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.

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 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.

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 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.

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.

To investigate the effect of HIF-1α on the migration and invasion of trophoblast cells in placenta of dairy cows affected by cobalt dichloride(CoCl₂),and reveal the formation mechanism of placenta of dairy cows under hypoxia conditions,bovine placental trophoblast cells(BTCs)were isolated and purified from the placenta of early pregnancy cows.The cell morphology was observed by giemsa staining method,and the expression of cell markers cytokeratin 7(CK7)and Thy/CD90 were detected by immunofluorescence method.pCI-neo-hTERT plasmid was then transfected into BTCs,qPCR and Western Blot methods were used to detect the TERT mRNA and protein expression in BTCs.Proliferation rate and placental lactogen(PL)secretory ability were analyzed with CCK8 method and ELISA,respectively.siRNA was further used to silence the expression of Hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)secretion ability in BTCs was detected by ELISA and the ability of migration and invasion of BTCs was detected by scratch test and transwell.According to the above all results,the isolated and purified BTCs were typical epithelioid cells with a certain number of binucleated cells and expressed epithelial marker protein CK7,but Thy/CD90 expression was not observed.The transfected cells could stably express telomerase reverse transcriptase(TERT)mRNA and protein.No cell aging was observed after 50 generations of continuous passage,and the proliferation rate was significantly higher than these primary BTCs(P<0.05),and the PL secretion ability showed no significantly different from that of primary BTCs(P>0.05).The cell viability of BTCs treated with CoCl₂ decreased with the increase of CoCl₂ concentration and time(P<0.05),and HIF-1α mRNA and protein expression decreased with the increase of CoCl₂ concentration(P<0.05).BTCs was treated with 400 μmol/L CoCl₂ for 24 h to establish a hypoxia model,and VEGFA secretion and invasion ability were significantly increased under BTCs hypoxia(P<0.05),while HIF-1α silencing decreased VEGFA secretion and invasion ability of BTCs(P<0.05).In conclusion,immortalized BTCs was established through exogenous TERT gene transduction.The immortalized BTCs had similar biological characteristics to the primary cells,and have stronger VEGFA secretion and invasion ability under hypoxia condition,and were associated with HIF-1α up-regulation.

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.

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.

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.

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.

To explore the biological function of cotton cytochrome P450 gene GhP450-94C1 in cotton Verticillium wilt response, and lay a foundation for cotton Verticillium wilt resistance gene mining and disease resistance breeding. A cytochrome P450 gene, GhP450-94C1, was cloned through transcriptome screening. The physicochemical properties of the gene were analyzed by bioinformatics methods. The expression pattern of GhP450-94C1 under Verticillium wilt induction was analyzed by Real-time quantitative polymerase chain reaction (qRT-PCR). Virus-induced gene silencing (VIGS) technology was used to preliminarily explore its biological function in cotton resistance to Verticillium wilt. The main results were as follows: upland cotton cytochrome P450 gene, GhP450-94C1, was obtained by cloning. The open reading frame (ORF) was 1 503 bp, encoding an acidic, hydrophilic and unstable transmembrane protein with 500 amino acids. The molecular formula was C₂₅₉₇H₄₀₂₅N₆₉₁O₇₂₅S₂₂ with a molecular weight of 57.23 ku, which was located in the endoplasmic reticulum membrane and contained a P450 domain. There was 86.45% probability of signal peptide; the secondary structure prediction showed that the protein contained 24 α-helixes and 8 β-sheets. This gene responds to Verticillium wilt infection, and after inhibiting its expression, the sensitivity of plants to Verticillium wilt is enhanced. GhP450-94C1 is a positive regulator of cotton resistance to Verticillium wilt.

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.

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.

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.

