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

In order to have a clearer understanding of the research progress on cold resistance of winter rapeseed in cold and arid regions of China,and to provide theoretical support for breeding and production.The research progress of cold-resistance and cold-resistance breeding of winter rapeseed were summarized respectively from six aspects such as selection index and method,genetic law,breeding method,screening effect,germplasm classification and molecular mechanism research,and four aspects such as breeding method,breeding varieties,cold resistance classification and adaptability screening of varieties.Based on the analysis of the existing problems,six measures were put forward,such as creating germplasm resources by multiple ways,screening out core germplasm by comprehensive evaluation,carrying out research on cold-resistance mechanism and construction of innovation system of molecular design breeding,constructing breeding system and carrying out cold-resistance breeding by multiple ways,improving the adaptability of varieties by comprehensive screening,promoting varieties yield level and improving benefits by carrying out matching cultivation techniques,in order to promote the development of rapeseed production in cold and arid regions of our country.

In order to investigate the reasons for the differences in plant height between SSSL-B50 and HJX74,an F₂ population was constructed by backcrossing SSSL-B50 with HJX74 for genetic analysis and gene mapping of the plant.The genetic analysis showed that F₁ plants exhibited tall plant height.And the separation ratio within F₂ was 3:1(χ²=0.18<3.84)between the tall plant individuals and the semi-dwarf ones,which indicated that the tall plant height of SSSL-B50 was a dominant trait.Based on the linkage analysis between the marker genotypes and phenotypes of plant height in the F₂ population,the gene controlling tall plant height was mapped between markers S18 and X161 within the 38.38—39.07 Mb interval of the substituted segment on chromosome 1.Moreover,it was found that the known gene of "Green revolution",SD1,fell to the mapping interval.Through amplification and sequence alignment of SD1 gene in Oryza glumaepatula,SSSL-B50,and HJX74,it showed that a 280 bp deletion in CDS in HJX74 compared with Oryza glumaepatula and SSSL-B50.The qRT-PCR results showed that the expression level of SD1 was significantly higher in the second,third,and fourth sections of SSSL-B50 stem than in HJX74.The comparison of the SD1 gene sequence of Oryza glumaepatula with the previous reported results showed that the base substitutions occurred at two sites (E100G, Q339R) in the encoded amino acid.The results revealed that the tall plant height of SSSL-B50 was controlled by SD1.Furthermore,a new allelic type of SD1, SD1^Glu,was identified in Oryza glumaepatula.

In order to further definite the genetic basis of black rot resistance in cabbage,and breed high-quality disease resistant cultivars,F₁ progenies were obtained by crossing the inbred line 4674(high resistance to race 1)as male parent,with the inbred line 4673(high susceptibility to race 1)as female parent.The F₂ population containing 152 individuals were obtained by F₁ self-crossing. The F₂ population was inoculated by spraying at seedling stage. After 12-14 days, the phenotype of F₂ population was identified according to the identification method of cabbage seedling stage.A total of 175 markers with good polymorphism and clear bands were selected from 404 pairs.Subsequently, the 175 molecular markers were used for genotyping of F₂ population and constructing a genetic linkage map. Finally, the QTL for black rot resistance of cabbage was mapped with phenotypic data and genetic map.The results showed that there were 154 molecular markers linked to nine chromosomes,including 110 pairs of InDel markers and 44 pairs of SSR markers and covering a length of 714.29 cM,and the average distance between markers was 4.64 cM.Seven QTLs were located,of which three major QTLs were qBR-7-2,qBR-7-3 and qBR-4-3,mapped on the genomic markers(CG842110-CG842482 and M29-M39)of chromosome 7,and on the genomic markers between CD838151 and BOE417 of chromosome 4,respectively.The explainable phenotypic variation of QTLs were 16.0%,9.2% and 10.0%,and their LOD values were 5.75,3.20 and 3.47,respectively.

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.

Auxin response factor(ARF)is a class of transcription factors with B3 domain,which is a direct molecule regulating auxin response and controlling gene expression.A gene,ZmARF10,which encoded ARF protein and actively participated in drought-rehydration stress response,was previously screened in maize by analyzed transcriptome data.To further research the molecular mechanism of ZmARF10 regulating drought resistance of maize,and also provide a new idea for molecular breeding of drought resistance,the gene was firstly analyzed by bioinformatics software.Secondly,Quantitative Real-time polymerase chain reaction(qRT-PCR)was used to detect the expression pattern of ZmARF10 in different tissues,under high temperature,drought,high salt,ABA and restoring treatments,and in different inbred lines.Finally,the function of ZmARF10 was analyzed using CRISPR/Cas9 technology.The results showed that ZmARF10 was located on chromosome 3 of maize,with a total length of 2 127 bp,and encoded 708 amino acids with a typical B3 domain.The upstream 2 kb region of ATG of this gene contained response elements related to methyl jasmonate,auxin,abscisic acid and low temperature.The phylogenetic tree showed that the protein encoded by the ZmARF10 gene was closely related to sorghum.qRT-PCR results showed that ZmARF10 was a constitutive expression gene,and the expression level of ZmARF10 was the highest in mature corn roots of maize.Under high temperature,drought,high salt and ABA treatments,the expression of this gene was significantly up-regulated,and the up-regulation ratio was up to 8.2 times after drought stress.After drought stress,the expression level of ZmARF10 gene was significantly higher in the drought-resistant inbred line Zheng 36 than that in the drought-sensitive inbred line B73.Investigation of Arabidopsis wild type and ARF10-deficient mutants showed that,compared with wild type,the mutant plants showed leaf wilting and even dry death,roots curled,root branch number decreased,and lateral root growth and development were hindered under drought conditions.Determination of physiological and biochemical indexes showed that the relative water content,chlorophyll content and net photosynthetic rate of the deficient mutants were significantly lower than those of the wild-type plants after drought stress,indicating that the drought resistance of Arabidopsis decreased after ARF10 gene was knocked out.

