Dataset Information

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a.

ABSTRACT: The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB, and dpoIII) were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v) ethanol). The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.

SUBMITTER: Peng S

PROVIDER: S-EPMC5946679 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a.

Peng Shuai S Liu Longxiang L Zhao Hongyu H Wang Hua H Li Hua H

Frontiers in microbiology 20180504

The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candid ...[more]

PMID: 29780378

Similar Datasets

Project description:Malolactic fermentation (MLF) positively influences the quality of the wine, and it occurs as a result of a lactic acid bacteria's metabolism, mainly of the Oenococcus oeni species. However, delays and halting of MLF are frequent problems in the wine industry. This is mainly because O. oeni's development is inhibited by different kinds of stress. Even though the sequencing of the genome of the PSU-1 strain of O. oeni, as well as other strains, has made it possible to identify genes involved in the resistance to some types of stress, all of the factors that could be involved are still unknown. With the aim of contributing to this knowledge, the random mutagenesis technique was used in this study as a strategy for genetic improvement of strains of the O. oeni species. The technique proved to be capable of generating a different and improved strain when compared to the PSU-1 strain (the parent from which it descends). Then, we evaluated the metabolic behavior of both strains in three different wines. We used synthetic MaxOeno wine (pH 3.5; 15% v/v ethanol), red wine (Cabernet Sauvignon), and white wine (Chardonnay). Furthermore, we compared the transcriptome of both strains, grown in MaxOeno synthetic wine. The specific growth rate of the E1 strain was on average 39% higher in comparison to the PSU-1 strain. Interestingly, E1 strain showed an overexpression of the OEOE_1794 gene, which encodes a UspA-like protein, which has been described as promoting growth. We observed that the E1 strain was able to convert, on average, 34% more malic acid into lactate than the PSU-1 strain, regardless of the wine being used. On the other hand, the E1 strain showed a flux rate of fructose-6-phosphate production that was 86% higher than the mannitol production rate, and the internal flux rates increase in the direction of pyruvate production. This coincides with the higher number of OEOE_1708 gene transcripts observed in the E1 strain grown in MaxOeno. This gene encodes for an enzyme fructokinase (EC 2.7.1.4) involved in the transformation of fructose to fructose-6-phosphate.

Project description:Chardonnay wine malolactic fermentations were carried out to evaluate the chemical transfers occurring at the wood/wine interface in the presence of two different bacterial lifestyles. To do this, Oenococcus oeni was inoculated into must and wine in its planktonic and biofilm lifestyles, whether adhering or not to oak chips, leading to three distinct enological conditions: (i) post-alcoholic fermentation inoculation in wine in the absence of oak chips, (ii) post-alcoholic fermentation inoculation in wine in the presence of oak chips, and (iii) co-inoculation of both Saccharomyces cerevisiae and O. oeni directly in Chardonnay musts in the presence of oak chips. Classical microbiological and physico-chemical parameters analyzed during the fermentation processes confirmed that alcoholic fermentation was completed identically regardless of the enological conditions, and that once O. oeni had acquired a biofilm lifestyle in the presence or absence of oak, malolactic fermentation occurred faster and with better reproducibility compared to planktonic lifestyles. Analyses of volatile components (higher alcohols and wood aromas) and non-volatile components (Chardonnay grape polyphenols) carried out in the resulting wines revealed chemical differences, particularly when bacterial biofilms were present at the wood interface. This study revealed the non-specific trapping activity of biofilm networks in the presence of wood and grape compounds regardless of the enological conditions. Changes of concentrations in higher alcohols reflected the fermentation bioactivity of bacterial biofilms on wood surfaces. These chemical transfers were statistically validated by an untargeted approach using Excitation Emission Matrices of Fluorescence combined with multivariate analysis to discriminate innovative enological practices during winemaking and to provide winemakers with an optical tool for validating the biological and chemical differentiations occurring in wine that result from their decisions.

Dataset Information

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a.

Publications

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in <i>Oenococcus oeni</i> SD-2a.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets