High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.
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.
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
Pu
P
Yan
Wentao
W
Shen
XinZheng
X
Gao
Xiaoying
X
Li
Peng
P
Zhou
Jun
J
Duan
D003001:Cloning, Molecular; D023061:Gene Order; D020868:Gene Silencing; D017343:Genes, Plant; D017930:Genes, Reporter; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D007438:Introns; D010944:Plants / Q000235:genetics*; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
D018749:RNA, Plant; D034741:RNA, Small Interfering