WRKY transcription factor is a family of transcription factors unique to plants.Studies have demonstrated that WRKY transcription factor played an important role in plant growth and development and in plant response to biological and abiotic stress.In order to reveal the function of tomato WRKY gene,two inbred lines of tomato with high resistance to bacterial wilt Hm 2-2(R)and high susceptibility to bacterial wilt BY 1-2(S)were used as experimental materials based on the preliminary transcriptome data,and a WRKY transcription factor SlWRKY75 gene(Solyc05g015850.3)was cloned.The structure,expression pattern and function of the gene and its encoded protein were analyzed by bioinformatics analysis,multiple alignment of amino acid sequences,phylogenetic tree construction,Real-time Quantitative PCR(qRT-PCR)and virus induced gene silencing(VIGS).The results showed that the full length of the cDNA of this gene was 653 bp,its maximum open reading frame was 519 bp,encoding 172 amino acids,the relative molecular weight of the protein was 19.878 51 ku,the theoretical isoelectric point was 9.32.The protein belonged to the hydrophilic non-secreted protein,and there was no transmembrane structure.Meanwhile,the protein had a highly conserved WRKY domain and a CX₄CX₂₃HXH zinc finger motif,which belonged to the Class Ⅱ family.Phylogenetic tree analysis showed that SlWRKY75 was closely related to Solanum pennellii SpWRKY75 and clustered into a group with other Solanaceae,while it was far related to Hevea brasiliensis HbWRKY75 and Gossypium hirsutum GhWRKY75 and was in different branches in the phylogenetic tree.The results of qRT-PCR analysis showed that the expression of SlWRKY75 gene were tissue-specific and could be induced by Ralstonia solanacearum,salicylic acid and jasmonic acid.VIGS result showed that silencing SlWRKY75 reduced plant resistance to bacterial wilt,indicating that SlWRKY75 positively regulated tomato resistance to bacterial wilt.These results suggested that SlWRKY75 gene played an important role in regulating tomato resistance to bacterial wilt.

In order to study the biological function of NbEHD1 in Nicotiana benthamiana,its gene structure,conserved domains,phosphorylation sites,subcellular localization and evolutionary relationship were predicted by bioinformatics methods.The encoding sequence of NbEHD1 was 1 638 bp,and its genomic sequence contained 16 exons and 15 introns.NbEHD1 protein was predicted located in the cytoplasm,which had 42 phosphorylation sites and had no signal peptide and transmembrane region.NbEHD1 belonged to the P-loop_NTPase superfamily and had a conserved domain specific to the EHD family.Phylogenetic relationship showed that NbEHD1 was closely related to EHD sequences of Solanum tuberosum and Solanum lycopersicum.Blast analysis against N.benthamiana sub-data base in SNG was performed to obtain the predicted full length of NbEHD1,then its full nucleic acid sequence was amplified by gene-specific primers.After obtaining NbEHD1 CRISPR/Cas9 gene editing construct,the vector was successfully transformed into the leaves of N.benthamiana by Agrobacterium-mediated genetic transformation.Eventually 16 T₀-generation plantlets were successfully identified,which provided materials for further research to determine the biological function of NbEHD1.

JAZ protein plays a key role in plant growth and development and stress signaling pathways.In order to explore the regulatory mechanism of JAZ protein in wheat late spring cold,TaJAZ6 gene was cloned from young spike of wheat,and its molecular characteristics,expression characteristics and subcellular localization were analyzed.The results showed that the full-length CDS sequence of the gene was 549 bp,encoding 178 amino acids.The predicted molecular weight of the encoded protein was 18.376 ku,the theoretical isoelectric point was 9.37,and the instability coefficient was 62.44,so it was an unstable protein.The protein encoded by this gene had a TIFY domain and a CCT_2 domain.Phylogenetic tree analysis showed that the protein had the closest relationship with TIFY 11b proteins of Triticum dicoccoides and Triticum urartu.In addition to the basic response elements such as CAAT-box and TATA-box,the promoter region of TaJAZ6 gene also contained hormone response elements,light response elements,low temperature response elements,defense and stress response elements.Real-time Quantitative PCR analysis showed that TaJAZ6 gene was expressed in roots,stems,leaves and young panicles,with the highest expression in roots.The expression of TaJAZ6 gene was also induced by low temperature and methyl jasmonate (MeJA).Under low temperature stress,the expression trends of TaJAZ6 in roots,stems and leaves of Aikang 58 (tolerant to late spring cold ) and Zhengmai 366 ( sensitive to late spring cold ) were the same trend,which significantly increased.After spraying 300,350 μmol/L MeJA,the expression of TaJAZ6 in plants treated with low temperature decreased significantly in both wheat varieties.The expression level of TaJAZ6 in the young panicles after low temperature stress showed an opposite trend.The expression level of TaJAZ6 in the young panicles of Aikang 58 decreased significantly,and increased significantly in the young panicles of Zhengmai 366.It was speculated that the gene might negatively regulate the defense response of wheat to late spring cold stress.By spraying MeJA,the relative expression of TaJAZ6 gene in young spikes of two wheat varieties under low temperature stress was significantly reduced,and the grain number of wheat was increased.Subcellular localization assay showed that TaJAZ6 protein was localized in the nucleus.The results above indicate that TaJAZ6 may play an important role in the response of wheat to late spring cold stress.

Grapes are often subjected to various abiotic stress during their growth and development,such as drought,salt,heat or chilling,among them,drought stress is one of the most important abiotic stresses inhibiting vegetative and reproductive growth of grape.Although grapes are more resistant to drought stress,about half of our country's main grape cultivation area belongs to arid and semi-arid climates,drought is one of the main factors restricting the development of our country's grape industry.To ensure the healthy development of our country's grape industry,the research on the effects of drought stress on grape,the formulation of rational irrigation system and the breeding of drought-resistant varieties have become the research hotspots in recent years.Due to the extensive effects of drought stress on grape,in older to cope with drought stress,grapes have evolved many regulatory mechanisms to balance the effects of drought stress in grape growth.Firstly,the water demand degree of grape in different growth period was analyzed according to the law of grape water consumption,and the effects of drought stress on main physical and chemical indexes were exhibited,including photosynthesis,osmotic regulation and reactive oxygen regulation.And then,the quality and yield of grape fruit were analyzed under drought stress,the effects of drought stress on grape fruit quality were reviewed.We give the prospect for how to use drought stress strategy to improve grape quality and how to further study the molecular mechanism of grape response to drought stress at last.

