An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA.
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 T(1) 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.
Yu
Jae-Gyeong
JG
Department of Horticultural Biotechnology, Kyung Hee University, Yongin, 446-701, Korea.
Lee
Gi-Ho
GH
Kim
Jung-Sun
JS
Shim
Eun-Jo
EJ
Park
Young-Doo
YD
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics; D029741:Brassica rapa / Q000235:genetics*; D002874:Chromosome Mapping; D032461:Chromosomes, Plant / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003199:Computer Systems; D004269:DNA, Bacterial / Q000235:genetics*; D030541:Databases, Genetic; D008040:Genetic Linkage; D018745:Genome, Plant; D016254:Mutagenesis, Insertional; D010641:Phenotype; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D018749:RNA, Plant / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018749:RNA, Plant; C036483:T-DNA