To eradicate Verticillium wilt of cotton,identification of resistant genes and breeding of resistant cotton varieties are the best methods at present.We used MEGA 5.2 and other related software to construct phylogenetic trees of the proteins encoded by WRKY7 genes in Gossypium hirsutum and Arabidopsis thaliana and Oryza sativa.The induction and response of GhWRKY7 gene to Verticillium dahliae and its mechanism were elucidate by subcellular localization,virus-induced gene silencing,qPCR and GUS reporting system analysis.The results showed that GhWRKY7 and AtWKRY7 were highly homologous and belonged to Group Ⅱ.GhWRKY7 was located in plant nucleus,and the relative expression of GhWRKY7 in cotton leaves and root organs was significantly higher than that in stems.GhWRKY7 gene expression was significantly upregulated after 24 h of inoculation.The GhWRKY7-silenced plants showed higher susceptibility to Verticillium dahliae infection compared to the control(Expression TRV empty vector plants),suggesting that GhWRKY7 gene positively regulated cotton disease resistance.Compared to the control,the expression levels of disease-resistance related genes,including GhPR1,GhPR3,GhPR4,GhPR5,GhPDF1.2,GhPAL1 and GhCYP71B36 in GhWRKY7-silenced plants significantly decreased after inoculation,indicating that GhWRKY7 improved the disease resistance of the plants due to increased the expression levels of disease-resistance related genes.Transient expression analysis of GhWRKY7 gene by constructing GUS reporter vector in tobacco cells revealed that GhCYP71B36 gene could specifically bound to cis-element of GhCYP71B36 promoter and transcriptionally activated downstream GhCYP71B36 expression,thus improved the disease resistance of cotton.In conclusion,GhWRKY7,as a transcription factor that positively regulates Verticillium wilt resistance in cotton,is involved in the expression of downstream disease-resistance related genes such as Camalexin synthesis,thereby improving plant disease resistance.Therefore,GhWRKY7 can be used as a candidate gene for cotton resistance breeding and provide security for cotton production.

It aimed to develop a polyclonal antibody to Avian leukosis virus (ALV)and to establish a simple and accurate method for the detection of ALV.pGEX-6p-1-p27 and pET-32a-p27 protein vectors were constructed by amplifying the target gene of ALV p27.The proteins were immunized in BALB/c mice and New Zealand white rabbits,and mouse-derived polyclonal antibodies and rabbit-derived polyclonal antibodies were prepared.The mouse-derived polyclonal antibody coupled fluorescent microspheres were prepared,and the mouse-derived polyclonal antibody IgG was sprayed onto the sample pads using a three-dimensional spray point platform;the purified rabbit-derived polyclonal antibody IgG and goat anti-rabbit antibody dilution were scribed onto the NC membrane using a scribing instrument as T and C lines for the assembly of test strips,and a preliminary Point-of-care testing(POCT)immunochromatographic assay was established.The test strips were assembled and a preliminary point-of-care testing(POCT)method was developed for the detection of ALV by fluorescent microsphere immunochromatography.The results showed that the prokaryotic expression vectors for pGEX-6p-1-p27 and pET-32a-p27 were successfully constructed and the expression of p27 recombinant protein was induced.After mixing the purified p27 fusion protein with the adjuvant as an immunogen immune BALB/c mouse and a New Zealand white rabbit,a mouse-derived and rabbit-derived polyclonal antibody of the p27 protein was successfully prepared,and the immunogen response of the multi-antibody was shown to be good by serum potency and Western Blot identification.The optimal pH of the fluorescent microsphere-coupled murine polyclonal antibody was 6.2.POCT strips showed that positive samples for ALV bound well to the fluorescent microsphere-coated murine polyclonal antibody,with higher T/C data than other samples,and that positive samples bound to the fluorescent microsphere-coated polyclonal antibody showed fluorescent bands in the C line(quality control line)and T line(detection line)under UV light,which the results were consistent with clinical diagnostic results.It can be concluded that the POCT fluorescent microsphere immunochromatographic detection method for ALV established provides a simpler method for the detection of Avian leukosis virus and facilitates clinical detection for primary veterinarians.

