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Lee J.,Korea Research Institute of Bioscience and Biotechnology | Kim J.,Korea Research Institute of Bioscience and Biotechnology | Choi J.-P.,Korea Research Institute of Bioscience and Biotechnology | Lee M.Y.,Korea Research Institute of Bioscience and Biotechnology | And 8 more authors.
BMC Genomics | Year: 2016

Background: One of the most important members of the genus Brassica, cabbage, requires a relatively high level of calcium for normal growth (Plant Cell Environ 7: 397-405, 1984; Plant Physiol 60: 854-856, 1977). Localized Ca2+ deficiency in cabbage leaves causes tip-burn, bringing about serious economic losses (Euphytica 9:203-208, 1960; Ann Bot 43:363-372, 1979; Sci Hortic 14:131-138, 1981). Although it has been known that the occurrence of tip-burn is related to Ca2+ deficiency, there is limited information on the underlying mechanisms of tip-burn or the relationship between Ca2+ and tip-burn incidence. To obtain more information on the genetic control of tip-burn symptoms, we focused on the identification of genes differentially expressed in response to increasing intracellular Ca2+ and K+ concentrations in B. oleracea lines derived from tip-burn susceptible, tip-burn resistant cabbages (B. oleracea var. capitata), and kale (B. oleracea var. acephala). Results: We compared the levels of major macronutrient cations, including Ca2+ and K+, in three leaf segments, the leaf apex (LA), middle of leaf (LM), and leaf base (LB), of tip-burn susceptible, tip-burn resistant cabbages, and kale. Ca2+ and K+ concentrations were highest in kale, followed by tip-burn resistant and then tip-burn susceptible cabbages. These cations generally accumulated to a greater extent in the LB than in the LA. Transcriptome analysis identified 58,096 loci as putative non-redundant genes in the three leaf segments of the three B. oleracea lines and showed significant changes in expression of 27,876 loci based on Ca2+ and K+ levels. Among these, 1844 loci were identified as tip-burn related phenotype-specific genes. Tip-burn resistant cabbage and kale-specific genes were largely related to stress and transport activity based on GO annotation. Tip-burn resistant cabbage and kale plants showed phenotypes clearly indicative of heat-shock, freezing, and drought stress tolerance compared to tip-burn susceptible cabbages, demonstrating a correlation between intracellular Ca2+ and K+ concentrations and tolerance of abiotic stress with differential gene expression. We selected 165 genes that were up- or down-regulated in response to increasing Ca2+ and K+ concentrations in the three leaf segments of the three plant lines. Gene ontology enrichment analysis indicated that these genes participated in regulatory metabolic processes or stress responses. Conclusions: Our results indicate that the genes involved in regulatory metabolic processes or stress responses were differentially expressed in response to increasing Ca2+ and K+ concentrations in the B. oleracea leaf. Our transcriptome data and the genes identified may serve as a starting point for understanding the mechanisms underlying essential macronutrient deficiencies in plants, as well as the features of tip-burn in cabbage and other Brassica species. © 2016 Lee et al. Source

Kim M.K.,Gyeongsangnam do Agricultural Research and Extension Service | Kang T.H.,Gyeongsang National University | Kim J.,Sunchon National University | Kim H.,Sunchon National University | Yun H.D.,Gyeongsang National University
Applied Biochemistry and Biotechnology | Year: 2012

This study was conducted to assess the gene duplication and diversification of tandem cellulase genes in thermophilic bacteria. The tandem cellulase genes cel5C and cel5D were cloned from Thermotoga maritima MSB8, and a survey of the thermophilic bacterial genome for tandem cel genes from the databases was carried out. A clone having 2.3 kb fragment from T. maritima MSB8 showed cellulase activity, which had two open reading frames in tandem (cel5C and cel5D). The cel5C gene has 954 bp, which encodes a protein of 317 amino acid residues with a signal peptide of 23 amino acids, and the other gene cel5D consisting of 990 bp encoding a protein of 329 amino acid residues. These two proteins have similarity with the enzymes of glycosyl hydrolase family 5. From the enzyme assay, it was observed that Cel5C was extracellular and Cel5D was intracellular cellulase. Phylogenetic and homology matrix analyses of DNA and protein sequences revealed that family 12 cellulase enzymes Cel12A and Cel12B displayed higher homology (>50 %), but Cel5C and Cel5D enzymes belong to family 5 displayed lower homology (<30 %). In addition, repeated and mirror sequences in tandem genes are supposed to show the existence of gene duplication and recombination. © 2012 Springer Science+Business Media, LLC. Source

