Gi-Ho Lee

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.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3–9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a hosts defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3-9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

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.

Transgene instability due to promoter hypermethylation and deletion in transgenic Nicotiana benthamiana

Epigenetics is the study of heritable alternations in gene expression resulting in phenotypic changes without modification of the nucleotide sequence, which could be called a successive evolution. In our study, a plant transformation strategy was employed by using Nicotiana benthamiana (Domin.) to study the heritable stability of the introduced transgenes through the T8 to T9 generations and represented transgene silencing phenomenon at the T9 generation. Through the introduction of the green fluorescent protein (GFP) reporter gene, characteristics of the gene-silencing phenomenon were analyzed. Individual plants were classified as follows according to their phenotype: GFP expression line, GFP mosaic expression line, and silenced line. This phenomenon resulted from by hypermethylation and nucleotide deletion in the cauliflower mosaic virus (CaMV) 35S promoter region. In particular, there was obvious differences in the levels of the expression of the introduced GFP gene and DNA methylation-related endogenous genes including domains rearranged methylase 2 (DRM2), SU(VAR)3-9 homolog 9 (SUVH9), and histone deacetylase 1 (HDA1). Our study demonstrates that the stability of transgenes was controlled by expression levels of genes involved in endogenous DNA methylations and hypermethylation on the introduced promoter, which triggered promoter region deletions or modifications.