Glyphosate is currently the most widely used broad-spectrum herbicide.Cultivating glyphosate tolerant crops will help improve the effectiveness of chemical control on weed in farmlands,reduce the use of pesticide,and simplify preventive and control measures.To fully detect the glyphosate tolerance(GT)loci in wheat,484 germplasm resources from the Huang-huai wheat region were used to identify glyphosate toxicity.Based on the wheat 15K SNP array data,genome-wide association analysis(GWAS)was used to explore QTL related to glyphosate tolerance in wheat.The main results were as follows:the trend of changes in glyphosate tolerance of wheat varieties cultivated in different eras was slow,and the glyphosate tolerance had not significantly improved;three glyphosate tolerant wheat germplasms(including Henong 130,Jimai 782 and Taishan 23)were selected based on the phenotypic identification results of pesticide damage;seven QTL associated with the level of wheat pesticide damage were detected by GWAS,including 19 significant SNPs,distributed on wheat chromosomes 1A(0.00—30.48 Mb),1B(6.57—30.57 Mb),1D(0.00—22.98 Mb),4A(656.09—680.09 Mb),5A(508.19—532.19 Mb),6A(54.56—85.09 Mb),and 6D(12.02—36.02 Mb);the two QTL qGlyT-1A and qGlyT-6A located on wheat chromosomes 1A and 6A were the main effector sites for glyphosate tolerance in wheat,containing a total of 16 genes that may be related to glyphosate tolerance in wheat.

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

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 transcription factor BnHY5-2 is associated with plant stress resistance.In order to reveal the response of Brassica napus L.transcription factor BnHY5-2 to salt alkali stress in B.napus,the response of BnHY5-2 to light and salt and alkali was analyzed by transient overexpression,qRT-PCR analysis and subcellular localization.The results revealed that under light conditions,the expression level of the BnHY5-2 gene in B.napus leaves and stems was 29.22 and 3.15 fold higher,respectively,compared to dark conditions.The higher sensitivity to light in leaves suggested that they were the primary site for light signal response.Under light conditions,the expression of BnHY5-2 in leaves and stems was significantly downregulated by 53.1% and 31.0%,respectively,when B.napus was planted in Dalian coastal saline-alkali soil;after applying saline-alkali treatment under dark conditions,the expression of BnHY5-2 was downregulated by 48.2% in the stem,while the difference in expression in the leaves was not significant,indicating organ differences,indicating that the leaves had stricter requirements for light conditions.In B.napus leaves with transient overexpression of BnHY5-2,two out of six genes related to saline-alkali stress(BnNAC32 and BnGS)showed upregulation by 1.25,3.28 fold,respectively,while the other four genes(Bnamy,BnAsp,BnNHX7,BnTPS)were downregulated by 24.8%,25.4%,71.0%,and 82.0%,respectively.Meanwhile,the content of the resistance substance betaine in B.napus increased from 0.256 to 0.573 mg/g,indicating an enhancement by 1.24 fold,suggesting that the overexpression of BnHY5-2 gene could improve the saline-alkali tolerance of B.napus.Subcellular localization results showed that the transcription factor BnHY5-2 was localized in the nucleus and regulates the expression of functional genes.Therefore,BnHY5-2 is not only related to light signaling but also participates in the saline-alkali resistance of Brassica napus L.

To investigate the role of calcium-dependent protein kinase (CDPK) in wheat growth and stress response,the TaCDPK17 gene was cloned from common wheat and its sequence structure,expression pattern,and stress resistance function were preliminarily analyzed.The results showed that the length of the TaCDPK17 gene coding region was 1 701 bp, encoding 566 amino acids and possessing typical structural features of the CDPK family, including one conserved serine/threonine kinase domain and four EF hand shaped domains. Evolutionary tree analysis of TaCDPK17 and CDPK17 from 12 other plants showed that TaCDPK17 had high homology with the CDPK17 sequence of gramineous crops,especially Aegilops tauschii and barley.The promoter region of TaCDPK17 gene contained multiple cis regulatory elements related to hormone signaling pathways,light response.Among them, there are more abscisic acid (ABA) responsive elements (ABRE) and methyl jasmonate responsive elements (CGTCA). The expression analysis based on Real-time Fluorescence Quantitative PCR showed that the expression level of TaCDPK17 increased to varying degrees after induced by 100 μmol/L ABA, 100 μmol/L methyl jasmonate, 20% PEG6000, and 250 mmol/L NaCl. Under stress conditions of 2 μmol/L ABA and 100 mmol/L NaCl, the germination rate of Arabidopsis seeds overexpressing TaCDPK17 was significantly higher than that of the wild type. Meanwhile, overexpression of TaCDPK17 alleviated the inhibitory effects of ABA or osmotic stress treatments on seedling root growth. During stomatal closure, transgenic plants overexpressing TaCDPK17 are more sensitive to ABA and exhibit a stronger stomatal closure trend compared to wild-type plants. These results indicated that TaCDPK17 plays an important role in stress response and hormone signaling in wheat.

