应用RNAi技术培育抗2种病毒病的转基因烟草

分别提取烟草普通花叶病(TMV)和烟草黄瓜花叶病（CMV）的病毒RNA。经反转录和外壳蛋白阅读框PCR扩增，获得TMV和CMV外壳蛋白基因cDNA, 分别进行两种病毒已知株系cDNA序列比对获得各自的保守序列，设计干涉序列，将干涉片段扩增产物连接到pMD18-T的相邻酶切位点，制备融合序列，并将其正向和反向序列插入pUCCRNAi载体,再转化到pCAMBIA2300-35S-OCS表达载体中。利用农杆菌LBA4404侵染烟草K326,获得3份含有TMV和CMV外壳蛋白基因干涉序列的转化材料，经分子鉴定证实干涉序列已导入烟草，并采用荧光定量PCR技术对其mRNA表达差异进行分析。抗病性调查表明转化烟株对TMV和CMV抗性都显著增强。

Study on tobacco virus resistance with RNAi method

Total RNA of tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were isolated and amplified using RT-PCR and ORF PCR methods.The conserved sequence of two virus among different strains were obtained using sequence alignment in NCBI GenBank; The fusion sequence for RNAi target sequence was built by inserting the interference sequence from CMV and TMV into the neighboring restriction site of pMD18-T.The PCR products with the forward and reverse primers was later subcloned into pUCCRNAi,and transferred into pCAMBIA2300-35S-OCS expression vector.Then introduced into tobacco varieties K326 by Agrobacterium mediated transformation.Three transformants which containing interference sequence of coat protein gene of TMV and CMV were obtained and approved by molecular identification.The results confirmed that RNA interference sequence from two virus have been introduced into Nicotiana tabacum successfully and the inhibition of RNA from virus in the infected tobacco were observed using real-time quantitative PCR.The data of disease screening found that the transformed tobacco showed resistance to TMV and CMV significantly.