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.

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

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

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Flavonoid 3'-hydroxylase (F3'H) plays important roles in flavonoids synthesis pathway.In previous research, f3'h gene from (Dendranthema morifolium) Chuju was cloned in our laboratory.For further elucidating the function,by using genome walking technology,its promoter of 1 348 bp in length was cloned.Sequence analysis showed that besides eukaryotic core promoter element TATA-Box, the promoter fragment also contains multiple cis-regulatory elements, such as the element involved in abscisic acid response (ABRE), G-Box participating in the optical response unit, plant hormone response unit, plant defense and stress response element, and environmental factors, etc. The cloning of gene promoter paves the way for the further research on the molecular mechanism of f3'h gene in the synthesis of flavonoids in Chrysanthemum,provides effective components for engineering Chrysanthemum to improve the content of flavonoids.

CorA/MRS2/MGT-type magnesium ion transporters play an important role in maintaining magnesium homeostasis in plants.In order to investigate the function of ZmMGT10 gene which is a maize CorA/MRS2/MGT-type magnesium ion transporter and expression is induced by magnesium deficiency,we constructed the overexpression vector of ZmMGT10 and transgenic Arabidopsis plants were obtained.Compared with wild-type plants,transgenic Arabidopsis plants grew more vigorously than wild-type plants under low Mg conditions,exhibited by longer root length,higher plant fresh weight and chlorophyll content.Further analysis indicated that the roots and shoots of transgenic plants had higher magnesium accumulation than wild-type plants under low magnesium condition.Furthermore,the roots of transgenic plants had enhanced magnesium uptake ability than wild-type plants.The results suggested that overexpression ZmMGT10 in Arabidopsis could enhance the resistance ability of transgenic plants to magnesium deficiency stress.

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.

This study provides a good foundation for further elucidating the function and mechanism of AhPLDα1 and AhPLDα2 in responding to drought stress in peanut.The full-length CDS sequences of AhPLDα1 and AhPLDα2 were cloned from Jihua 4 peanut leaf with ABA treatment using RT-PCR.The AhPLDα1 and AhPLDα2 gene fragments were inserted into the expression vector pBar-F3 by forward ways.These constructs were transformed into Agrobacterium tumefaciens GV3101 by freeze-thawing method and then introduced into wild-type Arabidopsis thaliana using modified floral-dip method.Under the condition of drought,the authors observed phenotypic changes of wild type and transgenic plants.Colony PCR and enzyme digestion results showed that,the plant overexpressing recombinant plasmid pBar-AhPLDα driven by CaMV35S promoter was successfully constructed.Glyphosate resistance screening,PCR detection and gene expression analysis showed that the positive transgenic plants were obtained.Under water deprivation,overexpression of AhPLDα1 and AhPLDα2 in transgenic Arabidopsis plants resulted in significantly enhanced tolerance to drought stress.In conclusion, AhPLDα1 and AhPLDα2 have a certain relation with drought stress signal transduction in transgenic Arabidopsis plants,and are potential candidate genes on the way to modified crop drought resistance.

In order to explore and identify more albino mutants for studying gene functions,a stage thermos-sensitive green-revertible albino mutant in rice,designated as stgra254,was isolated by treating the seeds of japonica variety Xiushui 09 using EMS.The phenotype of gra254 is thermos-sensitive.Under a constant temperature of 28℃,the white spots appeared on the fully expanded sixth leaves,and gradually enlarged and merged into one at maximum tillering stage.Then the leaves withered to death.The sixth leaf blades showed milder expression of the mutant phenotype under 32℃ condition than that under 28℃ condition,and gradually turned green from the third day onward.Under 24℃ condition,however,the gra254 plants produced normal green leaves.Histochemical staining analysis indicated that the albino trait of stgra254 was caused by the programmed cell death,which probably caused by oxidative burst accompanying by the accumulation of H₂O₂.Analysis by fluorescence analyzer presented that the maximal photochemical efficiency of PS Ⅱ decreased significantly.Genetic analysis indicated that the thermos-sensitive green-revertible albino trait was controlled by a single recessive nucleic gene.Based on the F₂ population derived from a cross between stgra254 and Zhenshan 97,the stgra254 was located between the SSR markers of RM17206 and RM17277 on chromosome 4 by bulked segregant analysis and linkage analysis.The genetic distance was 0.48,5.22 cM, respectively.

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.

