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
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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
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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)