Since the discovery that double-stranded RNA can cause RNA interference (RNAi), RNAi has become one of the important tools for analyzing gene function. With the wide application of RNAi technology in plant gene function analysis, there is an urgent need for a high-throughput method for constructing hairpin RNA (hpRNA) expression vectors.
Under the guidance of the tutor Duan Jun, Ph.D. student Yan Pu from the Key Laboratory of South China Agricultural Plant Genetics and Breeding, South China Botanical Garden, Chinese Academy of Sciences method. With this method, only the target gene sequence is amplified, and the corresponding BsaI recognition and cleavage sites are added to the two ends of the sequence by the primer. After a one-step digestion / ligation reaction, the target gene fragment can be simultaneously forwarded. And reverse position cloned into pRNAi-GG to form the ihpRNA expression vector structure; the entire construction process only needs to be completed in one reaction in a tube, the construction efficiency is high, and there is no background interference, and because pRNAi-GG is a plant binary expression Vector, the constructed ihpRNA expression vector can be directly used for Agrobacterium transformation research.
The vector construction method has the advantages of simplicity, speed, efficiency and low cost, and provides a high-throughput platform for large-scale plant functional gene research. Related research results have been published in the international academic journal PLoS ONE [2012, 5 (7): e38186].
A. Hairpin RNA expression box structure of pRNAi-GG. B. Schematic diagram of constructing plant ihpRNA expression vector using pRNAi-GG.
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