An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA
- First Online:
- 156 Downloads
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.
KeywordsAgrobacterium-mediated transformation Chinese cabbage functional genomics inverse PCR plasmid rescue
Unable to display preview. Download preview PDF.
- 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
- 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
- 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
- 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
- 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
- Tsuchiya, T., and Gupta, P.K. (1992). Chromosome engineering in plants: genetics, breeding, evolution, (Elsevier Press), pp. 161–180.Google Scholar
- 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