植物研究2011，Vol.31Issue(5)：537-542,6.
甘蔗黄叶病毒CP蛋白基因ihpRNA表达载体构建及烟草转化
Construction of ihpRNA Expression Vector of Sugarcane Yellow Leaf Virus CP Protein Gene and Genetic Transformation in Tabacco
摘要
Abstract
To improve the sugarcane resistance against disease, a suagrcane yellow leaf virus CP protein mediated RNA interference technology was set up. In the paper, a RNAi expression vector harboring CP protein gene fragment of sugarcane yellow leaf virus was constructed. On the basis of the complete genome sequence of sugarcane yellow leaf virus Hainan isolate ( GenBank accession no. HQ342888), two pairs of specific primers, CPsfl/CPsrl and Cpasfl/Cpasrl, containing different enzyme sites were designed. With the template of pMD19-T/CP plasmid constructed and middle vector of pRNAilO17, positive sense strand and antisense strand were obtained, which were separately inserted into the plant expressional vector pCAMBIA2300. The RNAi vector p2300-CP-F-R containing a hairpin structure was confirmed by the digestion of restriction enzyme Pst I . The p2300-CP-F-R was transformed into tobacco by Agrobacterium-medialed transformation system. The results revealed that 12 transgenic tobacco plants were obtained by PCR, Southern blot and semiquantitative RT-PCR. The CP gene was succeeded in being integrated into genome of tobacco and transcripted. This work laid the foundation for breeding of plant mediated RNAi technology in sugarcane against disease.关键词
甘蔗黄叶病毒/CP外壳蛋白/RNA干扰/ihp表达载体/烟草转化Key words
sugarcane yellow leaf virus/ CP protein/ RNA interference/ ihpRNA expression vector/ tobacoo transformation分类
农业科技引用本文复制引用
张雨良,熊国如,王健华,张树珍,官梅花,杨文君,刘志昕..甘蔗黄叶病毒CP蛋白基因ihpRNA表达载体构建及烟草转化[J].植物研究,2011,31(5):537-542,6.基金项目
国家科技支撑计划项目(2007BAD48B01)、中央级公益性科研院所基本科研业务专项(ITBBZD1051)和现代农业产业体系建设专项资金(nycytx-24) （2007BAD48B01）
