Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite
Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite
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DOI：
10.1016/j.ijfoodmicro.2015.08.012
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发表时间：
2015-12-23
影响因子：
5.4
通讯作者：
Corich, Viviana
中科院分区：
文献类型：
--
作者：
Nadai, Chiara;Campanaro, Stefano;Corich, Viviana
二氧化硫在工业发酵过程中被广泛使用，并与低pH值，高糖含量和乙醇浓度增加一起决定了酿酒的苛刻条件。因此，在酵母基因表达研究中必须考虑亚硫酸盐的存在，以正确理解酵母在酿酒等技术环境中的行为。一个可靠的表达模式，可以得到只有使用一个适当的参考基因组，是组成型表达，而不管扰动连接到实验conditions.In这项工作中，我们测试了15个候选参考基因，适用于分析必须在亚硫酸盐发酵过程中的基因表达。从全基因组表达实验中选择新的参考基因，该实验通过对在酿酒条件下生长的四种酿酒酵母葡萄酒菌株进行RNA测序获得。他们的表现与以前研究中使用的最常见的基因进行了比较。选择基于不同统计方法的最流行的软件（geNorm、Normalization和BestKeeper）来评估候选参考基因的表达稳定性。在15个参比基因中，ALG9、FBA1、UBC6和PFK1的可靠性最高，而ENO1、PMA1、DED1和FAS 2的可靠性最低。最常用的参考基因ACT1，广泛用于S.酿酒酵母基因表达的研究表明，稳定性水平明显低于我们选择的参考基因。最后，由于新的参考基因组的表达在整个发酵过程中保持恒定，而不考虑由于亚硫酸盐添加引起的扰动，因此当没有亚硫酸盐添加到果汁中时也可以考虑我们的结果。(C)2015爱思唯尔B.V.保留所有权利。
Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions.In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite.Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must. (C) 2015 Elsevier B.V. All rights reserved.