商陆抗病毒蛋白cDNA的植物表达载体构建及转化烟草的研究

以商陆抗病毒蛋白的cDNA序列为依据，设计特异性引物，应用PCR技术扩增出5′端含有BamHI，3′端含有KpnI限制性内切酶酶切位点的目的片段，且在序列的开始和结尾分别加入起始密码子ATG和终止密码子TAA，将该目的片段定向克隆于载体pROKII中与CaMV35S启动子和Nos末端构成植物表达载体pRPAP，通过菌落直接PCR法和酶切分析鉴定出阳性重组子，用直接导入法将其导入农杆菌EHA105，以此作为工程菌进行烟草转化，对筛选到的阳性植株随机取样进行Southern印迹分析，结果表明，PAP基因已经整合到烟草基因组中，证明载体构建成功。

Plant Expression Vector Construction and Tobacco Transformation of cDNA Encoding Pokeweed Antiviral Protein (PAP)

The specific primer was designed and synthesized for amplification of antiviral protein gene from pokeweed. The PAP cDNA fragment was amplified by PCR. The plant expression vector (pRPAP) was constructed by cloning the PAP cDNA fragment to pROKII. The positive recombinant was identified by direct PCR on colony and analysis of restrictive enzyme digestion. The engineered strain EHA105 (pRPAP) was constructed by directly conducting the pRPAP to Agrobacterium tumefaciens EHA105. The result showed that the PAP gene was integrated into the genomes of tobacco.