근류균의 세포벽 분해효소의 크로닝

윤한대,임선택,강규영 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.2

Rhizobium fredii USDA 193은 대두에 접종하면 대두(Peking)의 root hair 세포벽을 침투하여 근류를 형성하며 이 때의 세포벽 가수분해효소의 활성을 필요로 한다. 본 실험에서는 pLAFR_3 cosmid vector를 사용하여 R. fredii USDA 193 균주로부터 DNA를 분리하여 genomic library를 만들었다. 그 결과 1500개의 transductants를 얻었으며 그 중 한 clone(YA17)이 cellulase 활성을 가지고 있었으며 27kb insert를 함유하고 있는 것을 확인하였다. YA17을 pUC18vector에 subcloning하여 최종적으로 1.7kb의 cellulase 활성을 함유한 clone(YA300)을 얻었다. 다시 이들의 cellulase 활성을 검토하였으며 다른 cel gene과 활성을 비교검토하였다. Rhizobium fredii USDA193 infects and nodulates soybean root via root hairs by actively penetrating the root hair cell-wall. The penetration of Rhizobium into root hair cell-wall requires the activity of cell-wall hydrolyzing enzymes such as cellulases. We constructed a genomic library by cloning Sau 3A-digested genomic DNA from R. fredii USDA193 DNA into the BamHI site of the cosmid vector pLAFR_3. Out of 1500 Tc-resistance transductants of E. coli, one clone(YA17) had cellulase activity and contained pLAFR_3 cosmid with 27kb insert of R. fredii DNA. A 27kb BamHI fragment coding for cellulase was subcloned in pUC18 vector and a physical map of the resulting plasmid YA100 was constructed. Finally, we have subcloned 1.7kb Bgl II-HincII fragment DNA coding for cellulase in pUC18(YA300). The expression of rhizobial cellulase gene in E. coli was studied and compared with the products of cellulase genes from Clostridium thermocellum in E. coli.

폐섬유자원의 발효공학적 이용에 관한 연구 (제8보) 섬유소 자화세균의 혼합배양

윤한대,성낙계 ( Han Dae Yun,Nack Kie Sung ) 한국미생물생명공학회 1978 한국미생물·생명공학회지 Vol.6 No.2

SDS-Polyacryladmide gel 電氣泳動法에 依한 동부 蛋白質의 遺傳 分析

趙武濟,張權烈,李富永,尹漢大 慶尙大學校 1980 論文集 Vol.19 No.1

交配親 6個 品種과 이들을 二面交雜에 依해 얻은 F1 15個 組合의 동부種實을 SDS-polyacrylamide gel 電氣泳動法에 의하여 蛋白質패턴을 조사하고 品種間의 相互關係 및 各 蛋白質 band의 分子量을 測定한 結果를 要約하면 다음과 같다. 1) SDS-PAGE에 의해 분리된 동부의 공통적인 蛋白質 band는 11個가 나타났으며, parent D,E 그리고 F1 BD, CD, DE, DF 組合에서는 몇 개의 분리대가 더 나타났으며 특히 분자량 17,000 Dalton 부근에서는 parent D 그리고 F1 DE, DF組合에서 두개의 분리대로 나타났다. 2) 交配親 中에서 D,E 品種의 蛋白質패턴이 特異하였으며, 이들을 組合으로 한 F1 BD, CD, DE, DF組合에�� 特異性이 나타났다. 3) 분리된 各 蛋白質 band의 分子量은 17,000∼140,000 Dalton 사이인 것으로 推定되었다. Cowpea seed proteins of six parents and their 15 F1 hybrids were separated and the molecular weight of each protein bands were calculated by SDS-Polyacrylamide gel electrophoresis. The results obtained are as follows. 1. Eleven common protein bands were separated in all the parents and F1 hybrids, and three more bands were detected in parents D, E, and F1 BD, CD, DE, DF. Particularly in parent D, F1 DE and DF, two specific protein band were detected in 17,000 Dalton area. 2. Characteristic protein patterns were observed in parent D, E and F1 hybrids which have D and E as parents compared to other varieties. 3. The molecular weights of the each protein band in cowpea were ranged from 17,000 to 140,000 Dalton.

