Molecules and Cells

, Volume 29, Issue 3, pp 267–275

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA

  • Jae-Gyeong Yu
  • Gi-Ho Lee
  • Jung-Sun Kim
  • Eun-Jo Shim
  • Young-Doo Park
Article

Abstract

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, ’seoul’, using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T1 progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.

Keywords

Agrobacterium-mediated transformation Chinese cabbage functional genomics inverse PCR plasmid rescue 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Babiychuk, E., Fuangthong, M., Van Montagu, M., Inze, D., and Kushnir, S. (1997). Efficient gene ragging in Arabidopsis thaliana using a gene trap approach. Proc. Natl. Acad. Sci. USA 94, 12722–12727.CrossRefPubMedGoogle Scholar
  2. Butaye, K.M., Goderis, I.J., Wouters, P.F., Pues, J.M., Delaure, S.L., Broekaert, W.F., Depicker, A., Cammue, B.P., and De Bolle, M.F. (2004). Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J. 39, 440–449.CrossRefPubMedGoogle Scholar
  3. Chen, S., Jin, W., Wang, M., Zhang, F., Zhou, J., Jia, Q., Wu, Y., Liu, F., and Wu, P. (2003). Distribution and characterization of over 1000 T-DNA tags in rice genome. Plant J. 36, 105–113.CrossRefPubMedGoogle Scholar
  4. De Buck, S., Jacobs, A., Van Montagu, M., and Depicker, A. (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J. 20, 295–304.CrossRefPubMedGoogle Scholar
  5. De Buck, S., De Wilde, C., Van Montagu, M., and Depicker, A. (2000). T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol. Breeding 6, 459–468.CrossRefGoogle Scholar
  6. De Neve, M., De Buck, S., Jacobs, A., Van Montagu, M., and Depicker, A. (1997). T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J. 11, 15–29.CrossRefPubMedGoogle Scholar
  7. Denis, M., Renard, M., and Krebers, E. (1995). Isolation of homozygous transgenic Brassica napus lines carrying a seed-specific chimeric 2S albumin gene and determination of linkage relationships. Mol. Breeding 1, 143–153.CrossRefGoogle Scholar
  8. Ge, H., Walhout, A.J., and Vidal, M. (2003). Integrating ‘omic’ information: a bridge between genomics and systems biology. Trends Genet. 19, 551–560.CrossRefPubMedGoogle Scholar
  9. Goff, S.A., Ricke, D., Lan, T.H., Presting, G., Wang, R., Dunn, M., Glazebrook, J., Sessions, A., Oeller, P., Varma, H., et al. (2002). A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92–100.CrossRefPubMedGoogle Scholar
  10. Gustavo, A., Gonzalez-Cabrera, J., Vazquez-Padron, R., and Ayra-Pardo, C. (1998). Agrobacterium tumefaciens: A natural tool for plant transformation. Electronic J. Biotechnol. 1, 1–16.Google Scholar
  11. Holtorf, H., Guitton, M.C., and Reski, R. (2002). Plant functional genomics. Naturwissenschaften 89, 235–249.CrossRefPubMedGoogle Scholar
  12. Jin, Z.L., Hong, J.K., Yun, D.J., Lee, S.Y., Choi, Y.J., Bahk, J.D., Beachy, R.N., Cho, M.J., and Lim, C.O. (2002). Expression of the tobacco mosaic virus movement protein alters starch accumulation in Nicotiana benthamiana. J. Plant Biol. 45, 77–82.CrossRefGoogle Scholar
  13. Johnston, J.S., Pepper, A.E., Hall, A.E., Chen, Z.J., and Hodnett, G. (2005). Evolution of genome size in Brassicaceae. Ann. Bot. 95, 229–235.CrossRefPubMedGoogle Scholar
  14. Kim, H.S., Kim, S.H., and Park, Y.D. (2003). Development of rescue cloning vector with phosphinothricin resistant gene for effective T-DNA tagging. J. Kor. Soc. Hort. Sci. 44, 407–411.Google Scholar
  15. Kim, J.S., Chung, T.Y., King, G.J., Jin, M.A., Yang, T.J., Jin, Y.M., Kim, H.I., and Park, B.S. (2006a). A sequence-tagged linkage map of Brassica rapa. Genetics 174, 29–39.CrossRefPubMedGoogle Scholar
  16. Kim, S.Y., Park, B.S., Kwon, S.J., Kim, J.S., Lim, M.H., Park, Y.D., Kim, D.Y., Suh, S.C., Jin, Y.M., Ahn, J.H., et al. (2006b). Delayed flowering time in Arabidopsis and Brassica rapa by the overexpression of FLOWERING LOCUS C (FLC) homologs isolated from Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Cell Rep. 26, 327–336.CrossRefPubMedGoogle Scholar
  17. Kononov, M.E., Bassuner, B., and Gelvin, S.B. (1997). Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence or multiple complex patterns of integration. Plant J. 11, 945–957.CrossRefPubMedGoogle Scholar
  18. Krizkova, L., and Hrouda, M. (1998). Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J. 16, 673–80.CrossRefPubMedGoogle Scholar
