Molecules and Cells

, Volume 29, Issue 3, pp 267–275

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

Authors

  • Jae-Gyeong Yu
    • Department of Horticultural BiotechnologyKyung Hee University
  • Gi-Ho Lee
    • Department of Horticultural BiotechnologyKyung Hee University
  • Jung-Sun Kim
    • Agricultural Bio-resources, Functional Biomaterial DivisionRural Development Administration, National Academy of Agricultural Science
  • Eun-Jo Shim
    • Department of Horticultural BiotechnologyKyung Hee University
    • Department of Horticultural BiotechnologyKyung Hee University
Article

DOI: 10.1007/s10059-010-0013-3

Cite this article as:
Yu, J., Lee, G., Kim, J. et al. Mol Cells (2010) 29: 267. doi:10.1007/s10059-010-0013-3

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 transformationChinese cabbagefunctional genomicsinverse PCRplasmid rescue
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Copyright information

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