Clonging And Expression Analysis Of WRKY Family Genes In Betula Platyphylla

The WRKY transcription factor family is a category of transcription factors which is researched widely in recent years,it mainly exists in higher plants and rarely found in lower plants.It participates in various physiological processes of plant,including metabolic regulation,morphogenesis,seed germination,a variety of biological and abiotic stress factors induced WRKY gene expression in different extent,it also showed that the WRKY protein plays an important role in defense of plant reflect.This study rely on the NCBI site blastx to find 131 WRKY genes which is known,finally we got 37 pieces in a complete open reading frame finder(ORF)WRKY genes,obtain the whole part of the gene cDNA sequence,cloned the 20 pieces of the WRKY genes for further research.Real-time fluorescent quantitative technique was used to study the amount of gene expression under abiotic stresses in different time points or in different tissues of birch,the results showed that these genes expressed in different organizations specificly.under different abiotic stress presents express the different trend,under the same abiotic stress in different time point its expression also has certain differences.Integrated the above expression pattern to analysis results,analysis of the gene sequence and evolutionary tree etc,this article selects BplWRKY6 gene as the object of the follow-up study.Fuse BplWRKY6 gene with GFP gene,and transformated into onion epidermal cells by particle bombardmentgun to study the subcellular localization of BplWRKY6,the result shows that BplWRKY6 located in the cell nucleus.Cloned BplWRKY6 promoter,predict the possible cis-acting element associated with corresponding adversity stress on the promoter sequences by PLACE website,presumably the gene associated with adversity stress response.Replace 35S in expression vector pBI121-GUS with BplWRKY6 promoter directionaliy,build the plant expression vector which drive GUS gene,instantaneous infect the whole plant of wild type birch,result shows that the leaves,stems and roots of birch had different type of dyeing,BplWRKY6 promoter is not only drive the gene expression,but also has certain organization specificity.Build the target gene into the overexpression vector,pROK Ⅱ-BplWRKY6 overexpression vector was transformated into Arabidopsis by agrobacterium tumefaciens-mediated method,observe the phenotype of the wild-type plants and the overexpression plants.Results showed that the growth speed and development situation at an early stage has no significant differences,but eventually the height of over-expression strain plant express much lower than the wild type strains,drawing stem is also more delay.Under the abiotic stress of NaCl and Mannitol,seed germination rate of the over-expression strain plant is lower than the wild type.Physiological dyeing results of DAB,NBT and Evans Blue show that BplWRKY6 gene overexpression leads the release of reactive oxygen and the damage of cells.Instantaneous infection of wild type birch,the result of real-time fluorescent quantitative PCR showed that SOD,POD genes related to the resistance showed different degrees of down-regulated expression.We can indicate BplWRKY6 gene in plants is a negative regulatory factor.Built the target gene into yeast expression vector pGBKT7-BplWRKY6 to validate whether BplWRKY6 gene has self activation activity or not.Results show that the colony on SD/-Trp/-His/X and SD/Trp/-His/-Ade/X are blue,BplWRKY6 gene has self activation activity is proved.As there is no self activation activity in conservative district,according to the prediction of birch BplWRKY6 gene conservative district,it can be divided into four segments,respectively built into yeast expression vector pGBKT7,to confirm the location of the self-activation.The results showed that fragment in BplWRKY6 gene which has self-activation is located in the former 1-50 amino acid sequence.