  19. Krysan, P.J., Young, J.C., and Sussman, M.R. (1999). T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11, 2283–2290.CrossRefPubMedGoogle Scholar
  20. Lee, M.K., Kim, H.S., Kim, J.S., Kim, S.H., and Park, Y.D. (2004a). Agrobacterium-mediated transformation system for large-scale production of transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) plants for insertional mutagenesis. J. Plant Biol. 47, 300–306.CrossRefGoogle Scholar
  21. Lee, M.K., Kim, H.S., Kim, S.H., and Park, Y.D. (2004b). Analysis of T-DNA integration patterns in transgenic tobacco plants. J. Plant Biol. 47, 179–186.CrossRefGoogle Scholar
  22. Lee, S.Y., Ahn, J.H., Cha, Y.S., Yun, D.W., Lee, M.C., Ko, J.C., Lee, K.S., and Eun, M.Y. (2006). Mapping of quantitative trait loci for salt tolerance at the seedling stage in rice. Mol. Cells 21, 192–196.PubMedGoogle Scholar
  23. Lim, K.B., De Jong, H., Yang, T.J., Park, J.Y., and Kwon, S.J. (2005). Characterization of rDNAs and tandem repeats in heterochromatin of Brassica rapa. Mol. Cells 19, 436–444.PubMedGoogle Scholar
  24. Lim, K.B., Yang, T.J., Hwang, Y.J., and Kim, J.S. (2007). Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species. Plant J. 49, 173–183.CrossRefPubMedGoogle Scholar
  25. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T))method. Methods 25, 402–408.CrossRefPubMedGoogle Scholar
  26. Martineau, B., Voelker, T.A., and Sanders, R.A. (1994). On defining T-DNA. Plant Cell 6, 1032–1033.CrossRefPubMedGoogle Scholar
  27. Park, J.Y., Koo, D.H., Hong, C.P., Lee, S.J., and Jeon, J.W. (2005). Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kb gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5. Mol. Gen. Genet. 274, 579–588.Google Scholar
  28. Ramanathan, V., and Veluthambi, K. (1995). Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol. Biol. 24, 1149–1154.CrossRefGoogle Scholar
  29. Ryu, K.H., Lee, G.P., Park, K.W., Lee, S.Y., and Park, W.M. (1998). Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance. J. Plant Biol. 41, 255–261.CrossRefGoogle Scholar
  30. Szabados, L., Kovács, I., Oberschall, A., Abrahám, E., Kerekes, I., Zsigmond, L., Nagy, R., Alvarado, M., Krasovskaja, I., Gál, M., et al. (2002). Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome. Plant J. 32, 233–242.CrossRefPubMedGoogle Scholar
  31. Tsuchiya, T., and Gupta, P.K. (1992). Chromosome engineering in plants: genetics, breeding, evolution, (Elsevier Press), pp. 161–180.Google Scholar
  32. Tsuei, D.J., Chen, P.J., Lai, M.Y., Chen, D.S., Yang, C.S., Chen, J.Y., and Hsu, T.Y. (1994). Inverse polymerase chain reaction for cloning cellular sequences adjacent to integrated hepatitis B virus DNA in hepatocellular carcinomas. J. Virol. Meth. 49, 269–284.CrossRefGoogle Scholar
  33. Van Der Graaff, E., Den Dulk-Ras, A., and Hooykaas, P.J.J. (1996). Deviating T-DNA transfer from Agrobacterium tumetaciens to plants. Plant Mol. Biol. 31, 667–681.CrossRefGoogle Scholar
  34. Van Ooijen, J.W., and Voorrips, R.E. (2001). JoinMap® version 3.0: software for the calculation of genetic linkage maps, Wageningen, Plant Research International.Google Scholar
  35. Wenck, A., Czako, M., Kanevski, I., and Marton, L. (1997). Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol. Biol. 34, 913–922.CrossRefPubMedGoogle Scholar
  36. Yang, T.J., Kim, J.S., Kwon, S.J., Lim, K.B., and Choi, B.S. (2006). Sequence-level analysis of the diploidization process in the triplicated FLC region of Brassica rapa. Plant Cell 18, 1339–1347.CrossRefPubMedGoogle Scholar
  37. Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A., and Meyerowitz, E.M. (1990). The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35–39.CrossRefPubMedGoogle Scholar

Copyright information

© The Korean Society for Molecular and Cellular Biology and Springer Netherlands 2010

Authors and Affiliations

  • Jae-Gyeong Yu
    • 1
  • Gi-Ho Lee
    • 1
  • Jung-Sun Kim
    • 2
  • Eun-Jo Shim
    • 1
  • Young-Doo Park
    • 1
  1. 1.Department of Horticultural BiotechnologyKyung Hee UniversityYonginKorea
  2. 2.Agricultural Bio-resources, Functional Biomaterial DivisionRural Development Administration, National Academy of Agricultural ScienceSuwonKorea

Personalised recommendations