title: One-step, zero-background ligation-independent cloning intron-containing hairpin RNA constructs for RNAi in plants.
abstract: *The hairpin-based RNA interference (RNAi) technique plays an important role in exploring gene function in plants. Although there are several methods for making hairpin RNA (hpRNA) constructs, these methods usually need multiple relatively laborious, time-consuming or high-cost cloning steps. Here we describe a one-step, zero-background ligation-independent cloning (OZ-LIC) method for making intron-containing hpRNA (ihpRNA) constructs by our vector pRNAi-LIC. *To generate the ihpRNA constructs with zero-background, this method only requires treating two PCR products of target gene flanked with different LIC sequences and SmaI-linearized pRNAi-LIC vector by T4 DNA polymerase respectively, and then transforming these treated DNA mixture into Escherichia coli. *The ihpRNA constructs generated with our OZ-LIC RNAi vector can efficiently induce not only transient silencing of the exogenous marker genes and the endogenous resistance-related Nicotiana benthamiana SGT1 gene, but also stable transgenic suppression of Arabidopsis SGT1b gene. *Our new OZ-LIC method and RNAi vector will represent a powerful tool for gene knockdown in plants and may facilitate high-throughput determination of plant gene function.
authors.forename: Guoyong
authors.initials: G
authors.lastname: Xu
authors.forename: Ning
authors.initials: N
authors.lastname: Sui
authors.affiliation: Protein Science Laboratory of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Protein Science Laboratory of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Xie
authors.affiliation: Protein Science Laboratory of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yizhen
authors.initials: Y
authors.lastname: Lai
authors.affiliation: Protein Science Laboratory of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yule
authors.initials: Y
authors.lastname: Liu
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005819:Genetic Markers; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010641:Phenotype; D030821:Plants, Genetically Modified; D034622:RNA Interference; D018749:RNA, Plant / Q000737:chemistry* / Q000235:genetics*; D012231:Rhizobium / Q000502:physiology; D014026:Nicotiana / Q000235:genetics* / Q000382:microbiology
chemical_list: D029681:Arabidopsis Proteins; D005819:Genetic Markers; D018749:RNA, Plant; D049452:Green Fluorescent Proteins; D005964:Glucosyltransferases; C515668:SGT1 protein, Arabidopsis; D005966:Glucuronidase
references.citation: Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen HM, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657.
references.citation: Aslanidis C, de Jong P. 1990. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Research 18: 6069-6074.
references.citation: Aslanidis C, Dejong PJ, Schmitz G. 1994. Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. PCR Methods and Applications 4: 172-177.
references.citation: AzpirozLeehan R, Feldmann KA. 1997. T-DNA insertion mutagenesis in Arabidopsis: going back and forth. Trends in Genetics 13: 152-156.
references.citation: Broude NE, Zhang LG, Woodward K, Englert D, Cantor CR. 2001. Multiplex allele-specific target amplification based on PCR suppression. Proceedings of the National Academy of Sciences, USA 98: 206-211.
references.citation: Carvalho C, Santos A, Pires S, Rocha C, Saraiva D, Machado J, Mattos E, Fietto L, Fontes E. 2008. Regulated nuclear trafficking of rpL10A mediated by NIK1 represents a defense strategy of plant cells against virus. PLoS Pathogens 4: e1000247.
references.citation: Chen S, Songkumarn P, Liu J, Wang GL. 2009. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiology 150: 1111-1121.
references.citation: Conley E, Saunders V, Saunders J. 1986. Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules. Nucleic Acids Research 14: 8905-8917.
references.citation: Dieckman L, Gu M, Stols L, Donnelly M, Collart F. 2002. High throughput methods for gene cloning and expression. Protein Expression and Purification 25: 1-7.
references.citation: Dong Y, Burch-Smith T, Liu Y, Mamillapalli P, Dinesh-Kumar S. 2007. A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiology 145: 1161-1170.
