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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.

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A full length cDNA encoding the SAM synthetase gene was isolated from Chinese cabbage by RT-PCR and contains a 1,182 bp open reading frame encoding a putative 393 amino acid protein. This cDNA insert was subcloned into the pET15b vector to evaluate the expression and further characterize the SAMS gene. Recombinant SAMS was also generated in BL21 cells and showed a molecular weight of about 43 kDa. To elucidate the function of SAMS in the Chinese cabbage, overexpression and RNAi vectors for this gene were constructed and introduced into tobacco plants. For overexpression, the CaMV 35S promoter was introduced into the binary vector pBI121 and the full-length SAMS gene was subcloned into the resulting pCSAMS vector. To suppress SAMS, forward and reverse fragments from its ORF of 519 bp length was introduced into the RNAi vector, pJJSAMS. SAMS gene functions were subsequently evaluated by the phenotypic variation analysis and by observing the upregulation and/or downregulation of the ethylene and polyamine biosynthesis genes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and spermidine synthase (SPDS) in transgenic tobacco plants. The results of these experiments suggest that SAMS regulates ethylene and polyamine biosynthesis, at least in part, at the transcriptional level.Additional key words: 1-aminocyclopropane-1-carboxylate synthase (ACS), ethylene, polyamine, spermidine synthase (SPDS) Hort. Environ. Biotechnol. 53(1):57-65. 2012.

The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line 'Chiifu' under cold stress condition (4°C). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. T1 transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under 4°C treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.