Cloning, Expression Analysis And Genetic Transformation Of BdSnRk2.6 And BdSnRk2.7 Genes From Brachypodium Distachyon

Plants, the main members of the ecosystem, are always affected by some extreme environment in the process of growth, such as low temperature, drought, and high salinity, etc. However, plants have developed some resistance mechanisms to resist these adverse effects in the long process of evolution.There are many resistance mechanisms in plants and animals. Protein phosphorylation and dephosphorylation is one of the common regulatory mechanisms. It is catalyzed by protein kinase, and participates in many signaling transduction pathways. The SnRK2(sucrose non- fermenting1-related protein kinase 2) is a kind of Ser/Thr protein kinase existing widely in plants. SnRKs can participate in various signaling transduction pathways and improve plants abiotic stress tolerance. A large number of SnRK2 kinase has been found in Rice, Arabidopsis and some other plants, however, little is known about SnRK2 gene in Brachypodium distachyon is, a new model plant for monocotyledonous plants. In this study, two Sn RK2 genes, designated BdSn RK2.6 and BdSn RK2.7 were cloned successfully in B. distachyon. And the function of the two genes in resistance aspects has been conducted using molecular biology and genetics methods. The main research results are as follows:(1) Through the homology Brachypodium genome database, we searched the BdSn RK2 s sequence, and designed the specific primer, we cloned the full- length c DNA sequence of two Sn RK2 genes, which named as BdSnRK2.6 and BdSn RK2.7 according to phylogenetic tree.(2) The gene expression in different organs were analyzed by qRT-PCR, The results showed that BdSn RK2.6 expressed in stem and leaves, could not detect expression in root and spike; BdSn RK2.7 expressed in root, stem and spike, could not detect expression in leaves; Through the simulation of abiotic stress and signal molecules to handle B. distachyon, it was found that the two genes were up-regulated in a variety of treatments.(3) The yeast expression vectors pYES2- BdSnRK2.6 and p YES2- BdSn RK2.7 were constructed, and were transformed into IN VSC1 yeast, including empty vector plasmid. Results showed that yeast cells transformed BdSn RK2.6 had increased tolerance to 50? treatment among different stress treatments, however, BdSnRK2.7 had increased tolerance to NaCl treatment among different stress treatments.(4) In order to investigate the subcellular localization of BdSnRK2.6 and BdSnRK2.7, the pCAMBIA1304-BdSn RK2.6 and pCAMBIA1304-BdSn RK2.7 recombinant plasmid were constructed and transformed into onion epidermis. The results showed that BdSnRK2.6 and BdSnRK2.7 had no specific subcellular localization, distributed in the whole cel.(5) The recombinant vector plasmid and empty vector plasmid were transformed into Agrobacterium tumefaciens, and transgenic plants were obtained by Agrobacteriummediated transformation method.The results of these experiments have laid a foundation for further study on BdSn RK2.6 and BdSn RK2.7 in response to abiotic stress and functional study of this gene family.