glycinecin R에 내성인 트랜스포존 삽입 변이체 특성 연구

Alternative Title
Shin, Dong - Ho
Alternative Author(s)
Shin, Dong - Ho
일반대학원 생명과학과
The Graduate School, Ajou University
Publication Year
트랜스포존 삽입 변이체
Xanthomonas axonopodis pv. glycines 8ra는 glycinecin A, R, 그리고 P로 명명되어진 bacteriocin을 3종류 생산한다. 이들 bacteriocin에 모두 민감성을 나타내는 균주인 Xanthomonas campestris pv. vesicatoria 833의 민감성 관련 유전자 및 그 기작을 연구하기 위해서 transposon을 무작위로 삽입한 mutant library로부터 glycinecin R에 저항성을 나타내는 mutant가 분리되었다. 선별된 bacteriocin에 비민감성인 Xanthomonas campestris pv. vesicatoria 833 mutant의 transposon 삽입위치를 서열분석을 통하여 확인한 결과 세균의 표면에 존재하는 pilus의 구조 유전자인 pilA 유전자라는 것이 밝혀졌다. 본 연구에서는 glycinecin R에 저항성을 나타내는 Xanthomonas campestris pv. vesicatoria 833 mutant의 다양한 특성에 대하여 분석하였다. 비민감성인 mutant 균주는 민감성인 wild-type 균주들에 비하여 생장속도가 느렸으며 전자현미경으로 확인한 결과 표면의 구조 역시 차이점을 나타내었다. 또한 wild-type에 비해 더 활발한 이동성을 가진다는 차이점도 확인하였다. Xanthomonas campestris pv. vesicatoria 833 wild-type 균주로부터 PCR을 통하여 pilA gene을 증폭하고 발현벡터에 클로닝 하여 과발현을 확인하였다. 또한 Xanthomonas axonopodis pv. glycines 8ra로부터 분리된 glycinecin R 유전자와 상동성을 가지는 유전자를 포함한 Xanthomonas axonopodis pv. glycines 8ra의 cosmid library clone인 pG11을 선별하였다. 제한효소 처리 결과 pG11은 glycinecin R과는 다른 패턴을 나타내었으며 이를 통하여 Xanthomonas axonopodis pv. glycines 8ra 내에는 glycinecin R 유전자가 1개 이상 존재하는 것을 밝혔다.
Alternative Abstract
Previously, the mutant Xanthomonas campestris pv vesicatoria 833 resistant to glycinecin R was isolated from the mutant library constructed by random insertion of a transposon. The transposon in the mutant was determined to be inserted in the pil A gene. In this study I have characterized the transposon inserted mutant in detail. The mutant was first selected for resistance to glycinecin R, but inhibition assay revealed it is resistant to glycinecin A and to Xanthomonas axonopodis pv glycines 8ra. Based on the growth profile the mutant showed slower growth rate, and reached log phase slower and the population size at the stationery phase smaller than the wild type. The colony on the agar plate was less tight and spread out wider than the wild type. Observation through SEM and TEM revealed the mutant has less defined pili, and the colony contained slimy substance. To confirm the mutation in pilA is responsible for the resistance to the glycinecins I tried the wild type of pilA gene was introduced to the mutant. The optimum conditions for making competent cells and for electroporation still need to be determined. To study the interaction between PilA and Glycinecin A proteins both genes were cloned in pET vector for over-expression. Optimum condition for PilA was set up, and its over-expression was confirmed using gel electrophoresis. The cosmid clone, pG11, which contains a glycinecin R homolog was analyzed. The restriction pattern revealed that the glycinecin R gene in pG11 is not identical to the glycinecin R studied earlier, but there are some differences in the nucleotide sequence. However, the inhibition assay indicated that the glyR homolog in pG11 is also functional as a bacteriocin.

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