Cloning, Expression Analysis And Genetic Transformation Of Brachypodium Distachyon Genes BdWARKY2, BdWRKY7 And BdWRKY43

Abiotic stresses can seriously damage the growth and development of crops(such as wheat, barley, oats and so on), and severely reduce the production of them, so there is an urgent need to isolate abiotic stress response related genes and clarify their functions, then cultivate new stress-tolerant crops by genetic engineering methods. Brachypodium distachyon has a closely genetic relationship with wheat, which also belongs to cold-season Pooideae subfamily. The whole genome sequencing of Bd21 has been completed, and it is convenient for gene cloning, so it is an ideal model for wheat function gene study. The WRKY transcription factor is one of the top ten transcription factors in higher plants, which involves in many agronomic traits. The functions of WRKY related to plant growth, development, metabolism, senescence and so on as well as response to biotic or abiotic stresses. Therefore, the research about cloning, expression pattern analysis and genetic transformation of BdWRK Y genes is expected to provide support for genetic improvement of crops.In this study, BdWRK Y2/7/43 genes were cloned. Expression analysis, subcellular localization analysis, transformation of tobacco and the phenotypic analysis under drought stress of them were analyzed. The main results are as follows:(1) By searching for the databases of Brachy WRK Y and Eensembl Plants, the c DNA and DNA sequences of BdWRK Y2/7/43 genes were obtained, then designed specific primers by Primer Premier 5.0, at last the sequences of them were successfully cloned.(2) Results of Multiple sequence aligment by DNAMAN and phylogenetic analysis by MEGA 5.0 showed that BdWRK Y2 belongs to the subfamily IIc, BdWRK Y7 belongs to the subfamily I, BdWRKY43 belongs to the subfamily III.(3) By semi-quantitative RT-PCR to detect the expression levels of BdWRKY2/7/43 genes at different organs, under different abiotic stresses and signal molecules stimulus. The results showed that relatively high expression levels of BdWRK Y2/7/43 genes were at young leaves, and BdWRKY2/7/43 genes were responsive to various abiotic stresses.Expression levels of Bd WRK Y2 gene were upregulated by inducements with PEG, ABA, NaCl, H2O2 and heat; Expression levels of BdWRKY7 gene were upregulated by inducements with PEG, NaCl, H2O2, heat and cold; Expression levels of BdWRKY43 gene were upregulated by inducements with PEG and heat, but downregulated by inducements with ABA, NaCl, H2O2 and cold.(4) Overexpression vectors pCAMBIA1304-BdWRK Y2/7/43 were constructed by inserting BdWRKY2/7/43 genes into pCAMBIA1304 vector.(5) Bombardment of onion epidermal cells showed that transcr iption factors BdWRKY2, BdWRKY7 and BdWRKY43 were all located at the nucleus.(6) Thirteen BdWRK Y2, eight BdWRKY7 and twelve BdWRKY43 overexpression transgenic tobacco plants were obtained by Agrobacterium- mediated technology of leaf disc method. Meanwhile expression levels of a part of these transgenic plants were analyzed by semi-quantitative RT-PCR technology.(7) Phenotypic analysis of Bd WRK Y7 transgenic lines under drought stress indicated that BdWRKY7 transcription factor is a negative regulator in drought stress response.In this study, Bd WRK Y2/7/43 genes were successfully cloned and transformed into tobacco, and the expression levels of several BdWRK Y2/7/43 transgenic plants were analyzed. In addition, the expression profiles of Bd WRK Y2/7/43 genes under different abiotic stresses and signal molecules stimulus were analyzed. At last, the phenotypes of different BdWRKY7 transgenic lines under drought stress were analyzed. This work can lay a foundation for demonstrating functions of the three WRKY genes in response to abiotic stresses and for breeding new stress-tolerant varieties crops.