Kim M.K.,Gyeongsangnam do Agricultural Research and Extension Service | Kang T.H.,Gyeongsang National University | Kim J.,Sunchon National University | Kim H.,Sunchon National University | Yun H.D.,Gyeongsang National University
Journal of Microbiology and Biotechnology | Year: 2012

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) was 1,026 bp in length, encoding a protein of 366 amino acid residues with a calculated molecular mass of 40,168 Da. The molecular mass of the enzyme was estimated to be 40,000 Da. The Est5S protein contains the Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic serine hydrolases. However, the Asp or Glu necessary for the catalytic triad [Ser-Asp-(Glu)-His] was not present, indicating Est5S represents a novel member of the GHSQG family of esterolytic enzymes. BlastP in the NCBI database analysis of Est5S revealed homology to hypothetical proteins and it had no homology to previous known lipases and esterases. Est5S was optimally active at pH 7.0 and 40°C. Among the p-nitrophenyl acylesters tested, high enzymatic activities were observed on the short-chain p-nitrophenyl acylesters, such as p-nitrophenyl acetate, etc. The conserved serine residue (Ser190) was shown to be important for Est5S activity. The primers that amplified the est5S gene did not show any relative band with 49 species of culturable rumen bacteria. This implies that a new group esterase gene, est5S, may have come from a noncultured cow rumen bacterium. Source

Barman D.N.,Gyeongsang National University | Haque M.A.,Gyeongsang National University | Islam S.M.A.,Patuakhali Science and Technology University | Yun H.D.,Gyeongsang National University | Kim M.K.,Gyeongsangnam do Agricultural Research and Extension Service
Ecotoxicology and Environmental Safety | Year: 2014

Chlorpyrifos is an organophosphate pesticide that has adverse effect on animals and plants. We isolated endophytic bacterial strain, Pseudomonas sp. BF1-3, from balloon flower root which can hydrolyze chlorpyrifos. A gene (ophB) encoding a protein involved in chlorpyrifos degradation from this strain was cloned into Escherichia coli DH5α for confirming enzyme activity. After sequencing, total 1024bp nucleotide sequences were found in the open reading frame of ophB. The chlorpyrifos degradation patterns by E. coli DH5α (ophB) were observed. During incubation in minimal salt (M9) medium supplemented with chlorpyrifos (100mgL-1), the E. coli DH5α harboring ophB degraded about 97% initial chlorpyrifos (100mgL-1) and accumulated 86mgL-1 3,5,6-trichloro-2-pyridinol (TCP) within 9 days. In addition, optical density (OD) of E. coli DH5α (ophB) culture at 600nm was increased from 0.172 to 1.118 within 2 days of inoculation in the chlorpyrifos supplemented M9 medium. The estimated molecular weight of purified OphB protein was determined to be 31.4kDa by SDS-PAGE. The OphB enzyme was most active at pH 8 and an optimal temperature around 35°C. These results indicate that endophytic bacteria are supposed to be useful for biological control of environments contaminated with pesticides. © 2014 Elsevier Inc. Source

Haque M.A.,Gyeongsang National University | Haque M.A.,Gyeongnam National University of Science and Technology | Barman D.N.,Gyeongsang National University | Kim M.K.,Gyeongsangnam do Agricultural Research and Extension Service | And 2 more authors.
Journal of the Science of Food and Agriculture | Year: 2016

BACKGROUND: Imperata cylindrica is being considered as a biomass candidate for bioethanol. This work aimed to evaluate a mild alkali pretreatment effect on the Imperata recalcitrant structure. Therefore, varied concentrations of NaOH (0, 7.5, 15, 20, and 25gL-1) were applied as treatments to Imperata at 105°C for 10min. RESULTS: Scanning electron microscopy, atomic force microscopy, and Fourier transform infrared spectroscopy studies revealed that 20 to 25gL-1 NaOH-treated Imperata exposed amorphous cellulose on surface granules composed of lignin, waxes, and partly hemicelluloses were abolished due to the comprehensive disruption of the linkages between lignin and carbohydrates. The cellulose crystalline index was increased with 7.5 to 20gL-1 NaOH treatments and reduced with a 25gL-1 NaOH treatment. In fact, the cellulose content in solids increased with the increasing NaOH concentration and was estimated to be 720 and 740gkg-1 for the 20 and 25gL-1 NaOH treatments, respectively. The yield of the reducing sugar was obtained 805 and 813mgg-1 from 20 and 25gL-1 NaOH-treated Imperata, respectively. CONCLUSION: Considering the cost of pretreatment, the 20gL-1 NaOH treatment is judged to be effective for disrupting Imperata recalcitrance in this pretreatment regime. © 2015 Society of Chemical Industry. Source

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