建兰花叶病毒和齿兰环斑病毒RdRp基因hpRNAi表达载体的构建

Construction of hpRNAi expression vector of Cymbidium mosaic virus and Odontoglossum ringspot virus RdRp gene

为了构建建兰花叶病毒(CymMV)和齿兰环斑病毒(ORSV)复制酶RdRp基因hpRNAi的表达载体,根据GenBank中CymMV和ORSV RdRp基因的保守序列设计引物,以蝴蝶兰温室生产中两种病毒的分离物为模板,成功扩增了两种病毒的RdRp基因片段。扩增的这两种病毒的RdRp基因与数据库中病毒序列同源性均在98%以上。利用融合PCR将两种病毒片段串联在一起,然后通过Gateway技术的BP反应和LR反应将融合产物插入到基因沉默表达载体pHellsgate12中,成功构建了可同时抗建兰花叶病毒和齿兰环斑病毒的发夹结构RNA干扰载体,为兰花多抗基因工程育种奠定了基础。

In order to construct the hpRNAi expression vector of RNA-dependent RNA polymerase genes （RdRp） of Cymbidium mosaic virus and Odontoglossum ringspot virus, the primers of conserved sequences in the RdRp genes of the two viruses were designed and synthesized. With the two viruses separated from Phalaenopsis in greenhouse as templates, the RdRp genes of the two viruses were amplified successfully and had the homology to other RdRp genes in public database of above 98%. The RdRp genes of the two viruses were jointed by fusion PCR. The fusion products were inserted into gene silencing expression vector pHeUsgatel2 by a BP recombination reaction and an LR recombination reaction in Gateway technology. The hairpin structure RNA interference carrier resistant to both Cymbidium mosaic virus and Odontoglossum ringspot virus was constructed successfully, which provides a foundation for genetic engineering breeding in orchids.