The aim of this study was to clone the kinesin family member 2A gene(KIF2A),and explore the expression pattern of KIF2A gene in different tissues of yak,as well as the spatially and temporally expressed during the follicular development and oocyte meiotic maturation.The heart,liver,spleen,lung,kidney stomach,uterus and ovary tissues(n=5)were collected from 3—4 years old healthy female yak after slaughter.Yak KIF2A gene was amplified by RT-PCR with yak ovarian cDNA as template,and its protein were analyzed by bioinformatics software such as MAGA 7.0 and SWISS-MODEL etc.Quantitative Real-time PCR(RT-qPCR)was used to detect the expression level of KIF2A in yak various tissues and immunohistochemistry was used to analyze the expression and localization of KIF2A in follicles with different diameters.According to the diameter,the follicle were divided into three groups: small(diameter 1.0—2.9 mm),medium(diameter 3.0—5.9 mm),and large(diameter 6.0—9.0 mm)follicle.The expression characteristics of KIF2A gene in granulosa cells and oocyte meiosis in follicles were analyzed by RT-qPCR.The results showed that sequence length of KIF2A gene was 1 964 bp,and the CDS was 1 530 bp,which encoding 509 amino acids.KIF2A protein was negatively charged and belonged to hydrophobic protein.KIF2A gene was relatively conservative and widely expressed in various tissues of yaks, and KIF2A protein was mainly located in granulosa cells, its mRNA expression in follicular granulosa cells with different diameter and size increased with the growth and development of follicles. During the meiotic maturation of yak oocytes,the expression of KIF2A showed a sequential feature,and the G Ⅴ phase was significantly higher than that of M Ⅰ and M Ⅱ phase.The results showed that KIF2A might be involved in the regulation of follicular and oocyte maturation development in yaks,and the results of this study provided basic data for the functional study of KIF2A gene in yak follicular development.

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.

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.

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 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 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 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.

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.

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.

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.

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.

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.

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.

The purpose was to prepare polyclonal antibody against the recombinant double CP of Potato leafroll virus (PLRV)and Potato virus S (PVS),and apply the polyclonal antibody to indirect ELISA and DAS-ELISA detections of PLRV and PVS.Prokaryotic expression vector pET22b-LRCP/SCP of the fused double CP gene of PLRV and PVS was constructed.After replacement of lysozyme treatment by ultrasonic disruption,inclusion body protein was extracted from the recombinant strain BL21(pET22b-LRCP/SCP),the target protein(recombinant double CP)was purified by nickel ion affinity chromatography and high-purity target protein of 51.2 ku was obtained.The high-purity recombinant double CP was used as antigen to immunize rabbits to prepare an antiserum with a titer of 1∶128 k.Specific reactions were respectively observed between the purified polyclonal antibody (IgG) against the recombinant double CP and the positive standard of PLRV or PVS and no cross-reaction was found between the purified IgG and other four potato major viruses (PVX, PVY, PVA and PVM). The purified IgG against the recombinant CP with the diluted concentration of 1∶3 200 still positively reacted with PLRV or PVS in indirect ELISA detection.The purified IgG and the alkaline phosphatase-conjugated IgG both with the diluted contraction of 1∶100 also positively reacted with PLRV or PVS positive standard in DAS-ELISA detection.The results showed that one type of the prepared IgG against the recombinant double CP could detect two viruses of PLRV and PVS by DAS-ELISA or indirect ELISA.

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 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.

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.

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.

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.

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 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.

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.

In order to clarify the role of APOC3 gene in the differentiation of intramuscular adipocytes of goats,the APOC3 gene sequence was cloned by RT-PCR,and the biological information was analyzed by online software.Quantitative Real-time PCR(qPCR)was used to detect the expression of APOC3 gene in intramuscular adipocytes of goats at different tissues and differentiation stages.APOC3 overexpression vector was constructed by double-enzyme digestion method,and the effects of APOC3 gene overexpression on lipid droplet accumulation were determined by oil red O staining.At the same time,the mRNA relative expression level of marker gene of adipogenic differentiation was detected by qPCR.The results showed that the ORF region of APOC3 was 294 bp in length and encoded 97 amino acids with a functional domain of 23-88 aa. APOC3 was expressed in 14 tissues including heart,liver and spleen,and so on,which the highest level of APOC3 was found in liver.APOC3 expression was the highest at 48 h and extremely significantly higher than that before differentiation.After APOC3 was overexpressed,lipid drop accumulation increased,and the relative expression levels of marker genes SREBP1 and CEBPβ were extremely significantly up-regulated,PPARγ was significantly up-regulated,and Pref-1 was significantly down-regulated.APOC3 might promote the differentiation of intramuscular adipocytes by up-regulating SREBP1,CEBPβ,PPARγ and down-regulating Pref-1.

