Cloning And Function Analysis Of Grape WRKY Genes

In the processes of growth and development, grape often encounters biotic stresses such as pests, diseases and abiotic stresses such as drought, low temperature, mechanical damage, which can retard growth and productivity of plants. Pesticides and fertilizers used frequently cannot fundamentally solve the problems. The breeding of stresses-resistant crop is an only powerful technical countermeasure. Studies have shown that while the plants sense the stresses, the transcriptional factors will bind to specific sites of the gene and activate or inhibit the expression of the gene. The transcriptional factors play a regulatory role in plant defense response. Therefore, the understanding of plant-specific transcription factor, for instance, WRKY is a basis for genetic improvement of valuable commercial crops to resist to the stresses. This study focused on the molecular mechanisms of the WRKY transcription factors in the regulation of gene expression. Two members of WRKY family were cloned from the grape, and then bioinformatics analysis, expression analysis and resistance analysis of WRKYs overexpression were carried out to study the biological functions of WRKY genes. The main results are as follows:1. Homology analysis of56family members of grape WRKY transcription factor was carried out VvWRKY10and VvWRKY26, screened out for further study. The WRKYs were Cloned and the analysis of the open reading frame(ORF) of the WRKYs showed that the conserved WRKY domain was included in the sequences. Here are the results of the bioinformatics analysis:1). The protein compositions of amino acid, signal sequence and phosphorylation sites were analyzed by the bioinformatics-related Web sites and soft wares. The results indicated that VvWRKY10and VvWRKY26, which belong to the secondary group of the WRKY gene family, contained conserved WRKY domain, and multiple phosphorylation sites at serine、threonine and tyrosine residues, but no nuclear localization signal peptide.2). The homology of WRKY protein between grapes and other species was compared by using blastp and DNAMAN softwares. The results demonstrated that these homologous proteins shared similarities in a relatively higher extant, and the similarities were mainly concentrated in the conserved WRKY domain area. 2. Expression levels of VvWRKY10and VvWRKY26were measured by Real-time PCR after induction by SA、ET and mechanical damage. The results showed that the expression levels of both genes hadobvious changes under the stresses.3. The expression vectors of VvWRKY410and VvWRKY26genes were constructed and transfected Arabidopsis by Agrobacterium-mediation method.Transgenic Arabidopsis plants were screened out successfully.The preliminary studies prove that the two transcription factors, VvWRKY10and VvWRKY26, were regulated by some abiotic stresses.The results laid the foundation for the further research on molecular mechanism of the regulation of gene expression, and provide a theoretical basis for the breeding of high-resistant plants.