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.

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.

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.

알파파 근류로부터 분리한 Poly ( A+ ) - mRNA 에 의하여 합성된 Leghemoglobin 유전자의 cDNA Cloning 에 관한연구

조무제,윤한대,최용락,최영주,이경상,Winstion J . Brill ( Moo Je Cho,Min Suk Yang,Han Dae Yun,Yong Lark Choi,Young Ju Choi,Kyung Sang Lee ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.1

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 SalI 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 hybridarrested translation. One alfalfa leghemoglobin cDNA clone was selected, and restriction map and fingerprints of the cDNA from the selected clone were analyzed.

무름병균 Erwinia chrysanthemi PY35 의 CMCase isozymes 분리

박상렬,윤한대,조수정 한국농화학회 1999 Applied Biological Chemistry (Appl Biol Chem) Vol.42 No.3

Soft-rot bacterial pathogen, Erwinia sp., was isolated from Chinese cabbage tissue showing soft-rot symptom. This bacterial strain caused soft-rot to Chinese cabbage and potato, and identified as Erwinia chrysanthemi PY35(Ech PY35). Ech PY35 have extracellular CMCase, pectinase, pectate lyase, and protease activity, but not hemicellulase activity. The results of the microscopy showed that Chinese cabbage tissue and potato tissue were macerated by infection of Ech PY35. In analysis of the CMCases activity in the total protein of Ech PY35, three CMCases were detected as intracellular protein while two CMCases were as extracellular protein by CMC-SDS-PAGE direct stain method.

Molecular Approaches to Evaluate the Role of Some Genes Required for Plant Pathogenicity of Xanthomonas campestris pv. campestris

Bae, Dong Won,Yun, Han Dae,Kim, Hee Kyu 경상대학교 유전공학연구소 1997 遺傳工學硏究所報 Vol.16 No.-

십자화과 작물에 발생하는 검음썩음병(Black rot or Black vein of crucifer )의 병원성 세균인 Xanthomonas campestris pv. campestris 를 분리, 동정하고 병원성을 검정하였다. 이 X. c. pv. campestris는 3가지 종의 Chinese cabbage 에 병원성을 나타내었고, 병원성과 관련된 특성을 결정하기 위하여 Tn5 mutagensis 를 실시 cellulase negative mutant를 선발하여 병원성 검정하였다. 선발된 cellulase negative mutant를 배추에 분무 접종하여 광학 현미경과 전자현미경으로 관찰한 결과 cellulase negative mutant는 wild type 와 함께 기공표면과 기공하부조직에서 정착하였지만 그 밀도는 낮았다. 반면 접종 24시간 이후 wild type은 기공표면과 기공하부조직이 lysis 되기 시작하여 48시간 이후에는 병원성의 진전으로 보다 많이 lysis 되었다. 6일 후, wild type은 활성에 의해 식물체 조직에서 높은 증식력을 보이며 조직을 lysis시키고 또한 조직 깊숙이 침입, 정착하는 것을 관찰하였다. 이 결과로 X. c. pv.campestris의 cellulase 는 병원성에 관여하는 중요한 요인으로 생각된다. Xanthomonas campestris pv. campestris, causal agent of Black rot of crucifers, were isolated and identified from crucifer host. In order to determine the characters of X. c. pv. campestris associated with pathogenicity, Tn5 mutagensis was carried out by conjugating with E. coli pJB4J1. Transconjugants were plate-assayed for missing cellulase, protease and amylase activity. A cellulase negative mutant was selected and tested for pathogenicity. Light microscopy and Scanning electron microscopy revealed that substomatal tissues were colonized by mutant, but was far less extensive than those by wild type. Stomatal surface and substomatal tissue appeared to have degraded by only wild type in 24 hrs and progression of pathogenesis was distinct in 48 hrs. In 6 days, wild type proliferated well in the tissue facilitated by cellulase activity. As a result, cellulase was determined as the important factor in pathogenesis.