番木瓜环斑病毒海南株系植物RNAi表达载体的构建  

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

作　　者：赵辉[1,2] 张雨良[2] 孔华[2] 贾瑞宗 朱芸[3,4] 郭安平[2] 曾会才[2] 彭明[2] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所,农业部热带作物生物技术重点开放实验室,海南海口571101 [3]夏威夷农业研究中心,美国夏威夷96797 [4]夏威夷大学,美国夏威夷96822

出　　处：《热带作物学报》2015年第12期2210-2215,共6页Chinese Journal of Tropical Crops

基　　金：农业部948项目“广谱抗环斑病毒转基因番木瓜新品种培育技术的引进与利用(No.2013-Z15)”;“广谱抗番木瓜环斑病毒单/低拷贝转基因技术的引进和应用(No.2014-Z17)”;“番木瓜优质特异种质与抗PRSV转基因株系抗性和安全评价及后续育种技术的引进与应用(No.2015-Z23)”;海南省重点项目“广谱抗环斑病毒高产优质番木瓜新品种选育与应用(No.ZDXM2014052)”

摘　　要：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p UCCRNAi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p CAMBIA2300-35S-OCS植物表达载体,构建出抗PRSV植物表达载体p CAMBIA2300-35S-CP-RNAi-OCS、p CAMBIA230035S-Hc-Pro-RNAi-OCS、p CAMBIA2300-35S-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。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.

关 键 词：番木瓜 番木瓜环斑病毒 RNA沉默 基因转化 

分 类 号：S432.41[农业科学—植物病理学]