely T20A,R82K,Q226E,D366N and K407N,which were found for the first time in domestic strains.The growth curve showed that the mutant strain had a relatively fast growth trend,with a 10-fold difference in the highest titer compared with the PPV-QN strain.It reported the genomic characteristics of PPV strains containing novel mutant loci.

As part of flavone compounds,proanthocyanidins (PAs)play important roles in defense against pests and diseases,and they are also a kind of nutritionally valuable component of human diet.Anthocyanidin reductases (ANRs)are involved in the biosynthesis of PAs.We cloned two CtANR genes from Carthamus tinctorius L..Bioinformatic analysis showed that the full-length CDS sequences of CtANR2 and CtANR3 were 1 020 and 1 023 bp,respectively.Both of the genomic sequences contained five exons and four introns,the length of the first exon was different while the other four exons were the same,and the lengths of introns varied greatly.CtANR2 and CtANR3 genes encoded proteins with amino acid number of 339 and 340,respectively,and their secondary structures were mainly composed of α-helix and random curl.Both of them were hydrophilic proteins,CtANR2 protein was unstable,while CtANR3 was a stable hydrophilic protein.In addition,the two proteins both had no signal peptide sequence and transmembrane structure,and might be located outside the cells.Sequence alignment and phylogenetic analysis showed that CtANR2 and CtANR1 had the highest homology and the closest genetic relationship.The three CtANR proteins had the closest evolutionary relationship with ANR of Asteraceae,and also belonged to the same large branch with ANR of Solanaceae,Malvacede and Moraceae.Expression analysis showed that the tissue expression patterns of CtANR2 and CtANR1 were similar with the highest expression level in flowers and the lowest expression level in early fruit balls,while CtANR3 was highly expressed in bracts and almost not expressed in roots and early fruit balls.They displayed different expression patterns under different hormone treatments.The expressions of CtANR2 and CtANR1 were decreased after different hormone treatments,while the expression levels of CtANR3 were increased to different degrees after hormone treatments.These results suggest that CtANR genes might play different roles in different development stages and resistance to abiotic stresses of Carthamus tinctorius L..

In order to improve the blast resistance of Shuijing 3,an excellent food-flavor rice variety,CRISPR/Cas9 gene editing technology combined with gene chip technology were used to pyramid the R gene Pigm and the non-R gene bsr-d1 into Shuijing 3.Firstly,Bsr-d1 was selected as the target gene to construct a recombinant expression vector using the CRISPR/Cas9 gene editing system,and transformed into the excellent food-flavor rice Shuijing 3 by Agrobacterium-mediated method.The homozygous bsr-d1 mutant lines without T-DNA elements,including five mutation types as T insertion,G insertion,GA deletion,CGCA deletion and CGCAGA deletion,were screened out.The japonica line Jinyu 1 containing a broad-spectrum blast resistance gene Pigm was used as the gene donor parent to cross with the homozygous bsr-d1 mutant lines without transgenic components.The Pigm gene was introduced into bsr-d1 mutant lines by cross,backcross and self-cross combing molecular breeding chip to simultaneously perform Pigm gene and background-assisted selection.The improved lines SJ3-G1,SJ3-G2,SJ3-G3,SJ3-G4,SJ3-G5,which were homozygous for the disease resistance genes(carrying both bsr-d1 and Pigm genes)and whose background recovery rates were all above 96%,were finally obtained.The improved strains of Shuijing 3 displayed enhanced leaf blast resistance compared with the wild type in inoculated identification test using Magnaporthe grisea strain GUY11.After inoculation with M.oryzae,the POD activities in the improved strains of Shuijing 3 were significantly lower than that of the wild-type control,while the H₂O₂ contents were significantly higher than that of the wild-type control.The improved Shuijing 3 lines with blast resistance carrying both bsr-d1 and Pigm genes are obtained by CRISPR/Cas9 gene editing technology combined with gene chip technology.

In order to explore the molecular mechanism of Zoysia grass in resistance to abiotic stresses,in the present study we reported the function of a cold responding gene from Zoysia matrella designated as ZmCOR410. The sequence information of the gene was obtained on the base of transcriptome and genomic data. The expression profile of ZmCOR410 in response to low temperature was detected by qRT-PCR,and the functions of the gene under abiotic stresses were estimated in Arabidopsis and yeast by genetic transformation.The results showed that the CDS of the gene was 927 bp in length,encoding an acid dehydrin that contains 308 amino acids. In the polygenetic tree of COR410 homologues from gramineous grasses,ZmCOR410 had a close relationship with the homologues of Cleistogenes songorica and Eragrostis curvula,two species that were high tolerant to drought stress. In the genomic DNA,there were four copies of core sequence of DRE-cis element in the 1 700 bp region upstream of the CDS of ZmCOR410,and its mRNA was accumulated in leaves exposed to cold. Compared to WT plants,Arabidopsis plants over expressing ZmCOR410 showed reduced injury in leaves after a freezing-temperature exposure,and showed higher survival rates under drought and high temperature stresses. Yeast cells harboring ZmCOR410 were also more tolerant to high temperature stress than the control cells. The results indicated that the product of ZmCOR410 could enhance cells resistance to freezing cold,high temperature and drought stresses,which would help Zoysia matrella going through adverse environments.

