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为了获得既抗虫又抗病毒病,又对人体和环境安全的转基因辣椒(Capsicum annuum L.)材料,将启动子CaMV35S、终止子Nos-ter及缺失无毒型商陆抗病毒蛋白基因PamPAP一起插入到植物表达载体pCAMBIA1301-Cry2Aa2-PMI的多克隆位点上,构建双价表达载体,采用农杆菌介导法将其转入辣椒中,并用PMI/甘露糖筛选体系对转化植株筛选。经PCR扩增和Southern杂交检测,结果表明Cry2Aa2和PamPAP已经整合到辣椒基因组中。进一步经RT-PCR分析,证实Cry2Aa2和PamPAP在T0代转基因辣椒植株中共表达。经抗虫性、抗病毒病表型鉴定:T0代转基因辣椒植株对黄瓜花叶病毒(CMV)和斜纹夜蛾幼虫的抗性均显著高于非转基因植株。
In order to obtain a transgenic pepper (Capsicum annuum L.) material that is both insect-resistant and antiviral as well as safe for humans and the environment, the promoter CaMV35S, the terminator Nos-ter and the deletion of the nontoxic Pseudomonas aeruginosa protein PamPAP Inserted into the multiple cloning site of the plant expression vector pCAMBIA1301-Cry2Aa2-PMI to construct a bivalent expression vector, which was transformed into pepper by Agrobacterium-mediated method and the transformed plants were screened with a PMI / mannose screening system. PCR and Southern hybridization showed that Cry2Aa2 and PamPAP had been integrated into pepper genome. Further analysis by RT-PCR confirmed that Cry2Aa2 and PamPAP were co-expressed in T0 generation transgenic pepper plants. The anti-insect and anti-virus disease phenotype identification: T0 transgenic pepper plants cucumber mosaic virus (CMV) and Spodoptera lye larvae were significantly higher than non-transgenic plants.