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.
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.
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 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.
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.
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 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 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.
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 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 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 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.
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.
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 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 further reveal the decomposition process and mechanism of corn straw in the cold area under the application of low temperature straw degrading agent,and provide a new strategy and theoretical basis for the decomposition and comprehensive utilization of straw in the cold area.Corn straw was decomposed by low temperature straw degrading agent and its physical and chemical characters and microbial diversity were analyzed.According to the change of temperature in the fermentation process,the ripening process of corn straw was divided into five stages:initial stage(A1),heating stage(A2),ripening stage(A3),stable stage(A4)and cooling stage(A5).The monitoring results of the physical and chemical properties of corn straw in different maturity stages were as follows:with the extension of maturity time,the fiber bundles on the surface of corn straw were destroyed gradually.After maturity during the cooling stage,the relative content of cellulose and hemicellulose in corn straw decreased to 33.48% and 16.57%,respectively,while the content of total nitrogen and ammonium nitrogen in corn straw decreased at first and then increased,and the contents of organic matter decreased to 149.7 g/kg.The monitoring results of microbial diversity in the fermentation stacks were as follows:Guehomces were the most abundant and dominant species in the ripening process;Pseudomonas and Sphingobacterium were the main genera in the initial and ripening stages,and thermophilic microorganisms played a major role in the heating,stationary and cooling stages of ripening.The changes of physical and chemical properties and microbial diversity in fermentation stack of corn straw after the application of low temperature straw degradation agent in cold area were clarified.
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.
Wheat yellow dwarf disease,caused by Barley yellow dwarf viruses(BYDVs),is an important viral disease in wheat production.BYDV GAV has become the main pathogen causing wheat yellow dwarf disease.Till now the studies on the function of BYDV GAV encoded proteins P1,P2 and CP are lacking.We focued on the function of P1,P2 and CP,which could lay the foundation for the pathogenic mechanism of BYDV GAV.Phylogenetic analysis of BYDV GAV encoded P1,P2 and CP was conducted using Mega 7.0.We constructed the YFP expression vector of P1,P2 and CP,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.The subcellular localization of P1,P2 and CP was observed using confocal laser scanning microscopy(CLSM).We constructed the biomolecular fluorescence complementation assay(BiFC)vectors of five coding proteins,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.CLSM was used to observe the interaction of P1,P2 and CP and other viral proteins in vivo.Furthermore,we constructed the Potato virus X(PVX) expression vectors of P1,P2 and CP,transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.At 5 days post inoculation(dpi)the symptom formation of PVX infection was observed.The systemic leaves were collected for detection of viral accumulation to determine the effects of the pathogenicity of P1,P2 and CP.Results showed that BYDV GAV was most closely related to BYDV PAV at the nucleotide level.Subcellular localization of P1,P2 and CP was cytoplasm and nuclear.P1 interacted with itself in vivo using BiFC.In the pathogenicity assay,the systemic leaves of PVXCP infection showed chlorosis at 5 dpi,and PVX accumulation was detected,while PVX,PVXP1 and PVXP2 infection showed no symptoms in systemic leaves and PVX accumulation was undetectable,which was detected at 10 dpi,indicating that CP promoted the formation of PVX symptoms.In brief,P1 possibly involves in viral infection via self-interaction in vivo,and CP can promote viral infection.
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 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.
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 pFastBacTMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology,and T-Vector pMD19(simple)vector and pFastBacTMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBacTMHTB-COV19-N,and finally construct recombinant bacmid DH10Bac-pFastBacTMHTB-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-pFastBacTMHTB-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.
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.
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.
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%.
