Analysis and characterization of peptidyl-prolyl cis/trans isomerase (FklB) promoter region of Vibrio anguillarum
- Abstract
- Analysis and characterization of peptidyl-prolyl cis/trans isomerase (FklB) promoter region of Vibrio anguillarum
Abstract
When bacteria faced environmental stress, they produce specific gene products to response these environmental stressors. These products are the proteins defined as molecular chaperones. One of the molecular chaperone, peptidyl-prolyl cis/trans isomerase (FklB), a key enzyme involved with accelerating the refolding rate of proteins, catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. Recently, we reported that FklB expression was interestingly increased in alkaline conditions, pH 10 determined by two-dimensional electrophoresis (2-DE) analysis.
In this study, using LacZ as a reporter of gene expression, β-galactosidase activity assay was performed in different pH condition. And then, quantitative reverse transcription (qRT)-PCR revealed that it is highly induced by alkaline pH, and the expression level achieved twice than normal condition. The structure and function of the promoter region of the FklB of V. anguillarum was analysed. There are six hypothetical promoter elements predicted in the 235bp upstream of the open reading frame (ORF) of FklB. Through deletion analysis of the promoter, it was demonstrated that two active promoters were located between –235 and -110 of the ORF of FklB. One of those promoter was very similar to that of the rpoD (TTGACA), and the other promoter is similar with rpoH (GAACTT) of E. coli. These results suggest that the promoter of FklB is an efficient and strong alkaline-inducible promoter.
- Issued Date
- 2015
- Awarded Date
- 2015. 8
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Alternative Author(s)
- Gyuyou Noh
- Affiliation
- 부경대학원 수산과학대학 생물공학과
- Department
- 대학원 생물공학과
- Advisor
- 공인수
- Table Of Contents
- CONTENTS
List of Tables iii
List of Figures iii
Abstract iv
1. Introduction 1
2. Materials and Methods 4
2.1. Bacterial strains, media, culture conditions and plasmids 4
2.2. Constructions of the recombinant vector 4
2.3. Bioinformatics analysis 7
2.4. Bacteria cultures in pH stress and β-galactosidase assay 7
2.5. Total RNA extraction and cDNA synthesis 8
2.6. qRT-PCR analysis of expressed genes 8
2.7. Identification of the active promoter through deletion analysis 9
3. Results 11
3.1. Cloning of FklB promoter and reporter gene lacZ 11
3.2. Comparison of the nucleotide sequences 11
3.3. Bioinformatics analysis 12
3.4. Bacteria cultures in pH stress and β-galactosidase assay 12
3.5. Gene expression patterns by RT-PCR & real time PCR 12
3.6. Cloning of deleted FklB promoter region and β-galactosidase assay 13
3.7. Heat-shock stress and β-galactosidase assay 14
4. Discussions 25
5. References 29
Acknowledgments 31
- Degree
- Master
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