To clarify the function of the receptor gene for Gossypium hirsutum L. cytokinin receptor (GhCRE1) in the regulation of plant growth and development, and to provide material for studying the molecular mechanism of the GhCRE1 gene in regulating seed germination in Gossypium hirsutum, ultimately improving seed germination rates at the molecular level. The GhCRE1 gene was cloned and the fragment was ligated to the pCUbi1390 vector containing the Ubiquitin promoter using gene overexpression technology to construct GhCRE1 overexpression vector, while the sgRNA fragment of the Arabidopsis CRE1 gene was designed by CRISPR/Cas9 gene editing technology and the sgRNA nucleotide sequence was synthesized to construct the Arabidopsis CRE1 gene targeting knockout vector. The above two vectors were transformed into Agrobacterium and the inflorescence dip method was used to create GhCRE1 overexpression strains and gene editing loss-of-function mutants using Arabidopsis thaliana as the recipient material. Screening and identification by PCR and qPCR techniques, then, two strains of GhCRE1 Arabidopsis over-expressed and the CRE1-deficient Arabidopsis strains were obtained. After comparing the germination potential of harvested seeds with that of wild-type seeds on 1/2 MS medium, further clarify the function of GhCRE1 gene in affecting seed germination of Gossypium hirsutum. The results showed that the Arabidopsis GhCRE1 overexpression strains and CRISPR gene editing loss-of-function mutant strain were successfully obtained, after compiling the data related to seed germination potential, it was found that on the 2nd day, the germination potential of the two overexpression transgenic strains increased by about 15% and 11%, respectively, compared with the wild type, while the Arabidopsis CRE1 loss-of-function mutant germination potential decreased by about 15%. This study revealed that GhCRE1 gene overexpression significantly increased germination speed in Arabidopsis.

It explored the ratio of organic fertilizer replacing nitrogen fertilizer in the piedmont plain of Hebei Province,in order to provide a basis for reducing the amount and increasing the efficiency of nitrogen in wheat in this area.Field experiments were carried out in Boyuan farm in Yongnian,Hebei Province for two consecutive years,and five organic and inorganic fertilizer combination treatments were set up.The results showed that organic fertilizer instead of 20% and 40% chemical fertilizer could significantly improve the number of grains per spike and yield.Compared with the high nitrogen and saving nitrogen treatment of single chemical fertilizer application,the yield increased by more than 4.0%,and the number of grains per spike increased by 3.6—5.6.Most of the grain quality indexes for organic fertilizer instead of 20% and 40% chemical fertilizer treatment,and saving nitrogen treatment were better,and the stabilization time increased by 2.2—2.7 min,the tensile area increased by 10.5—17.5 cm²,and the maximum tensile resistance increased by 28.0—75.5 EU.Various nitrogen efficiency indicators of treatment for organic fertilizer instead of 20% were higher.The nitrogen fertilizer efficiency,nitrogen utilization efficiency,and nitrogen harvest index increased 109.3%,9.3% and 11.3% respectively compared with high nitrogen treatment,and 6.9%,8.5% and 8.3% respectively compared with the saving nitrogen treatment.When organic fertilizer replaced chemical fertilizer in different proportions,nitrate nitrogen in 0—20 cm soil appeared "surface accumulation",and the content of nitrate nitrogen increased,which was more than 38.5% higher than that of the saving nitrogen treatment.The nitrate nitrogen in 20—40 cm soil was significantly higher for the saving nitrogen treatment and the high nitrogen application treatment.Organic fertilizer instead of 20% nitrogen fertilizer treatment had the best yield and grain quality,significantly improve the nitrate nitrogen content in 0—40 cm soil,improve the nitrogen absorption and utilization of wheat,and finally obtain higher environmental benefits.

This study aims to screen the differentially expressed genes in perirenal fat of Simmental cattle and Angus cattle,and analyze the differences in serum indexes between the two breeds,in order to determine the lipid metabolism and endocrine function of perirenal fat,so as to lay a foundation for cattle breed selection.Three Simmental cattle and three Angus cattle of the same age were selected,and fasting blood was collected before feeding to separate and for determination of serum biochemical indexes.After slaughter,perirenal fat of the same size was sampled,and total RNA was extracted for transcriptome sequencing analysis.The differentially expressed genes(DEGs)were identified using the DEGseq method,and GO and KEGG analysis were performed.The sequencing results were verified using Real-time fluorescence quantitative PCR.The results showed that:the serum contents of albumin(ALB2),urea nitrogen(UREAL)and glucose(GLUC3)in Angus cattle were significantly lower than that of Simmental cattle(P<0.05),and the contents of triglyceride(TRIGL)were significantly higher than that of Simmental cattle(P<0.05).Transcriptome results showed that 1 743 genes were differentially expressed between the perirenal fats of two breeds.Among that,1 361 and 382 genes were up regulated and down regulated,respectively.GO analysis showed that DEGs were significantly enriched to 52 biological functions,which were mainly related to cellular processes and cells.KEGG analysis showed that DEGs were significantly enriched mainly in fat digestion and absorption,cAMP,PPAR and Rap1 signaling pathways.Five genes(SCD5,APOA4,PTX3,OLR1 and PRKCZ)related to lipid metabolism were identified,and the sequencing results of these five genes were verified by Real-time fluorescence quantitative PCR,these results indicated that the sequencing results were reliable and the obtained differential genes could be used as basic data for breeding beef cattle breeds.