To further understand the role of the promoter in the regulation of the expression of the Brassica napus FIL gene(BnaFIL),it used the DNA extracted from the leaves of Brassica napus as a template based on the genomic data of Brassica napus.For cloning,the length was 1 326 bp.Using PlantCARE online analysis software to perform bioinformatics sequence analysis on the promoter sequence,the results showed that the sequence contained some conserved DNA modules involved in the photoreaction and essential elements for core promoters such as CAAT-box and TATA-box.Expression related cis-acting regulatory elements CAT-box and photosensitive response elements.Replace the CaMV35S promoter on the pBI121 plant vector with the promoter sequence,fused the promoter with the GUS gene to obtain the pBnaFIL-GUS expression vector,and transfer the vector into Arabidopsis thaliana by the method of Agrobacterium inflorescence.Flower promoter recombinant plasmid-positive transgenic lines and late flower promoter recombinant plasmid-positive transgenic lines.After that,GUS staining analysis was performed on the transgenic Arabidopsis plants,and the expression effect of the promoter was tested.Finally,the GUS gene expression was found in different transgenic Arabidopsis plants.The results showed that there were differences in promoter expression between early-flowering materials and late-flowering materials.The promoters of early-flowering materials could drive gene expression better than those of late-flowering materials.It was inferred from this that the driving effect of the promoter regulates the early and late flowering of rape,resulting in different expression effects of the FIL gene in different materials.

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.

To clarify the lignin accumulation ways in the stem of soybean variety,and further understand the relation between the lignin accumulation and lodging resistance of soybean plant,which could give the theory evidence for plant phenotype regulation and new soybean variety breeding under different planting densities,Charleston,a representative variety with determinate growth habit,was selected in current study and were planted with two densities of 20,35 plants/m².The lignin content in the elongation zone(EZ)and the secondary cell wall maturation zone(MZ)at the third internode of Charleston's stem were measured and the activities of key enzymes such as 4-Coumarate:coenzyme A ligase(4CL)and phenylalanine aminotransferase(PAL)and cinnamate dehydrogenase(CAD)involved in the lignin synthesis were determined at two different densities.The results showed that lignin content in the stem of Charleston under low density was higher than that under high density.The content of lignin in single internode(stem 3)of Charleston under both high and low density treatments was MZ>EZ.4CL,PAL and CAD activities in low density Charleston stem were higher than those in high density of Charleston and in accordance with gene expression levels.The key genes for lignin synthesized were distributed in soybean genome in different gene families.Moreover,different enzyme gene in one gene family showed different expression style in the same section.Same results could be observed in different organ for one gene.

In order to promote the breeding of long-grain japonica rice varieties,two knock-out vectors,pYLCRISPR/Cas9-GS3-RNA and pYLCRISPR/Cas9-GS3-GS9-RNA,were constructed with japonica rice varieties Dongfu 139,Longjing 31 and Dongnong 427,the genes of GS3 and GS9 were edited by Agrobacterium tumefaciens in vitro.In the end,GS3,GS9 and GS3,GS9 double-gene mutations without T-DNA elements were obtained in the T₂.The results showed that the grain length and 1000-grain weight of gs3 mutant plants of three varieties were significantly increased compared with those of wild type,grain width,seed setting rate and grain number per panicle did not change significantly,grain length of gs9 mutant increased significantly,and grain width decreased significantly,while 1000-grain weight,seed setting rate and grain number per panicle did not change significantly,and grain length of gs3gs9 mutant increased more than gs3 and gs9,at the same time,grain width decreased significantly,1000-grain weight increased significantly,while seed setting rate and grain number per ear did not change significantly.To sum up,three japonica rice varieties,Dongfu 139,Longjing 31 and Dongnong 427 were improved by using CRISPR/Cas9 technique,which accelerated the breeding process of new long-grain japonica rice varieties.

