title: Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC.
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.
authors.affiliation: Institute of Molecular Biology, Faculty of Life Science, Hubei University, Wuhan, China. yanhong@hubu.edu.cn
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
authors.forename: Xing
authors.initials: X
authors.lastname: Zhong
authors.forename: Sijing
authors.initials: S
authors.lastname: Jiang
authors.forename: Chao
authors.initials: C
authors.lastname: Zhai
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D035683:MicroRNAs / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D035683:MicroRNAs