In order to improve blast resistance of the maintainer line Ruanhua B,to carry rice blast resistance genes Pi46 and Pi2 high-quality Indica H281 as the donor parent,Ruanhua B as recurrent parent,using marker-assisted selection(MAS)technology combined with pedigree breeding method,polymerization of two foreign genes with improved maintenance line Ruanhua B resistance,Ruanhua B was carried out on the characteristics of stable strain identification of resistance to rice blast,rice quality analysis,etc.Two BC₁F₆ populations,two BC₂F₅ populations and two BC₃F₄ populations with two homozygous target genes were obtained by backcrossing,multi-generation self-crossing and molecular marker detection.Field naturally induced identification showed that the improved lines of different backcrossing generations were resistant to rice blast.The sterility of backcross generation to sterile lines ranged from 52.7% to 100.0%.Agronomic traits and rice quality analysis showed that the improved lines basically conserved the main agronomic characters and rice quality characteristics of Ruanhua B.The results of SNP gene chip analysis showed that the background response rate of BC₁F₆ was 74.42%—77.77%,that of BC₂F₅ was 86.42%—87.75%,and that of BC₃F₄ was 92.27%—92.59%.Multiple resistance genes can be effectively polymerized by continuous backcross,self-cross and marker-assisted selection techniques to obtain a new maintainer line resistant to rice blast,and achieve rapid molecular improvement of maintainer line Ruanhua B.

In order to investigate the optimal staining conditions for soybean seed viability determination and the changes of important physiological indexes during seed germination stage,single factor and orthogonal tests were used to design and screen the optimal combination of Triphenyltetrazolium chloride(TTC)for soybean seed viability determination.The changes of germination rate and endogenous cell wall hydrolase of soybean seeds under different germination conditions were also analyzed.Univariate analysis showed that the presence or absence of imbibition treatment was critical for seed staining,and low concentration(0.1%)TTC solution significantly reduced the seed staining rate.The results of multi-factor orthogonal test showed that the optimal dyeing conditions of soybean seeds were as follows:imbibition time 6 h,TTC concentration 1%,dyeing temperature 35 ℃,dyeing time 60 min.The dyeing rate of seeds in this combination was 98.5%.Germination rate test showed that light and dark conditions had no significant effect on germination process.Seed germination was inhibited under more water and promoted under less water.In a certain temperature range,low temperature delayed the germination process of seeds,but increased the final germination percentage of seeds.The activities of cellulase,polygalacturonase and β-galactosidase did not change significantly when seeds germinated under light and dark conditions,while the activities of xyloglucan endoglycosylase under light were significantly higher than those under dark conditions.The activities of cellulase and polygalacturonase did not change significantly when seed were imbibed in various water content.The activity of β-galactosidase increased gradually with the increase of water,while the activity of xyloglucan endoglycosylase showed an opposite trend.It is noteworthy that the activities of four cell wall hydrolases in soybean embryos were significantly elevated with the increase of germination temperature.In conclusion,TTC staining results have important practical significance for the rapid determination of soybean seed viability and the improvement of soybean seed quality testing technical regulations.Exploring the activity of cell wall hydrolase in soybean seeds under different germination conditions will help to reveal the mechanism of seed germination in dicotyledons.

In order to explore the important role of SBP1(S-RNase-binding protein 1)gene in plant self-incompatibility,a diploid wild potato was used as the material to obtain the full-length cDNA sequence of potato SpSBP1 (GenBank: MZ803088)by using RT-PCR cloning technique,and performed bioinformatics analysis and CRISPR/Cas9 vector construction for SpSBP1 gene.The results showed that the full length cDNA of SpSBP1 gene was 1 176 bp,containing 92 bp 5' non-coding region and 163 bp 3' non-coding region.The maximum open reading frame(ORF)of SpSBP1 gene was 921 bp,which encoding 306 amino acids.Protein domain analysis showed that SpSPB1 protein included Smc superfamily domain,RING finger domain and Zinc finger domain.The homologous sequence alignment and phylogenetic tree analysis showed that SpSBP1 had the highest homology with S.chacoense,followed by Nicotiana alata.Bioinformatics analysis showed that the molecular weight of SpSBP1 was 34.731 44 ku,with a theoretical isoelectric point of 5.10,and it was speculated that SpSBP1 was an acid unstable hydrophilic protein. The secondary structure predicted that the protein was mainly composed of α helix and random curling. And the protein was a non-secretory protein and had no transmembrane structure. SpSBP1 gene was cloned from wild diploid potato,and CRISPR/Cas9 vector was successfully constructed,and also the genetic transformation was carried out.

Discuss the difference and change rule of texture characteristics of Huahong and Huayue apples after different shelf temperature combined with 1-MCP treatment during the shelf life,in order to determine the appropriate shelf temperature and time for apples.Using Huahong and Huayue as the test materials,the physical property analyzer texture multi-faceted analysis(TPA)and puncture test were used to determine the pulp elasticity,gummy stickiness, chewability, pulp hardness, rupture work, rupture force, rupture displacement, yield work, yield force and yield displacement.Factor analysis was applied to these indicators,and the original indicators were reduced in dimensionality,so that a comprehensive index was used to evaluate 1-MCP(1.0 μL/L)treatment,it was placed at shelf temperatures of 5,10,15,20 ℃ to maintain the texture properties of Huahong and Huayue apples.The shelf life of Huahong and Huayue fruit texture characters could be prolonged by 1-MCP treatment,especially for Huahong fruit.Under the same treatment temperature,the fruit decline of the two varieties treated with 1-MCP was significantly less than that of the untreated group,which could be reduced by about 10%.Correlation analysis showed that there was a very significant positive correlation between the texture indexes of apple fruit,but there were some differences in the degree of tightness.After factor analysis and based on the principle that the eigenvalue was greater than 1,two main factors were extracted,and the cumulative variance contribution rate was 91.194%.According to the representative indexes of each main factor,they were named puncture factor F₁ and TPA factor F₂,and the variance contribution rates were 79.870% and 11.324%,respectively.According to the score of the main factor,the most suitable shelf temperature of untreated Huahong fruit was 5 ℃.The temperature of the treated fruit was 15 ℃.The optimum shelf temperature of Huayue fruit without treatment was 10 ℃,and the suitable shelf temperature of 1-MCP treated fruit was 5,10 ℃.The CK group of Huahong fruit could be stored for 8 d at 5 ℃,and the shelf temperature of the treatment group could be extended to 16 d at 15 ℃;the CK group and the treatment group of Huayue fruit could be stored at 10 ℃,could be stored for 16 d.

