Cloning And Transformation Of Cucumber Calcium-dependent Protein Kinase

Nitrogen fertilizer is very important for plants. Cucumber (Cucumis sativus L.) is one of themost important vegetables worldwide, which growth needs more nitrogen fertilizers. With theincreasing demand of cucumber yield, the demand of nitrogen fertilizer was also increased. Thisdoes not only cause a reduction of cucumber fruit quality and food safety, but also cause harm tohuman health; therefore, nitrogen is lost to the atmosphere or leached into groundwater, lakes andrivers, which causes increasingly severe pollutions to the environment. Furthermore, fertilizerapplication has now become the major cost in crop production, which greatly affects the income offarmers. Therefore, to breed a new cucumber cultivar with a high capability of low-nitrogenresistance is essential to reduce the cost of cucumber production as well as environmental damageand harm to human health.Calcium-dependent Protein Kinases which phosphorylated a key enzyme of sucrose synthesispathway phosphorylation-sucrose phosphate synthase (SPS) and the rate-limiting enzyme of nitrateassimilation Nitrogen-nitrate reductase(NR) involved in plant carbon and nitrogen metabolism andstress response. A previous study of our team revealed that CDPK gene expression in cucumberleaves was up-regulated in low nitrogen stress conditon. This may be involved in signaltransduction of abiotic stresses to improve the resistance of cucumber to resistant low nitrogen.Therefore, cloning and expression analysis of CDPK under low nitrogen onditions can explore itsfunction and help improving cucumber resistance under low nitrogen stress in the futher.In this study, the full-length cDNA of CDPK gene was cloned in cucumber by PCR, and itssequence characteristics, expression pattern was also investigated. The construction of plantexpression vector and establishment of Cotyledon regeneration system of Cucumis sativus L. invitro. CDPK gene was transformed into Cucumis sativus L. and Arabidopsis thaliana viaAgrobacterium tumefaciens.The main results were as follows.(1) CDPK was amplified by RT-PCR from a cucumber cultivar with low-nitrogen resistance‘Jinyan No.4’ leaves under low nitrogen conditions. The gene is1584bp and encoding527aminoacids, estimated molecular weight was60KD and isoelectric point was5.195. The protein encodedby this gene was a stable and hydrophilic protein, no transmembrane structure and no signalpeptide, and with EF-hand calcium-binding domain, and serine/threonine protein kinase activesite,and so on. Blast on NCBI indicated that protein encoded byCDPK of cucumber had more than70％similarity with the Vitis vinifera, Populus trichocarpa and Ricinus communis, was aconserved gene.（2）Construction of plant expression vectors pBI121-CDPK was successed.(3)8positive transgenic cucumber plants were obtained by PCR which contains3excessiveexpression plants and5antisense expression plants. Got the22Arabidopsis transformation seedlings, including12excessive expression plants and10antisense expression plants.