To reveal the mechanism of drought resistance of different resistant rice during germination period,Rice drought-sensitive materials(Calrose,Jingning 10,Shanxing 86)and drought resistance materials(Farry,Songjing 3,Ningjing 36)were studied on the effects of simulated drought stress(15% PEG-6000)on the growth index,physiological indexes and corresponding gene expression of different rice seeds.The results showed that under normal conditions,there were no significant differences in the expression levels of growth indicators and stress-related genes between drought-sensitive and drought-resistant cultivars.However,changes in physiological indicators were shown that there were no significant differences in the activities of superoxide dismutase(SOD) and peroxidase(POD),the contents of soluble sugar(SS) and hydrogen peroxide(H₂O₂) among different genotypes.The contents of malondialdehyde(MDA) and superoxide anion($\mathrm{O}_{2}^{\bar{.}}$) in the drought-sensitive cultivar Shanxing 86 were significantly higher than those in other materials,and the contents of catalase(CAT),proline(Pro) and soluble protein(SP) of drought resistant Ningjing 36 were significantly higher than those of other materials as well.Under drought stress,the relative germination potential(RGP),relative bud length(RSL),germination drought resistance index(GDRI)and vitality index(VI)of germinating seeds increased by 0.03—0.07 percentage,0.32—0.39 percentage,0.12—0.18 percentage and 92.41%—108.39%,respectively;MDA and reactive oxygen species($\mathrm{O}_{2}^{\bar{.}}$,H₂O₂) contents in germinating seeds of drought-resistant cultivars decreased by 2.54%—61.64%,19.60%—46.30% and 35.61%—62.02% respectively compared with drought-sensitive cultivars.The contents of osmotic regulating substances(Pro,SS,SP) increased by 5.93%—18.29%,1.08%—7.97% and 3.47%—6.03% respectively.The activities of antioxidant enzymes(SOD,POD, CAT) were increased by 17.29%—33.12%,15.24%—76.06% and 14.68%—18.61% respectively.The relative expression levels of OsP5CS,antioxidant enzyme synthesis genes (OsALM1, OsPOX1, OsCATC) were up-regulated by 2.66%—182.31% and 57.14%—513.27%,0.38%—109.06% and 63.39%—184.25% respectively.Comprehensive analysis showed that drought stress inhibited the germination of rice seeds and affected the physiological characteristics of seeds and the expression of corresponding genes during germination.Under drought stress,vigor index(VI),peroxidase(POD)and peroxidase synthesis gene(OsPOX1)are the key indicators affecting rice seed germination,whether it is drought-resistant or drought-sensitive materials.In addition to the above indicators,soluble protein(SP),proline synthesis gene(OsP5CS)and catalase gene(OsCATC)are other key indicators affecting drought-resistant materials.Relative shoot length(RSL),hydrogen peroxide(H₂O₂)and superoxide dismutase gene(OsALM1)are other key indicators affecting drought-sensitive materials.

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

Soybean P34 protein mainly exists in soybean seeds,and its upstream promoter was likely to regulate the high expression of downstream genes in seeds.In order to further study the tissue expression pattern of soybean P34 protein gene and the regulatory activity of soybean P34 protein gene promoter,qRT-PCR was used to detect the expression of soybean P34 protein gene in soybean tissues.The 5'upstream sequence of soybean P34 protein gene(GmP34P)was cloned.The transcription initiation sites and cis-elements were analyzed by bioinformatics.The expression vector was constructed and the tobacco was transformed by Agrobacterium-mediated leaf disk method to detect GUS expression in transgenic tobacco.The results showed that the expression of P34 protein gene in soybean seeds was significantly higher than that in roots,stems,leaves and flowers.The length of GmP34P sequence obtained by cloning was 1 380 bp.Predictive analysis showed that the transcriptional start site of this sequence was base A at position 1 342,and the sequence contained a variety of cis-acting elements related to high seed expression,such as RY element,Skn-1 motif,2S seed protbanapa,etc.The plant expression vector pCAM-GmP34P containing GUS gene driven by GmP34P promoter was obtained.The positive transgenic plants were screened by hygromycin,PCR and RT-PCR.The results showed that GUS gene expression was extremely significant in transgenic tobacco seeds compared with other tissues by qRT-PCR with positive pCAM-GmP34P transgenic tobacco plants.GUS histochemical staining showed that the GmP34P promoter could regulate the high expression of downstream GUS gene in seeds.

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

Leaf color mutant is one of the ideal materials to explore the development mechanism of chloroplast and the utilization of C4 light energy in foxtail millet. To study the molecular mechanism of leaf color mutant in foxtail millet,we screened and identified a stripe leaf mutant wsl2 from the EMS mutagenesis library of the main variety Changnong 35. The mutant was studied through phenotype identification,genetic background detection and genetic analysis,location of the mutant gene rapidly and precisely with MutMap method and developing co-isolated molecular markers according to the mutation site. The results showed that wsl2 showed stripe leaf phenotype at seedling stage,but returned to normal leaf phenotype from jointing stage. wsl2 had the same genetic background as the wild type by detecting the genetic background. And wsl2 was controlled by a recessive nuclear gene with genetic analysis. The association interval contained 9 non-synonymous mutant genes by MutMap method. Seita.9G561800 encoded a PsbP-like protein associated with chloroplast,containing 6 exons and 5 introns. A G/T base mutation occured at 77 bp in the first exon,resulting in an arginine(R)becoming leucine(L). The mutation site of candidate gene in wsl2 mutant was further verified by the dCAPS marker MRI498-1(Cac8 Ⅰ)and the co-isolation marker MRI501-3. This study identified a new stripe leaf mutant wsl2, which laid a theoretical foundation for further study on the mechanism of photosynthesis reaction of PsbP gene,enriched the resources of leaf color mutant,and verified the effectiveness of MutMap method in cloning mutant gene in foxtail millet.

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.

Phytosulfokine receptor(PSKR)plays an important role in promoting plant cell proliferation and is involved in plant response to abiotic stresses.To explore the sequence characteristics and the function of wheat PSKR genes,the cDNA sequences of three homologous genes of TaPSKR1 were cloned from wheat variety Jinmai 47 by homologous cloning technology,named TaPSKR1-6A,TaPSKR1-6B and TaPSKR1-6D because of their locations on chromosome 6A,6B and 6D,respectively.The gene structure,protein physical and chemical properties,cis acting elements,functional domains and evolutionary relationships were analyzed by bioinformatics analysis.The expression patterns of TaPSKR1 genes in different tissues and under different stresses were detected by qRT-PCR.The results showed that TaPSKR1-6A,TaPSKR1-6B and TaPSKR1-6D all contained one exon.The open reading frame(ORF)of the three TaPSKR1 genes were 3 153,3 132,3 156 bp,respectively,which encoded 1 050,1 043 and 1 051 amino acid residues.Bioinformatics analysis showed that TaPSKR1 proteins were located on the cell membrane,containing signal peptide,transmembrane domains,eight LRRs type domains and intracellular kinase domain,which belonged to PSKR gene family.Phylogenetic analysis showed that TaPSKR1 proteins had closely relationship with its related species and rice,which were clustered into the same subgroup.The results of expression analysis showed that TaPSKR1 genes were expressed in roots,stems,leaves and seeds,and the expression levels in roots were the highest.Under drought and salt stress treatments,the expressions of three homologous copies of TaPSKR1 genes were sharply upregulated in leaves,suggesting that TaPSKR1 might play an important regulatory role in wheat defense to abiotic stresses.

