马铃薯卷叶病毒缺失突变CP基因酿酒酵母表达载体的构建  

Construction of Expression vector Vector of Deletion Mutation CP Gene of Potato Leafroll Virus for Saccharomyces cerevisiae

作　　者：左玉玲[1] 王晓杰[1] 樊连梅[1] 王晶珊[1] 郭宝太[1] 

机构地区：[1]青岛农业大学生命科学学院,山东青岛266109

出　　处：《青岛农业大学学报（自然科学版）》2009年第4期305-308,共4页Journal of Qingdao Agricultural University(Natural Science)

基　　金：山东省自然科学基金(Y2008D43);青岛市自然科学基金(05-1-JC-91)资助

摘　　要：以重组质粒pBAD-LRCP-126为模板,经PCR扩增获得了PLRV缺失突变CP基因的特异性条带。目的片段与pYES2.1/V5-His/-TOPO的连接产物转化大肠杆菌TOP10F′获得了氨苄青霉素抗性菌落,酶切与DNA测序结果确认了酵母表达载体的正确性,该表达载体命名为pYES-LRCP-126。在30℃,酿酒酵母工程菌株INVSc1(pYES-LRCP-126)经2%半乳糖诱导培养16h,SDS-PAGE显示蛋白图谱上有一条23kDa的诱导表达的蛋白条带,其大小与预期的重组CP大小相符,表明PLRV缺失突变基因在酵母菌中实现了表达。Using plasmid pBAD -LRCP - 126 as template, specific band of PLRV deletion mutation CP gene was obtained by PCR amplification. The target DNA fragment was recovered and ligated into the initial expression vector pYES2.1/V5 -His/-TOPO. The recombinant plasmid was transformed into E. coli TOP10F＇. Single digestion of restriction endonuclease and DNA sequencing confirmed the accuracy of the CP gene expression vector named pYES -LRCP- 126. The engineered strain INVScl （pYES- LRCP- 126） was induced with 2% galactose at 30℃ for 16 hours, and specific 23 kDa protein band was found in the SDS - PAGE pattern. Deletion mutation CP gene of PLRV was expressed correctly in Saccharomyces cerevisiae.

关 键 词：马铃薯卷叶病毒 CP基因 缺失突变 酿酒酵母 表达载体 

分 类 号：Q786[生物学—分子生物学] S532[农业科学—作物学]