Introduction of Ti Plasmid into Bradyrhizobium japonicum by Spheroplast Transformation

YUN, HAN DAE,CHO, MOO JE 경상대학교 유전공학연구소 1986 遺傳工學硏究所報 Vol.5 No.-

Bradyrhizobium japonicum spheroplasts were prepared by culturing cells in the presence of glycine, followed by treatment with lysozyme. The cells were examined by electron microscopy during the formation of spheroplast. Then Ti plasmid from Agrobacterium tumefaciens 15955 was introduced into Bradyrhizobium japonicum by glycine-lysozyme induced spheroplast transformation. After cell wall regeneration, transformants were selected by the ability of utilization of octopine. Transformation were received at a frequency of 1 × 10^-7. Tumors induced on Petunia hybrida by transformants were small size of gall compared with that of gall by Agrobacterium tumefaciens 15955.

Molecular Cloning of Genetic Determinants of Pectate Lyases from Rhizobium fredii USDA 193

YUN, HAN DAE 경상대학교 유전공학연구소 1990 遺傳工學硏究所報 Vol.9 No.-

Rhizobium fredii USDA193 is a microsymbiont of Glycine max(soybeans) which was originally isolated from People's Republic of China^19). It infects and nodulates soybean roots via hairs by actively penetrating the root hair cell-wall. The penetration of Rhizobium into the root hair cell-wall requires the activity of cell-wall hydrolyzing enzymes such as cellulases and pectinases. Evidence is presented that R. fredii USDA193 produces pectate lyase(pel) enzymes and that their genetic determinants are borne on the resident symbiotic plasmid(pSym) DNA. The pectate lyase genes pelB and pelE of Erwinia chrysanthemi EC16 show homology to a 4.0 Kb EcoR1 fragment of pSym DNA. The pelB and pelE homologues in R. fredii USDA193 are tandemly located on a 0.7 Kb Sal1-HindⅢ and 1.3 Kb HindⅢ- EcoR1 fragment, respectively. These two subfragments carrying the homologous RpelB and RpelE(for Rhizobium pel genes) genes, have been subcloned into high-express pectate lyase enzyme activity in an Escherichia coli background.

Molecular Cloning of the cel Gene from Xanthomonas campestirs pv. oryzae

YUN, HAN DAE,LIM, SUN TECH,CHUNG, MIN HWA,PARK, YONG WOO,KIM, HEE KYU,KANG, KYU YOUNG 경상대학교 유전공학연구소 1991 遺傳工學硏究所報 Vol.10 No.-

Xanthomonas campestris pv. oryzae(Ishiyama) Dye is a cacusal orginism of bacterial leaf blight of rice(Oryzae sativa). It infects the rice through hydathod or wounds. No report on infection through stomata is available. The penetration of Xanthomonas campestris pv. oryzae into the plant requires the activity of cell wall hydrolyzing enzymes. Enzymatic process of infection was evidenced by smooth surfaces without trichomes upon inoculation and many penetrating bacterial cells through stoma by scanning eletron microscopy. The presence of pathogens in the mestome sheath of rice leaf was also confirmed by transmission electron microscopy. Furthermore, Xanthomonas campestris pv. oryzae exhibited the activities of extracellular cellulase and proteases as cell wall degrading enzymes, but not polygalactronase, pectate lyase, and hemicellulase by the agar diffusion method. Thus, we constructed the genomic libary of Xanthomonas campestris pv. oryzae using the cosmid vector pSAFR₃to assess the contribution of the cell wall degrading enzymes to the pathogenicity of Xanthomonas campestris pv. oryzae. Out of one thousand transductants, one extracellular cellulase and several proteases clones were isolated. This clone, designasted pXC3-43 as cel gene plasmid, independently expresses cellulase activity in an Escherichia coli background, and was mutagenized with Tn5 to make a cel^- mutant.

