title: Universal vectors for constructing artificial microRNAs in plants.
abstract: Universal amiRNA vectors (pUAs) for constructing plant amiRNAs in Arabidopsis and rice have been developed. By using type IIg restriction enzyme, BaeI, a single amiRNA construct can be produced using only one PCR and one ligation reaction. Thus, only one pair of primers is required for each amiRNA vector and these can be designed to be compatible with existing or newly developed methods. Because the BaeI recognition sequence is completely digested, there is no modification to the miRNA backbone, therefore avoids the risk of sequence changes that may affect downstream analysis. Based on these vectors, specific amiRNA constructs were created and verified. With optimized parameters, 38-45% colonies for each amiRNA construct contain insertions with the expected orientation, and approximately 80% of these colonies have the correct sequences.
authors.affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, MOA Key Laboratory for Plant Protection and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.forename: Feibo
authors.initials: F
authors.lastname: Yu
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.forename: Xuming
authors.initials: X
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.forename: Ye
authors.initials: Y
authors.lastname: Cheng
authors.forename: Chengqi
authors.initials: C
authors.lastname: Yan
authors.forename: Jianping
authors.initials: J
authors.lastname: Chen
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D018744:DNA, Plant; D035683:MicroRNAs; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D000072481:DNA Ligase ATP