In order to provide a theoretical basis for rational application of organic fertilizers and ensure the healthy development of soil ecosystems in corn fields,the effects of soil fungal communities under different levels of organic fertilizer application for the composition,diversity,and functional characteristic were studied.In the meantime,a field experiment was used,with a total of 4 treatments were set up,and the high-throughput sequencing and FUNGuild method were used to analyze the soil fungal diversity,community structure and functional groups in different fertilization rates.The results showed that the increased application of organic fertilizer could increase the content of soil organic matter,available phosphorus,available potassium,urease,catalase,etc.,and it was positively correlated with the application amount of organic fertilizer.Applied organic fertilizers could increase the diversity of soil fungal communities and reduce their richness.The dominant fungal communities in the soil under different fertilization rates were Ascomycota,Mortierellomycota,Basidiomycota,and Olpidiomycota,the relative abundance of Ascomycota and Basidiomycota was significantly higher in the treatment with a medium amount of cattle manure than in the control without organic fertilizer;from the perspective of fungal genera,the dominant genera include Fusarium,Humicola,Olpidium,and Microidium.The application of organic fertilizer increased the relative abundance of symbiotic and saprophytic nutrients,and with the increase of organic fertilizer application,the abundance of pathological nutrients showed a decreasing trend;the number of woody saprophytic fungi in the treatment with increased organic fertilizer application was significantly higher than that in the treatment without organic fertilizer application,while the number of plant and animal pathogens was lower than the control.Therefore,it is believed that increasing a certain amount of organic fertilizer application could optimize the soil microbial environment and promote the increase of corn plant yield.

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

In order to determine the suitable sowing date of green manure for multiple cropping rape in Hetao irrigation area,field experiments were carried out in Hangjinhou Banner,Bayannur City,Inner Mongolia Autonomous Region in 2019 and Dalad Banner,Ordos City in 2022.There were five sowing dates in Hangjinhou Banner experimental site from July 26th to August 15th,2019,and one sowing date was set every five days,which were sown on July 26th,July 31st,August 5th,August 10th and August 15th,respectively.Similarly,from July 22nd to August 11th,2022,the experimental site in Dalad Banner was set up with five sowing dates,and one sowing date was set every five days to study the effects of different sowing dates on the green manure biomass,nutrient content and nutrient accumulation of rape.The results showed that although the biomass level and climate conditions of the two experimental sites were different,the overall trend showed a decline with the delay of sowing date.Compared with the first sowing stage,the rape biomass in the fifth sowing stage decreased by 90.3% and 75.4% respectively,and the average active accumulated temperature,effective accumulated temperature and sunshine hours in the two-point growth period decreased by 469.9 ℃,409.9 ℃ and 179.1 h,respectively.At the same time,when sowing in the first,second and third stages,compared with the nutrient input (N 27.3 kg/ha,P₂O₅ 34.5 kg/ha),the nutrient input of rape was higher than the input,and the amount of carbon and potassium returned to the field was at least C1 800 kg/ha and K₂O 200 kg/ha.Therefore,considering the biomass of rape green manure and the amount of nutrients returned to the field,rape could be sown early after wheat harvest in Hetao irrigation area,making full use of light and temperature resources to promote rape growth and nutrient accumulation.In order to achieve more than 5 t/ha of rape returning to the field and obtain a higher amount of nutrients returning to the field,at least 1 300 ℃ of active accumulated temperature,1 100 ℃ of effective accumulated temperature and 640 h of effective sunshine hours should be accepted during the rape growth period.

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

To analyze the same protein of whey and milk fat globule membrane (MFGM)of jersey yak crossbreeds milk and yak milk by proteomics method. The milk yield of jersey yak crossbreeds was higher than that of yak. If the function of jersey yak crossbreeds milk is like that of yak milk,it will improve the economic level of local herdsmen. Whey and milk fat were separated by centrifugation,and proteins were extracted respectively. The same protein was obtained by qualitative and quantitative analysis of protein by tandem mass tag (TMT)technique. GO functional annotation,KEGG metabolic pathway,protein interaction and other bioinformatics analysis were performed on the same protein. Result analysis indicated that 651 whey proteins and 990 MFGM proteins were identified in the peak lactation period of jersey yak crossbreeds milk and yak milk by label quantitative proteomics,and 298 whey proteins and 283 MFGM proteins were screened. GO annotation analysis showed that the same proteins of jersey yak crossbreeds milk and yak milk whey were mainly involved in the negative regulation of endopeptidase activity,immune response and immunoglobulin production. The same proteins of MFGM were mainly involved in the negative regulation of endopeptidase activity and complement activation. The cell components of the same proteins of whey and MFGM in the classical pathway were mainly extracellular space and extracellular region. The molecular functions involved were mainly guanosine 5'-triphosphate (GTP)enzyme activity and GTP binding. KEGG enrichment analysis showed that the main enrichment pathways of the same proteins in jersey yak crossbreeds milk and yak milk whey and MFGM included complement and coagulation cascades,phage and protein processing in endoplasmic reticulum. During the peak lactation period,the proteins with higher abundance in the milk of jersey yak crossbreeds and yak showed excellent performance in immunity and promoting nutrient absorption. However,the milk yield of jersey yak crossbreeds was higher than that of yak,which would increase the economic income of local herdsmen and provide more valuable plateau dairy products for people.

Wheat yellow dwarf disease,caused by Barley yellow dwarf viruses(BYDVs),is an important viral disease in wheat production.BYDV GAV has become the main pathogen causing wheat yellow dwarf disease.Till now the studies on the function of BYDV GAV encoded proteins P1,P2 and CP are lacking.We focued on the function of P1,P2 and CP,which could lay the foundation for the pathogenic mechanism of BYDV GAV.Phylogenetic analysis of BYDV GAV encoded P1,P2 and CP was conducted using Mega 7.0.We constructed the YFP expression vector of P1,P2 and CP,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.The subcellular localization of P1,P2 and CP was observed using confocal laser scanning microscopy(CLSM).We constructed the biomolecular fluorescence complementation assay(BiFC)vectors of five coding proteins,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.CLSM was used to observe the interaction of P1,P2 and CP and other viral proteins in vivo.Furthermore,we constructed the Potato virus X(PVX) expression vectors of P1,P2 and CP,transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.At 5 days post inoculation(dpi)the symptom formation of PVX infection was observed.The systemic leaves were collected for detection of viral accumulation to determine the effects of the pathogenicity of P1,P2 and CP.Results showed that BYDV GAV was most closely related to BYDV PAV at the nucleotide level.Subcellular localization of P1,P2 and CP was cytoplasm and nuclear.P1 interacted with itself in vivo using BiFC.In the pathogenicity assay,the systemic leaves of PVXCP infection showed chlorosis at 5 dpi,and PVX accumulation was detected,while PVX,PVXP1 and PVXP2 infection showed no symptoms in systemic leaves and PVX accumulation was undetectable,which was detected at 10 dpi,indicating that CP promoted the formation of PVX symptoms.In brief,P1 possibly involves in viral infection via self-interaction in vivo,and CP can promote viral infection.

