大豆花叶病毒HC-Pro基因保守序列克隆及其RNAi载体的构建  

Cloning of Conserved Sequence of HC-Pro Gene of Soybean Mosaic Virus and Construction of Its RNAi Vector

作　　者：高乐[1] 宋英培 李凯[1] 章红运[1] 沈颖超 翟锐[1] 智海剑[1] 

机构地区：[1]作物遗传与种质创新国家重点实验室/南京农业大学国家大豆改良中心/农业部大豆生物学与遗传育种重点实验室,江苏南京210095

出　　处：《大豆科学》2013年第6期744-749,共6页Soybean Science

基　　金：转基因生物新品种培育专项(2008ZX08004-004);国家自然科学基金(31171574;31101164);现代农业产业技术体系(CARS-004);南京农业大学青年科技创新基金(KJ2010002);江苏高校优势学科建设工程资助项目

摘　　要：大豆花叶病毒(soybean mosaic virus,SMV)病是全球最主要的大豆病害之一,严重危害大豆的产量和品质。RNA干扰(RNA interference,RNAi)能够在mRNA水平特异性沉默同源靶基因,为抗病毒作物的培育提供了新的途径。本研究对国内外11个不同SMV流行株系的HC-Pro基因进行了核苷酸序列比对,并以SC3株系的cDNA为模板克隆出高度保守的268 bp的基因片段,进而利用GATEWAY技术构建了适合农杆菌介导大豆转化的RNAi表达载体pB7GWIWG2(II)-HC-Proi。并对BP反应和LR反应的纯化产物进行了测序鉴定,测得序列在NCBI上进行比对,匹配度为100%;以35S启动子和终止子设计引物,扩增得到464和478 bp两个片段,说明HC-Proi基因与pB7GWIWG2(II)重组形成反向重复结构,载体构建成功。结果为利用RNA干扰技术改良大豆对大豆花叶病毒的抗性奠定了基础。Soybean mosaic virus（SMV） is one of the major viral diseases in soybean, which severely affects the soybean production and quality. RNA interference（RNAi） can specifically silence the homologous target genes in mRNA level,which provides a new strategy for virus-resistant crop breeding. In this study, we made the nucleotide sequence alignment of HC-Pro genes from 11 popular strains of SMV in the world and cloned 268 bp the most conserved gene fragment using the cDNA of SC3 strain. Using the GATEWAY technology, the RNAi vector pB7GWIWG2 （II）-HC-Proi suitable for agrobacterium-mediated transformation of soybean was constructed. Identification of the sequence and the direction of HC-Proi in the vector through the primers of 35S promoter and terminator showed that the recombination of HC-Pro and pB7GWIWG2 （II）formed the inverted repeat. It may lay the foundation for improvement of soybean resistance to SMV using RNA interference technology.

关 键 词：大豆花叶病毒 HC—Pro基因 克隆 RNAI GATEWAY技术 载体构建 

分 类 号：S435.651[农业科学—农业昆虫与害虫防治]