Serine hydroxymethyltransferase(SHMT),as an important enzyme involved in basic metabolism,plays an important role in plant cell metabolism,photorespiration and defense activities.To understand the bioinformatics function of the SHMT gene family in watermelon,explore its gene expression characteristics under abiotic stress,and provide a basis for the functional development of watermelon SHMT and the breeding of watermelon stress-resistance genes.Bioinformatics methods were used to identify SHMT family,and RT-qPCR was used to analyze the expression patterns of ClSHMTs in different tissues and abiotic stresses.The results showed that 8 ClSHMTs gene family members were identified in the whole genome of watermelon,which were unevenly distributed on 6 chromosomes and named ClSHMT1—ClSHMT8 in turn.There were some differences in the physical and chemical properties of each gene family member,such as the number of amino acids,molecular weight,isoelectric point.The protein contained 471—585 amino acids,with molecular weight of 51.87—65.00 ku and isoelectric point of 6.57—8.52,all of which were hydrophilic proteins.The subcellular localization prediction was mainly distributed on mitochondria.Gene structure and protein conserved motifs analysis showed that the ClSHMTs structure consisted of 4—15 exons and 3—14 introns,and all ClSHMTs contained conserved SHMT domains.Furtherly,phylogenetic analysis with 6 species such as cucumber and wheat showed that 50 SHMTs were divided into 3 sub-families,Group Ⅰ—Ⅲ.Promoter of ClSHMTs contained cis-acting elements related to light response,plant hormone response and stress response.The expression pattern analysis showed that 6 ClSHMTs were expressed in different tissues of watermelon,and the expression levels of ClSHMT1,ClSHMT4,ClSHMT5,ClSHMT8 in leaves were significantly higher than those in other tissues.Under low temperature,drought and salt stress,the expression abundance of ClSHMTs varies,but the expression was mainly up-regulated.In conclusion,this study systematically analyzed the SHMT gene family in watermelon,and will provide a reference for the further study of the biological functions of ClSHMTs.

In order to study the function of GhAOP2-like in cotton,based on the previous proteomic data from the laboratory of Institute of Cotton, Hebei Academy of Agriculture and Forestry Sciences, this study obtained the gene sequence of GhAOP2-like using homologous cloning strategy.The physicochemical properties,structure and subcellular localization of GhAOP2-like protein were analyzed by bioinformatics method.The tissue-specific expression of GhAOP2-like and the change of expression level under drought,salt and hormone treatment were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The results showed that the CDS sequence length of GhAOP2-like was 972 bp and encoded 323 amino acids.GhAOP2-like was located on chromosome D13.GhAOP2-like protein did not contain signal peptides and transmembrane domains,and was located in the cytoplasm with the molecular formula of C₁₆₅₄H₂₅₂₅N₄₂₉O₄₇₈S₁₉ and the theoretical isoelectric point was 5.20.The clustering analysis results showed that the AOP sequences in cotton and other species were clearly divided into two groups,but had the closest relationship with the AOP sequences of Hibiscus syriacus.qRT-PCR results showed that GhAOP2-like was expressed in roots,stems,leaves,and developing seeds,but the expression level of GhAOP2-like was the highest in roots.GhAOP2-like was up-regulated under drought,salt,GA₃,MeJA and ABA treatments,but the expression level changed most in MeJA treatment,indicating that GhAOP2-like might improve the resistance of upland cotton by participating in JA signaling pathway.

Drought stress has a serious effect on the growth and development of maize,which leads to a decrease of maize yield.Purple acid phosphatase is a phospholipase protein involved in many physiological and biochemical functions of plants.In order to further study the role of purple acid phosphatase family genes in the process of stress resistance of maize,this paper explored the response mode of ZmPAP26b gene under drought stress,and Real-time fluorescence Quantitavive analysis was used to analyze the relative gene expression in different maize inbreeding lines under simulated drought conditions;ZmPAP26b(GenBank:NC_050104.1)was cloned from maize,and PAP genes in Zea mays,Arabidopsis thaliana,Oryza sativa L.,Triticum aestivum L.,Sorghum bicolor and Brachypodium distachyon were identified and bioinformatic analysis was performed.Meanwhile,prokaryotic overexpression strains were constructed for functional verification.The results showed that the expression of this gene decreased in drought tolerant materials and increased in drought sensitive materials under drought stress.The CDS length of this gene was 1 431 bp,encoding 476 amino acids.A total of 228 PAP genes were found in six species,divided into 4 subfamilies by phylogenetic analysis.The 19 PAP genes in maize were distributed on 9 chromosomes and had similar conserved domains.Analysis of promoter cis-acting elements showed that they contained elements responding to drought and hormones.Prokaryotic expression experiments showed that the growth of strains containing the recombinant plasmid pET28a-ZmPAP26b was inhibited compared with non-loaded strains under 10% PEG-6000 and 15%PEG-6000 simulated drought stress.In summary,it is speculated that ZmPAP26b is negatively regulated under drought stress.

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 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.

To elucidate the mechanisms of cold response and explore superior cold-tolerance gene resources in wheat,this study employed transcriptome sequencing to uncover key regulatory networks underlying wheat cold response,and performed functional validation of the candidate gene TaGGCT18-6A.The results demonstrated that 4 ℃ cold treatments induced 10 893 and 18 784 differentially expressed genes(DEGs)in wheat seedlings after 6 h and 24 h cold treatment,respectively.KEGG analysis revealed significant enrichment of DEGs in pathways including MAPK signaling transduction and glutathione metabolism.A γ-glutamyl cyclotransferase gene TaGGCT18-6A was cloned through screening key genes in glutathione metabolism.This gene had a length of 1 772 bp,encoding 218 amino acids with conserved GGCT-like superfamily and ChaC core domain.Promoter cis-acting element analysis identified stress-responsive elements such as low-temperature-responsive(LTR)and dehydration-responsive (DRE)elements; consistently,expression pattern analysis showed sustained upregulation of TaGGCT18-6A under 4 ℃ cold treatments. Functional validation in transgenic rice revealed that overexpression lines OE#1, OE#2 and OE#3 exhibited significantly enhanced survival rate,plant height,and biomass. Overexpression lines OE#1 and OE#2 exhibited significantly enhanced plant height, while overexpression lines OE#1, OE#2 and OE#3 exhibited significantly reduced relative electrolyte leakage under -4 ℃ freezing treatments.After 4 ℃ treatment,overexpression lines accumulated higher levels of osmolytes(proline and soluble sugars),decreased malondialdehyde(MDA)content,and increased activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT).These findings collectively demonstrated that TaGGCT18-6A enhanced plant cold tolerance by regulating glutathione metabolism to improve antioxidant capacity.This study will provide a theoretical foundation and valuable genetic resources for molecular breeding of cold-tolerant wheat.