The Role of the Extracellular Cellulase Gene in Xanthomonas oryzae pv.oryzae on Bacterial Leaf Blight

YUN, HAN DAE,PARK, YOUNG WOO,LIM, SUN TECH,KIM, HEE KYU,KANG, KYU YOUNG 경상대학교 유전공학연구소 1992 遺傳工學硏究所報 Vol.11 No.-

A plasmid, designated pXC3-43, encoding an extracellular cellulase activity from Xanthomonas oryzae pv. oryzae(Ishima) was reported previously by authors^1). The cellular distribution of the endoglucanase activities of pXC3-43 and X. o. pv. oryzae was almost exculusively extracellular. Total endoglucanase activity expressed by the pXC3-43 was about 80% of X. o. pv. oryzae k1 wild type. Tn5 mutagenesis of pXC3-43 revealed that extracellular cellulase structural gene is located in a ca. 5 kb KpnI fragment. To examine the role of the extracellular cellulase in disease process, three cellulase-minus mutants of X. o. pv. oryzae were constructed by marker exchange of negative Tn5 insertions into the chromosome of the wild type. These cellulase -minus mutants were as virulent as the wild type in pathogenicity test on leaves of rice(Oryzae sativa L.). Furthermore, When investigated under transmission electron microscopy (TEM), bacterial leaf blight(BLB) symptoms by the cellulase-minus mutants, X. o. pv. oryzae K1::Tn5 were similar to those by wild type. These results suggest that the extracellular cellulase of X. o. pv. oryzae is not primary mechanism of pathogenesis for the pathogenesis of BLB, but mightbe involved in an early disease developmental stage.

식물 내생균 Bacillus sp. CY22가 생성하는 iturin isoform의 분리 및 특성

조수정,윤한대,Cho, Soo-Jeong,Yun-Han-Dae Korean Society of Life Science 2005 생명과학회지 Vol.15 No.6

Endophytic Bacillus sp. CY22 was previously isolated from the interior of balloon flower root and showed strong antifungal activity against phytopathogenic fungi such as Rhizoctonia solnni, Fusarium oxysporum, and Phythium ultimum. Many Bacillus strains produce antifungal compound such as iturin, fengycin, and mycosubtilin. We isolated and identified antifungal compound from cell supernatant of the endophytic strain. By the MALDI-TOF mass result, the antifungal compound was similar to the known antifungal lipopeptide iturin. It was found that the purified iturin had three isoforms with protonated masses of m/z 1,043.39, 1,057.42, and 1,071.42 and different structures in combination with $Na^{+}$ ion using MALDI-TOF MS. The ita22 gene, which transacylase gene is associated with production of antifungal iturin, had an open reading frame (ORF) of 1,200 bp encoding 400 amino acids. Results of deduced amino acids sequence homology search, Ita22 was homologous with FenF (BAB69697) of Bacillus subtilis 168.

Molecular Cloning of Alfalfa(Medicago Sativa L. Vernal) Leghemoglobin Structural Genes Synthesized by $Poly(A)^+$-mRNA from Alfalfa Root Nodules

조무제,윤한대,최용락,최영주,이경상,Cho, Moo-Je,Yun, Han-Dae,Choi, Yong-Lark,Choi, Young-Ju,Lee, Kyung-Sang,Brill, Winston J. 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.1

