Xanthomonas axonopodis pv. glycins 8ra가 생산하는 박테리오신 glycinecin에 대해 내성을 보이는 트랜스포존 삽입 돌연변이체 분리 및 특성 분석
Isolation and analysis of a transposon-inserted Xanthomonas campestris pv. vesicatoria 833 resistant to glycinecin R
- 주제(키워드) bacteriocin+pilA+Xanthomonas
- 발행기관 아주대학교
- 지도교수 문은표
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 석사
- 학과 및 전공 일반대학원 생영과학과
- 본문언어 영어
초록/요약
The glycinecin R (glyR) is one of bacteriocins produced by Xanthomonas axonopodis pv. glycines 8ra. It shows strong bactericidal activity on number of major crop pathogens including Xanthomonas oryzae pv. oryzae and Xanthomonas campestris pv. vesicatoria. To identify the receptor on the X. campestris pv. vesicatoria 833 responsible for glyR recognition a mutant library was constructed using transposon-insertion mutagenesis, and a mutant X. campestris pv. vesicatoria 833 clone which is resistant to glyR was isolated. The transposon-inserted genomic region was cloned, and the gene inactivated by transposon insertion was identified as the pilA gene by nucleotide sequence analysis. The pilA gene is 429bp long and it encodes protein associated with type Ⅳ fimbriae. The data implies that the pili on the surface of X. campestris pv. vesicatoria 833 interact with bacteriocin glyR, and as the result the growth of X. campestris pv. vesicatoria 833 is inhibited. For further studies on the protein interaction between the glyR and the PilA as the receptor, the wild type pilA gene was cloned.
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Abstract
Contents
List of Figures
List of Tables
Ⅰ. Introduction
Ⅱ. Material and Methods
A.Bacterial strains and media
B.General experimental protocols of molecular biology
1.Plasmid DNA isolation
2.Agarose gel electrophoresis
3.DNA elution from electrophration gel
4.DNA ligation
5.Competent cell and transformation
C.Preparing bactericins for assay
1.Crude extract of glycinecin R/A
2.Supernatant of Xag 8ra
D.Assay for bacteriocin activity
1.Spot on lawn method
2.Agarose diffusion method
E.Construction of Tn5 inserted mutant library
1.EZ::TN™
2.Mating method
F.Mutant library screening
G.Tn5 clone rescue
H.Nucleotied sequencing of Tn5 insertion site
I.Database-search to indentify Tn5 insertion site(NCBI blastx)
J.PCR pilA gene from wild Xcv 833
Ⅲ. Results and Discussion
A.Bactericidal activity of Xag 8ra
B.Confirmation for Tn5 insertion
C.A mutant colony is resistant to glycinecin R
D.Nucleotide sequence of Tn5 inserted site
E.BlastX program was used for sequence analysis
F.pilA gene of Xcv 833 was compaired to other pilA genes
G.Isolation of the wile-type pilA gene
H.Cloning for over-expression
Ⅳ. Conclusion
Ⅴ. Bibliography
국문 초록