In order to clarify the sequence characteristics of the MYB62 transcription factor,expression changes after stress,and further explore the biological function of the MYB62 transcription factor,kohlrabis were used as plant materials.MYB62 transcription factors were obtained by homologous cloning method and bioinformatics was analyzed.Spatiotemporal expression analysis of MYB62 and its expression after stress were performed by Real-time PCR.Results of gene cloning showed that gDNA length of BocMYB62 gene was 1 353 bp,the length of CDS was 837 bp,which contained four exons and three introns and encoded 278 amino acids.The sequence structure analysis showed that BocMYB62 was a hydrophilic protein with two SANT-MYB domains,belonging to the R2R3-MYB type in the MYB transcription factor family.The spatial structure prediction revealed a typical α-helix structure.Phylogenetic analysis indicated that BocMYB62 was closely related to MYB62 in Brassica napus.The spatiotemporal expression results showed that BocMYB62 was consistently higher in green kohlrabi than that in purple with clear tissue specificity.The expression of BocMYB62 was significantly increased during drought stress with the highest expression under 12 h.The BocMYB62 expression was significantly lower than control and the lowest at 4 ℃ cold stress.These results inferred that BocMYB62 may be involved in the regulation of anthocyanin biosynthesis and the regulation of stress.It provides a theoretical basis for the subsequent functional identification of MYB62 transcription factors.

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.

In order to clarify the effects of high temperature stress during grain filling stage on leaf senescence and yield,a field experiment from 2019 to 2020 was conducted with employing heat-sensitive variety(XY335)and heat-resistant variety(ZD958). Simple plastic greenhouse was constructed for heat treatment(HT)during 12-25 days after silking,the natural temperature was set as control(CK). The two years results showed that from 12 to 25 days after silking,high temperature stress significantly reduced the grain weight and yield of summer maize. For ZD958,the grain yield of HT was reduced by 18.8% and 19.3% than CK and thousand kernels weight(TKW)was decreased by 23.4% and 9.1%,respectively.For XY335,the grain yield and TKW of HT were reduced by 29.4%,26.3% and 20.1%,14.2%,respectively.High temperature during grain-filling stage accelerated leaf senescence. The two years results showed that leaf senescence rate(LSR)of ZD958 and XY335 under HT were increased by 76.4%,140.6% and 135.1%,139.6%,respectively.Leaf area duration(LAD)after silking with HT was reduced by 17.7%-36.5% compared with CK. Ear leaf photosynthesis under HT was lowered by 20.0%-42.9% than CK.Correlation analysis results indicated that accelerated LSR under HT had negative correlation with grain yield but without statistical significance(P=0.064 7).LAD after silking,photosynthesis,and SPAD significantly reduced under HT,which showed significant positive correlation with grain yield(P< 0.05). Taken together,heats tress during grain filling significantly accelerated leaf senescence,thus lowering kernel weight and resulting in yield loss. Under high temperature stress,heat-resistant maize variety had higher regulation ability than high temperature-sensitive maize variety,which could significantly mitigate yield loss caused by high temperature stress.

In order to study the response mechanism of pepper CaWRKY30 transcription factor and Tomato spotted wilt orthotospovirus,it was experimental materials with pepper Xiangyan 11.The CaWRKY30 coding sequence was obtained by RNA extraction,RT-PCR,split gel and cloning.Biological information analysis results showed that CaWRKY30 full length was 1 122 bp,encoding 373 amino acids,the gene encoded protein contains 1 WRKY conservative domain and 1 C₂H₂ domain,belonged to a typical Ⅱ(e)subfamily member.System evolution analysis showed that the relative relationship with the potato StWRKY22 amino acid sequence was recently.It was found that CaWRKY30 was positioned in the nucleus and cell membranes in its cigarette seedlings,and leads to cell membranes.The results of Real-time fluorescence quantitative PCR analysis showed that the viral accumulation of Tomato spotted wilt orthotospovirus mechanical friction-vaccination was found that the viral accumulation was gradually increased from 1 to 14 days after inoculation,and virus accumulation reached its maximum in 14 days,after inoculation 14 days,viral accumulation gradually declined.At the same time,CaWRKY30 was induced by Tomato spotted wilt orthotospovirus,when the inoculation 1—14 days,the CaWRKY30 expression was raised,and the peak was reached in 14 days,the expression in 14 days gradually decreased.In summary,it obtained the CaWRKY30 transcription factor gene sequence,which was located in the nucleus and cell membrane,and preliminarily explained the expression trend of CaWRKY30 transcription factors under the stress of Tomato spotted wilt orthotospovirus.

In order to clarify the relationship between the morphological and structural characteristics of stem basal node 2 and lodging resistance in wheat,we explored key stem morphological indicators and Quantitative trait loci(QTL)sites for lodging resistance.120 RILs families were selected as research materials,and stem strength,basal second internode length,stem diameter,wall thickness,cellulose content and lignin content were measured in 2020 and 2021,respectively.Multiple regression analysis and QTL locations were performed by combining 55K SNP data.The results showed that the stem strength was significantly or extremely significantly positively correlated with the stem diameter and wall thickness of the second basal internode,and was extremely significantly positively correlated with the cellulose content and lignin content of the second basal internode.Multiple regression analysis showed that cellulose content in basal second internode was the key index affecting stalk strength of wheat.A total of 19 QTLs related to stem traits were detected on chromosomes 1A,1D,2B,2D,4D,5A,5B,5D and 7B,explaining 7.67% to 65.33% of the phenotypic variation.On chromosome 1D,the QTL linked to AX-110771095 and AX-109431570 simultaneously controlled the basal second internode length,wall thickness and cellulose content,explaining the phenotypic contribution of 7.96%-10.76%.