알팔파 근류에서 분리 정제한 $poly(A)^+$-mRNA로부터 leghemoglobin message 함유비율이 높은 9S, 18S 및 28S mRNA를 이용하여 cDNA를 합성하였다. 이들 cDNA를 Sal I linker에 연결 pBR322의 Sal I site에 크로닝하여 cDNA library를 만든 후 이들 library로부터 colony hybridization, Southern blot hybridization, hybrid-released 및 hybrid-arrestes translation에 의하여 leghemoglobin cDNA clone을 동정하였으며, 동정된 clone으로부터 alfalfa leghemoglobin cDNA의 제한효소 지도를 작성하였다. Complementary DNAs were synthesized by the leghemoglobin message rich 9S, 18S and 28S-mRNA fractions prepared from the $poly(A)^+$-mRNA of alfalfa root nodules. The synthetic Sal I linkered double stranded cDNA was inserted into the Sal I site of the pBR322, and transformed in E. coli HB101. The leghemoglobin cDNA clones were screened by colony hybridization and Southern blot hybridization with the soybean leghemoglobin cDNA as a probe, and further screened by hybrid-released and hybrid-arrested translation. One alfalfa leghemoglobin cDNA clone was selected, and restriction map and fingerprints of the cDNA from the selected clone were analyzed.

규산시용량(珪酸施用量)이 수도(水稻)의 지방산(脂肪酸) 및 Sterol 조성(組成)에 미치는 영향(影響)

양민석,윤한대,허종수,Yang, Min-Suk,Yun, Han-Dae,Heo, Jong-Soo 한국토양비료학회 1983 한국토양비료학회지 Vol.16 No.4

A pot experiment was conducted to find out the effect of application amounts of silicates on the fatty acid and sterol composition of paddy rice at various growing stages. The results obtained were as follows; 1. At maximum tillering stage and heading stage, significant positive correlations were obtained between the ratio of unsaturated fatty acid to saturated fatty acid and silica absorption, dry matter or grain yield, respectively. But significant correlation was not observed between them at harvesting time. 2. At maximum tillering stage and heading stage, the ratio of unsaturated fatty acid to saturated fatty acid in paddy rice was increased with increasing silica application from 80 to 180 ppm of soil, but these ratio was decreased at the plot of 200Kg/10a of silicate. 3. The ratio of sitosterol to stigmasterol in paddy rice at maximum tillering stage was increased with increasing silica application of soil, but these ratio was increased with increasing silica application up to 180 ppm of soil at heading stage, and was decreased at the plot of 200Kg/10a (410 ppm in soil) application of silicates. 1. 식물체(植物體) 중(中) 전생육기간(全生育期間)동안 검출(檢出)된 지방산(脂肪酸)의 종류(種類)는 $C_{12:0}$, $C_{14:0}$, $C_{16:0}$, $C_{16:1}$, $C_{16:2}$, $C_{18:0}$, $C_{18:1}$, $C_{18:2}$, $C_{18:3}$, $C_{20:2}$와 몇종(種)의 미동정(未同定)된 지방산(脂肪酸)으로 구성(構成)되어 있었다. 2. 규산흡수양(珪酸吸收量), 건물중(乾物重) 및 수량(收量)은 unsaturated fatty acid/saturated fatty acid와 최고분얼기(最高分蘖期) 및 출수기(出穗期)에는 유의적(有意的)인 정(正)(+)의 상관(相關)이 있었으며 수확기(收穫期)에는 유의적(有意的)인 상관(相關)은 없었으나 전반적(全般的)으로 역(逆)(-)의 관계(關係)가 있었다. 3. 식물체중(植物體中) UFA/SFA 비(比)의 평균치(平均値)는 최고분얼기(最高分蘖期) 및 출수기(出穗期)에서는 토양중(土壤中) 규산함량(珪酸含量)이 180ppm까지는 규산함량(珪酸含量)이 높을수록 증가(增加)하였고, 규산자원(珪酸資源) 200kg/10a 시비구(施肥區)(약(約) 410ppm)에서는 감소(減少)하였다. 4. 최고분얼기(最高分蘖期)의 sitosterol/stigmasterol 비(比)는 토양중(土壤中) 규산농도(珪酸濃度)가 높을수록 계속 증가(增加하였으나, 출수기(出穗期)에 있어서는 그 비(比)가 토양중(土壤中) 규산농도(珪酸濃度)를 180ppm 조절시(調節時)까지는 증가(增加)하다가 200kg/10a 시용(施用)(410ppm)에서는 감소(減少)하였다.