利用Ⅱ型启动子转录的U6 RNA提高植物病毒表达载体在植物中表达外源基因的水平  

Improvement of Plant Virus RNA Vector- mediated Heterologous Gene Expression in Plant by PolⅡ Promoter- derived U6 RNA

作　　者：高丁梅[1] 马婷[1] 丁向真[1] 李志英[2,1] 王盛[2,1] 

机构地区：[1]宁夏大学生命科学学院,银川750021 [2]西部特色生物资源保护与利用教育部重点实验室,银川750021

出　　处：《中国生物化学与分子生物学报》2015年第8期883-890,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.31160032)资助~~

摘　　要：Ⅱ型启动子转录的外源短链RNA可以竞争性抑制细胞内源mRNA的核质转运,因而可能会提高植物RNA病毒载体表达的外源基因在植物中的积累.为了验证这一假说,利用OE-PCR技术合成拟南芥U6-1核内小RNA序列,并构建其Ⅱ型启动子转录的植物表达载体.以农杆菌渗滤技术,与烟草花叶病毒(Tobacco mosaic virus,TMV)表达载体共接种寄主植物本氏烟,通过对报告基因绿色荧光蛋白(green fluorescence protein,GFP)的荧光观察,并以Western印迹和ELISA测定GFP在烟草中的表达情况,分析共表达Ⅱ型启动子转录的U6 RNA对外源基因在植物中表达的作用效果.结果表明,共接种Ⅱ型启动子转录的U6 RNA对TMV病毒表达载体表达外源基因的水平有明显的增效作用,推测RNA核质转运干扰是提高外源基因表达的可能机制.Short RNAs regulated by Pol II promoters interfere the nuclear export of cellular mRNAs,which need to be considered for over-expressing exogenous transgene in plants with viral vectors. We constructed a plant expression vector of PolⅡ promoter-directed U6 RNA,using synthesized Arabidopsis thaliana U6-1 RNA gene by OE-PCR（ overlap / extension PCR） in vitro. The vectors were introduced into Nicotiana benthamiana plants with Tobacco mosaic virus（ TMV） vector through agro-infiltration. The expression levels of green fluorescence protein（ GFP） in inoculated Nicotiana leaves were then examined by Western blotting and ELISA. The data were collected for the assessment on PolⅡpromoter-driven U6 RNA expression. The results showed that the production of exogenous PolⅡ-directed U6 RNA resulted in high level of plant virus-based foreign gene expression in the infiltrated plant tissues.. We speculated that the competition of nuclear export resulted in decreased the export of protective antiviral mRNAs into the cytoplasm,and eventually enhanced foreign gene expression in plants.

关 键 词：烟草花叶病毒 表达载体 U6核内小RNA RNA核质转运 RNA聚合酶Ⅱ 

分 类 号：Q781[生物学—分子生物学]