Jae-Gyeong Yu

Genome-wide characterization of the CBF/DREB1 gene family in Brassica rapa

The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREBs) are important proteins in involved in responses to abiotic stress in plants. We identified ten BrDREB1 genes belonging to the CBF/DREB1 gene family in the Brassica rapa whole genome sequence, whereas six genes are found in the Arabidopsis thaliana genome. The deduced amino acid sequences of the B. rapa genes showed conserved motifs shared with other known plant CBF/DREB1s. Comparative analysis revealed that nine of the BrDREB1 genes were derived from the recent genome triplication in the tribe Brassiceae and the other one was translocated. The nine genes were located in seven of the 12 macrosyntenic blocks that are triplicated counterparts of four Arabidopsis macrosyntenic blocks harboring six CBF/DREB1 genes: one gene on each of three blocks and three tandemly arrayed genes on another block. We inspected the expression patterns of eight BrDREB1 genes by RT-PCR and microarray database searches. All eight genes were highly up-regulated during cold (4 °C) treatment, and some of them were also responsive to salt (250 mM NaCl), drought (air drying), and ABA (100 μM) treatment. Microarray data for plant developmental stages revealed that BrDREB1C2 was highly expressed during a period of cold treatment for vernalization, similar to abiotic stress-inducible genes homologous to Bn28a, Bn47, Bn115, and BoRS1, but almost opposite of BrFLC genes. Taken together, the number of BrDREB1 genes increased to 10 by genome triplication and reorganization, providing additional functions in B. rapa abiotic stress responses and development, as distinct from their Arabidopsis homologs.

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 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.

Characterization of Brassica rapa S-adenosyl-L-methionine synthetase gene including its roles in biosynthesis pathway

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.

Physiological role of endogenous S-adenosyl-L-methionine synthetase in Chinese cabbage

S-adenosyl-L-methionine synthetase (SAMS) catalyzes the synthesis of S-adenosyl-L-methionine, a molecule which functions as the methyl group donor in the biosynthesis of nucleic acids, proteins, lipids, polysaccharides, and secondary products. To analyze the physiological role of endogenous S-adenosyl-L-methionine synthetase, Chinese cabbage was transformed with pCSAMS vector for SAMS over-expression and pJJSAMS vector for SAMS down-regulation, respectively. From the results of both quantitative real-time PCR and northern hybridization, SAMS showed a 2.5-fold greater expression in the pCSAMS line and approximately 2-fold suppression in the pJJSAMS line. T1 progenies of these transgenic lines and a wild type control were analyzed by microarray to evaluate genes that are functionally related to SAMS. Expression level changes of SAMS strongly affected not only genes related to defense response to abiotic stress but also protein, jasmonic acid, and ethylene synthesis. Based on these results, we conclude that SAMS plays an important role in plant metabolic pathways and in the biosynthesis of phytohormones related to plant growth. By phenotype analysis, the SAMS over-expression lines were found to grow rapidly with flattened and serrated leaf margin. The down-regulated SAMS lines, however, could be characterized by stunted growth and the appearance of thick and asymmetric leaves.

Current status of Brassica rapa functional genome research in Korea

The purpose of functional genome research is to identify biological function of useful gene and to give an agricultural value in plant biotechnology. Brassica rapa is an economic crop which recorded 1,000 billion won of domestic market and 100 million dollar of exports and it produces 2.5 million ton in 50,000 ha as a major ingredient of representative Korean food, Kimchi. Furthermore, it is very important crop economically and commercially because Korea is major seed exporter. The fact that Multinational Brassica Genome Project (MBGP) was launched and Arabidopsis thaliana, affiliated to same genus with B. rapa, has been fully sequenced activated functional genome research of B. rapa. Besides new technologies related to gene function analysis keep developing, many results are reporting every year by international research including Korea. This review paper introduces development of Chinese cabbage mutants which is a first step in functional genome research, variant phenotypes of mutants, flanking DNA analysis in B. rapa genome, gene identification, gene analysis using microarray, and representative researches.