In order to find out the cytoplasm types of some sterile line and maintainer line materials in the process of sugar beet breeding, it provides a guiding direction for the next breeding work. Molecular marker primer TR1 was used to identify the molecular level of 30 test materials cytoplasm types, and to compare the fertility traits in the field. The results showed that the PCR electrophoresis bands of maintainer line 960767 and sterile line N9848 were 750,500 bp.We named these cytoplasm types as N1 and S,respectively.And 750 bp bands were detected by T152,T328,T334,T306,T360,as N1 cytoplasm.The 500 bp bands detected were T766,T154,N122,I-1,B-3,Z-1,41419,10320,B-3-23,S865,S301,S151,S327,S333,S115,S305,S153,S359,S137,S163,and S313,inferred to be S type cytoplasm.The materials for the bands detected between 500 bp and 750 bp were T302,and T116.Because T302 also had the ability to maintain the sterility of S301 in field investigations,T302 and T116 were classified into another type,N2 cytoplasm type.Some plants with 500, 750 bp of materials were T302, I-1 and B-3, which included both normal and Owen cytoplasm were characteristic materials. The results of field pollen fertility survey T766, T154, N122, I-1, B-3, Z-1, 414191, 10320 and B-3-23 were all fertile, but their cytoplasm type was identified to belong to S type cytoplasm. According to molecular identification and field fertility trait survey, the results of most materials were consistent with the results of fertility trait survey. Some materials had inconsistent molecular identification and fertility trait results, requiring further adjustment of the selection direction.

In order to clarify the effects of drought stress on the photosynthetic characteristics in flag leaf in the afternoon during grain filling stage and grain yield of winter wheat with different drought resistance,an experiment with two winter wheat cultivars and four water levels was conducted under the condition of rainproof pond cultivation in 2018—2019 and 2019—2020.The two winter wheat cultivars were Jinmai 47(JM47,strong drought resistance)and Yanzhan 4110(YZ4110,weak drought resistance).The four water treatments included severe drought(W1:65% MFC(maximum field water capacity)before sowing + 45%—55% MFC after jointing),moderate drought(W2:75% MFC before sowing + 55%—65% MFC after jointing),mild drought(W3:75% MFC before sowing+65%—75% MFC after jointing),suitable water supply(W4:75%MFC before sowing+75%—85% MFC after jointing).The net photosynthetic rate(Pn),stomatal conductance(Gs),intercellular CO₂ concentration(Ci),transpiration rate(Tr),instantaneous water use efficiency(IWUE),maximum photochemical efficiency of PS Ⅱ (Fv/Fm)and actual photochemical efficiency of PS Ⅱ(ΦPS Ⅱ)in flag leaf from 14:00 to 16:00 during the early,medium and medium-late grain filling stage and the grain yield and its components at maturity were investigated.The results showed that both water and cultivars had significant effects on the photosynthetic and fluorescence characteristics in flag leaf in the afternoon during the grain filling stage and the grain yield at maturity of winter wheat.From the two-year average,compared with W4,the Pn,Gs and ΦPSⅡ in flag leaf in the afternoon during the grain filling stage under drought stress(W1,W2 and W3)respectively decreased by 2.07%—68.92%,-3.23%—50.00% and -1.89%—30.19% in JM47,and decreased by 7.71%—80.19%,11.11%—59.26% and 0—73.47% in YZ4110;the flag leaf Tr values in the afternoon during the medium grain filling stage in JM47 and YZ4110 respectively decreased by 6.30%—32.87% and 6.49%—41.74%,and the flag leaf Fv/Fm values in the afternoon during the medium-late grain filling stage decreased by 1.20%—18.52% and 2.50%—30.00%.In general,for all the above indexes,the decreasing amplitude for the same index was JM47<YZ4110.Compared with YZ4110,the Pn,Gs,ΦPSⅡ and Fv/Fm in flag leaf in the afternoon during the grain filling stage under drought stress(W1,W2 and W3)of JM47 respectively increased by 0.86%—64.89%,8.33%—36.36%,1.96%—184.62% and 1.25%—17.86%,and the grain yields of JM47 were respectively increased by 28.91%,8.06% and 5.40%.Except for IWUE,the flag leaf photosynthetic parameters in the afternoon during the grain filling stage were significantly and extremely significant correlated with grain yield,but the correlation indexes varied with variety and grain filling stage.For JM47,the correlation indexes between the grain yield and flag leaf photosynthetic parameters were highest for Pn,Gs and Fv/Fm during the medium-late grain filling stage,for ΦPSⅡ during the medium grain filling stage,and for Tr during the early grain filling stage.For YZ4110,the correlation indexes were highest for Pn,Gs and Tr during the early grain filling stage,for ΦPSⅡ during the medium grain filling stage and for Fv/Fm during the medium-late grain filling stage.In summary,drought stress decreased the photosynthetic function of flag leaf in the afternoon during grain filling stage and thus decreased the grain yield of winter wheat,the strong drought resistance variety could maintain better flag leaf photosynthetic characteristics in the afternoon during the grain the filling stage under drought stress condition,and significantly improved the ΦPSⅡ of flag leaf in the afternoon during the medium grain filling stage and the Pn,Gs and Fv/Fm of flag leaf in the afternoon during the medium-late grain filling stage,thereby increasing the grain yield.