Gibberellin oxidase gene(GAox)is a key enzyme in the synthesis and regulation of gibberellin,and effect on plant height through regulating active GA level.To analyze the gene of gibberellin oxidase in castor,we identified gibberellin oxidase gene(RcGAox)from the castor bean whole genome by bioinformatics,and analyzed physicochemical properties,conserved domain,phylogeny,promoter cis-acting element.The RcGAox gene expression pattern was analyzed by tissue specific expression of online database and apical tender stem transcriptome sequencing.A total of 30 gibberellin oxidase genes were identified from the castor genome,including 7 RcGA2ox,4 RcGA3ox,and 19 RcGA20ox.The molecular weight was ranged from 26.12 to 44.31 ku,and the isoelectric point was ranged from 5.06 to 7.82.Gene structure analysis showed that the number of introns was ranged from 1 to 2.Protein conserved domain analysis showed that all the genes shared conserved Motif 1,Motif 2 and Motif 4.Phylogenetic analysis showed that RcGAox genes were clustered into five subfamilies Ⅰ,Ⅱ,Ⅲ,Ⅳand C20 GA2ox,and subfamiliesⅠ,Ⅱ,Ⅲ correspond to GA2ox,GA3ox and GA20ox respectively.Promoter cis-acting element prediction showed that light-related elements had largest number and uniform distribution in predicting region,and 18 genes had 1 to 2 gibberellin-related element.There were 7,2,and 1 RcGAox genes specifically expressed in endosperm,male flowers and leaves respectively.Transcriptome sequencing showed that 5 genes were expressed in tender stems.It was supposed that RcGA2ox7,RcGA20ox1 and RcGA20ox14 might be the main gene involved in gibberellin synthesis pathway to regulate castor plant height.These results might provide theoretical basis for further studies on the RcGAox gene function in castor bean.

In order to explore the interaction between ScPRT1 and Sporisorium scitamineum genes in sugarcane,this study used Saccharum spontaneum and Saccharum as research objects.The ScPRT1(GenBank Accession Number:MT747433)gene was cloned by RT-PCR and was analyzed by bioinformatics,quantitative Real-time PCR and subcellular localization.Bioinformatics analysis showed that the full-length cDNA of the ScPRT1 gene was 1 621 bp,including a complete open reading frame with 1 260 bp in length and encodes 419 amino acids.The ScPRT1 protein with two RING domains and one ZZ domain,was 46.56 ku in molecular weight and was an acidic and unstable hydrophilic protein with nuclear localization signal and no signal peptide.The higher structural elements of protein were mainly random coil.The results of qRT-PCR analysis showed that ScPRT1 was higher expressed in stem pith than in bud,epidermis and leaf.And ScPRT1 was significantly up-regulated under abscisic acid(ABA)stress and in the smut-resistant variety but significantly down-regulated in the smut-susceptible variety under Sporisorium scitamineum stress.The result of subcellular localization showed that ScPRT1 was localized in the nucleus.In the co-expression network between ScPRT1 and Sporisorium scitamineum genes,three genes that encoded effectors with aromatic amino acid residues at the N-terminal were found co-expressing with ScPRT1,and their proteins may interact directly.It is speculated that ScPRT1 plays an important role in sugarcane hormone signal transduction and response to smut infection.

In order to further mine the enzyme gene related with methyl-eugenol synthesis and to elucidate the secondary metabolism pathway of Asarum heterotropoides. According to the transcriptome database information of Asarum heterotropoides,AhOMT1 gene was screened and cloned,and the corresponding bioinformatics analysis and prokaryotic expression analysis were carried out. The results showed that AhOMT1 contained an open reading frame of 1 089 bp,encoding 362 amino acids,with a theoretical molecular weight of 40.23 ku and an isoelectric point of 5.34. AhOMT1 protein was a hydrophilic protein without transmembrane structure and signal peptide sequence. AhOMT1 gene had dimerization domain and methyltransferase domain. AhOMT1 was close with the O-methyltransferase catalyzing flavonoids and eugenols. The prokaryotic expression vector pET-32b-AhOMT1 was successfully constructed and the recombinant protein of about 60 ku was successfully induced. The optimal induction condition was 0.2 mmol/L IPTG and induced at 16 ℃ for 16 h. Most of the recombinant proteins existed in the form of soluble proteins. AhOMT1 protein was separated and purified with Ni²⁺ resin,and the maximum amount of protein was obtained by eluting with 200 mmol/L imidazole. AhOMT1 gene of Asarum heterotropoides was cloned for the first time,prokaryotic expression vector was constructed,and the best induction conditions of the protein were screened.