In order to study the function of pollen lethal gene ZmAA1 and create tansgenic male sterile materials. ZmAA1 and its specific promoter Pg47 were found according to GenBank,designed to plus restriction endonuclease on the downstream and inserted them into an cloning vector puc57,construction of vector pCAMBIA3300-Pg47-ZmAA1-35S-bar used traditional construction methods digested connection.A maize inbred line Zheng 58 was infected by Agrobacterium tumefacfaciens with the vector pCAMBIA3300-Pg47-ZmAA1-35S-bar, 94 PPT-resistant seedlings were obtained and 47 plants were PCR positive.The pollen sterility in greenhouse RT-PCR assay for gene bar in the transgenic plant leaves and immuno strip test suggested that pollen lethal gene ZmAA1 had been integrated into the maize genome,and protein was expressed.

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

MYB transcription factor proteins are ubiquitous in plants and play an important role in biological and abiotic stresses. To explore the function of cotton MYB gene, the paper cloned a MYB gene from cotton leaves using homologous cloning technology, and analyzed its bioinformatics and expression under different stresses. The results showed that a new MYB transcription factor gene GhMYBPA1 (gene entry site XM_016869420) was successfully cloned from Gossypium hirsutum cv. Zhongmian 35, the full of cDNA was 825 bp in length with a 630 bp ORF, which encoded a 210 amino acids peptide. Bioinformatics analysis results showed that the molecular weight of GhMYBPA1 was 20.183 ku, GhMYBPA1 contained two conserved DNA-binding domains at N-terminal, which belonged to R2R3-MYB transcription factors. Amino acid homology analysis showed that GhMYBPA1 had higher identification with GaMYB12-like from Gossypium arboreum. Based on qRT-PCR analysis, GhMYBPA1 was constitutively expressed in cotton roots, stems, leaves, and it was dominantly expressed in flowers and then was leaves. Moreover, the results of plant treated with various stresses showed that the expression of GhMYBPA1 gene changed under high salt, low temperature and drought stress, it was suggested that GhMYBPA1 might play an important regulatory role in the abiotic stress process of cotton. The results could lay a theoretical foundation for further researches on the function of GhMYBPA1 gene.

The effect of different concentrations salt stress on seedling growth and expression of related genes were explored in this study. Firstly, salt tolerant variety Yura Hama Daikon and susceptible variety Wujinhong were selected according to germination under salt stress conditions among 11 cultivars.And then the seedling height(SH) and the leaf scorch index(LSI) of Yura Hama Daikon and Wujinhong were investigated under salt stress treatment.The results showed that SH decreased and LSI increased under salt stress in both varieties.Compared with the salt-sensitive variety Wujinhong,the salt-tolerant variety Yura Hama Daikon had a smaller decrease in SH and lower LSI.Under 200 mmol/L salt stress,SH and LSI of salt-tolerant Yura Hama Daikon were 46.18% and 20.56,respectively,while those of salt-sensitive Wujinhong were 75.25% and 56.11.The transcription of RsCAT and RsSOD genes was studied in salt-tolerant and susceptible varieties under different salt concentrations by qPCR.The expression of RsCAT gene was first increased and then decreased under low salt concentration treatment,and reached the maximum at 7 day.When treated with high salt concentration,the transcription of RsCAT in Wujinhong was the highest at 48 h,while the expression level in salt-tolerant varieties increased gradually and maintained for a longer time,reaching the highest at 7 day.After high salt concentration treatment,the transcription of RsSOD gene reached the highest expression level at 24 hours,and then maintained a higher level in salt-tolerant variety.In salt-sensitive varieties,the maximum expression level of RsSOD appeared at 14 day.The above results will lay foundation for revealing the mechanism of salt stress in radish and provide technical support for radish salt-tolerant breeding.

WRKY is a unique class of transcription factors in plants,which plays an important role in plant abiotic stress response,seed dormancy and germination,growth and development,etc.In order to reveal the function and underlying molecular mechanism of GmWRKY44 gene in soybean WRKY transcription factor family,bioinformatics analysis and biology function verification of soybean Williams 82 GmWRKY44 were performed.GmWRKY44 gene was 1 077 bp in length and encoded 358 amino acids;the results of structural prediction and evolutionary analysis showed that,the secondary structure was composed of 23.46% α-helix,4.75% β-fold,58.94% irregular coil and 12.85% extended chain,and the tertiary structure was unified with the secondary structure;It contained a conserved WRKY domain, the zinc finger structure was of the C₂H₂ type, and it belonged to the WRKY IIc subfamily; GmWRKY44 is a homologous gene of Arabidopsis thaliana AtWRKY71 with a similarity of 35.56%, and the two genes had similar gene structures. RT-qPCR analysis showed that GmWRKY44 responded to salt stress and its expression level first decreased and then increased.Under salt stress,the germination rate and root length of wild-type(Col-0)and GmWRKY44 overexpressing Arabidopsis lines were inhibited to a certain extent,but GmWRKY44 overexpressing lines were significantly better than Col-0.In addition,under salt stress,the growth inhibition of GmWRKY44 overexpressing lines was lower than that of Col-0.Physiological index analysis revealed that under salt stress,the overexpression lines of GmWRKY44 exhibited significantly higher activities of superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT)than Col-0,while the content of malondialdehyde(MDA)was significantly lower than Col-0.These data indicated that overexpression of GmWRKY44 could improve salt tolerance in transgenic Arabidopsis.

