Optimal reference genes for real-time quantitative PCR and the expression of sigma factors in Acidithiobacillus caldus under various conditions

Abstract

Aims: Acidithiobacillus caldus is an important sulphur-oxidizing bacterium that plays crucial roles in the bioleaching industry. This study aims to analyse the optimal reference gene for real-time quantitative PCR (RT-qPCR) under different conditions and investigate the transcription levels of the sigma factor genes in the stress response.

Methods and results: We selected six housekeeping genes and analysed them via RT-qPCR using two energy resources, under four stress conditions. Three statistical approaches BestKeeper, geNorm, and NormFinder were utilized to determine transcription stability of these reference genes. The gapdH gene was the best internal control gene using elemental sulphur as an energy resource and under heat stress, map was the best internal control gene under pH and osmotic stress, era was the best internal control gene for the K₂ S₄ O₆ energy resource, and rpoC was the best internal control gene under Cu²⁺ stress. Furthermore, the expressional levels of 11 sigma factors were analysed by RT-qPCR in the stress response.

Conclusions: Stable internal control genes for RT-qPCR analysis of A. caldus were determined, and the expression patterns of sigma factor genes of A. caldus were investigated.

Significance and impact of the study: The identification of the optimal reference gene and analysis of transcription levels of sigma factors in A. caldus can provide clues for reference gene selection and the study of sigma factor function.

Keywords: Acidithiobacillus caldus; RT-qPCR normalization; reference gene; sigma factor; stress response.