To explore the genetic relationship of alfalfa germplasm resources and provide theoretical basis for the identification of alfalfa varieties and the breeding of new varieties.Genetic diversity of 30 alfalfa materials was analyzed by ISSR and SSR markers.A total of 125 bands and 117 polymorphic bands were amplified by 12 ISSR labeled primers.The polymorphic band ratio(PPB)was 93.01%.The average effective allele number(Ne),Nei's gene diversity index(H)and Shannon diversity index(I)were 1.465 9,0.281 3 and 0.431 4,respectively.A total of 152 bands and 144 polymorphic bands were amplified by 12 SSR markers,and the polymorphism band ratio(PPB)was 94.05%.The average effective allele number(Ne),Nei's gene diversity index(H)and Shannon diversity index(I)were 1.542 7,0.313 9 and 0.470 1,respectively.The genetic similarity coefficient and cluster analysis of the two markers showed that Zxy2010p-7900 and Zxy2010p-7740 could be divided into a separate category and were far related to other materials.Qingshui alfalfa,Longdong alfalfa and Gannong No.4 mixed alfalfa can be divided into one group,the genetic similarity coefficient between the varieties is large,and the relationship is close.The phylogenetic relationship of the tested alfalfa germplasm was objectively determined by the two labeling methods.

Genomic-wide identification of systematic work on the SKS gene family in cotton has not been reported.Identifying SKS (Skewed5 similar) gene family members and analyzing evolutionary relationship in Gossypium hirsutum,and analyzing the mechanism of GhSKS13 gene regulating cotton disease resistance are expected to provide a new direction for the cultivation of resistant plants.Here,the members of upland cotton SKS family were identified by bioinformatics at the genome-wide based on published genomic data of upland cotton genetic standard line TM-1,and G.hirsutum variety CRI-14 was used as the material.And chromosome distribution,evolutionary relationships,gene structure,and collinearity of SKS family members were predicted.Then,the expression pattern of GhSKS13 was analyzed by Real-time Quantitative PCR and virus-induced gene silencing was employed for preliminary investigation of the function of GhSKS13 in cotton resistance to Verticillium dahliae.A total of 48 upland cotton SKS genes were identified,unevenly distributed on 19 chromosomes,clustered into 5 subgroups with highly conserved gene sequences.Collinearity analysis revealed that the upland cotton SKS gene family was subject to purifying selection.Expression pattern analysis showed that GhSKS13 was predominantly expressed in upland cotton root tissues and significantly up-regulated by V.dahliae infection. GhSKS13-silenced reduced plants resistance to V.dahliae and suppressed the expression of the pathogen-related genes including GhPR1, GhPR2,GhPR3 and GhPR5 compared to control plants. GhSKS13-silenced plants invaded by V.dahliae showed significantly lower hydrogen peroxide (H₂O₂) deposition compared to the control,suggesting that GhSKS13 promotes the formation of reactive oxygen species (ROS).In conclusion,this study clarified the phylogenetic relationships,chromosomal distribution characteristics and gene structure characteristics of upland cotton SKS family members,and elucidated the involvement of GhSKS13 in upland cotton plant resistance to V.dahliae.

The plant NADPH oxidase Rbohs(Respiratory burst oxidase homologs) is the main source of reactive oxygen species (ROS),which participate in various physiological processes such as plant growth,development,stress resistance and plant-microorganism interaction.In order to explore the function and mechanism of Rbohs in symbiotic nitrogen fixation,GmRbohL,a member of soybean Rbohs gene family,was cloned in this study.The gene expression pattern,protein subcellular localization and gene function were studied by molecular biology,cell biology and genetics,respectively.The results revealed that: GmRbohL gene was induced by rhizobia and expressed specifically in soybean roots and nodules.Subcellular localization analysis indicated that the gene-encoded protein GmRbohL was a membrane protein.The plant gene silencing (RNAi) vector of GmRbohL was constructed,and the transgenic hairy roots were obtained by the transformation of soybean hairy root mediated by Agrobacterium rhizogenes K599.Gene silencing of GmRbohL resulted in a significant reduction in the number of nodules of transgenic hairy roots,and the production of ROS was also inhibited. Gene silencing of GmRbohL reduced the infection of rhizobia at the stage of root nodule organogenesis,and the expression level of nodulation marker genes also decreased with the decrease of GmRbohL expression.The root nodule tissue sections showed that gene silencing of GmRbohL significantly reduced the number of symbionts in the infected area of root nodules,and the nitrogenase activity of root nodules also decreased accordingly.The above data indicated that gene silencing of GmRbohL significantly inhibits the symbiotic nodulation process of soybean by reducing the production level of ROS.It is speculated that GmRbohL may play an important positive regulatory role in organogenesis of soybean nodules and regulation of nitrogen fixation function.

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.

Photoperiod is one of important environmental factors affecting plant bolting and flowering which are regulated by plant phytochrome proteins.The signal network mediated by PHYB has an important inhibitory effect on plant bolting and flowering.Previous studies revealed that there were large segment insertion/deletion differences between the PHYB gene promoter of the Chinese cabbage late-bolting line 06-247 and the easy-bolting line He102.In order to further investigate the impact of promoter mutation on PHYB and the key genes of its downstream pathways,this study was conducted.Based on bioinformatics methods,the redundancy characteristics of phytochrome genes in genome Chinese cabbage were firstly analyzed.It was found that the Chinese cabbage genome contained six phytochrome genes,of which PHYA had two copies,and PHYB,PHYC,PHYD,and PHYE all had only one copy.Then amino acid sequence alignment was used for screening of the specific sequence of PHYB.Antigenic determination clusters were designed based on the specific sequence and the antibodies against PHYB was prepared.The fluorescence quantitative RT-PCR technology and Western Blot technology were used to study the relative content of PHYB in 06-247 and He102.At the same time,the dynamic changes of key regulatory genes such as CCA1,FLC,CO and FT which in the downstream of PHYB pathway were also compared.The results showed that the promoter mutation caused significant differences both in level of mRNA in 06-247 and then significantly increased the protein level of PHYB.At the same time,the downstream regulatory genes such as CCA1,FLC,CO and FT were highly expressed in 06-247,which had an important impact on bolting resistance of 06-247.

