黄芩苷铝胁迫下产肠毒素大肠杆菌内参基因的筛选

Reference gene selection for real-time quantitative PCR normalization in enterotoxigenic Escherichia coli exposed to baicalin-aluminum complexes

摘要:

【背景】产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli，ETEC)是导致仔猪腹泻的主要致病菌之一，黄芩苷铝对ETEC诱导的仔猪腹泻具有良好的治疗效果，但作用机制尚不明了。【目的】筛选出黄芩苷铝胁迫下ETEC中表达水平最为稳定的内参基因。【方法】采用qPCR技术测定了12个内参基因16S rRNA、mdh、recA、gapA、gyrA、gyrB、rpoA、rpoB、mdoG、dnaG、secA和fusA在不同浓度(250、500和1 000 μg/mL)黄芩苷铝胁迫下培养不同时间后(4.5 h和6 h)的表达水平，并采用比较Ct值法、geNorm、BestKeeper和NormFinder软件4种方法对12个候选内参基因的表达稳定性进行评估。【结果】 rpoA表达丰度适中，并在多个分析方法中表达最稳定。【结论】确定了rpoA为ETEC qPCR的最佳内参基因，此研究为后续利用qPCR法研究黄芩苷铝胁迫ETEC后目的基因功能的表达奠定了基础。

Abstract:

[Background] Diarrhea due to enterotoxigenic Escherichia coli (ETEC) is one of the most frequent diseases in piglets. Baicalin-aluminum has a good therapeutic effect on ETEC-induced intestinal injury in piglets, but the mechanism of action remains to be clarified. [Objective] Screening out the most stable internal reference genes in ETEC under the stress of baicalin-aluminum is the basis for studying the effect of baicalin-aluminum on the expression of ETEC target genes. [Methods] In this study, 12 internal reference genes 16S rRNA, mdh, recA, gapA, gyrA, gyrB, rpoA, rpoB, mdoG, dnaG, secA and fusA were determined at different concentrations (250, 500 and 1 000 μg/mL) baicalin-aluminum using qPCR technology. The expression level after different culture time (4.5 h and 6 h) under baicalin-aluminum stress. Four methods including comparative Ct value method, geNorm, BestKeeper and NormFinder software were used to evaluate the stability of the expression of 12 candidate internal reference genes. [Results] The results show that rpoA expression is moderately abundant and the most stable expression among multiple analysis methods. [Conclusion] It was determined that rpoA is the best internal reference gene for ETEC qPCR. This study laid the foundation for the subsequent use of qPCR to study the expression of target gene function after baicalin-aluminum stress ETEC.