In order to study the molecular mechanism of sodium silicate enhancing risistance of potato to Rhizoctonia solani,a gene StWRKY11 with high expression level in potato transcriptome induced by sodium silicate was cloned and its bioinformatics was analysed.Total RNA was extracted from potato,amplified by RT-PCR and cloned.Through bioinformatics related software,the structure prediction and prediction analysis were carried out.The results showed that StWRKY11 gene with a open reading frame of 1 005 bp was cloned from potato Atlantic,encoding 334 amino acids.The molecular formula of the expressed protein was C₃₀₁₃H₅₀₂₃N₁₀₀₅O₁₂₆₀S₁₉₉,the molecular weight was 81.867 94 ku,the theoretical isoelectric point(pI)was 5.09,and the total number of atoms was 10 500.The expressed protein contained a typical WRKYGQK conserved domain,and the zinc finger structure was CX₅CX₂₃HXH,belonging to the second Ⅱ d subfamily.The secondary structural elements were α-helix,extended chain,β-folding and random coiling,among which the proportion of random curl was the highest,up to 61.68%.There were 29 phosphorylation sites in total,which might be located in the nucleus.There were cis-regulatory elements upstream of the promoter that might related to resistance stress response and cis-regulatory elements related to growth and development and hormone response.The gene was closely related to potato StWRKY5 gene,and the amino acid homology of the coding protein reached 95%.

Nitrate nitrogen is one of the two main form of nitrogen uptaken by plants.Nitrate transporter 2(NRT2)plays a leading role in nitrogen uptake under nitrate deficiency conditions.In order to explore characteristics and expression pattern of NRT2 gene in Chenopodium quinoa,in the present study,fifteen members of C.quinoa NRT2s were identified based on bioinformatics analysis,and their encoding protein,chromosome location,gene structure,systematic evolution,promoter elements,tissue expression specificity and expression profile under different nitrogen levels were analyzed.The results showed that 15 NRT2s of C.quinoa were distributed on 7 chromosomes,each member contained 1-9 introns and 2-10 exons,respectively.These C.quinoa NRT2 proteins were predicted to be located on plasma membrane and belong to hydrophobic proteins.The evolutionary analysis found that the NRT2 protein family of quinoa belonged to two subfamilies,and they showed closer relationship to Arabidopsis thaliana.The cis-acting element analysis exhibited that the promoter regions of NRT2 had elements related to growth and stress responses.NRT2s showed varied expression profile in different C.quinoa tissues where more CqNRT2 members were higher expressed in roots,stems and leaves than those in other organs.The expression of CqNRT2.4,CqNRT2.7 and CqNRT2.15 was up-regulated under low nitrogen conditions where the expression levels of CqNRT2.7 and CqNRT2.15 were up-regulated sharply with 0 and 25% nitrogen treatment.The study might provide a reference basis for subsequent cloning,functional and nitrogen stress analysis of CqNRT2s gene.

In order to study the effects of nano-selenium on serum antioxidant and immune indexes of rainbow trout under heat stress,600 8-month-old rainbow trout with an average body weight of 80 g were selected and randomly divided into 3 groups:the control group was fed with the basal diet,and the experimental groups were fed with 5,10 mg/kg nano-selenium diets.The rainbow trout was fed at the suitable growth water temperature(18 ℃)for 9 days,At 24 ℃ (heat stress for rainbow trout)for 48 h.10 fish were randomly selected from each group before heat stress(18 ℃,0,3,6,9 d)and after heat stress(24 ℃,4,8,12,24,48 h)and serum was collected to determine antioxidant and immune indexes.The results showed that compared with the control group,the experimental group significantly increased the contents of TP,ALB and GLB in serum(P<0.05);the activity of GSH-Px and SOD were significantly decreased,as well as CAT and MDA were significantly increased in heat stress,but the addition of nano-selenium significantly increased the activity of GSH-Px,SOD,CAT and decreased MDA activity;the contents of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in serum of the control group were gradually increased by heat stress. The contents of TNF-α and IL-1β in serum of the 5 mg/kg nano-selenium group were significantly increased at 4 h, and the contents of IL-1β were significantly decreased at 12 h compared with the control group.The content of IgM decreased at 4 h and increased at 12 h after heat stress,and nano-selenium supplementation significantly increased the activity of IgM;after heat stress,the content of C3 decreased gradually,but the addition of nano-selenium significantly increased the activity of C3.In conclusion,dietary nano-selenium supplementation can improve the antioxidant capacity and immune capacity of rainbow trout under heat stress.Compared with 10 mg/kg nano-selenium,the antioxidant effect and immune effect of 5 mg/kg nano-selenium are more stable.