references.citation: Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.
references.citation: Gray W, Muskett P, Chuang H, Parker J. 2003. Arabidopsis SGT1b is required for SCF(TIR1)-mediated auxin response. Plant Cell 15: 1310-1319.
references.citation: Helliwell C, Waterhouse P. 2003. Constructs and methods for high-throughput gene silencing in plants. Methods 30: 289-295.
references.citation: Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM. 2002. High-throughput vectors for efficient gene silencing in plants. Functional Plant Biology 29: 1217-1225.
references.citation: Hsiao KC. 1993. Exonuclease-III induced ligase-free directional subcloning of PCR products. Nucleic Acids Research 21: 5528-5529.
references.citation: Jefferson R, Kavanagh T, Bevan M. 1987. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6: 3901-3907.
references.citation: Johansen L, Carrington J. 2001. Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiology 126: 930-938.
references.citation: Kumar A, Hirochika H. 2001. Applications of retrotransposons as genetic tools in plant biology. Trends in Plant Science 6: 127-134.
references.citation: Liu Y, Schiff M, Marathe R, Dinesh-Kumar S. 2002a. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant Journal 30: 415-429.
references.citation: Liu Y, Schiff M, Serino G, Deng X, Dinesh-Kumar S. 2002b. Role of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to tobacco mosaic virus. Plant Cell 14: 1483-1496.
references.citation: Llave C, Kasschau K, Rector M, Carrington J. 2002. Endogenous and silencing-associated small RNAs in plants. Plant Cell 14: 1605-1619.
references.citation: Martienssen RA. 1998. Functional genomics: probing plant gene function and expression with transposons. Proceedings of the National Academy of Sciences, USA 95: 2021-2026.
references.citation: Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH. 2006. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nature Biotechnology 24: 1420-1428.
references.citation: Ossowski S, Schwab R, Weigel D. 2008. Gene silencing in plants using artificial microRNAs and other small RNAs. Plant Journal 53: 674-690.
references.citation: Palauqui JC, Elmayan T, de Borne FD, Crete P, Charles C, Vaucheret H. 1996. Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiology 112: 1447-1456.
references.citation: Palauqui J-C, Vaucheret H. 1995. Field trial analysis of nitrate reductase co-suppression: a comparative study of 38 combinations of transgene loci. Plant Molecular Biology 29: 149-159.
references.citation: Peart J, Cook G, Feys B, Parker J, Baulcombe D. 2002. An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant Journal 29: 569-579.
references.citation: Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D. 2006. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18: 1121-1133.
references.citation: Smith N, Singh S, Wang M, Stoutjesdijk P, Green A, Waterhouse P. 2000. Total silencing by intron-spliced hairpin RNAs. Nature 407: 319-320.
references.citation: Stuttmann J, Parker J, Noël L. 2008. Staying in the fold: the SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins. Plant Signal and Behavior 3: 283-285.
references.citation: Voinnet O. 2005. Non-cell autonomous RNA silencing. FEBS letters 579: 5858-5871.
references.citation: Wang H, Buckley K, Yang X, Buchmann R, Bisaro D. 2005. Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins. The Journal of Virology 79: 7410-7418.
references.citation: Wesley S, Helliwell C, Smith N, Wang M, Rouse D, Liu Q, Gooding P, Singh S, Abbott D, Stoutjesdijk P et al. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant Journal 27: 581-590.
references.citation: Xiao Y, Yin M, Hou L, Pei Y 2006. Direct amplification of intron-containing hairpin RNA construct from genomic DNA. BioTechniques 41: 548, 550, 552.
references.citation: Yan P, Shen W, Gao X, Duan J, Zhou P. 2009. Rapid one-step construction of hairpin RNA. Biochemical and Biophysical Research Communications 383: 464-468.
references.citation: Zhang X, Henriques R, Lin S, Niu Q, Chua N. 2006. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols 1: 641-646.