In order to promote the breeding of high quality grain shape Japonica rice varieties,a natural population consisting of 295 Japonica rice varieties collected from around the world was used as test material for phenotypic analysis of five grain shape-related traits,including grain length,grain width,grain thickness,aspect ratio and thousand grains weight,between 2018 and 2019,combined with 788,396 SNPs obtained by resequencing,and genome-wide association analysis using the MLM model of TASSEL 5.0.Haplotype analysis was performed on all the genes in the QTL intervals that were jointly detected during the two years and controlled multiple shape-related traits, and candidate genes for high quality rice grains were mined by combining the results of previous studies and functional annotation of the genes. The results showed that there was extensive phenotypic variation for the five grain shape-related traits,all of which were approximately normally distributed,and most of the grain shape-related traits were significantly or highly significantly correlated with each other. And a total of 221 QTLs significantly associated with rice grain shape-related traits were detected under the threshold condition of P≤5.46×10^-6,which were distributed on all 12 chromosomes of rice,and phenotypic contribution was 8.62%-20.73%, of which, seven QTLs were jointly detected in two years, and combined with the haplotype analysis and functional annotation of the genes, it was hypothesized that LOC_Os12g 44290 was a new candidate gene for rice grain shape. In summary,this study used genome-wide association analysis for QTL localization and candidate gene analysis for grain type-related traits in 295 Japonica rice varieties,providing a theoretical basis for breeding Japonica rice varieties with high quality grain types.

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

In order to reveal the influence mechanism of sowing date adjustment on crop yield formation, a field experiment was conducted to adjust the sowing date of the same variety of summer maize from 2019 to 2021 at Hebei Gucheng Agricultural Meteorology National Observation and Research Station in Northern north China, which was set up in four sowing date, as 10 d early sowing, 10 d late sowing, 20 d late sowing and control. By monitoring the growth period changes, plant dry matter accumulation, leaf photosynthetic characteristics and grain filling characteristics, and sampling at maturity to determine the yield components, the results showed that:the sowing date of summer maize was appropriate to be advanced,the seedling stage,ear stage and the whole growth period were extended,especially,the duration of effective filling was extended with the advance of sowing date.The effective grain filling duration days were extended by 4.7 days for every 10 days ahead of the sowing date.The average filling rate of winter wheat was 4.04% higher on the sowing date was advanced by 10 days, compared with the sowing date was deferred by 10 or 20 days and the control.Grain filling accumulation increased,and 100-grain weight increased by 5.459 g.The main yield components of summer maize,such as grain number per ear,grain weight per ear and 100-grain weight showed significant differences during sowing,and increased with the sowing date.The theoretical yield increased by 1 395.4 kg/ha with 10 days ahead of the sowing date during the experimental period from June 8 to July 8.The average net photosynthetic rate(Pn)of leaves in the critical development period of summer maize increased by 0.764 μmol/(m²·s)with every 10 days ahead of sowing date,and the average Pn of sowing date 10 days earlier was increased by 7.31% compared with the control and late sowing 10 and 20 days.With the increase of photosynthetic rate,dry matter production,accumulation and transfer to grain were increased,and the grain weight per ear and 100-grain weight increased by an average of 24.01% and 18.00%,respectively,compared to the control and late sowing 10 and 20 days.Advancing sowing date resulted in low plant height,strong stems,lodging resistance,large individual green leaf area,high leaf area index(LAI),and high leaf photosynthesis ability,above-ground dry matter allocation rate at maturity stage every 10 days in advance of sowing date:grain weight improved by 2.26%,the source—pool distribution of dry matter between plant vegetative organ and ear changed,ear weight per ear,100-grain weight and grain yield increased.The research showed that the double cropping area of winter wheat—summer maize in north China could effectively improve the yield per unit area by making full use of the heat resources increased by climate warming,reasonably allocating stubble,sowing summer maize early at the appropriate time,and extending the development period and grain filling time.

In order to investigate the structural characteristics of Brassica juncea AHK family proteins and their regulatory functions related to seed weight traits,it employed bioinformatics methods to perform whole-genome identification,physicochemical properties analysis,phylogenetic tree construction,protein structure analysis,promoter cis-elements analysis,and differential tissue expression profiling of the BjAHK family members.The main findings were as follows:a total of 19 BjAHK protein sequences were identified from the B.juncea genome,and phylogenetic analysis classified the BjAHK family members into four branches:G1 (BjAHK2—BjAHK4),G2 (BjAHK5),G3 (BjAHK1),and G4 (BjCKI1).Analysis of functional domains revealed the presence of six conserved motifs (Motif1—Motif6) in BjAHK family members,corresponding to the HisKA,HATPase_c,and REC conserved domains.The N-terminal regions of BjAHK1—BjAHK4 and BjCKI1 proteins exhibited relatively conserved transmembrane domains,which might be associated with their transmembrane function.Differential tissue expression profiling revealed that the genes encoding cytokinin receptor proteins (BjAHK2—BjAHK4) were constitutively expressed genes,with the highest expression levels observed in root tissues.Through integration with existing transcriptomic data on seed weight traits in B.juncea,four candidate genes potentially involved in the regulation of seed weight traits were identified:BjA07.AHK3,BjB03.AHK3,BjA10.AHK5,and BjB05.AHK5.BjA07.AHK3 and BjB03.AHK3 might positively regulate seed development,while BjA10.AHK5 and BjB05.AHK5 might have the opposite effect.Based on the integration of gene family analysis and transcriptome result,it was inferred that BjAHK3 and BjAHK5 genes play an important role in the regulation of seed development in B.juncea.

To explore the function of the TCP gene family in the growth and development of Jasminum sambac,bioinformatics methods were used to identify and analyze the whole genome of J.sambac TCP(JsTCP)gene family.Moreover,the expression levels of TCP gene family in different stages of flower development and pollen-stigma interaction were analyzed.A total of 27 TCP gene family members were identified from the whole genome of J.sambac,named JsTCP1—JsTCP25.The protein contained 208—539 amino acid residues,with molecular weight of 22.95—56.96 ku and isoelectric point of 5.70—9.97,and all of them were unstable hydrophilic proteins.The subcellular localization prediction showed that all JsTCPs were located in the nucleus.JsTCPs were unevenly distributed on 10 chromosomes.Gene structure analysis showed that JsTCPs had 1—5 exons and 0—4 introns.Protein conserved motifs and phylogenetic analysis showed that JsTCPs contained conserved TCP domain,and they were divided into two subclasses:Class Ⅰ and Class Ⅱ.Promoter of JsTCPs contained cis-acting elements related to plant hormone response,stress response,and growth and development.The expression pattern analysis showed that 24 JsTCPs were expressed at different stages of flower development,and 22 genes were expressed in pollen-stigma interactions.In conclusion,27 TCP gene family members were identified from J.sambac,and it found that they expressed specifically at different stages of flower development and different stages after pollination.