The objective of this study is to identify interacting proteins of resistance-related gene T1N6_22 in Arabidopsis thaliana, and to provide a basis for clarifying the regulation mechanism of the T1N6_22 gene in Arabidopsis resistance. The yeast two-hybrid vectors of the T1N6_22 gene and its candidate interacting protein gene,including AD-T1N6_22, AD-AT1G06050, AD-AT1G21400, AD-AT2G19480, BD-T1N6_22, BD-AT1G06050, BD-AT1G21400, and BD-AT2G19480 were constructed by Gateway method. In order to detect self-activation of T1N6_22, AT1G06050,AT1G21400, and AT2G19480 genes,the AD vector was combined with BD-T1N6_22, BD-AT1G06050, BD-AT1G21400, and BD-AT2G19480 respectively,and transformed into yeast competent cells. The results showed that AT1G06050 had self-activation activity and T1N6_22, AT1G21400, AT2G19480 didn't have self-activation activity. For yeast two hybrid, the AD-T1N6_22 vector was combined with BD-AT1G06050, BD-AT1G21400, BD-AT2G19480 vectors,respectively, and the BD-T1N6_22 vector was combined with AD-AT1G06050, AD-AT1G21400, AD-AT2G19480 vectors,respectively. It was found that yeast colonies which were co-transformed with AD-T1N6_22 and BD-AT1G06050, BD-AT1G21400 or BD-AT2G19480, BD-T1N6_22 and AD-AT1G06050, AD-AT1G21400 or AD-AT2G19480 grew well in both -Leu/-Trp and -Leu/-Trp/-His and -Leu/-Trp/-His/-Ade media.Yeast two-hybrid results indicated that the T1N6_22 gene directly interacted with AT1G06050,AT1G21400 and AT2G19480 in yeast.

To explore flower development molecular mechanism of the Double lily,the AGL6 homologous gene was cloned from Double lily cv. Belonica. The open reading frame of LiAGL6 gene was 744 bp,encoded a protein of 247 amino acids. The LiAGL6 protein molecular weight was 28.3 ku,the grand average of hydropathicity was -0.748 and the theoretical pI was 8.23. The protein structure analysis showed that LiAGL6 protein had a typical MADS-box domain,a K-box region and two AGL6 motifs. Phylogenetic tree analysis showed that LiAGL6 belonged to monocotyledon group,AGL6 branch of the AP1/AGL9 subfamily,and had the highest similarity with Hyacinthus orientalis which was also belonged to monocotyledons, the similarity reached 78%. All the results indicated LiAGL6 was the homologous gene of AGL6, so named it LiAGL6. Cellular localization assay revealed LiAGL6 expressed in the nucleus of onion epidemical cells, which was the basic characteristics of the transcription factors. The qRT-PCR result showed the highest expression of LiAGL6 was in flowers while no expression occurred in leaves and stems. LiAGL6 was expressed most in the seventh whorl petals followed by the sixth and third whorl petals,however,there was almost no expression in the second whorl petals. The research showed the LiAGL6 gene might play a regulatory role on formation of Double lily flowers.

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.

This study was aimed to improve the resistance of Wuyunjing 29196.Blast resistant gene Pigm(t) showed a broad spectrum of strong resistance.Wuyunjing 29196 is an excellent conventional Japonica rice,which has good yielding but sensitive to rice blast.This study was aimed to improve the resistance of Wuyunjing 29196 to blast by MAS and anther culture.GM4,a variety carrying a resistant gene Pigm(t),was used as the donor parent.Two closely linked InDel markers,S95477 and S29742,were employed to select the Pigm(t) gene in each generation.Finally,185 DH (Double haploid) lines were developed from the anthers of BC₂F₁ plants.Among them,82 improved lines with homozygous Pigm(t) were selected.Multiple agronomic traits including grain yield and resistance to rice blast and the rice quality were characterized.Two fine lines,DH036 and DH158 screened out,finally which had increased resistance level and enhanced yield production.More importantly,they had similar plant architecture and other traits.Simultaneously had high resistance to rice blast and quality.The combination of anther culture and marker-assisted selection successfully improved the resistance to rice blast,in a short breeding period.The improved Wuyunjing 29196 with high blast resistance level provided important genetic resources in breeding.

In order to further excavate the related genes of wheat vernalization,explored the regulation mechanism of wheat vernalization,this study screened and cloned an EST sequence from a high-throughput RNA sequencing analysis comparing the transcriptomes with wheat varieties with different developmental characteristics under vernalization and non-vernalization treatment,and named Trx59. The open reading frame (ORF) of Trx59 gene was 372 bp,ecoding 124 amino acids,and contained one thioredoxin domain structure. Expression analysis was conducted by qRT-PCR and the gene function was verification by using VIGS technique. The result showed that the expression level of Trx59 was gradually up-regulated during vernalizaton process;the expression level and time of Trx59 gene in spring variety LC10 was higher and earlier than that in winter variety J841. BSMV:Trx59 recombinant vector was built and inoculated wheat plants of J841. After 14 days of inoculation,the leaves appeared visible light bleaching phenomenon,and the expression level of Trx59 reduced sharply,indicating that Trx59 gene had been significantly suppressed;the spike differentiation process of plants inoculated by BSMV:Trx59 was later than that of the negative control group,deducing that the Trx59 genes may be related to the wheat developmental characteristics.