In order to explore the mechanism of AcWRKY70 transcription factor in response to kiwifruit canker stress in different resistant varieties,AcWRKY70 gene was cloned from the leaf cDNA of resistant variety Xuxiang and highly susceptible variety Hongyang kiwifruit.The sequence structure characteristics of Hongyang AcWRKY70 gene,subcellular localization and evolutionary relationship of Hongyang AcWRKY70 protein were analyzed. The expression patterns of AcWRKY70 gene,including the tissue expression and different expression in resistant kiwifruit varietie and susceptible variety under Pseudomonas syringae pv. Actinidiae(Psa),salicylic acid(SA)and methyl jasmonate(MeJA)treatment were analyzed by RT-qPCR. Results showed that the full length of the Hongyang AcWRKY70 gene was 906 bp(GenBank accession number was MW881147). AcWRKY70 gene contained 885 bp of open reading frame(ORF)and encoded 294 amino acids. AcWRKY70 protein had typical WRKYGQK domain and the zinc finger structure was C2-HC. It belonged to class Ⅲ group of WRKY family and islocated in the nucleus. Phylogenetic tree analysis showed that it had the closest genetic relationship with tea plant. Both Hongyang and Xuxiang AcWRKY70 genes had the highest expression level in leaves. Under Psa treatment, the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang, the expression of Hongyang reached the maximum at 48 h.Under the co-treatment of salicylic acid and Psa(SA+Psa),Methyl jasmonate and Psa(MeJA+Psa),the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang. Under SA+Psa and MeJA+Psa treatment,the expression of Hongyang reached the maximum at 72,24 h,respectively. AcWRKY70 gene played a certain role in resistance stress of kiwifruit,and the response mechanism to pathogens in different resistance kiwifruit varieties may vary considerably.

To explore the immune function and expression pattern of C-type lectin family members in Pinctada fucata,a cDNA sequence of C-type lectin gene PfCLEC17A in P.fucata was cloned through RACE technique.The molecular characteristics of PfCLEC17A were determined by bioinformatics analysis and its mRNA expression level was detected in different tissues including gill,mantle,adductor muscle,gonad,blood,foot and hepatopancreas,as well as expression level in hepatopancreas after Vibrio alginolyticus challenged using the Real-time fluorescent quantitative PCR.The results showed that a new C-type lectin gene PfCLEC17A was successfully cloned from P.fucata,the full cDNA was 699 bp in length with an open reading frame(ORF)of 534 bp,which encoded 178 amino acids.The deduced amino acid sequence was predicated containing a CTL domain.Phylogenetic tree analysis showed that the PfCLEC17A of P.fucata and other shellfish proteins were clustered together in a branch.The Real-time fluorescent quantitative PCR results showed that PfCLEC17A was expressed in all tissues detected in P.fucata,and the highest was found in hepatopancreas,which was significantly higher than the other tissues.The high expression level in hepatopancreas,one of the most important immune tissues in mollusc,suggested that PfCLEC17A gene might be involved in the body's immune reaction.The expression level of PfCLEC17A increased significantly and reached the peak at 1 h after V.alginolyticus challenge,indicating that the gene expression was induced by microbial stimulation at early stage.Therefore,based on the above findings,it could be concluded that the PfCLEC17A gene participates in the immune activation response of P.fucata and functions in immune system.

In order to solve the problem of the reduction of the migration of young rural labor force and the aging of the population,the enthusiasm for food crop production has been greatly reduced,and it is urgent to explore rice planting methods with less labor input and matching high-yield varieties.Through field trials from 2017 to 2019,two common and easy-to-promote planting methods were compared,and 14 early rice varieties and 12 late rice varieties were screened for easy purchase.The planting methods were simulated machine transplanting and machine direct seeding.Effects of different planting methods on the growth period,yield and dry matter weight of different early and late rice varieties.The results showed that direct seeding could shorten the growth period compared with transplanting.The average growth period of early rice direct seeding was shortened by 7 d compared with transplanting,and the average growth period of late rice was shortened by 8 d.The growth period of direct seeding had a smaller fluctuation range and more stable performance than transplanting.The annual average yield was significantly higher than that of transplanting,which was 29.71% higher in 2017,12.37% higher in 2018,and 7.15% higher in 2019,and it was found that the yield and dry matter accumulation at different periods were affected by the variety,the planting method and year had a very significant effect;through the linear regression covariance test,there was a positive correlation between the yield and the dry matter accumulation,which showed that the yield increases with the increase of the dry matter accumulation,and the coefficient of determination of the linear regression of the direct seeding method(R²)were higher than the transplanting method.A comprehensive comparison showed that the direct seeding method performs better in both the early and late rice planting methods,and the stable and high-yield(high-yield performance in the field in three years)that is matched with the direct seeding method is screened.The early rice variety Zhuliangyou 829(the yield fluctuation range of 6.02—10.90 t/ha),Yuliangyou 4156(6.49—10.22 t/ha),and stable and high-yielding late rice variety Wuyou 308(10.43—12.65 t/ha),the selected early and late rice varieties had moderate growth periods and can be effectively connected achieve high-yield planting of early and late rice.

During the growth and development stages,tuber mustard is always affected by abiotic stresses.As a plant stress hormone,ABA plays an important role in regulating plant response to abiotic stress.BjuEAR1-1 was a homologue of negative regulator EAR1 in ABA signaling pathway,and its mutant ear1 exhibited an ABA sensitive phenotype.Therefore,to explore the biological function of BjuEAR1-1 will provide a direction for the regulation mechanism of resistance to adverse environment stresses,and also provide an important theoretical basis for the effective use of gene resources to cultivate new varieties of BjuEAR1-1 with resistance to adverse stresses and stable yield.The BjuEAR1-1 coding sequence was cloned by PCR method and the plant overexpression vector 35S∷PTF101-GFP-BjuEAR1-1 was constructed by enzyme digestion and ligation method.The subcellular localization of BjuEAR1-1 was analyzed by transient transformation technology of tobacco leaves.The cis-element of BjuEAR1-1 gene promoter was analyzed,and the gene expression patterns of BjuEAR1-1 under different abiotic stresses and different tissues and organs were detected by fluorescence quantitative PCR.The result of subcellular localization assay showed that BjuEAR1-1 was localized in the nucleus and cytoplasm of tobacco.BjuEAR1-1 was expressed in root,stem,leaf,flower,seed pod and swollen stem of tuber mustard,with the highest expression in root.The expression level of BjuEAR1-1 was significantly induced under low temperature and ABA stresses,and there was no significant difference under salt stress. BjuEAR1-1 was localized in the nucleus and cytoplasm and regulated the response of tuber mustard to low temperature and ABA stresses.

