| Chrysanthemum(Chrysanthemum grandiflorum Ramat.)is one of is one of the four major cut flowers in the world,and the ornamental and economic value are extremely high,but the soil salinization is common in chrysanthemum anniversary production,that affecting the cut flower productivity and quality seriously.WRKY transcription factor plays an important role in responding to various abiotic stresses.In this study,leaves of chrysanthemum 'Jinba' under salt stress were used as experimental materials,and the transcriptome data were analyzed by Illumina HiSeqTM 2000 double-terminal sequencing technique.One WRKY gene was cloned and then genetically transformed it into chrysanthemum.This experiment provides a theoretical basis for improving the salt tolerance of cultivated chrysanthemum by the means of molecular biology.The main findings are as follows:1.The cDNA library was constructed by using the leaves of chrysanthemum under salt stress.Transcriptome sequencing was performed using the Illumina HiSeqTM 2000 system,A total of 9.4 million effective sequences were obtained with an average length of 94.37 bp.We obtained 365,323 transcripts greater than 100 bp in length,161,522 Unigenes were obtained after further stitching.All 161,522 Unigenes were matched to the public databases,and the Unigenes matched to the GO and KOG databases were 12.153 and 23,477 respectively.A total of 25.173 Unigenes were annotated by KEGG referred to 306 pathways.And then we screened the salt stress response genes according to differentially expressed genes.2.One WRKY gene sequence was filtered from the chrysanthemum transcriptome database.The full length primers were designed and we obtained the full-length sequence of the WRKY gene named DgWRKY2 by cloning.The length of ORF was 978 kb,encoding 325 amino acids,the molecular weight of amino acids was 36.55 kDa and the PI=6.66,that contains 1 typical WRKY domains(WRKYGQK)and zinc finger(C-X4-5-C-X22-23-H-X1-H),We speculated the gene is a member of the WRKY transcription factor ? subfamily.The DgWRKY2 protein was analyzed with the WRKY protein of other plants such as grape,carrot etc.The protein was clustered with carrot DcWRKY71,it suggested that they have a close genetic relationship.3.The plant expression vector was constructed,and then DgWRKY2 was transferred to chrysanthemum by agrobacterium tumefaciens method.The DNA of anti-kanamycin transgenic chrysanthemum was extracted by CTAB method and as a template for PCR detection.And the expression level of DgWRKY2 in chrysanthemum was detected by qPCR.4.The overexpressing DgWRKY2 transgenic chrysanthemum plants had no significant difference in phenotype with wild type plants.The DgWRKY2 overexpressing chrysanthemum and wild type chrysanthemum were subjected to salt stress treatment and the related physiological indexes were determined.The results showed that when the transgenic chrysanthemum was under salt stress,the activities of antioxidant enzyme,proline content,soluble sugar content,soluble protein content and chlorophyll content were higher than that of wild type,while MDA content,H2O2 content and O2-lower,and DAB and NBT staining were also lighter,the survival rate was higher than that of wild type.It was proved that the salt tolerance of transgenic chrysanthemum was higher than that of wild type chrysanthemum.5.The expression of CuZnSOD,CAT,APX,P5CS,DREBIA and DREB2A were studied by qPCR.The overexpression DgWRKY2 through induction genes up-regulated related to ROS signaling pathway,proline biosynthesis signal pathway and ABA signaling pathway to improving the salt tolerance of transgenic chrysanthemum.In this study,we obtained the overexpression DgWRKY2 chrysanthemum,and the salt tolerance of the transgenic plants was increased,and the salt stress response genes were up-regulated in transgenic chrysanthemum,it indicated that the DgWRKY2 gene could regulate the expression of stress-resistant genes to improve plant salt tolerance through proline biosynthetic signal transduction and ABA signal transduction. |
PDF Full Text Request