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 whether 1-deoxygen-D-xylose-5-phosphate reductisomerase(DXR)participates in the environmental resilience of Dryopteris fragrans.It used polymerase chain reaction(Polymerase Chain Reaction,PCR)methods to clone and identify Dryopteris fragrans DfDXR gene,the protein sequences were analyzed by bioinformatics through online prediction website,the relative expression levels of DfDXR gene under different chemical substances and stress were studied by Real-time fluorescence quantitative PCR(qRT-PCR).The experiment further explored how DfDXR gene responded to the stress-resistance process of Dryopteris fragrans,and laid a foundation for studying its mechanism.Experimental results:The full-length CDS sequence of DfDXR gene 1 434 bp was successfully cloned,encoding 477 amino acids,and the secondary structure of DXR protein was alpha-beta.Multi-sequence alignment and phylogenetic tree analysis of the proteins suggested that DfDXR was closely related to Adiantum capillus-Veneris AcDXR,but far related to DXR protein of apple and peach.Motif analysis showed that the protein contained PLN02696 domain,which was the conserved domain of DXR protein.Subcellular localization predicted that DXR protein was located in chloroplast.The analysis of qRT-PCR data showed that the relative expression of DfDXR showed an upward trend after treatment with methyl 3-oxo-2-(pent-2-enyl)cyclamate(MeJA)and polyethyleneglycol(PEG),and reached the highest level at 1,12 h respectively.Under NaCl treatment,there was a "down—up—down" trend,but the expression levels under each treatment time were lower than those in the control group.The relative expression of DfDXR also changed obviously under the treatment of salicylic acid(SA),abscisic acid(ABA),ethephon(ETH),high teperature(HT)and low temperature(LT).DfDXR plays a regulatory role in the response of Dryopteris fragrans to different abiotic stresses,and its response time to different chemical substances and stress is different.

To study the sequence and function of rice SAND domain protein, the gene encoding SAND was identified by Blast against the rice genome, and then over-expression vector was constructed to study its gene function by genetic transformation.The results found that the rice genome contains a gene LOC_Os01g57240 (named as OsULT1), which encoded amino acid sequence containing SAND domain and ULT B-box consensus sequence.The amino acid sequence similarity between OsULT1 and ULTs of other plants varied from 49.3% to 92.3%, they were highly conservative. The 35S::OsULT1 transgenic lines generated some mutant spikilets, which included under-developed or degenerated paleas, over-developed locidules, the increased number of locidules, the variant number of stamen, two pistils, or extra floret.The results indicates OsULT1 should play important roles in regulation of floral meristem identity in rice.

Application of some hormonal signaling compounds,as brassinolides and their derivatives,could significantly improve the salt stress resistance in plants.The purpose of present work was to test whether the over-expressing of Methylsterol monooxygenase gene(SMO),a key gene coding a bio-synthesizing enzyme of sterol in plants,could promote the salt-stress tolerance of target plants.PnSMO1.1,gene encoding of methylsterol monooxygenase in the plant species of Pharbitis nil was firstly cloned and then used as target gene for following genetic transformation process,while wild-type Pharbitis nil seedlings were used as the receptor plants for constructing of the transgenic lines which over-expressed PnSMO1.1 genes.In this work,the PnSMO1.1 gene transformed Pharbitis nil lines were constructed via an ovary injection transformation method.Plantlets from individual PCR identified transgenic plant lines were used as materials to detect vegetative growth figures and some pivotal physiological indicators,for instance,contents of malondialdehyde (MDA),relative conductivity,as well as castasterone and 6-deoxo-castasterone contents in cells under stresses with gradient NaCl conditions varied from 0—200 mmol/L.The results showed that the over-expression PnSMO1.1 significantly improved the relative growth of roots and hypocotyls in transformants than in wild-type (WT) or vacant plasmid transformed control (BL) plants under 100—250 mmol/L NaCl stresses.Compared to WT and BL seedlings,significantly higher accumulation of 6-deoxo-castasterone,but lower relative conductivity (rEC) values,castasterone accumulation or MDA contents were found in transgenic lines under various NaCl stresses.Stresses such as salinity,drought and freezing temperatures,had severely suppressed the vegetative growth of plants,as well as their yields.These results highlighted that over-expression of the PnSMO1.1 gene could significantly improve the salinity-stress resistance of transgenic plants by delicately adjusting the dynamic homeostasis of brassinolides in cells,and protecting the structural integrity of plasma membrane.

Analysis of the expression pattern of florigen gene (Hd3a)under different photoperiod conditions,aims at revealing the role of florigen gene that played in photoperiod-regulated flowering process of foxtail millet. First,a SiHd3a gene sequence(Seita.4G067600,named SiHd3a)on Chr.4 of foxtail millet was obtained by bioinformatics method and according to the transcribed sequence of Seita.4G067600,a pair of specific primers were designed successfully. Then the total RNA of millet land race Huangmaogu was extracted and after reverse transcription the cDNA sequence of SiHd3a gene was obtained by RT-PCR technology. The cDNA sequence was 792 bp which included a 537 bp CDS region that coding 178 amino acids. The molecular weight,isoelectric point of SiHd3a protein were 19.74 ku and 6.82 respectively,which was preliminarily judged as a hydrophilic protein. The proportion of irregular curl was the highest in protein secondary structure(43.26%),following it were extended chain(β-sheet,29.21%),α-helix(16.29%)and β-turn(11.24%). Subcellular localization analysis showed that SiHd3a protein was located in the cytoplasm and intercellular substance. Phylogenetic analysis based on Hd3a protein sequences showed that there was a close relationship between foxtail millet and crown,maize,sorghum,but a distant relationship between foxtail millet and rice. Semi-quantitative PCR showed that under short-day condition, SiHd3a gene gave a circadian rhythm expression pattern,with two expression peaks at six am,and twelve am respectively. While under long-day condition, SiHd3a gene gave almost stable expression level in 24 hours. It could be speculated that the diurnal expression pattern difference of SiHd3a gene between long-day and short-day might be the cause leading to photoperiod sensitivity of foxtail millet.

