南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (01): 70-.

• • 上一篇    下一篇

副溶血弧菌基因回补实验方法的建立与应用

陈振鸿，王 丽，张义全，冯 娇，杨瑞馥，常 德，安 莉，刘长庭，周冬生   

  • 出版日期:2014-01-20 发布日期:2014-01-20

Establishment of a method for gene complementation in Vibrio parahaemolyticus

  • Online:2014-01-20 Published:2014-01-20

PDF

2526

摘要： 目的建立以pBAD33质粒为载体的副溶血弧菌（Vibrio parahaemolyticus, VP）基因回补实验平台。方法PCR扩增aphA
和opaR的整个ORF区序列，并将其直接克隆入pBAD33质粒中，构建重组质粒。分别将重组质粒转入到ΔopaR 和ΔaphA中（分
别代表opaR 和aphA的突变株），以构建出相应的回补株C-ΔaphA和C-ΔopaR。分别在aphA和opaR基因内设计特异性引物，采
用RT-PCR 方法，验证在相应的回补突变株中aphA 和opaR 是否转录。利用引物延伸实验研究野生株（WT）、ΔopaR 、ΔaphA、
C-ΔaphA 和C-ΔopaR中mfpA（aphA负调控，opaR正调控其表达）的相对RNA水平。结果aphA和opaR在相应的回补株中发生
转录；且mRNA水平与野生株一致。结论成功建立了以pBAD33质粒为载体的VP基因回补方法，并得到应用。

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
Amethod has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.