玉米磷酸烯醇式丙酮酸羧化酶基因高效表达载体构建及其导入小麦的研究  

Construction of a High-efficient Expression Vector for Maize Phosphoenolpyruvate Carboxylase Gene and Its Transformation in Wheat

作　　者：李艳[1,2] 许为钢[2] 胡琳[2] 张磊[2] 齐学礼[2] 张庆琛[1] 王根松[2] 

机构地区：[1]河南农业大学农学院,河南郑州450002 [2]河南省农业科学院小麦研究中心,河南郑州450002

出　　处：《麦类作物学报》2009年第5期741-746,共6页Journal of Triticeae Crops

基　　金：国家转基因生物新品种培育重大专项项目(2008ZX08002-003);河南省杰出人才计划项目(084200410005);河南省基础与前沿技术研究计划项目(082300430090)

摘　　要：为获得具有C4光合特征的高光效转基因小麦材料,利用从玉米中克隆的磷酸烯醇式丙酮酸羧化酶(PEPC)基因(pepc,GenBank接受号为FJ415327)构建高效双元表达载体,通过基因枪介导法将其导入小麦品种(系)中,利用Real-time PCR方法分析外源基因在转基因植株中的拷贝数。PCR和双酶切鉴定表明,玉米PEPCcDNA序列已插入双元表达载体pCAMBIA3301中,命名为p3301-pepc;对获得的抗性再生植株进行PCR扩增,其中有342株扩增出目的条带;在选取的15株转基因植株中7株拷贝数为1,3株拷贝数为2,2株拷贝数为3,拷贝数为5、8和17的分别为1株。初步证明玉米pepc基因已整合到小麦基因组中,且外源基因在转基因植株中既有单拷贝插入,也有多拷贝插入,这为进一步研究该基因在小麦基因组中的功能表达提供了试验材料。Transferring of key enzymes gene of C4 photosynthetic character is an important pathway to improve the photosynthesis efficiency and seed yield of C3 crop wheat. Phosphoenolpyruvate carboxylase （PEPC） plays an important role in C4 photosynthetic pathways. In order to obtain high photosynthetic efficiency transgenic wheat germplasm with C4 photosynthetic characteristics, a full-length cDNA sequence coding for maize PEPCase gene was cloned into the high-efficiency binary expression vector pCAMBIA3301, and was introduced into wheat by particle bombardment mediated transformation. The copy number of different transgenic lines were analyzed by real-time fluorescence quantitative PCR. PCR and double enzymes digestion determined that maize C4-specific PEPLase gene was cloned into the high-efficiency binary expression vector pCAMBIA3301, named as p3301-pepc; the expected 164 bp fragment was present in 342 of the 400 resistant plants; Among the fifteen positive transgenic lines, seven had one copy three had two copies, two had three copies, one had five copies, one had eight copies and one had seventeen copies. The results preliminary proofed that maize C4-specific PEPLase gene was integrated into wheat genome, and the copy number was different in transgenic lines, which would provide experimental materials for further function study of pepc gene in wheat genome.

关 键 词：小麦 玉米磷酸烯醇式丙酮酸羧化酶(PEPC) 高效表达载体 转基因 拷贝数 

分 类 号：S512.1[农业科学—作物学] S336