title: One-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system.
abstract: Hairpin RNA-based RNA interference (hpRNAi) has become a powerful tool for exploring gene function in reverse genetics. Although, several methods are available for making constructs that express hpRNAi, multiple time-consuming cloning steps are usually involved. Here, we introduce an efficient and flexible hpRNAi vector construction method via the isothermal in vitro recombination system (IR-hpRNAi). For an IR-hpRNAi reaction, two PCR products of a target gene sequence are generated, which containS complementary ends (~20 bp) to each other and to the ends of linearized vector, are fused in a way of head-to-head or tail-to-tail into the vector. This IR-hpRNAi method offers two options to construct the RNAi vectors. Using this method, we created a IR-hpRNAi construct for the Arabidopsis PDS3 gene,and verified the silencing effect via Agrobacterium-mediated transformation. The IR-hpRNAi system rules out the requirement of engineering restriction enzyme cutting sites in target DNA fragments, and is ligation-independent. Thus, this method has advantages over the other hpRNAi construction methods.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China. jiangyanxiang67@163.com
authors.forename: Yanxiang
authors.initials: Y
authors.lastname: Jiang
authors.forename: Min
authors.initials: M
authors.lastname: Xie
authors.forename: Qinlong
authors.initials: Q
authors.lastname: Zhu
authors.forename: Xinliang
authors.initials: X
authors.lastname: Ma
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.forename: Yaoguang
authors.initials: Y
authors.lastname: Liu
authors.forename: Qunyu
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D007438:Introns / Q000235:genetics; D008027:Light; D010088:Oxidoreductases / Q000235:genetics*; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects; D030821:Plants, Genetically Modified; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011995:Recombination, Genetic; D036226:Seedlings / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects
chemical_list: D017931:DNA Primers; D018749:RNA, Plant; D034741:RNA, Small Interfering; D010088:Oxidoreductases; C022398:phytoene dehydrogenase