摘要
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。
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.
出处
《中国农学通报》
CSCD
2012年第3期233-239,共7页
Chinese Agricultural Science Bulletin
基金
国家十二五科技支撑计划"南方优质饲草高效生产加工利用关键技术研究与集成示范"(2011BAD17B03)
四川省十二五牧草育种攻关和国家牧草产业技术体系"阿坝综合实验站"(CARS-345)
关键词
绿色荧光蛋白
zeolin基因
载体构建
瞬时表达
green fluorescent protein （GFP）
zeolin gene
vector construction
transient expression
