Abstract
The central goal of current genomics research in plants, as in other organisms, is to elucidate the functions of every gene. Insertional mutagenesis using known DNA sequences such as T-DNA is a powerful tool in functional genomics. Development of efficient methods for isolating the genomic sequences flanking insertion elements accelerates the systematic cataloging of insertional mutants, and thus allows functions to be assigned to uncharacterized genes via reverse genetic approaches. In our current study, we report a rapid and efficient inverse PCR (iPCR) method for the isolation of gene tags in T-DNA mutant lines of rice (Oryza sativa), a model monocot plant.
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References
Arabidopsis Genome Initiative. (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815.
Yu, J., Hu, S., Wang, J., Wong, G. K., Li, S., Liu, B., et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. Indica). Science 296, 79–92.
International Rice Genome Sequencing Project. (2005) The map-based sequence of the rice genome. Nature 436, 793–800.
Krysan, P. J., Young, J. C., and Sussman, M. R. (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11, 2283–2290.
Weigel, D., Ahn, J. H, Blazquez, M. A., Borevitz, J. O., Christensen, S. K., Fankhauser, C., et al. (2000) Activation tagging in Arabidopsis. Plant Physiol. 122, 1003–1013.
Jeon, J. -S., and An, G. (2001) Gene tagging in rice: a high throughput system for functional genomics. Plant Sci. 161, 211–219.
Østergaard, L., and Yanofsky, M. F. (2004) Establishing gene function by mutagenesis in Arabidopsis thaliana. Plant J. 39, 682–696.
Krishnan, A., Guiderdoni, E., An, G., Hsing, Y. I., Han, C. D., Lee, M. C., et al. (2009) Mutant resources in rice for functional genomics of the grasses. Plant Physiol. 149, 165–170.
Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
Triglia, T., Peterson, M. J., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16, 81–86.
Liu, Y. G., and Whittier, R. (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25, 674–681.
Liu, Y. G., Mitsukawa, N., Oosumi, T., and Whittier, R. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 8, 457–463.
Balzergue, S., Dubreucq, B., Chauvin, S., Le-Clainche, I., Le Boulaire, F., de Rose, R., et al. (2001) Improved PCR-walking for large-scale isolation of plant T-DNA borders. Biotechniques 30, 496–504.
Cottage, A., Yang, A., Maunders, H., de Lacy, R. C., and Ramsay, N. A. (2001) Identification of DNA sequences flanking T-DNA insertions by PCR-walking. Plant Mol. Biol. Rep. 19, 321–327.
Parinov, S., Sevugan, M., De, Y., Yang, W. C., Kumaran, M., and Sundaresan, V. (1999) Analysis of flanking sequences from dissociation insertion lines. A database for reverse genetics in Arabidopsis. Plant Cell 11, 2263–2270.
Hanley, S., Edwards, D., Stevenson, D., Haines, S., Hegarty, M., Schuch, W., et al. (2000) Identification of transposon-tagged genes by the random sequencing of mutator-tagged DNA fragments from Zea mays. Plant J. 23, 557–566.
An, S., Park, S., Jeong, D. -H., Lee, D. -Y., Kang, H. -G., Yu, J. -H., et al. (2003) Generation and analysis of end sequence database for T-DNA tagging lines in rice. Plant Physiol. 133, 2040–2047.
Alonso, J. M, Stepanova, A. N., Leisse, T. J., Kim, C. J., Chen, H., Shinn, P., et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657.
Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, C. J., et al. (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302, 842–846.
Hirochika, H., Guiderdoni, E., An, G., Hsing, Y. I., Eun, M. Y., Han, C. D., et al. (2004) Rice mutant resources for gene discovery. Plant Mol. Biol. 54, 325–334.
Kolesnik, T., Szeverenyi, I., Bachmann, D., Kumar, C. S., Jiang, S., Ramamoorthy, R., et al. (2004) Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences. Plant J. 37, 301–314.
An, G., Jeong, D. H., Jung, K. H., and Lee, S. (2005) Reverse genetic approaches for functional genomics of rice. Plant Mol. Biol. 59, 111–123.
Jeong, D. -H., An, S., Park, S., Kang, H. G., Park, G. G., Kim, S. R., et al. (2006) Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J. 45, 123–132.
Chern, C. G., Fan, M. J., Yu, S. M., Hour, A. L., Lu, P. C., Lin, Y. C., et al. (2007) A rice phenomics study-phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population. Plant Mol. Biol. 65, 427–438.
Hsing, Y. I., Chern, C. G, Fan, M. J., Lu, P. C., Chen, K. T., Lo, S. F., et al. (2007) A rice gene activation/knockout mutant resource for high throughput functional genomics. Plant Mol. Biol. 63, 351–364.
Kumar, C. S., Wing, R. A., and Sundaresan, V. (2008) Efficient insertional mutagenesis in rice using the maize En/Spm elements. Plant J. 44, 879–892.
Wan, S., Wu, J., Zhang, Z., Sun, X., Lv, Y., Gao, C., et al. (2009) Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Mol. Biol. 69, 69–80.
Jeon, J. S., Lee, S., Jung, K. H., Jun, S. H., Jeong, D. H., Lee, J., et al. (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22, 561–570.
Kim, S. R., Lee, J., Jun, S. H., Park, S., Kang, H. G., Kwon, S., et al. (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol. Biol. 52, 761–773.
Acknowledgments
The methods described in this report were developed with the support of the Crop Functional Genomic Center, the 21st Century Frontier Program (Grant CG1111); from the Biogreen 21 Program, Rural Development Administration (20070401-034-001-007-03-00); from the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund, KRF-2007-341-C00028); and from Kyung Hee University.
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Kim, SR., Jeon, JS., An, G. (2011). Development of an Efficient Inverse PCR Method for Isolating Gene Tags from T-DNA Insertional Mutants in Rice. In: Pereira, A. (eds) Plant Reverse Genetics. Methods in Molecular Biology, vol 678. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-682-5_11
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DOI: https://doi.org/10.1007/978-1-60761-682-5_11
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