Expression Analysis Of OsCPK9, Its Expression Vector Construction And Genetic Transformation In Rice

Rice is one of the staple food crops and also a model plant in molecular biology. Therice blast caused by pathogenic bacterium Magnaporthe grisea is one of the seriousdiseases to affect the quality and yield of rice, however, frequent application of pesticide isdifficult to work and pollutes the environment easily, while the basic measures to solve theproblem of crop diseases are to strengthen the resistance function and cultivate highresistant varieties. The family of calcium dependent protein kinase （CDPK） gene in ricehas been proved to play important roles in response to various biotic and abiotic stress.OsCPK9from rice belongs the CDPK gene family, and can be specifically up-regulated byinfection of rice blast pathogen. This paper focus on the research of OsCPK9gene in rice,including semi-quantitative expression analysis of different resistant varieties underdifferent treatments, construction of overexpression and RNAi vectors, transientexpression for subcellular localization, genetic transformation in rice. The main results areas follows:(1) Three different resistant varieties were treated with Magnaporthe grisea, salicylicacid (SA) and methyl jasmonate (MeJA), and it was found that the OsCPK9gene wasinvolved in the SA-and MeJA-mediated signal transduction pathway of diseaseresistance, and it locates in the upstream of the signal transduction pathway, because of itsearlier response to rice blast treatment;(2) GFP fusion expression vector of OsCPK9, pBI121-OsCPK9-GFP wasconstructed for the subcellular localization successfully. By using the biolistictransformation technology, fusion plasmid was transformed into onion epidermal cells,and the result indicates the protein of target gene was mainly located in the nucleus andplasma membrane;(3) Overexpreesion and RNA interfence vectors of OsCPK9gene, pSN1301-OsCPK9and pSN1301-RNAi were successfully constructed;(4) Transgenic rice of overexpression, RNAi and vacant vector were obtained viaAgrobacterium mediated transformation method;(5) Using molecular identification and GUS histochemical staining method,10 vacant vector control,30overexpression and10RNAi transgenic lines were gotten. Bysemi-quantitative PCR method for further identification, we finally obtained:2vacantvector transgenic lines having similar expression level of OsCPK9with wild-type,2overexpression transgenic lines having obvious higher expression level of OsCPK9thanwild-type, and2RNAi transgenic lines having obvious lower expression level of OsCPK9than wild-type. The results of present study provided with usuful materials andinformation for further studying the function of OsCPK9in rice against rice blastinfection.