As one of the main products of photosynthesis,sucrose has a variety of physiological functions in plants.As a group of glycosyltransferase enzymes,sucrose synthase plays an important role in the process of sucrose metabolism.In plants,sucrose synthase reversibly catalyzes the decomposition of sucrose and nucleoside diphosphate(NDP)to produce nucleoside diphosphate glucose(NDPG)and fructose.This reaction is the most important rate-limiting step in sugar metabolism.Sucrose synthase exists in the form of a gene family in plants.Phylogeny shows that there are three subgroups of sucrose synthase,and different subgroups have different expression patterns.Recent studies have shown that sucrose synthase has several major physiological functions in plants,such as participating in the synthesis of macromolecular sugars,response to adversity,participating in reproductive growth and regulating fruit quality.This article reviews the research progress of sucrose synthase in recent years,and looks forward to future research directions in order to provide a reference for future research.

Fruit development is the key stage which determines the yield and quality of grape.WRKY family transcription factors play important roles in regulating plant development and environment adaptation.Ethylene is the important plant hormone which participates in regulation of fruit development,and ACC synthase is the key enzyme that limits ethylene synthesis.Using grape variety Zuoyouhong, VvWRKY13 overexpressing grape callus,as well as VvWRKY13 heterologous over-expressed tomato lines as materials,the role of VvWRKY13 from grape in fruit development and its relationship with ethylene were studied though plant physiological and biochemical methods as well as molecular biological techniques.The results displayed that the expressions of VvWRKY13 as well as ACC synthase genes VvACS2 and VvACS7 significantly upregulated at early stage of fruit development,and in VvWRKY13 over-expression grape callus,the expression of VvACS7C was significantly higher than control,but the expression of VvACS2 displayed no significant difference compared with control.Yeast one hybrid experiment showed that VvWRKY13 could directly bind to the VvACS7 promoter,VvWRKY13 had no direct interaction with VvACS2.We also found that the ethylene content and the expression of ACS family members,such as SlACS1b,SlACS4 and SlACS6,the ethylene synthesis genes,were significantly induced in VvWRKY13 heterologous over-expressed tomato,and the time from flowering to breaking of tomato was 3—6 days shorter than wild type;the above results indicated that,VvWRKY13 could regulate ethylene synthesis by promoting the expression of ACC synthase gene to participate in regulation of fruit development.

Breeding resistant varieties is the most economical and feasible method to control wheat leaf rust.In order to further explore the resistance genes,50 wheat varieties were selected in this study,including wheat varieties from Shandong,Henan,Hebei,Shandong and other 8 provinces.Firstly,16 physiological races of leaf rust fungus (THFS,TGTS,THJS,FHKT,FGJN,KHKS,FCJQ,RFKS,THFM,MHGT,KHGS,KBGT,FHGT,PHHT,FHJT,FCJT) were inoculated to 36 vector cultivars containing known leaf rust resistance genes,and 50 wheat varieties tested during the seedling stage.Due to the different virulence of each strain,specific molecular markers closely linked to known disease resistance genes can be combined and analyzed based on the differences in phenotype,and it can be inferred that 50 wheat materials may contain leaf rust resistance genes.The leaf rust resistance genes were identified by genetic deduction,molecular markers and pedigree analysis.The results showed that a total of 9 known resistance genes (Lr1,Lr2c,Lr10,Lr16,Lr26,Lr34,Lr37,Lr45 and Lr46) and a few unknown genes were detected in 50 cultivars.There were 22 varieties including Zimai 12 that contained the Lr1 gene;there were 10 varieties including Lumai 14 that contained the Lr2c gene;Laizhou 9361 only contained the Lr10 gene;there were 25 varieties including Kenong 199 that contained the Lr16 gene;there were 15 varieties including Xuzhou 24 that contained the Lr26 gene;Baomai 3 and Jingdong 8 contained the Lr34 gene;The Lr37 gene was contained in Zimai 12,He 0927,and He 9946;there were 11 varieties including Lianmai 2 that contained the Lr45 gene;there were 38 varieties including Shannong 19 that contained the Lr46 gene.

In order to explore the gene expression regulation mechanism in the formation of reactive oxygen species (ROS) induced by heat stress,heat stress treatments were set at seedling,heading and filling stage to investigate the dynamic change of ROS accumulation in rice,respectively.And the Quantitative Real-time PCR (qRT-PCR) was used to analyze the expression pattern of nine respiratory burst oxidase homologue (Rboh) encoding genes (OsRboh1-OsRboh9) in rice under different growth stages.Results showed that the ROS accumulation in rice leaves and grains significantly increased with the extension of heat stress.The ROS content increased slowly after 7 days of heat stress at seedling stage, while increased continuously after heat stress during the heading stage and early filling stage (1-10 d) in rice grains. Expression levels of the nine OsRboh family genes continuously increased with the extension of heat stress at seedling and heading stages,and OsRboh7 and OsRboh5 showed higher expression levels under heat stress.The expression levels of OsRboh1,OsRboh5 and OsRboh9 were continuously increased,while other genes showed a change tendency of increasing initially and then decreasing under heat stress at filling stage.The expression levels of OsRboh7 and OsRboh5 were all reached to a high level at seedling,heading and filling stage under heat stress.Furthermore,higher expression levels of OsRboh7 and OsRboh5 were showed in various tissues and organs of rice such as seedling leaves,flag leaf,floret,lemma,palea,stamen,pistil and grain.The higher induced range of gene expression levels in OsRboh7 and OsRboh5 by heat stress was shown in seedling leaves,floret,stamen,pistil and grain.Taken together,OsRboh7 and OsRboh5 were mostly remarkably responsive to heat stress at different growth stages among the OsRboh family genes in rice,which indicated that OsRboh7 and OsRboh5 played a vital role in the regulation of ROS formation pathway in rice under heat stress condition.

