玉米Opaque-2基因在毕赤酵母中的表达  

Maize Opaque-2 Gene Expression in Pichia pastoris

作　　者：周志强[1] 宋丽雅[2] 颜娜[1] 张世煌[1] 李新海[1] 郝转芳[1] 翁建峰[1] 白丽[1] 李明顺[1] 

机构地区：[1]中国农业科学院作物科学研究所/农业部遗传育种重点实验室,北京100081 [2]北京工商大学/北京市植物资源研究开发重点实验室,北京100048

出　　处：《玉米科学》2014年第5期6-12,19,共8页Journal of Maize Sciences

基　　金：国家现代农业产业技术体系建设专项(CARS-02-01);中央级公益性科研院所基本科研业务费专项;北京市自然科学基金项目(6112003)

摘　　要：从玉米自交系辽2345授粉后18 d的胚乳中提取总RNA,经反转录后得到第一链c DNA,利用Opaque-2基因的特异引物克隆出该基因的全编码区,将该编码区的全序列以正确的阅读框架利用Infusion同源重组的方法亚克隆到毕赤酵母表达载体p PIC9的α因子下游,并置于AOXⅠ启动子控制下,利用p PIC9表达载体上的特异引物进行PCR鉴定,鉴定后的重组质粒用Bgl II酶切线性化后,电转化导入甲醇型毕赤酵母菌株GS115中,利用MM、MD和菌落PCR的方法筛选出Mut+表型,得到的重组子以甲醇诱导表达蛋白。经SDS-PAGE结果显示,Opaque-2蛋白得到表达,其相对分子质量(Mr)约为47 KD。The total RNA extracted from the endosperm of Liao2345 18 days after pollination, then the first chain eDNA had been done reverse transcription. In order to get the correct reading frame,using specific primers of the Opaque-2 gene to clone the full coding region, infusion homologous recombination could be utilized, so the cod- ing sequence was following the alpha factor of pPIC9 and placed in the downstream of AOX1 promoter by this way. The specific primers of pPIC9 was used to identify the PCR production. After the recombinant plasmid digest with Bgl II, the linear plasmids are conversed into Pichia Pastoris strain GS115. By using the medium of MM, MD and colony PCR screening to select the Mut＋ phenotype, the recombinant strains are reduced to express protein by methanol. SDS-PAGE results showed the Opaque-2 protein expression and its relative molecular mass was about 47 KD.

关 键 词：玉米 OPAQUE-2 毕赤酵母 蛋白表达 

分 类 号：S513.035.3[农业科学—作物学]