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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.
Ervinia carotovora subsp . carotovora LY34에서 pelCI 유전자 클로닝
임선택,박용우,윤한대 ( Sun Tech Lim,Yong Woo Park,Han Dae Yun ) 한국응용생명화학회 1997 Applied Biological Chemistry (Appl Biol Chem) Vol.40 No.5
Phytopathogenic Erwinia carotovora subsp. carotovora (Ecc) LY34 causes plant tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL) existed as multiple isoenzyme form. Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript Ⅱ SK^+. Among them, a clone hydrolyzing polypectate was selected and its DNA was digested with BamHI. Through the subsequent subcloning the resulting 3.1 kb fragment, corresponding to a pelCI, was subcloned into pLYPA 100. The structural organization of a pelCI gene encoding a 374 amino acid residues consists of an open reading frame (ORF) of 1,122 by commencing with a ATG start codon and followed by a TAA stop codon. PelCI contained a typical prokaryotic signal peptide of 22-amino acid. Since the deduced amino acid sequences of PelCI protein was very similar to those of PelIII of Erwinia carotovora subsp. carotovora, and to those of Pel3 of Erwinia carotovora subsp. atroseptica, and to those of PelC of Erwinia carotovora subsp. carotovora, it belong to the same family PLbc group. The 374-amino acid PelCI had a calculated Mr of 40,507 and pI of 7.60.
E. coli에서 근류균 섬유소 분해효소 유전자의 발현 및 생화학적 특성조사
윤호종,박용우,임선택,강규영,윤한대 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3
본 실험은 대두 근류균(Rhizobium fredii USDA193)의 세포벽 분해효소 유전자를 조사하기 위해 Rhizobium fredii USDA 193 균주의 genomic DNA library로부터 선별한 1.7 kb CMCase gene을 함유한 섬유소 분해효소 유전자의 발현특성을 E. coli에서 조사하였다. 이 유전자의 크로닝 방향을 달리하기 위해 pBluescript II KS^+(pYA500)와 pBluescript II SK%(pYA600)에 각각 재크로닝하여 CMCase 활성을 조사한 결과 pYA500에서 효소 활성이 증가되었으며 이 유전자를 probe로 하여 같은 fast growing group에 속하는 R. melioti와 상동성 실험을 한 결과 서로 상동성이 관찰되었다. 이 효소의 생화학적 특성으로 최적 효소활성 온도는 37℃ 부근이었으며 최적 pH는 7 근방이었다. 또한 Tn5(pJB4JI)를 pYA500에 삽입하여 CMCase mutant를 얻었으며 이들의 SDS-PAGE에 총 단백질의 양상을 비교한 결과 이 유전자 산물의 단백질 분자량은 약 45 kDa에 달하는 것으로 추정하었다. From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and aYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37℃. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase-negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS-PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.
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.
The Role of the Extracellular Cellulase 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 1993 遺傳工學硏究所報 Vol.12 No.-
A plasmid, designated pXC3-43, encoding an extracellular cellulase activity from Xanthomonas oryzae pv. orzae(Ishiyama) was reported previously(Yun et al, 1992). The cellular distribution of the cellulase actinities of pXC3-43 and X.o.pv.oryzae was almost exclusively extracellular. Total cellulase activity expressed by the pXC3-43 was about 80% of that expressed by o. pv. oryzae K1 wild type. Tn5 mutagenesis of pXC3-43 revealed that the extracellular cellulase structural gene is located in about 5 kb KpnI fragnebt, Three cellulase-minus mutants of as virulent as the wild type in pathogenicity tests on leaves of rice(Oryzae sativa L.). Furtherblight(BLB) symptoms used by the cellulase-minus mutants, X.o.pv. oryzae K1::Tn5, were similar to those used by the wild type. These results suggest that the extracellular cellulase of X.o.pv. oryzae is minor factors for the pathogenesis of BLB, but might be involved in an early stage of disease development.