副溶血性弧菌毒力基因表达时内参基因的选择

Selection of reference genes for virulence gene expression in Vibrio parahaemolyticus

摘要:

摘要:【目的】筛选出合适的内参基因用于分析不同环境条件下副溶血性弧菌毒力基因的表达情况。【方法】本研究以虾样品中、海水样品中、过滤海水样品中以及TSB 培养条件下的副溶血性弧菌为材料，利用qRTPCR技术评价了GAPDH、pvuA、pvsA 和rpoS 4种常用管家基因在不同条件下的表达稳定性。【结果】4种管家基因均能特异扩增，表达稳定性排列顺序为pvuA(2.906)＞pvsA(3.197)＞GAPDH(3.746)＞rpoS(6.512)，进一步通过geNorm软件分析，最终选择两个表达最为稳定的内参基因即pvuA和pvsA，以二者的几何平均值作为参照可更为准确地校正目的基因的表达。【结论】pvuA和pvsA可作为环境样品中副溶血性弧菌毒力基因表达变化研究的内参基因。

关键词:关键词:副溶血性弧菌，环境样品，qRT-PCR，内参基因

Abstract:

Abstract:［Objective］ We selected reference genes for virulence gene expression of Vibrio parahaemolyticus under different environmental conditions． ［Method］Using qRT-PCR，we evaluated the expression stability of four housekeeping genes (GAPDH，pvuA，pvsA and rpoS) of V．parahaemolyticus cultivated separately in seawater，filtered seawater，shrimp and Tryptone Soya Broth.［Result］The result shows that all the candidate reference genes could be amplified specifically in qRT-PCR reaction． The expression stability of the four reference genes ranked pvuA (2.906)＞ pvsA (3.197)＞GAPDH(3.746)＞rpoS(6.512)．Further analysis with geNorm program reveals that the highest stability was observed in pvuA and pvsA．The geometric average score of the two genes was considered as the most appropriate reference gene for normalizing the expression of virulence genes of V．parahaemolyticus.［Conclusion］The pvuA and pvsA genes could be used as reference genes to study the virulence gene expression of V．parahaemolyticus.

Key words:Keywords: Vibrio parahaemolyticus，qRT-PCR，reference gene