Grain specific promoter with high transcription activity can regulate the specific and high-level expression of target genes in plant grains.In order to explore specific promoters of maize grain,the dominant expression gene GRMZM2G006585 was screened from the published data of maize expression profile microarray.The DNA sequence about 2 000 bp upstream of its coding region was cloned and named PZm2G006585.By using the online websites New PLACE and PlantCARE to analyze its promoter elements,we found that it contained multiple grain specific related elements such as E-box and P-box.It was preliminarily considered that the upstream sequence of the cloned coding region was a grain specific promoter from maize.In order to verify its function,the expression vector of GUS gene was constructed and transformed into plants.GUS histochemical staining results of transgenic rice showed that the expression pattern of exogenous genes driven by the promoter was grain specific and embryo dominant.The results of GUS activity detection in T₃ seeds of transgenic Arabidopsis thaliana single copy line showed that the GUS activity driven by PZm2G006585 was 909.52 nmol/(min·mg).The discovery and functional verification of the grain-specific promoter PZm2G006585 can provide candidate promoter resource for specific expression of target genes in maize,rice and other monocotyledons.

In order to explore the role and expression patterns of members of the Calmodulin-like proteins gene family in castor, the family gene CML42 was extracted from castor, and its cloning and expression were analyzed. Zhebi No.3 plant was used as the material to clone and obtain the complete coding region sequence (CDS) of RcCML42, bioinformatics method was used to analyze RcCML42 gene sequence, included amino acid coding, protein molecular weight and isoelectric, transmembrane domain, amino acid sequence consistency and so on. Using qRT-PCR to analyze the changes of expression of castor RcCML42 gene in root, stem and leaf tissues at different time points.The results showed that RcCML42 CDS was 597 bp in length, encoded 198 amino acids, contained three EF-hand calcium-binding domains, the protein molecular weight was 22.27 ku, the isoelectric point was 4.55, and there was no transmembrane domain. Analysis of amino acid sequence identity showed that RcCML42 had the highest identity with Hevea brasiliensis, reaching 86.84%.The results of qRT-PCR analysis showed that the expression of RcCML42 in the roots after NaCl treatment showed a trend of first decreasing and then rising. The expression level of RcCML42 was highly expressed at 12 h in the stem and at 8 h in the leaf. And expression in stems was significantly higher than its expression in roots and leaves CML is one of the most important calcium receptors in plants. It regulates plant cells by binding with calmodulin binding protein. When plants are affected by NaCl in the environment, CML can participate in the regulation of calcium ions in the body, thus reducing the damage to plants caused by salt stress.In summary, RcCML42 plays an important role in the signal transduction of castor under salt stress.

To clarify the coding sequence and molecular characteristics of the CDS1 gene in Qinchuan cattle and its expression characteristics in different tissues.The full coding region sequence of CDS1 gene was cloned by PCR amplification technique in Qinchuan cattle,and the sequence characteristics of its full coding region were analyzed by bioinformatics.qRT-PCR was used to detect the expression levels of CDS1 gene in brain,testis,pancreas,lung,spleen,kidney,rumen,longest dorsal muscle,liver,intramuscular fat and heart tissues using GAPDH as an internal reference gene.The results showed that the coding region of CDS1 gene in Qinchuan cattle was 1 392 bp,encoding 464 amino acids,and the CDS1 gene was highly conserved among different species and had the highest homology with rumen cattle;the protein encoded by CDS1 gene was a hydrophobic unstable transmembrane protein,mainly composed of α-helix and irregular convolutions,and the subcellular localization analysis showed that CDS1 protein was mainly distributed with endoplasmic reticulum and mitochondria.Protein interaction analysis showed that it interacted with PGS1,PPAPDC1 series,CDIPT,PLD series,CRLS1 and other riboproteins related to phospholipid synthesis,suggesting that CDS1 gene was involved in regulating phospholipid anabolic process;tissue expression analysis showed that CDS1 gene was widely expressed in various tissues,and the highest expression was in testis,and the expression level in brain was also higher.The tissue expression analysis showed that CDS1 gene was widely expressed in all tissues,and its expression level was highest in testis and higher in brain,both of which were significantly higher than that of other tissues,and the expression level was lowest in heart.