The overexpression of Serine/Threonine protein kinase gene AtSTK can significantly enhance the salt tolerance of Arabidopsis thaliana. In order to study the regulation mechanism of AtSTK gene expression,the promoter sequences of AtSTK were amplified from genomic DNA and constructed into the reporter vector pAbAi. The recombinant plasmid pAbAi-HYT was linearized by Bstb Ⅰ and transformed into the yeast strain Y1H Gold,the linear pAbAi-HYT integrated into the genomic DNA. Then,the purified double strand cDNA of Arabidopsis and the linearized library vector pGADT7-Rec were co-transformed into the yeast cell which containing reporter vector pAbAi-HYT. The positive clones were screened on the SD/-Leu medium containing 100 ng/mL AbA by the yeast one hybrid method. Through the identification,it was found that four genes might be involved in the regulation of AtSTK gene expression. The sequencing results indicated that the Arabidopsis thaliana gene AT3G32090,which contained a conserved domain of WRKY transcription factor,was a member of the WRKY transcription factor. Therefore,the expression level of AtSTK might affect the salt tolerance of Arabidopsis plants through the MAPK signaling pathway transcription factor which coded by the gene AT3G32090.

OFP is a class of plant-specific transcription factors that play important roles in the regulation of plant organ morphogenesis and response to abiotic stresses.In order to study the characterization of soybean OFP transcription factor family members and their roles in drought stress and salt stress,bioinformatics methods were applied to identify and analyze soybean OFP family members.The results showed that:a total of 41 GmOFPs,named GmOFP-1—GmOFP-41,were identified in soybean;these genes were unevenly distributed on 19 chromosomes of soybean,encoding 152—414 amino acids;subcellular localization predicted that soybean OFP proteins were mainly localized in nucleus,chloroplasts,and mitochondria;a total of 10 conserved motifs were identified in soybean OFP proteins,conservative Motifs 1 and 2 were present in all OFP members.Phylogenetic analysis classified soybean and Arabidopsis OFP proteins into five subfamilies ClassⅠ—Class V,of which soybean OFP family genes were mainly distributed in ClassⅠ and Class Ⅲ.The collinearity analysis revealed that 75 pairs of genes in the soybean genome had collinearity,four pairs of genes had tandem duplications,and only two genes,GmOFP-2 and GmOFP-39,did not have collinearity,which indicated that gene fragment duplication was the main reason for the increase in the number of soybean OFP family members.The expression patterns of GmOFP gene family members under drought stress and salt stress treatments were analyzed by qRT-PCR,and the results showed that,compared with the control,16 members out of 41 GmOFP genes exhibited significant differences in gene expression levels after drought treatments,with significant up-regulation of the expression of GmOFP-15,GmOFP-17,and GmOFP-32,while the GmOFP-4,GmOFP-5,GmOFP-6,GmOFP-9,GmOFP-12,GmOFP-21,GmOFP-23,GmOFP-25,GmOFP-26,GmOFP-27,GmOFP-38,GmOFP-39,and GmOFP-40 were significantly down-regulated after drought treatment.Eight members of GmOFPs showed significant differences in gene expression levels after salt treatment,among which GmOFP-7,GmOFP-14,GmOFP-31,GmOFP-32,GmOFP-36,and GmOFP-40 were significantly up-regulated,and GmOFP-1 and GmOFP-15 were significantly down-regulated.The above results suggest that the soybean OFP gene family may have important functions in response to drought stress and salt stress.

To further understand the function of MADS-box family gene,the genome-wide of beet MADS-box genes were first identified by bioinformatics,and chromosomal localization,phylogenetic relationship,gene structure,conserved domain,expression pattern and proteins function connections were predicted and analyzed.The results showed that there were 34 MADS-box genes in beet,among which 7 members belong to type Ⅰ and 27 members belong to type Ⅱ.Type Ⅰ was further divided into three class:Mα (3),Mβ (1) and Mγ (3),type Ⅱ was further divided into two class:MIKC^C (22) and MIKC^* (5),and MIKC^C class were further divided into ten subclass:AG (2),AGL12 (2),AP3-PI (4),Bs (2),SOC1 (1),SVP (1),SEP (3),AGL17 (5),AP1-FUL (1) and FLC (1).The MADS-box genes showed uneven distribution on the chromosome,and genes cluster distribution on one chromosome.The most distributed was on chromosome 6,none on chromosome 7.The structure of the MADS-box genes was different,but the protein sequences were conserved relatively.The expression profile showed that most MADS-box genes were expressed highly in meristem,and some MADS-box genes were expressed advantageously in seeds,roots,young leaves,and so on.Some MADS-box genes are slightly up-regulated in response to salt or heat stress and may be involved in the physiological regulation under stress.

In order to explore new approaches for high-oil peanut breeding and establish a new method for directly developing high-oil peanut germplasm,this study employed in vitro mutagenesis breeding technology to create new high-oil peanut germplasm.Jihua 9 embryo leaflet was used as mutagenic test materials,Jihua 9 and Jihua 54 were used as control test materials,and bleomycin was used as mutagenic agent.The ovules were sterilized and placed in gradient mutagenesis medium and screened for semi-lethal concentrations of bleomycin.After somatic embryos germinated into seedlings,sterile peanut seedlings were used as rootstocks,and transplanted to the field.Bioinformatics analysis of two known regulated peanut fat synthesis genes WRI1 and experimental feasibility validation by the correlation of WRI1 gene expression in grain and crude fat content of mutagenic plants were conducted.The results were best when the bleomycin was 3 mg/L.The crude fat content of IM13-3 was higher than that of Jihua 9(CK₁,test variety control)and Jihua 54(CK₂,high oil variety control).Two WRI1 genes,WRI1X2 and WRI1X1,encoding 366 and 357 amino acids,respectively,were both unstable hydrophilic proteins. WRI1 gene expression and crude fat content were significantly positively associated in grain.Bleomycin was first used as a peanut vitro mutagenesis agent,and IM13-3 was obtained with a crude fat content of 56.64%.It further proves the authenticity of Jihua 9 high oil mutant and the feasibility of peanut in vitro mutagenesis method. The gene expression level of the high-oil mutant WRI1 was determined and was significantly different from the control varieties. Demonstrate the feasibility of breeding methods for in vitro mutagenesis of peanut.