The Pineal-hypothalamus-pituitary-thyroid axis(PHPTA)could significantly regulate reproductive activities of animals.In order to investigate the expression of Kiss1/GPR54 system on PHPT axis and its regulation on reproductive activities of Tibetan sheep,24 healthy and non-pregnant Tibetan sheep in estrus cycle were selected as experimental group and 6 non-breeding season Tibetan sheep as control group.The dynamic changes of plasma Kisspeptin were detected by ELISA,and the mRNA and protein expression of Kiss1 and GPR54 in optic nerve,pineal body,hypothalamus,pituitary body and thyroid gland were detected by Real-time quantitative PCR,Western Blot and immunohistochemistry.The results showed that the plasma Kisspeptin secretion in late estrus was significantly higher than that in other periods,and that in each period of breeding season was significantly higher than that in non-breeding season.The mRNA and protein of Kiss1 and GPR54 were expressed in optic nerve and PHPT axis.The relative expression of mRNA and protein of Kiss1 and GPR54 in optic nerve were significantly higher in early estrus than in other periods(P<0.05).The mRNA and its protein of Kiss1 of the pineal body reached its maximum value in estrus,significantly higher than that in reproductive cycle.The mRNA relative expression of Kiss1 and GPR54 of hypothalamus and thyroid gland were significantly increased in late estrus(P<0.05).The mRNA relative expression levels of the two in pituitary body were significantly higher in estrus than in other periods.Immunohistochemical results showed that Kisspeptin and GPR54 were mainly distributed in the glial nucleus and cytoplasm,respectively.Both were strongly positive in pineal cell cytoplasm.Kisspeptin was expressed in neuroendocrine small cells and glial cells in hypothalamus,GPR54 was expressed in cytoplasm of neuroendocrine small cells.Kisspeptin and GPR54 were mainly expressed in the cytoplasm of basophils in pituitary body.In thyroid gland,they were mainly expressed in the cytoplasm of follicular cells.The dynamic changes of plasma Kisspeptin and the differential expressions of Kiss1 and GPR54 in different tissues during estrus cycle of Ganjia Tibetan sheep indicated that Kiss1/GPR54 system was involved in regulating reproductive physiological activities of Ganjia Tibetan sheep.
In order to clarify the expression pattern and physiological function of Kiss element (KISS1)and Tyrosine protein kinase A(TrkA) in the reproductive axis of ovulation inducing animals,and analyze the regulatory mechanism of KISS1 and TrkA on the reproduction of ovulation inducing animals.Domesticated rabbits were taken as an ovulation-inducing model.The reproductive axis tissues including hypothalamus,pituitary,ovary and uterus were collected from the pregnant Oryctolagus cuniculus f. domestica(Gestation lasts about 14—18 days).The expression patterns and distribution of KISS1 and TrkA mRNA and proteins was detected using hematoxylin-eosin(H&E)and immunohistochemistry(IHC)staining,semi-quantitative interpretation(RT-PCR),Real-time fluorescent quantitative PCR (qRT-PCR)and Western Blot(WB)in the HPG tissues.The results of IHC showed strongly positive reaction of KISS1 protein was presented in the pituitary basophils and zona pellucida of ovary while the positive expression of TrkA protein presented in the neuroendocrine cells of hypothalamus,basophils of pituitary,granular lutein cell of ovary and lamina propria of uterus.The results of RT-PCR KISS1 was expressed in the reproductive axis of pregnant rabbits,and there was significant difference between the two groups.The expression abundance of TrkA mRNA in hypothalamus,pituitary and ovary was significantly different between the two groups.The qRT-PCR results of KISS1 and TrkA genes were consistent with the RT-PCR results.The results of WB suggested that the relative expression levels of KISS1 protein was only observed in pituitary tissue while the relative expression levels of TrkA protein was presented in the HPG tissues and with a down-regulated trend.The results indicating that KISS1 and TrkA are participated in the regulation of pregnancy in Oryctolagus cuniculus f. domestica,and TrkA has a wider scope of action.
Keywords: Pregnant Oryctolagus cuniculus f. domestica;Reproductive axis;Reproductive hormone;Tyrosine protein kinase A(TrkA);Kiss element(KISS1)
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.
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.
Bimonthly, Started in 1962
CN 13-1101/S/S; ISSN 1000-7091
Responsible Institution: Hebei Academy of Agriculture and Forestry Sciences
Sponsored by: the Academy of Agricultural Sciences and Agricultural Association of Hebei, Beijing, Tianjin, Shanxi, Henan and Inner Mongolia.
Editor-in-chief: SUN Shigang
Edited and Published by: Editorial Department of Acta Agriculurae Boreali-Sinica
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