Abstract
Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or transposable elements, but the former has been more frequently employed in Arabidopsis. A primary concern for working with insertional mutant lines is whether the respective insertion results in a complete or rather a partial loss of gene function. The effect of the insertion largely depends on its position with respect to the structure of the gene. In order to quickly identify and obtain knockout lines for genes of interest in Arabidopsis, more than 325,000 mapped insertion lines have been catalogued on indexed libraries and made publicly available to researchers. Online accessible databases provide information regarding the site of insertion, whether a mutant line is available in a homozygous or hemizygous state, and outline technical aspects for plant identification, such as primer design tools used for genotyping. In this chapter, we describe the procedure for isolating knockout lines for genes of interest in Arabidopsis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ma H, Yanofsky MF, Meyerowitz EM (1991) AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes Dev 5(3):484–495
Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky MF (2004) The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Curr Biol 14(21):1935–1940
Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405(6783):200–203
Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF (2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404(6779):766–770
Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF (2003) Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424(6944):85–88
Wang YH (2008) How effective is T-DNA insertional mutagenesis in Arabidopsis? J Biochem Tech 1(1):11–20
Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11(12):2283–2290
Parinov S, Sevugan M, De Y, Yang WC, Kumaran M, Sundaresan V (1999) Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis. Plant Cell 11(12):2263–2270
Woody ST, Austin-Phillips S, Amasino RM, Krysan PJ (2007) The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline. J Plant Res 120(1):157–165
O’Malley RC, Ecker JR (2010) Linking genotype to phenotype using the Arabidopsis unimutant collection. Plant J 61(6):928–940
Weigel D, Ahn JH, Blázquez MA, Borevitz JO, Christensen SK, Fankhauser C, Ferrándiz C, Kardailsky I, Malancharuvil EJ, Neff MM, Nguyen JT, Sato S, Wang ZY, Xia Y, Dixon RA, Harrison MJ, Lamb CJ, Yanofsky MF, Chory J (2000) Activation tagging in Arabidopsis. Plant Physiol 122(4):1003–1013
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301(5633):653–657
Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53(1–2):247–259
Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14(12):2985–2994
Gusmaroli G, Feng S, Deng XW (2004) The Arabidopsis CSN5A and CSN5B subunits are present in distinct COP9 signalosome complexes, and mutations in their JAMM domains exhibit differential dominant negative effects on development. Plant Cell 16(11):2984–3001
Okushima Y, Mitina I, Quach HL, Theologis A (2005) AUXIN RESPONSE FACTOR 2 (ARF2): a pleiotropic developmental regulator. Plant J 43(1):29–46
Bouche N, Bouchez D (2001) Arabidopsis gene knockout: phenotypes wanted. Curr Opin Plant Biol 4(2):111–117
Daxinger L, Hunter B, Sheikh M, Jauvion V, Gasciolli V, Vaucheret H, Matzke M, Furner I (2008) Unexpected silencing effects from T-DNA tags in Arabidopsis. Trends Plant Sci 13(1):4–6
Ulker B, Peiter E, Dixon DP, Moffat C, Capper R, Bouche N, Edwards R, Sanders D, Knight H, Knight MR (2008) Getting the most out of publicly available T-DNA insertion lines. Plant J 56(4):665–677
Carles CC, Choffnes-Inada D, Reville K, Lertpiriyapong K, Fletcher JC (2005) ULTRAPETALA1 encodes a SAND domain putative transcriptional regulator that controls shoot and floral meristem activity in Arabidopsis. Development 132(5):897–911
Samson F, Brunaud V, Balzergue S, Dubreucq B, Lepiniec L, Pelletier G, Caboche M, Lecharny A (2002) FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants. Nucleic Acids Res 30(1):94–97
Robinson SJ, Tang LH, Mooney BA, McKay SJ, Clarke WE, Links MG, Karcz S, Regan S, Wu YY, Gruber MY, Cui D, Yu M, Parkin IA (2009) An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches. BMC Plant Biol 9:101
Sundaresan V, Springer P, Volpe T, Haward S, Jones JD, Dean C, Ma H, Martienssen R (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev 9(14):1797–1810
Ito T, Motohashi R, Kuromori T, Mizukado S, Sakurai T, Kanahara H, Seki M, Shinozaki K (2002) A new resource of locally transposed dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome. Plant Physiol 129(4):1695–1699
Tissier AF, Marillonnet S, Klimyuk V, Patel K, Torres MA, Murphy G, Jones JD (1999) Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell 11(10):1841–1852
Acknowledgments
Work in our laboratory is supported by grants from Spanish Ministerio de Economía y Competividad (BFU2011-22734; and Programa Consolider-Ingenio, CSD2007-00036), and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (grant SGR2009-GRC476). T. M. was supported by a fellowship from the European Molecular Biology Organization (EMBO), and T. F. by a fellowship from the Center for Research in Agricultural Genomics (CRAG).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Matus, J.T., Ferrier, T., Riechmann, J.L. (2014). Identification of Arabidopsis Knockout Lines for Genes of Interest. In: Riechmann, J., Wellmer, F. (eds) Flower Development. Methods in Molecular Biology, vol 1110. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4614-9408-9_20
Download citation
DOI: https://doi.org/10.1007/978-1-4614-9408-9_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4614-9407-2
Online ISBN: 978-1-4614-9408-9
eBook Packages: Springer Protocols