Salt Tolerance Identification And Transcriptome Sequencing Of Chrysanthemum DgWRKY5 Gene

Chrysanthemum is one of the ten famous traditional flowers in China and one of the four cut flowers in the world.Its ornamental and economic value is very high.At present,cut chrysanthemum is mainly cultivated continuously through intensive facilities.The secondary salinization of soil in the facility seriously affects the chrysanthemum production and quality.Therefore,cultivating new varieties of salt-tolerant chrysanthemum has become an important goal of the cut chrysanthemum breeding.Studies have shown that WRKY overexpressing plants have strong resistance to salt and the phenotype of plants is not defective.Therefore,in this study,DgWRKY5 gene was isolated and functionally identified.At the same time,the screening of different genes of DgWRKY5 chrysanthemum and non transformation chrysanthemum can provide a new way for breeding new varieties of chrysanthemum with good salt resistance and no phenotypic defects through genetic engineering.The mechanism of salt resistance of DgWRKY5 is discussed.The main results are as follows:1.The expression of DgWRKY5 transgenic chrysanthemum was analyzed by Real-time PCR under various stresses,including NaCl,ABA,GA,and H₂O₂ stress;and the expression level of transgenic chrysanthemum in different tissues.The results showed that DgWRKY5expression was induced under various stresses.And the expression level of DgWRKY5observed higher expression levels in leaves compared to other tissues.2.Overexpressing DgWRKY5 gene chrysanthemum,selecting OE-17 and OE-56 lines and wild-type chrysanthemum for NaCl treatment and salt tolerance identification.The results showed that the leaves of transgenic chrysanthemum had no obvious wilt and yellowing under salt stress,and the survival rate of OE-17 and OE-56 in DgWRKY5 chrysanthemum was significantly higher than that of wild type?P<0.05?.Under salt stress,the activities of superoxide dismutase?SOD?,peroxidase?POD?and catalase?CAT?enzymes in transgenic chrysanthemum were significantly higher than those in WT,whereas the accumulation of H₂O₂,O₂^-and malondialdehyde?MDA?was reduced in transgenic chrysanthemum.DAB and NBT staining were also lighter than those of the wild type.Several parameters including root length,root length,fresh weight,chlorophyll content and leaf gas exchange parameters in transgenic chrysanthemum were much better compared with WT under salt stress.3.The relative expression levels of eight stress-related genes were determined by qRT-PCR,and the molecular mechanism of DgWRKY5 on salt stress was preliminarily explored.The expression of stress-related genes DgAPX,DgCAT,DgNCED3A,DgNCED3B,DgCuZnSOD,DgP5CS,DgCSD1 and DgCSD2 was up-regulated in DgWRKY5 transgenic chrysanthemum compared with that in WT,thus improving the salt tolerance of transgenic chrysanthemum.4.Illumina HiSeq was used for transcriptome sequencing of transgenic DgWRKY5chrysanthemums and wild chrysanthemum.A total of 309,068 transcripts with a length greater than 200 bp and 153,887 Unigenes were generated,the total amount of data is 14.26G.The sequenced Unigene samples were compared with major public databases for functional annotation.There were a total of 1078 differential genes in DgWRKY5 transgenic chrysanthemum and wild type chrysanthemum?qvalue<0.005 and|log2?foldchange?|?1?,of which 593 genes were up-regulated and 485 genes were down-regulated.Through the KEGG metabolic pathway enrichment analysis of differential genes,the annotated metabolic pathways associated with abiotic stress were found:glutathione metabolism,carotenoid biosynthesis,plant hormone signal transduction,arginine and proline metabolism and so on.Validation of Gene Expression Levels of 8 Differential Genes by qRT-PCR.The above results showed that DgWRKY5 transgenic chrysanthemum was more salt-tolerant than wild type chrysanthemum,and DgWRKY5 gene could increase the content of osmotic regulators,regulate the content of active oxygen scavenging enzymes,and increase leaf photosynthesis and regulation stress response gene expression to enhance salt tolerance of chrysanthemum.