Establishment of a method for gene complementation in Vibrio parahaemolyticus
- VernacularTitle:副溶血弧菌基因回补实验方法的建立与应用
- Author:
Zhenhong CHEN
1
;
Li WANG
;
Yiquan ZHANG
;
Jiao FENG
;
Ruifu YANG
;
De CHANG
;
Li AN
;
Changting LIU
;
Dong-Sheng ZHOU
Author Information
1. 解放军总医院南楼呼吸科
- Keywords:
Vibrio parahaemolyticus;
gene complementation;
pBAD33;
aphA;
opaR
- From:
Journal of Southern Medical University
2014;(1):70-74
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33. Methods The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into ΔopaR and ΔaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-ΔaphA and C-ΔopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, ΔopaR, ΔaphA, C-ΔaphA, and C-ΔopaR. Results opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains. Conclusion A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.
