黄瓜Rubisco活化酶基因CsRCA表达载体构建与遗传转化  

Expression Vector Construction of Rubisco Activase Gene CsRCA and Genetic Transformation to Cucumber

作　　者：刘培培[1] 姜振升 王美玲[1] 毕焕改[1] 艾希珍[1] 

机构地区：[1]山东农业大学园艺科学与工程学院,作物生物学国家重点实验室,山东泰安271018 [2]山东金正大生态工程股份有限公司,山东临沂276700

出　　处：《园艺学报》2012年第5期869-878,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30972032);国家‘973’项目(2009CB119000)

摘　　要：核酮糖–1,5–二磷酸羧化/加氧酶（Rubisco）是光合碳循环中的关键酶,为了探明其在植物体内的活化机制,用农杆菌介导法将Rubisco活化酶（RCA）基因CsRCA导入黄瓜,分别用PCR、real-timePCR和Western杂交法进行分子检测,分析转基因植株的表达量。酶切鉴定结果显示,CsRCA正向插入植物表达载体pCAMBIA1301的CaMV35S启动子和NOS终止子之间,成功构建CsRCA的正义表达载体。将pCAMBIA1301-CsRCA导入黄瓜自交系‘08-1’,获得7株转基因植株,拷贝数均为2（非转基因植株的拷贝数为1）,转化率约为3.5%。表达分析结果表明,7株T0代转基因植株叶片的CsRCA mRNA表达量为野生型（WT）的1~1.98倍,在蛋白水平的表达信号显著强于WT。T1代转基因植株的叶片叶绿素和类胡萝卜素含量、光合速率（Pn）及可溶性糖和淀粉含量均显著高于WT。研究结果表明,利用农杆菌介导法获得了稳定遗传的黄瓜CsRCA转基因植株,CsRCA过量表达能显著提高黄瓜叶片的Pn,增加干物质积累。Ribulose-1,5-bisphosphate carboxylase/xygenase（Rubisco）is a key enzyme in the photosynthetic carbon cycle. In order to elucidate the activating mechanism of Rubisco in plants,the Rubisco activase gene CsRCA was introduced into inbred line of cucumber with the Agrobacterium-mediated method. The transgenic plants were screened by PCR,and their expression in mRNA and protein level were analysed by real-time PCR and western blot respectively. The restriction enzyme result showed that the CsRCA was inserted into the binary vector pCAMBIA1301,a sense expression vector containingCsRCA gene was constructed. The resulting plasmid was introduced into cucumber inbred lines‘08-1’,and seven transgenic plants which contain two CsRCA copies were obtained（The wild type plants contain one CsRCA copy）. The transformation rate was about 3.5%. The seven T0 transgenic cucumber plants showed 1–1.98 folds in CsRCA mRNA abundance and a significant increase in expression of protein level compared with the wild type（WT）plants. CsRCA over expression led to significant increase in the pigment content, photosynthetic rate Pn）,soluble sugar and starch contents in T1 transgenic plant leaves. These data （ indicated that stable genetic CsRCA transgenic cucumber plants were obtained with Agrobacterium-mediated method. CsRCA over expression increased the Pn and carbohydrate significantly in cucumber leaves.

关 键 词：黄瓜 核酮糖-1 5-二磷酸羧化/加氧酶(Rubisco) Rubisco活化酶(RCA) 遗传转化 过量表达 

分 类 号：S642.2[农业科学—蔬菜学]