Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

AMB Express, Nov 2016

Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species.

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Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Che et al. AMB Expr Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1) Jin‑xin Che 1 Jun‑ling Shi 0 Yao Lu 0 Yan‑lin Liu 1 0 Key Laboratory for Space Biosciences and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University , Xi'an 710072, Shaanxi , People's Republic of China 1 College of Food Science and Engineering, Northwest A & F University , 28 Xinong Road, Yangling 712100, Shaanxi , People's Republic of China Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT‑ PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT‑ PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species. qRT‑ PCR; Reference genes; α‑ Tubulin; Elongation factor 1; Alternaria sp - Many important bioactive compounds are widely used in medical services and health care (Khan 2016; Larsen and Matchkov 2016; Morata et  al. 2015). Many of these compounds are either microbial metabolites or their semi-synthetic derivatives (Golinska et al. 2015; Sessitsch et al. 2013; Stepniewska and Kuzniar 2013). In the microbial population, endophytes are a large group which may contain millions of different species, but only a minority of them have been studied (Sessitsch et  al. 2013). Several endophytic fungi (Alternaria sp.) were identified previously that are capable of independent resveratrol production (Shi et  al. 2012). Although fundamental physiological research has been performed (Zhang et al. 2013a, b), the metabolic pathways and cellular processes remain to be elucidated. Gene expression profiling is an informative technique to investigate biological systems (Li et  al. 2015). The method of qRT-PCR (quantitative real time PCR) can measure gene expression across different sample populations (Derveaux et  al. 2010; Wong and Medrano 2005). However, there are many factors that can influence the accuracy of the results such as the quality and quantity of mRNA templates or amplification efficiency. Generally, normalizing expression of the target genes to one or © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. several reference genes provide an efficient way to reduce these effects and increase the relevance of the results (Huggett et al. 2005; Marabita et al. 2016). However, the use of inappropriate reference genes that change expression levels under different conditions can cause interpretation errors. Thus, the choice of appropriate reference genes for normalization is a prerequisite for qRT-PCR assay. In recent years, validation of reliable reference genes before their use for normalization has been performed for many species, such as Talaromyces marneffei (Dankai et  al. 2015), Staphylococcus aureus (Sihto et  al. 2014), Beauveria bassiana (Zhou et  al. 2012), Oenococcus oeni (Sumby et al. 2012) and others. Commonly used reference genes for these fungi include the genes encoding the 18S ribosomal RNA (18S), ubiquitin fusion degradation protein (UFD), ribosomal protein (RPS), elongation factor (EF), β-actin (ACTB), α-tubulin (TUBA), ubiquitin-conjugating enzyme (UBC), and glyceraldehyde (...truncated)


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Jin-xin Che, Jun-ling Shi, Yao Lu, Yan-lin Liu. Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1), AMB Express, 2016, pp. 106, Volume 6, Issue 1, DOI: 10.1186/s13568-016-0283-z