In order to perform transcription kinetics,eukaryotic expression and subcellular localization of ORFV113 protein.DNAStar software was used to perform alignment analysis of ORFV113 gene.In the presence or absence of cytarabine,Hela cells were harvested and the total RNA were extracted at various time points(2,4,6,12,24 h)post ORFV infection.RT-PCR was used to amplify ORFV113 gene to determine the dynamic transcription level of ORFV113 gene.ORFV113 gene was amplified by PCR and subcloned into pEGFP-N1 vector to construct pEGFP-ORFV113 recombinant plasmid.After being correctly identified by restriction enzyme digestion and Sanger sequencing,the pEGFP-N1 plasmid and pEGFP-ORFV113 plasmid was transiently transfected into HEK293 cells with Lipofectamine 3000.The expression of ORFV113-EGFP fusion protein in HEK293 cells was detected by Western Blot.Hela cells were transiently transfected with pEGFP-N1 plasmid and pEGFP-ORFV113 recombinant plasmid.After 24 h,the nucleus was stained with Hoechst 33342,and the subcellular localization of ORFV113 protein was observed under an inverted fluorescence microscope.The results revealed that ORFV113 gene was 627 bp in length,which was highly conserved among ORFV strains.It belonged to an early gene of ORFV.The pEGFP-ORFV113 recombinant plasmid was successfully constructed.The ORFV113-EGFP fusion protein with a molecular mass between 60 and 70 ku,approximately 10—20 ku larger than the predicted molecular mass,was successfully expressed in HEK293 cells,indicating that ORFV113 protein had undergone post-translational modification.ORFV113 protein mainly localized to the cytoplasm of Hela cells.In conclusion,the transcription kineics,eukaryotic expression and subcellular localization of ORFV113 protein were successfully performed in the present study.

Under the double cropping pattern in the North China Plain,based on the conventional application level of phosphorus,a phosphorus-deprived level without phosphorus fertilizer was adopted at the same time for three previous crops,corn,soybean and peanut,and then the soil nutrient concentration after previous crop harvesting and the grain yield and nutrient accumulation of winter wheat,the subsequent crop,were examined,to provide a proposal for crops planting in the North China Plain.The results showed that,for the soil nutrients after the previous crop harvesting,the soil total phosphorus concentration was higher in the previous soybean treatment without phosphorus fertilizer,and the concentration in peanut previous treatment was similar to that under conventional fertilizers;the soil available phosphorus content was higher overall in the previous peanut treatment;the soil nitrate and ammonium nitrogen concentrations were both highest in the previous soybean treatment.For post-crop wheat,wheat thousand grains weight,yield,seed N and P accumulation and N fertilizer bias productivity were all higher in the previous peanut treatment without phosphorus fertilizer.The previous peanut treatment increased by 0.60%,6.19%,15.46%,18.11% and 6.21%,respectively,compared to the previous maize treatment,and increased by 2.18%,7.30%,17.66%,13.40% and 7.30%,respectively,compared to the previous soybean treatment.In summary,in order to ensure soil nutrient balance,peanut was selected as a suitable previous crop in summer before winter wheat as a better model for crop mix in the southern two maturity zones of the North China Plain at low phosphorus levels.

In order to reduce the labor costs and guarantee the seed purity, male sterility gene was applied to sweet corn hybrid seed production. We used male sterility 2020 (ms2020), a spontaneous male sterility mutant derived from sweet corn inbred line K78, as the experimental material to construct F₁ and corresponding F₂ populations of ms2020 and sweet corn inbred line M08. We performed phenotypic identification, genetic analysis, and gene mapping for ms2020 mutant. Phenotypic identification showed that the F₁ population was fertile, and the F₂ population was partially sterile. The sterile plants could be tasseled normally, but the anthers were not exposed, no pollen shed, anthers small, and pale yellow. Employing 1% I₂-KI staining showed that the anthers of sterile plants contained abortive pollen grains that could not stain normally. The results of genetic analysis showed that the ratio of fertile normal plants to sterile plants was 3∶1, indicating that ms2020 male sterile mutant was a recessive mutant controlled by a single gene. The target gene was initially located on the short arm of chromosome 7 by the BSA method. Subsequently, 20 pairs of SSR markers in the initial interval were used to locate the sterile gene, and gene was finely located between markers S1 and W10, with a physical distance of 11.30 kb. Two genes, Zm00001d018802 and Zm00001d018803 were included in this region. Based on the functional analysis of candidate genes, it is speculated that Zm00001d018802 (ZmMs22/ZmMSCA1) encoding gludodoxin, which has been reported as a male sterility gene in maize, may be the key candidate gene for ms2020.The abortion characteristics and genetic regularity of ms2020 male sterility mutant were identified, which provided the material for male sterility hybridization seed production of sweet corn. The key candidate gene of the mutant were located, which laid a foundation for further analysis of its molecular mechanism.

This study aims to use high-throughput sequencing technology to perform mRNA sequencing and differential analysis of longissimus dorsi muscle tissue samples from Songliao black pigs and Landrace pigs,and screen out key genes that affect pig muscle growth,meat quality and fat deposition,so as to provide pork quality research provides new reference information.The longissimus dorsi muscle tissue samples of 6 Songliao black pigs and 6 Landrace pigs were collected,their RNA was extracted,and the mRNA was sequenced by Illumina HiSeq 2500 high-throughput sequencing technology,and the obtained reads were compared,annotated and differentiated.For expression analysis,NOISeq was used to screen out differentially expressed genes and perform enrichment analysis of related biological functions.The results showed that 664 differentially expressed genes were screened from the two pig species,of which 364 genes were highly expressed in Songliao black pigs and 300 genes were highly expressed in Landrace pigs.Through the biological function analysis of differentially expressed genes,LPIN1,FADS1,FADS2,PLIN2,PPARGC1A,PRKAG2 and ACSL1 were screened to participate in the regulation of lipid metabolism and muscle development.The related pathways were fatty acid metabolism,PPAR signaling pathway,AMPK signaling pathway,insulin signaling pathway and adipocytokine signaling pathway and so on.