Abstract
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.
Highlights
After double-stranded RNA was discovered as the trigger of RNA interference (RNAi) [1], RNAi has become one of the most powerful tools for the analysis of gene function [2,3,4]
Our results suggest that this novel method provides a high-throughput and reliable platform for making intron-containing hairpin RNA (ihpRNA) constructs, thereby, may facilitate a large-scale functional genomics analysis in plants
To make the ihpRNA construct, the target region of the gene of interest is amplified with BsaI recognition sites flanked at both ends, and the cleavage site sequences are complementary to the appropriate sequences on the vector
Summary
After double-stranded RNA was discovered as the trigger of RNA interference (RNAi) [1], RNAi has become one of the most powerful tools for the analysis of gene function [2,3,4]. Chen et al used a similar strategy to construct ihpRNA cassettes which were immediately inserted into the final plant expression vectors by TA cloning [12] These PCR-based methods are simpler and faster than the conventional ligase-based system, and more cost-effective than the GATEWAY system. This method is simple and fast, as it can generate ihpRNA constructs for plant RNA silencing by one-step transformation It still needs two rounds of PCR for amplifying the two inverted repeats with different adaptors and a step of restriction enzyme digestion for vector linearization before using the ligation- independent cloning
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