AUTHOR=Zhao Ning , Xu Junnan , Jiao Lingxia , Qiu Mengzhen , Zhang Jie , Wei Xinyuan , Fan Mingtao 

TITLE=Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.731205

DOI=10.3389/fmicb.2021.731205

ISSN=1664-302X

ABSTRACT=<p>Alicyclobacillus <italic>acidoterrestris</italic> is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of <italic>A. acidoterrestris</italic> to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of <italic>A. acidoterrestris</italic> under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene <italic>ctsR</italic> showed that <italic>dnaG</italic> and <italic>dnaN</italic> were the optimal multiple RGs for normalization at pH 4.0; <italic>ytvI</italic>, <italic>dnaG</italic>, and 16s rRNA at pH 3.5; <italic>icd</italic> and <italic>dnaG</italic> at pH 3.0; and <italic>ytv</italic>I, <italic>dnaG</italic>, and <italic>spoVE</italic> at pH 2.5. This study revealed for the first time that <italic>A. acidoterrestris</italic> had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.</p>