番木瓜环斑病毒海南株系植物RNAi表达载体的构建

为研究广谱抗环斑病毒（PRSV）海南株系转基因番木瓜育种方案，采集海南番木瓜各主要种植区的PRSV株系，以外壳蛋白（CP）、辅助成分－蛋白酶（HC-Pro）和复制酶（Nib）基因作为靶标基因，通过序列比对和运用Clustalx软件分析，得到干扰基因的保守区域和挑选出海南PRSV典型株系的代表样品。以pUCCRNAi为骨架质粒，利用代表样品的CP、NIb、Hc-Pro基因片断保守序列构建RNAi发夹结构，将发夹结构导入pCAMBIA2300-35S-OCS植物表达载体，构建出抗PRSV植物表达载体pCAMBIA2300-35S-CP-RNAi-OCS、pCAMBIA230035S-Hc-Pro-RNAi-OCS、pCAMBIA2300-35S-NIb-RNAi-OCS。实验将PRSV不同株系保守序列的分析与RNAi的高效抗病设计理念相结合，可用于创造高效、广谱抗PRSV病毒病的新种质。

Construction of RNAi Plant Expression Vectors with Broad Spectrum Resistance to Hainan Papaya Ringspot Virus

The PRSV strains of main papaya planting areas in Hainan were collected for breeding transformation papaya resistant to papaya ringspot virus. The conserved interference regions of target CP, Hc-Pro and Nib gene were analyzed and the typical representative sample strain of Hainan PRSV was selected among them by sequence alignment and by Clustalx software. On the basic of pUCCRNAi vector, the conserved regions of target genes of typical sample was PCRed to construct RNAi hairpin structures. Then the RNAi hairpin structures were jioned into the plant expression vector pCAMBIA2300-35S-OCS. The broad spectrum PRSV resistant plant expression vector of pCAMBIA2300-35S-CP-RNAi-OCS, pCAMBIA230035S-Hc-Pro-RNAi-OCS, pCAMBIA2300-35S-NIb-RNAi-OCS were constructed. Hanging the analysis of PRSV conservative sequence together with the design concept of RNAi high resistance, new germplasms with high efficient and broad virus resistant to Hainan PRSV could be created.