中国商陆抗病毒蛋白基因的克隆及其转化辣椒的研究

本研究的目的是获得抗病毒病的辣椒材料，为选育抗病毒病辣椒品种奠定基础。根据美洲商陆抗病毒蛋白基因序列设计引物，从中国商陆（Phytolacca acinosa）叶片基因组DNA扩增克隆缺失无毒型中国商陆抗病毒蛋白基因（PacPAP1，构建该基因的植物表达载体，采用农杆菌介导的叶盘法转化辣椒，对转基因植株进行分子检测、TMV及CMV的人工接种鉴定，接种25d后，统计发病情况。结果表明：克隆得到缺失无毒型PAP基因PacPAP，大小为714 pb，与美洲商陆抗病毒蛋白基因（PamPAP）的同源性为99.7%；得到了PacPAP1整合入辣椒基因组中的阳性植株；接种TMV、CMV阴性对照植株都明显发病，转PacPAP1基因植株未出现症状，说明转PacPAP1辣椒植株可获得抗TMV和CMV材料。

Cloning of Pokeweed Antiviral Protein Gene from Phytolacca acinosa and its Transfer to Pepper(Capsicum annuum L)

The aim of this study is to obtain transgenic pepper plants resistant to TMV and CMV that could be used in breeding new pepper variety with virus-resistant traits. The primers were designed based on the sequences of PAP in Phytolacca Americana to amplify non-virulent defective PAP gene , PacPAP1 from genomic DNA of leaves in Phytolacca acinosa. The expression vectors was constructed and the PacPAP1 was transferred into pepper (Capsicum annuum L.) by flamingo bill mediated-Agrobacterium. In the transgenic plants , the molecular test were done and resistance assay was performed at 25d following artificial inoculation with TMV or CMV. The results showed that the size of the isolated non-virulent defective PacPAP is 714bp, bearing 99.7% identity with PamPAP from Phytolacca american. The accession numbers of the gene in GenBank is AY603353. The transgenic pepper plants were obtained with PacPAP. The non-transgenic pepper plants were susceptible to virus, no symptoms were observed in the positive transgenic plants. This suggests that transgenic pepper plant resistant to TMV and CMV can be obtained by transferring PacPAP1.