Cloning, Expression Analysis And Genetic Transformation Of BdCDPK4 And BdCDPK14 From Brachypodium Distachyon

During the growth and development processes, plants are constantly challenged by various bio- and abiotic stresses such as drought, salinity, extreme temperatures and pathogen attack. In long time of evolution, plants have developed sophisticated mechanisms to respond and combat with these environmental stresses. Ca2+, as a second messenger in signal transduction pathway, plays important roles during the growth and development processes of plants and in response to stresses. Calcium dependent protein kinases(CDPKs) are vital parts in the calcium signaling pathway. An array of evidences has shown that CDPKs participate in the regulation of plant growth and in response to environmental stresses.Brachypodium distachyon, the genome of which shares high homology with wheat, barley and other Poaceae, has been used as new model species of Gramineae. Researches on CDPKs in B. distachyon are rare. Studies on two numbers of BdCDPK, BdCDPK4 and BdCDPK14 in B. distachyon were carried out, and the main results are as follows:(1) The cDN As encoding two numbers of BdCDPK from B. distachyon, designed as BdCDPK4 and BdC DPK14 respectively, were successfully cloned using specific primer pairs designed according to the retrieval results of NCBI. Multiple sequence alignment results suggest that BdCDPK4 and BdCDPK14 are truly members of CDPK family.(2) Results of semi-quantitative RT-PCR showed that BdCDPK4 and BdCDPK14 were expressed in all tissues including root, stem, leaf and caryopsis, with higher expression level in root and leaf, and lower expression in stem and caryopsis; the expressions of BdCDPK4 and BdCDPK14 were upregulated by 20% PEG, cold and heat, while their expressions were downregulated by hydrogen-peroxide treatment. NaCl treatment obviously upregulated BdCDPK14 and salicylic acid treatment downregulated BdCDPK4.(3) Transient expressions of p BI121-35S::BdCDPK4-GFP and pBI121-35S:: BdCDPK14-GFP constructs in onion epidermal cells showed that both BdCDPK4-GFP and BdCDPK14-GFP were mainly localized to the plasma membrane.(4) In order to clarify the functions of these two genes, overexpression vectors of pCAMBIA1304-BdCDPK4 and pCAMBIA1304-BdCDPK14 were constructed and were transferred to the tobacco via Agrobacterium mediated transformation method. A total of 12 and 9 PCR-positive BdCDPK4 and BdCDPK14 overexpressing transgenic tobacco plants have been successfully obtained.Results of present study provided with useful information and materials for further studying the function of these two genes in response abiotic stresses.