In order to explore the genetic basis of drought resistance and guide drought resistance breeding, mapping of QTL associated with drought resistance at germination stage of foxtail millet was done. An F₂ segregating population was constructed using 100 individuals derived from a cross between Shanxi 2010 and K359×M4-1. A genetic linkage map was constructed based on 2b-RAD sequencing, which was then combined with the phenotypic trait of drought resistance for QTL mapping. The results showed that drought resistance at germination stage of foxtail millet was a complex quantitative trait and controlled by multiple genes. A genetic map containing 583 SNP makers was constructed by 2b-RAD of parents and F₂ population. The map covered 9 chromosomes of foxtail millet, with an average number of 64.8 and an average genetic distance of 0.97 cM between markers. A total of 3 QTLs were identified:qSIDR-5a, qSIDR-6a and qSIDR-6b, located in foxtail millet chromosome 5 and 6, respectively, which explained 12.4%-14.3% of phenotypic variation. Among them, phenotypic contribution rate of qSIDR-5a was the highest, which explained 14.3% of phenotypic variation. These QTLs were not included in the same chromosome interval as the identified drought resistance related QTLs of foxtail millet, and thus were described as new candidate gene loci associated with germination stage drought resistance.These QTLs can be used for fine mapping and gene cloning, as well as molecular regulation mechanism of drought resistance of foxtail millet.

In order to explore the role of citrus MYB96 gene in the process of citrus stress resistance,four MYB transcription factor genes were cloned from sweet orange,lemon,kumquat and ponkan,and named CsMYB96,ClMYB96,FmMYB96 and CrMYB96,respectively.Their bioinformatics and expression patterns under different abiotic stress treatments were analyzed.The results showed that the open reading frame of CsMYB96,ClMYB96,FmMYB96 and CrMYB96 were 1 032,1 035,1 035 and 1 032 bp,respectively.The citrus MYB96 proteins were composed of 343,344,344 and 343 amino acids,respectively,with a molecular weight of about 38.16, 38.27, 38.22 and 38.13 ku and an isoelectric points of 6.31,6.35,6.35 and 6.31,these proteins were all unstable hydrophilic proteins and the prediction results of subcellular localization were all located in the nucleus.The secondary structures of four MYB96 proteins were similar,mainly composed of α-helix and random curls.And all of four MYB96 proteins had a highly conserved R2 and R3 domains.In terms of evolutionary relationship,the proteins encoded by these four genes are most closely related to Citrus clementine CcMYB96 protein.The promoter of CsMYB96 gene contained abscisic acid response element(ABRE),low temperature response element(LTR),anaerobic response element(ARE),MYB binding site(MBS)involved in drought induction and other abiotic stress response related cis acting elements.Real-time quantitative PCR analysis showed that CsMYB96 could be induced by low temperature and drought stress,while ClMYB96 and FmMYB96 were induced under high salt stress.The expression of CrMYB96 was down-regulated in different degrees under low temperature,drought and high salt stress.

The objective of this study was to investigate the transcriptome differences after and before high temperature stress during maize anthesis stage, and to explore the key genes and proteins which are contributed to the high temperature resistant. The pollen activities of maize inbred lines Chang 7-2 and Zheng 58 were detected by Ampha^TM Z30 pollen activity analyzer (Amphays, Switzerland), firstly. RNA-sequencing, construction library and quality assessment were carried out for two periods before and after high temperature treatment using the Illumina Hiseq2000^TM sequencing platform, then the sequencing results were analyzed by bioinformatics. The differential expression genes (DEGs) were analyzed using edegR software. Key different expression genes and proteins with high temperature resistance were analyzed by VENN mapping, GO functional enrichment, KEGG pathway method and transcription factors analysis.The pollen activity of Chang 7-2 grown in normal condition was 42.89% and the pollen activity which was treated by high temperature during anthesis stage was 24.37%. The pollen activity of Zheng 58 which was grown in normal condition was 64.83% and the pollen activity treated by high temperature was 35.57%. The results showed that 4 176 significant DEGs were detected in Chang 7-2 inbred under normal growth condition and high temperature treatment. 5 487 significant DEGs were detected in Zheng 58 inbred under normal growth condition and high temperature treatment. KEGG pathway enrichment analysis showed that 2 062 differentially expressed genes in Zheng 58 were enriched in 399 related pathways. KEGG pathway enrichment analysis showed that 1 943 DEGs in Chang 7-2 were enriched in 352 related pathways. A total of 55 transcription factor families were obtained by the transcription factor analysis, of which 45 transcriptional factors contained more than 10 differentially expressed genes. In conclusion, Comparing the RNA-sequencing after and before high temperature treatment of Chang 7-2 and Zheng 58, many DEGs were obtained. The differences of response mechanisms between Chang 7-2 and Zheng 58 to anthesis high temperature stress were comparatively analyzed. The three gene families (Hsfs, IAA and PIP₂) and the Hsps proteins are playing significant roles in the regulation of high temperature stress in maize.

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.

Temperature stress is one of the main nonbiological stresses that affect the quality and yield of spinach.Investigating the molecular response mechanism of spinach to temperature stress is crucial for spinach stress tolerance breeding.To provide a theoretical basis for the research of the mechanism of spinach resistant to cold stress and heat stress,this study used the cold-tolerant inbred line D3 and the heat-tolerant inbred line M10 of spinach as experimental materials and analyzed their transcriptomes and metabolomes under cold and heat stress to explore the transcriptional and metabolic mechanisms underlying spinach tolerance.Transcriptomic analysis showed that the pathways in which the DEGs were the most enriched in D3 and M10 were essentially the same under cold stress and heat stress.Metabolomics analysis showed that under cold stress,they were coenriched in the pyrimidine metabolism and lysine degradation in KEGG pathways.Under heat stress,these were mainly enriched in the tryptophan metabolism,toluene degradation,biosynthesis of various other secondary metabolites,and glycine,serine and threonine metabolism in KEGG pathways.The joint transcriptomic and metabolomic analysis indicated that through data analysis and gene annotation.SpADH(sov2g036390),SpSHMT(sov1g001130)and SpALDH-1(sov4g007150)were identified as the candidate genes for cold stress tolerance in spinach.SpALDH-1(sov4g007150),SpALDH-2(sov1g043320)and SpNPC(sov1g040610)were identified as candidate genes for heat stress in spinach.Among them,SpALDH-1(sov4g007150),which may be a regulatory gene for spinach stress tolerance,was significantly expressed under both cold and heat stress.

