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Molecular analysis of T-DNA insertion mutant population for transgenic rice plants
Hyemin Lim,A-Ram Kim,Jung-Il Cho,Hyeon-So Ji,Ung-Han Yoon,Tae-Ho Kim,Gang-Seob Lee 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained GPD, UtrCSP, BrTSR15 and BrTSR53 genes overexpression constructs under the control of the constitutive CaMV 35S promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also FSTs analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 95 FSTs, among which 37 (38.9%) were integrated into genic regions and 58 (61.1%) into intergenic regions. About 27 homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S::BrTSR15 and 35S::BrTSR53 transgenic plants were displayed higher levels of transgene protein than WT plants. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.
Hyemin Lim,Sung Han Park,A-Ram Kim,Hyeon-So Ji,Ung-Han Yoon,Tae-Ho Kim,Soo-Chul Park,Jang-Ho Hahn,Gang-Seob Lee 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07
In plants, the Dof (DNA binding with One Finger) proteins are plant-specific transcription factors with a particular class of zinc-finger DNA-binding domain. The Dof genes have been predicted 30 different Dof genes in the rice Oryza sativa genome by phylogenetic analysis. The mostly Dof proteins contain a conserved region of 50 amino acids with a C2-C2 zinc finger motifs that binds a cis-regulatory element sequence 5’-T/AAAAG-3’. We found that a member of the DOF transcription factor family, Dof1 gene of rice, was expressed to wound from Ds insertion mutant population. Sequencing of the flanking regions of the transposon insertion site indicated that the gene-trap had been inserted near the front of the second exon of OsDof1 gene in chromosome 7. Genomic southern analysis revealed that mutant line contained a single copy of Ds gene trap. The Ds tagged rice mutant line, OsDof1::Ds, wound-inducible GUS expression was identified. To analyze the cis-acting elements, we constructed fusion genes with the OsDof1 promoter fused to the β-glucuronidase (GUS) reporter gene and transformed Arabidopsis and rice plants with these constructs. Wound-induced GUS expression was observed in the leaves of transgenic OsDof1::GUS rice and Arabidospsis plants. These results showed that, OsDof1 protein might be involved in stress responses and growth regulation in plant, might plays a role as a transcription regulator in stress response signal transduction pathways of plant.
Hyemin Lim,조만호,전종성,Seong Hee Bhoo,Yong-Kook Kwon,Tae-Ryong Hahn 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.6
Pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP subunit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO2 assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes.
Lim, Hyemin,Cho, Man-Ho,Jeon, Jong-Seong,Bhoo, Seong Hee,Kwon, Yong-Kook,Hahn, Tae-Ryong Springer-Verlag 2009 Molecules and cells Vol.27 No.6
<P>Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO(2) assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPbeta affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes.</P>
Generation and characterization of T-DNA insertion population for genetically-modified rice
Hyemin Lim,A-Ram Kim,Hyun-Ju Hwang,Jung-Il Cho,Hyeonso Ji,Chang-Kug Kim,Soo-Chul Park,Gang-Seob Lee 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes over-expression constructs under the control of the constitutive (CaMV 35S) promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also flanking sequence tags (FSTs) analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 157 flanking sequence tags (FSTs), among which 58 (36%) were integrated into genic regions and 97 (62%) into intergenic regions. About 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants were displayed higher levels of transgene protein. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.
( Tae-lim Kim ),( Kyungmi Lee ),( Wonwoo Cho ),( Danbe Park ),( Il Hwan Lee ),( Hyemin Lim ) 한국육종학회 2021 Plant Breeding and Biotechnology Vol.9 No.2
Water deficit is a critical factor obstructing the growth and survival of plant. Therefore, researchers have been trying to develop drought-resistant varieties. To find indicators of drought stress-tolerance of cypress (Chamaecyparis obtusa), we analyzed the response of cypress seedlings from six provenances of Korea (Jeju, Suwon, Seoul, Seongnam, Yong-in, and Osan) to drought stress. Additionally, the genetic diversity of C. obtusa from the six provenances were determined using microsatellite markers. We confirmed that populations from Suwon and Seongnam were relatively separated from other populations through genetic distance and cluster analysis. We examined their physiologic and metabolic responses after drought treatment for five weeks. Almost all of the cypress seedlings showed a reduced shoot growth rate under drought treatment compared to controls. In addition, temperature of drought treated cypress seedling leaves was 1.2-2℃ higher than that of the controls. Almost all of the drought stress-treated cypress showed increased carbon metabolite contents and pigments. In particular, the cypress seedlings from Osan showed the highest increase in all of the measured metabolites. Therefore, it is suggested that the seedlings from Osan are susceptible to drought stress. Conversely, the seedlings from Jeju, Suwon, and Yong-in showed a lower sensitivity to drought treatment. These results indicate that the cypress trees from the six provenances have a different response to drought stress. In addition, it is confirmed that previously identified indicators of drought stress, especially those that measure total soluble sugar, carotenoid, and H2O2, can be used in the selection of drought resistance cypress. These findings may useful in studies concerned with the metabolic and physiological responses of young cypress to drought.