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High-Throughput Construction of Intron-Containing Hairpin RNA Vectors for RNAi in Plants

文献类型： 外文期刊

作者： Yan, Pu 1 ; Shen, Wentao 2 ; Gao, XinZheng 3 ; Li, Xiaoying 2 ; Zhou, Peng 2 ; Duan, Jun 1 ;

作者机构： 1.Chinese Acad Sci, S China Bot Garden, Guangzhou, Guangdong, Peoples R China

2.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Haikou, Peoples R China

3.Hainan Med Coll, Dept Basic Med Sci, Haikou, Peoples R China

4.Chinese Acad Sci, Grad Univ, Beijing, Peoples R China

期刊名称：PLOS ONE （ 影响因子：3.24； 五年影响因子：3.788 ）

ISSN： 1932-6203

年卷期： 2012 年 7 卷 5 期

页码：

收录情况： SCI

摘要： With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

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