pCB-zeolin-GFP表达载体的构建及瞬时表达

为利用荧光蛋白基因GFP检测外源基因在转基因植株中的表达和定位，构建含有GFP基因的植物表达载体pCB-zeolin-GFP。在目的基因的开放阅读框（ORF）两端设计引物，并引入酶切位点和保护碱基，用PCR方法从pDHA扩增得到zeolin基因的全长，克隆到中间载体pMD18-T，分别用NcoⅠ和BglⅡ2种限制性内切酶酶切重组质粒和经过改良的pCAMBIAI1302植物表达载体，经回收、连接、转化、鉴定后，利用基因枪转化法将重组载体转入洋葱表皮细胞，通过共聚焦显微镜检测绿色荧光蛋白在洋葱表皮细胞中的瞬时表达。构建了zeolin基因与绿色荧光蛋白（GFP）融合的植物表达载体pCB-zeolin-GFP，并在洋葱中得到了表达。构建的融合植物表达载体pCB-zeolin-GFP正确，该载体的成功构建为今后进行基因转移、基因功能研究及培育新品种奠定了基础。

Construction of Plant Expression Vector pCAMBIAl302-zeolin-GFP and the Transient Expression

To utilize fluorescin gene GFP to detect the express and localization of exogenous gene, plant expression vector pCAMBIAl302-zeolin with a green fluorescent protein gene would be constructed. The total length sequence of zeolin gene in pDHA plasmid was amplified by PCR. The fragment was cloned into pMD18-T middle vector, and a new recombined vector named pMD18-T-zeolin was obtained. A new plant expression vector named pCB-GFP-zeolin was constructed after cutting two vector pMD18-zeolin and pCAMBIAI1302 with restriction enzymes, subsequently reclamation, ligation, transformation and identification. Then the recombined plant expression vector pCAMBIAl302-zeolin with a green fluorescent protein gene was transformed into epidemic cells of onion by gene gun method and was detected by confocal microscopy. The recombined plant expression vector pCAMBIAl302-zeolin with a green fluorescent protein gene had been constructed and the fusion gene was transient expressed. It was suggested that the recombined plant expression vector pCAMBIAl302-zeolin with a green fluorescent protein gene was successful. Construction of the plant expression vector might play an important role in genetic transformation, the gene functional identification and breeding of new varieties.