Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC |
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Authors: | Yan Hong Zhong Xing Jiang Sijing Zhai Chao Ma Lixin |
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Institution: | (1) Institute of Molecular Biology, Faculty of Life Science, Hubei University, Wuhan, 430062, China |
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Abstract: | Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions
in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by
PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The
amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an
amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which
was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants. |
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