Obtaining the LaeA gene sequence of Ganoderma lucidum through electronic cloning,analyzing its gene sequence information and preliminarily exploring its regulatory function.This study adopts the method of electronic cloning,using the known LaeA protein sequence of Penicillium citrinum as a template,perform sequence similarity search and alignment(Blast)in the EST database of Ganoderma lucidum and obtain the cDNA sequence of the LaeA gene of Ganoderma lucidum through electronic cloning methods such as sequence splicing,sequence validation and sequence extension.Some characters of amino acids encoded by LaeA gene,including the physical and chemical properties,hydrophobicity/hydrophilicity,subcellular localization,secondary and tertiary structure of protein,and phylogenetic relationship were analyzed by bioinformatics tools.The length of LaeA gene from G.lucidum was 1 134 bp,encoded 378 amino acids.The protein encoded molecular weight of 42.895 3 ku.The protein was an instability protein that was present in the cytoplasm and not secreted to the extracellular.Furthermore,the structure of LaeA protein was mainly composed by 47.88% random coil,33.33%α-helix and 18.78% extension strand,contained SAM binding site,belonged to AdoMet_MTases superfamily proteins.Phylogenetic analysis showed that LaeA was closely related to white rot basidiomycetes such as Trametes versicolor and Dichomitus squalens;the Real-time Fluorescence Quantitative PCR results showed that the expression level of LaeA in Ganoderma lucidum cells during liquid static culture was significantly higher than in oscillatory culture.It is speculated that the LaeA protein as a methyltransferase protein that participates in histone methylation modification,thereby affecting the expression level of gene clusters.

By analyzing different materials of Medicago sativa L. and Medicago falcata L.,this study aims to observe the leaf morphology and tissue anatomical structure of alfalfa under drought stress,and explore the adaptive changes in leaf morphology and anatomical structure of alfalfa seedlings under natural drought stress.This study used three alfalfa and two Medicago falcata L. as research materials,and set four different soil relative moisture contents(70%,55%,40%,and 25% of the maximum soil moisture capacity)for experiments. After 30 days of treatment,the morphological and anatomical structures of the leaves were determined. To investigate the response of alfalfa seedling leaves to natural drought stress by observing the leaf morphology and anatomical structure of purple and sickle alfalfa seedlings through electron microscopy. Drought stress would inhibit the growth of alfalfa leaves. With the increase of drought stress,the soil moisture decreased,the leaf length,leaf width,and leaf area of alfalfa and sickle alfalfa decreased,the thickness of upper epidermis increased,the thickness of leaf,mesophyll,and lower epidermis decreased,the thickness of palisade cell and sponge tissue decreased,the thickness of midrib center decreased,the palisade sea ratio gradually decreased,and the compactness of leaf tissue structure increased,the degree of looseness decreased and the degree of leaf vein protrusion increased. The intensity of drought stress was negatively correlated with leaf length,leaf width,leaf area,leaf thickness,mesophyll thickness,lower epidermis thickness,palisade cell thickness,sponge tissue thickness,midvein center thickness,palisade sea ratio,and leaf tissue structure porosity.It was positively correlated with the thickness of the upper epidermis,the tightness of leaf tissue structure,and the degree of leaf vein protrusion.

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

Calmodulin is an important Ca²⁺ receptor protein in plants,which plays an important role in calcium signaling pathway and stress resistance.It is of great significance to study the mechanism of CaM protein in tomato under low temperature stress for cold tolerance breeding of tomato.Tomato calmodulin genes SlCaM3, SlCaM4 and SlCaM5 were cloned from tomato varieties Heinz1706,LA3969,Jifen 2,Jifen 3 and Nongbofenba 15,and their sequences and protein sequences were analyzed by bioinformatics;the cis acting elements in the promoter regions of SlCaM3,SlCaM4 and SlCaM5 were analyzed by plantcare;Quantitative Real-time PCR was used to analyze the expression patterns of SlCaM3,SlCaM4 and SlCaM5 under 15,5 ℃ temperature stress in different tomato varieties,and the specific expression of SlCaM3,SlCaM4 and SlCaM5 in different tissues was analyzed in combination with RNA-seq data.The results showed that the coding sequence of SlCaM3,SlCaM4 and SlCaM5 were 450 bp,and their similarity was 93.63%;the encoded amino acid sequences were identical,belonging to acidic stable hydrophilic protein with typical conservative domain of cam protein.The analysis of cis acting elements showed that the promoter regions of the three genes contained not only the necessary core elements,but also a variety of biotic and abiotic stress response elements,and showed a complementary pattern.The analysis of the expression patterns of different degrees of low temperature stress showed that the expression patterns of SlCaM3,SlCaM4 and SlCaM5 in five tomato materials showed a trend of first decreasing and then increasing at 15 ℃,and the expression of SlCaM5 increased more significantly.At 5 ℃,SlCaM3 and SlCaM4 did not change significantly,but the expression level of SlCaM5 gene increased significantly in the later stage of treatment.The high expression of SlCaM5 gene in the process of tomato resistance to low temperature indicated that it maintained the translation level of cam protein under low temperature and ensured that the function of CaM protein was not affected.The analysis of SlCaM3,SlCaM4 and SlCaM5 specific expression in different tissues of Heinz1706 showed that SlCaM3 and SlCaM4 were highly expressed in meristem,while the expression of SlCaM5 different tissues was not significantly different.

To further investigated the effect of different allelic variants of TaGAMyb-B genes in wheat on stem elongation, used the rice Agrobacterium transformation system,RT-qPCR,tissue section and cell tissue specific analysis to systematically study the function of the 84 bp InDel of TaGAMyb-B.The results showed that,in the over-expressed transgenic rice lines,transcript expression of TaGAMyb-B was detected in seeds,roots,stems and leaves;TaGAMyb-Bb-GFP transgenic seeds were more sensitive under the treatment of NaCl,GA and mannitol compared with TaGAMyb-Ba-GFP;the diameters of first,second and third stem inter-nodes,spike length and tiller number of TaGAMyb-Ba-GFP were significantly larger than that of TaGAMyb-Bb-GFP;and the analysis of cell tissue sections showed that the average thickness of the thick-walled tissue cells in the transverse sections of the transgenic TaGAMyb-Ba-GFP rice was significantly greater than that of the transgenic TaGAMyb-Bb-GFP event,the average length of thick-walled cells was extremely significantly shorter than that of transgenic TaGAMyb-Bb-GFP event.The above mentioned results indicated that the 84 bp deletion in TaGAMyb-B increased not only abiotic stress resistance and plant lodging resistance,but also spike length and tiller number in transgenic rice.