This study investigated the accumulation and distribution of dry matter,nitrogen(N)and zinc(Zn)of summer maize under N and Zn fertilization,to provide a basis for reasonable application of zinc fertilizer and combined fertilization.Split-split design was used with three N rates(90,180,225 kg/ha N)as the main factor,two Zn rates(0,4.5 kg/ha ZnSO₄·7H₂O)as the second factor,two varieties(ZD958 and GSY66)as the third factor.The effect of N and Zn application on the grain yield,dry matter accumulation dynamic and N and Zn absorption,accumulation and distribution of each organ of different maize varieties by field experiment.The results showed that the grain yield achieved 9.77,10.42 t/ha under the N rates of 180,225 kg/ha,respectively,increased by 18.0% compared with 90 kg/ha.The treatment of 225 kg/ha N had the highest plant dry matter accumulation after silking,while the treatment of 90 kg/ha had higher ratio of cob and grain dry matter at the mature stage.Highest N concentration in each organ,Zn concentration in the stem and N and Zn accumulation in the leaf and grain were obtained in the treatment of 225 kg/ha,while highest Zn concentration in the sheath,bract and grain and ratio of grain N and Zn were found in the treatment of 90 kg/ha.No effect was found in the grain yield and dry matter accumulation and distribution under two Zn treatments.Zn application significantly increased N and Zn concentration and accumulation but decreased distribution ratio of grain N and Zn by 6.93,6.86 percentage points,respectively.Relative to GSY66,ZD958 had higher grain yield and dry matter ratio,and increased dry matter by 29.2% at maturity.Besides this,ZD958 decreased grain N and Zn concentration by 8.9% and 5.3%,respectively,but improved grain accumulation and distribution ratio.Correlation analysis showed that grain yield and N concentration in the stem,leaf and grain were significantly positively correlated.There was also a significantly or extremely significantly positive correlation between Zn concentration in the leaf and N concentration in the sheath and grain,between Zn concentration in the cob and N concentration in the sheath,cob and bract.These results suggested that combination of N and Zn fertilizers could increase yield and plant dry matter accumulation,enhance the N and Zn absorption and accumulation in each organ especially grain,but decrease distribution ratio of N and Zn in the grain.