Characterization of a drought tolerance-related gene of Chinese cabbage in a transgenic tobacco plant

Drought is a major abiotic stress that affects plant growth and productivity in many regions of the world. As climate change has increased the incidence of drought, research of the genes related to drought stress and development of drought-tolerant plants is necessary now more than ever. In this study, genes related to drought tolerance were screened from Brassica rapa 135k microarray data and a gene with full-length sequence and unknown function was selected and named BrDSR28 (B. rapa drought stress resistance 28). The expression of BrDSR28 was over 4-fold higher in drought-tolerant Chinese cabbage than in wild-type controls. This gene contains a 483 bp open reading frame encoding a 160 amino acid polypeptide and it contains a senescence regulator domain. To characterize the function of BrDSR28, Nicotiana tabacum was transformed with over-expression and down-regulation vectors of the gene. Transgenic tobacco plants were confirmed by PCR, Southern hybridization, and RT-PCR analyses. The expression levels and phenotypes of the transgenic tobacco plants were analyzed under drought stress. The BrDSR28 over-expression lines showed higher expression of BrDSR28 in all stages of drought treatment and showed significant tolerance to drought when compared to the non-transgenic lines and BrDSR28 down-regulated tobacco plants.

Gene expression and phenotypic analyses of transgenic Chinese cabbage over-expressing the cold tolerance gene, BrCSR

The goal of this study is to characterize the BrCSR (B. rapa Cold Stress Resistance) gene and to analyze the expression pattern of genes related to cold response under low temperatures in Chinese cabbage. A transgenic Chinese cabbage line was generated using expression vector pSL101 containing the BrCSR full length CDS. Five transgenic plants of T1-progeny that were selected by PCR and southern hybridization showed approximately 2-fold higher expression than the wild-type control under cold stress conditions. These transgenic plants showed less susceptibility to chilling injury compared to the control. To evaluate genes that are functionally related to BrCSR and cold-responsive genes, a gene co-expression network had been preferentially constructed, and then B. rapa 135K cDNA microarray was subsequently analyzed. BrCSR was strongly associated with PDP1, NYC1, and CYP72A11, which are involved in the cold stress tolerance response. Expression levels of genes related to the biosynthesis of succinate and thiamine, which have reported to be associated with cold tolerance, significantly changed in the gene co-expression network. Taken together, these results indicated that BrCSR plays a significant role in the adaptation of plants to low temperature conditions.

Correlation Network Analysis of Abiotic StressrelatedGenes Reveals the Coordinated Regulationof Transcription in Chinese Cabbage

Plant responses to abiotic stresses such as drought, cold, and salt stress include altered expression of genes involved in metabolic processes, including growth, development, and physiological changes. Non-biological stress can lead to changes in the growth and morphology of crops, as well as reduced harvest volume. Plants must respond simultaneously to multiple stresses in the environment; therefore, research on abiotic stress should focus on the interactions of these stress responses. In the present study, we constructed a co-expression network for multidirectional analysis of cold, drought, and salt stress response genes in Chinese cabbage (Brassica rapa L. ssp. pekinensis). We constructed the co-expression network using abiotic stress-related data from the KBGP-24K microarray in the B. rapa Expressed sequence tag data and microarray database (BrEMD) and performed abiotic-stress specific gene expression analyses of B. rapa. The core mechanism underlying abiotic stress tolerance in B. rapa is the inactivation of abscisic acid metabolism, which triggers proline biosynthesis. We also characterized unknown genes possibly related to abiotic stress tolerance by producing transgenic Chinese cabbage lines overexpressing these genes. Additional key words: co-expression network, functional genomics, hydrogen peroxide, microarray, proline

Drought tolerance induction in transgenic tobacco through RNA interference of BrDST71, a drought-responsive gene from Chinese cabbage

Because drought is a major environmental factor that causes serious agricultural problems, understanding the mechanisms and genetic bases underlying plant responses to drought stress is essential. Using the Brassica rapa 135 K microarray, BrDST71 gene was identified. BrDST71 expression in drought-tolerant Chinese cabbage showed an eight-fold decrease than in wild-type, and encodes a 362 amino acid protein containing a secretory peroxidase domain. Overexpression and RNAi vectors of BrDST71 were constructed and each vector was transformed into Nicotiana tabacum by the Agrobacterium-mediated transformation method. The expression level of BrDST71 and the phenotype were analyzed under drought condition. Transgenic lines with suppressed expression of BrDST71 showed more tolerance to drought stress compared to wild-type and overexpression transgenic lines. It showed that suppressing BrDST71 expression is correlated to better growth under drought conditions. Based on these results, we suggest that down-regulation of BrDST71 improves drought tolerance.