高粱花叶病毒Hcpro、CP和PIPO基因酵母双杂交载体构建及自激活验证  

Construction of Bait Vectors Containing Hcpro,CP and PIPO Genes from Sorghum mosaic Virus in Yeast Two-hybrid System and Identification of Their Autonomous Activation

作　　者：张雨良[1,2] 黄启星[1,2] 张树珍[1,2] 王健华[1,2] 余乃通[1,2] 刘志昕[1,2] 

机构地区：[1]中国热带农业科学院热带生物技术研究所,海南海口571101 [2]农业部热带作物生物学与遗传资源利用重点实验室,海南海口571101

出　　处：《热带作物学报》2012年第12期2252-2256,共5页Chinese Journal of Tropical Crops

基　　金：中央级公益性科研院所基本科研业务专项(ITBB110303);现代农业产业体系建设专项资金(nycytx-24);国家科技支撑计划课题(2007BAD48B01)

摘　　要：采用同源克隆技术获得侵染甘蔗高粱花叶病毒(Sorghum mosaic virus,SrMV)的Hcpro、CP、PIPO基因片段。将该基因片段连接至pGBKT7载体,分别构建酵母双杂交诱饵载体pGBKT7-Hcpro、pGBKT7-CP和pGBKT7-PIPO,经PCR、酶切及测序鉴定,证明重组诱饵载体构建成功后,将重组质粒导入Y2H Gold酵母菌株,检测其表达产物对酵母细胞有无毒性及对报告基因有无激活作用。结果表明,含重组诱饵质粒的酵母菌株在SD/-Trp营养缺陷平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性。含重组诱饵质粒的酵母菌株在SD/-Leu、SD/-Trp/-Ade和SD/-Trp/-His营养缺陷平板上不能生长,表明重组诱饵质粒表达产物对ADE2、HIS3报告基因无自激活作用。研究结果为下一步利用酵母双杂交系统检测与高粱花叶病毒Hcpro、CP、PIPO蛋白相互作用的甘蔗蛋白提供基础。The HcPro, CP and PIPO genes encoding fragments from sorghum mosaic virus （SrMV） were obtained by homologous cloning and then fused with the pGBKT7 vector to construct bait vectors pGBKT7-Hcpro, pGBKT7-CP and pGBKT7-PIPO. After confirmation with PCR, enzyme restriction and sequencing analysis, the plasmid was transformed into the yeast cell Y2H Gold, and its toxicity and transcriptional activation was tested by color assay. The yeast strain Y2H Gold transformed with bait plasmids grew well on SD/.--trp plate, without toxicity effect, whereas, they could not grow on SD/-Leu, SD/-Trp/-Ade and SD/-Trp/-His plates, which indicating there were no autonomous activities between the bait proteins and ADE2, HIS3 report genes. These results lay the foundation for using yeast two-hybrid system to screen the sugarcane proteins interacting with HcPro, CP and PIPO genes.

关 键 词：高粱花叶病毒 Hcpro CP PIPO 酵母双杂交 诱饵载体 

分 类 号：S432.4[农业科学—植物病理学] Q78[农业科学—农业昆虫与害虫防治]