To reveal the function of rice polypeptide release factor eRF1 in protein synthesis,the full length cDNA and promoter (2 120 bp) of rice eRF1 gene,named OseRF1-3,was isolated by PCR.Sequence analysis indicated that the OseRF1-3 contained a 1 308 bp CDS sequence that encoded 436 amino acids with three conserved N,M and C domains.qRT-PCR results indicated that the transcript of OseRF1-3 was detected in all of the tissues examined,but the highest level of expression was detected in spikes.The genome DNA from leaves of rice was used as the template, the 2 120 bp promoter sequence of the initiation codon upstream of OseRF1-3 gene were cloned using the PCR method. The promoter of OseRF1-3 gene was fused with GUS reporter gene and transferred into Oryza sativa L.callus by Agrobacterium-mediated method.Histochemical staining of different organs of the transgenic plants showed that histochemical GUS expression could be found in all of the tissues examined,and a low GUS reporter gene expression in root and higher expression in flowers and rice husk.The results would provide the basis for the understanding of the OseRF1-3 gene function.The results of GUS gene expression driven by the promoter of OseRF1-3 gene were in good agreement with the results from qRT-PCR analysis. So the results will provide the basis for the further explore of the OseRF1-3 gene function.

Tiller-related traits are important characteristics of wheat plant type,which determine plant structure and affect grain yield.In order to understand the inheritance and drought resistance of tiller-related traits in wheat under different water conditions,and to excavate the loci related to tiller-related traits,240 wheat varieties (lines) were selected as the subjects of this study,based on the phenotypic identification of tiller angle,effective tiller number and yield per unit area under normal irrigation (NI) and drought stress (DS) conditions,and the comprehensive evaluation of drought resistance,combined with 90K gene chip,genome-wide association study (GWAS) was to identify genetic loci for tiller-related traits and to screen for superior germplasm.The tiller angle,effective tiller number and yield per unit area showed significant difference,and the coefficient of variation ranged from 0.07 to 0.33.According to D-value,the drought resistance of Zhongyou 206 was the best.A total of 54 stable genetic loci significantly associated with tiller angle and other traits were detected, distributed on all chromosomes except 3D, 4D and 5D. Three identical stable loci were commonly detected under both treatments, located on chromosomes 2B, 4B, and 6B. Additionally, four pleiotropic loci were commonly detected in different traits, located on chromosomes 2B, 2D, and 5B.At the same time,the haplotype analysis of Ra_c491_902 (R²=5.45%—17.91%),which was significantly correlated with tiller angle on chromosome 2B,showed that there were three haplotypes:TA-Hap1,TA-Hap2 and TA-Hap3,the haplotypes (lines) containing TA-Hap1 were mainly derived from Huanghuai winter wheat regain.Five candidate genes related to tiller angle were screened by screening the stable genetic loci detected under different treatments.Gene annotation of the genes selected on Ra_c491_902 showed that the genes encoding cytochrome P450 family protein can be used as important genes such as regulating tillering angle,plant drought resistance and defense,to explore the association between genes and phenotype,and lay the foundation for the genetic improvement of tiller-related traits in wheat.

A Receptor-like kinase(RLK)gene, OsSIK1,plays important roles in drought stress-tolerance in rice,through the activation of the antioxidative system.To make maize plants have much more drought resistance genes and further to obtain drought tolerance maize germplasm.In this study, OsSIK1 gene of rice was transformated into maize inbred Zheng 58 plants by pollen-mediated method.First,transgenic plants in T₁,T₂ and T₃ were detected by Kanamycin resistance screening,PCR and Southern Blotting,transgenic plants were obtained from T₁ and pure transgenic lines was obtained from T₃.Next,drought resistance analysis to transgenic maize plants and non-transformation control plants were conducted under the condition of 16.1% PEG drought stress.The results showed that compared with non-transgenic plants,the seedling leaf relative water content,chlorophyll content and SOD activity of transgenic plants were increased by 7.4%-19.8%,11.3%-106.9% and 45.8%-93.4%,respectively;furthermore,the relative conductivity and MDA content were decreased by 35.4%-58.1%,and 25.7%-50.4%,respectively.All the physiological indexes under the drought stress proved that transgenic OsSIK1 gene improved the drought resistance of transgenic maize plants,further analysis found that there were significant differences on drought tolerance between 5 transformed lines and their control groups,and their performance of field were superior to that of non-transgenic maize seedlings.At last,5 transgenic maize inbred lines were obtained,which suggested that genetically modified maize has improved the drought resistance by introducing foreign OsSIK1 gene of rice.

In order to study the tolerance of heterologously overexpressed Atvip1 gene in sorghum to defense saline-alkali stress and the corresponding growth, NaHCO₃:Na₂CO₃ of 5:1 solution with 75 mmol/L and pH 9.63 was used in sorghum at the stage of three leaves and one heart. The root growth index, chlorophyll content, antioxidant enzyme activity and MDA content were measured at 0, 4, 12, 24, 72, and 120 h of stress. The results indicated that the heterologous overexpression of Atvip1 gene could alleviate the damage of saline-alkali stress on the growth of sorghum seedlings, increase the root surface area and root volume, the number of root tips and branches, and also cause the browning of sorghum main roots to appear later and mild symphonys, and the earlier and more lateral roots occurrence. The new leaves could still be normally extended at 72 h and present little effect on the growth of aboveground. Overexpression of Atvip1 gene could increase the activity of O₂^-· resistance, decrease the content of MDA and enhance the activities of antioxidant enzymes in transgenic sorghum roots. SOD, CAT and GR had obvious effects at 4-12 h during the early stage of stress, respectively. All enzymes played roles during the middle of stress at 24-72 h. CAT and GSH-PX played important roles at the later stage of stress at 120 h. On the base of differential transcriptome analysis of saline-alkali stress, COG analysis of differentially expressed genes(DEGs) showed that defense mechanisms accounted for a relatively large proportion during various periods, and 42 DEGs related to antioxidant enzymes were obtained. Heterologous overexpression of Atvip1 gene can improve the resistance of transgenic sorghum to saline-alkali stress by alleviating the effects on photosynthesis, growth and development, reducing the damages of reactive oxygen species and membrane damage.