苹果抗 ASPV 病毒 RNAi 载体构建及转化砧木 M26的研究

摘要

In order to develop novel disease-resistant transgenic plant materials against ASPV,a virus resistant RNAi plant expression vector was constructed by the method of homologous recombination.With the Malus rootstock of M26,genetic transformation was conducted and positive transgenic plantlets were obtained successfully.Firstly,a conserved segment of coat protein gene of ASPV (489 bp)was cloned by PCR from cDNA of the virus-infective leaves of Royal gala.Then the fragment was inserted into the vector pENTR /SD /D by TOPO cloning technology to form the intermediate vector of pEN-ASPV.After this fragment was replaced and connected into the destination vec-tor pHellsgate12 by LR reaction,the RNAi plant expression vector Phe-ASPV was obtained.And the engineering bacteria EH-ASPV for plant transformation was obtained after the constructed RNAi vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.By the Agrobaterium mediated method, transformation was conducted with the leaf discs of the Malus rootstock of M26.After culture and subculture for anti-biotics screening,bacteria elimination and rooting,compact kanamycin resistant plantlets formed from buds were ob-tained.After total RNA extracted from the well rooted plantlets,RT-PCR test was conducted and the result showed that the gene fragment of 489 bp carried on the RNAi structure had been transformed into the material of M26, which was expressed in plants on the level of RNA.Through this study,novel transgenic materials were obtained successfully,which laid foundation for disease-resistance evaluation by pathogen inoculation and virus-resistant mo-lecular breeding in the near future.%为获得抗病毒的转基因植物新材料，采用同源重组法构建了抗苹果茎痘病毒的植物表达载体，并对苹果砧木材料 M26进行了遗传转化，成功获得了转基因阳性植株。首先以感病的皇家嘎拉叶片为材料，反转录合成 cDNA第一链，采用 PCR 方法克隆获得了489 bp 的 ASPV 外壳蛋白基因的保守片段，通过 TOPO 克隆技术将该基因片段连接进入载体 pENTR ／SD ／D，构建了入门克隆载体 pEN-ASPV，再通过 LR 反应置换该片段连接进入目标载体 pHells-gate12，构建了 RNAi 植物表达载体 Phe-ASPV，并采用冻融法转入根癌农杆菌菌株 EHA105中，获得了可用于植物遗传转化的工程菌 EH-ASPV。采用农杆菌介导的方法转化苹果砧木 M26叶盘，经抗生素筛选和脱菌培养获得了 Kan 抗性芽，进一步进行生根培养后获得了完整的 Kan 抗性植株，对生根植株提取总 RNA 后 RT-PCR 检测结果显示，RNAi表达载体上携带的外源基因片段（489 bp）已成功转入苹果砧木材料 M26中，并在转基因植株内 RNA 水平上得到表达。获得的转基因新材料，为进一步通过病毒接种进行抗病性评价及抗病毒分子育种奠定了基础。