Reducing fertilizer application and increasing efficiency is of great significance to ensuring the sustainable development of agriculture.Research on the impact of new-type synergistic compound fertilizers on rice nutrient absorption and yield will help ensure the food security.Four treatments which were conventional compound fertilizer(CG),synergistic compound fertilizer(ZZ),conventional compound fertilizer reduction by 20%(80% CG),and synergistic compound fertilizer by 20%(80%ZZ)was set.The results showed that:Compared with the conventional compound fertilizer treatment,the synergistic compound fertilizer treatment could increase the fresh weight,plant height,SPAD value and root volume of rice at tillering stage,jointing stage,heading stage,filling stage and maturity stage.At the maturity stage,the fresh weight,plant height,SPAD value and root volume of rice plants treated with the synergistic compound fertilizer increased by 11.71%,1.29%,8.02% and 46.48%,respectively.The rice plant treated by the synergistic compound fertilizer reduced by 20% treatment increased by 2.29%,0.31%,3.70% and 4.09%,respectively.Compared with the conventional compound fertilizer treatment,the synergistic compound fertilizer treatment could increase the dry matter weight of stems and leaves and the dry matter weight of grains by 24.46% and 21.39%,respectively.The synergistic compound fertilizer treatment to increase the dry matter weight of rice stems and leaves and the dry matter weight of grain was 6.07% and 8.15%,respectively.Compared with the conventional compound fertilizer treatment,the nitrogen content of rice stems and leaves and the nitrogen content of grains under the treatment of synergistic compound fertilizer increased by 10.68% and 36.96%,respectively.The phosphorus content of rice stems and leaves increased by 26.51% and 11.24%,respectively.The phosphorus content of rice grains treated with synergistic compound fertilizer increased by 10.39%,and the phosphorus content of rice grains treated with synergistic compound fertilizer reduced by 20% decreased by 5.41%.Compared with the conventional compound fertilizer treatment,the potassium content in the stems and leaves of rice treated with the synergistic compound fertilizer increased by 19.91%,the potassium content in the rice grains decreased by 15.83%,and the potassium content in the stems and leaves of the synergistic compound fertilizer reduced by 20% treatment decreased 6.56%,the potassium content of grains increased by 0.62%.Compared with conventional compound fertilizer treatment,the yield of rice treated with synergistic compound fertilizer increased by 22.02% and the difference was significant.The yield of rice treated with synergistic compound fertilizer reduced by 20% increased by 1.12%.The rice panicle length,panicle number,grain number per panicle,1 000-grain weight and seed setting rate of rice of synergistic compound fertilizer were the highest among all the treatments.In summary,the synergistic compound fertilizer can improve the growth index of rice in each growth period,improve the nitrogen absorption,phosphorus absorption and potassium absorption of rice,and increase crop yield.Compared with conventional compound fertilizer,the 20% reduction treatment of synergistic compound fertilizer can realize the reduction of chemical fertilizer without reducing production,and it is worthy of further popularization and application.

In order to reveal the effect and molecular mechanism of salicylic acid-induced resistance to black spot disease of Yali,the leaves and fruits of Yali were used as experimental material which were collected from the experimental orchard of Hebei Agricultural University.Tissue isolation method was used to isolate and purify the pathogen of black spot disease and detected its pathogenicity.Morphological observation and multi-genes of ITS,HIS,RPB2 and ACT were used to identify the pathogen of black spot disease.The detached leaves of Yali were inoculated with conidia suspension of Alternaria alternata by acupuncture for 0,6,12,24,48,72,96 and 120 h,respectively.Real-time RT-PCR was used to analyse the expression of related genes which involved in salicylic acid signaling,and high-performance liquid chromatography (HPLC) was used to detect the content of endogenous SA at 0 and 72 h,respectively.Exogenous SA treatment with different concentrations (0,0.002,0.02,0.2,2.0,10.0,20.0 mmol/L) were used to identify the effect to the mycelial growth of A.alternata;Exogenous SA treated Yali fruit with different concentrations (0,0.02,0.2,2.0 mmol/L),respectively,and inoculated with A.alternata to detect the effect of disease resistance.The results showed that the pathogen of Yali black spot disease was A.alternata. Detached leaves of Yali were inoculated with A.alternata for 72 h,the free state SA content increased from 0 mg/g to 0.02 mg/g and the binding state SA content increased from 0.47 mg/g to 1.55 mg/g,and for 96 h compared with 0 h,the expression of Pbrgene12425,Pbrgene6286, increased 5.48,4.66 times,respectively,the expression of Pbrgene8895 and Pbrgene43605 increased 7.90 and 10.0 times for 120 h.Exogenous 0.2 mmol/L SA significantly increased the resistance of Yali fruit to black spot disease.In the process of Yali resistance to black spot disease,the free state SA content of Yali leaves significantly increased,and the related genes in SA signaling were induced to up-expressed,exogenous 0.2 mmol/L SA significantly increased the resistance of Yali to black spot disease.