title: Rapid construction of a plant RNA interference expression vector for hairpin RNA-mediated targeting using a PCR-based method.
abstract: Here we describe a rapid and efficient PCR-mediated ligation protocol for constructing a plant RNA interference vector to express long hairpin RNA (hpRNA). In the protocol, four oligonucleotide primers were used and three rounds of PCRs performed. The product of the first PCR was used as a megaprimer for the second PCR to generate a chimeric molecule with a gene-specific sequence and a spacer spliced together. The chimeric product could be used as another megaprimer for the third PCR to ligate another gene-specific sequence to the other end of the spacer, but in the reverse orientation. Thus, within a few days, two gene-specific sequences could be ligated to a spacer in the antisense and sense orientations using the PCR-mediated ligation method, without reliance on restriction cleavage and DNA ligation. The ligated product could be inserted into the plant expression vector for plant transformation. The transcribed RNA formed hpRNA constructs containing sense/antisense arms for specific gene targeting. Overexpression of hpRNA constructed by a Medicago truncatula xyloglucan endotransglycosylase gene retarded the growth of transgenic M. truncatula roots.
authors.affiliation: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, People's Republic of China.
authors.forename: Ji-Ren
authors.initials: JR
authors.lastname: Chen
authors.forename: Xingyao
authors.initials: X
authors.lastname: Xiong
authors.forename: Tian-Xiang
authors.initials: TX
authors.lastname: Wang
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Lü
authors.forename: Shou-Yi
authors.initials: SY
authors.lastname: Chen
authors.forename: Hua-Fang
authors.initials: HF
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D021901:DNA, Intergenic / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D055029:Inverted Repeat Sequences; D046913:Medicago truncatula / Q000235:genetics / Q000254:growth & development; D018517:Plant Roots / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics* / Q000378:metabolism*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D021901:DNA, Intergenic; D016372:RNA, Antisense; D018749:RNA, Plant