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

Che, Jing; Shi, Junling; Lu, Yao; Liu, Yanlin

doi:10.1186/s13568-016-0283-z

Cited by 11 publications

(11 citation statements)

References 60 publications

(82 reference statements)

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“…Moreover, it may be a better choice to select a combination of two or more RGs as an effective internal control to further improve the accuracy and reliability of gene expression normalization under different stresses. In conclusion, this study provides a guideline to select a valid RG combination that can ensure more accurate qRT-PCR-based gene expression quantification and basic data to facilitate future molecular studies on gene expression in S. chamaejasme and other Thymelaeaceae species ( Che et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

Liu

Guan

Song

et al. 2018

PeerJ

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BackgroundStellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported.MethodIn this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.ResultOur results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization.DiscussionThis work is the first attempt to comprehensively estimate the stability of RGs in S. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

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Section: Discussionmentioning

confidence: 99%

Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

Liu

Guan

Song

et al. 2018

PeerJ

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“…MG1 [37] and the sequences are shown in Table 4. For each gene, the expression value was normalized with respect to the reference gene EF1 [76]. The reaction volume was set to 20 μL in accordance with the operation manual of the TransStart Tip Green qPCR SuperMix Kit (Transgene Biotech Co., Ltd., Beijing, China).…”

Section: Methodsmentioning

confidence: 99%

“…All reactions were conducted in triplicate. The program and qRT-PCR analyses were performed as previously described [76, 77].…”

Section: Methodsmentioning

confidence: 99%

Genomic sequencing, genome-scale metabolic network reconstruction, and in silico flux analysis of the grape endophytic fungus Alternaria sp. MG1

Che

et al. 2019

Microb Cell Fact

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BackgroundAlternaria sp. MG1, an endophytic fungus isolated from grape, is a native producer of resveratrol, which has important application potential. However, the metabolic characteristics and physiological behavior of MG1 still remains mostly unraveled. In addition, the resveratrol production of the strain is low. Thus, the whole-genome sequencing is highly required for elucidating the resveratrol biosynthesis pathway. Furthermore, the metabolic network model of MG1 was constructed to provide a computational guided approach for improving the yield of resveratrol.ResultsFirstly, a draft genomic sequence of MG1 was generated with a size of 34.7 Mbp and a GC content of 50.96%. Genome annotation indicated that MG1 possessed complete biosynthesis pathways for stilbenoids, flavonoids, and lignins. Eight secondary metabolites involved in these pathways were detected by GC–MS analysis, confirming the metabolic diversity of MG1. Furthermore, the first genome-scale metabolic network of Alternaria sp. MG1 (named iYL1539) was reconstructed, accounting for 1539 genes, 2231 metabolites, and 2255 reactions. The model was validated qualitatively and quantitatively by comparing the in silico simulation with experimental data, and the results showed a high consistency. In iYL1539, 56 genes were identified as growth essential in rich medium. According to constraint-based analysis, the importance of cofactors for the resveratrol biosynthesis was successfully demonstrated. Ethanol addition was predicted in silico to be an effective method to improve resveratrol production by strengthening acetyl-CoA synthesis and pentose phosphate pathway, and was verified experimentally with a 26.31% increase of resveratrol. Finally, 6 genes were identified as potential targets for resveratrol over-production by the recently developed methodology. The target-genes were validated using salicylic acid as elicitor, leading to an increase of resveratrol yield by 33.32% and the expression of gene 4CL and CHS by 1.8- and 1.6-fold, respectively.ConclusionsThis study details the diverse capability and key genes of Alternaria sp. MG1 to produce multiple secondary metabolites. The first model of the species Alternaria was constructed, providing an overall understanding of the physiological behavior and metabolic characteristics of MG1. The model is a highly useful tool for enhancing productivity by rational design of the metabolic pathway for resveratrol and other secondary metabolites.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1063-7) contains supplementary material, which is available to authorized users.

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“…Alternaria sp. MG1 was prepared as spore suspensions of 1 × 10 6 spores/mL from the 5-day PDA (Che et al 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Strategies to enhance the production of pinoresinol and its glucosides by endophytic fungus (Phomopsis sp. XP-8) isolated from Tu-chung bark

Zhu

Lü

et al. 2018

AMB Expr

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To improve the production yield of (+)-pinoresinol (Pin), (+)-pinoresinol monoglucoside (PMG), and (+)-pinoresinol diglucoside (PDG), different methods were conducted, including co-culture with resveratrol-producing Alternaria sp. MG1 spores and addition of Tu-chung in a medium at the start of cultivation, ultrasound treatment (40 kHZ, 10 min) on 5-day culture, and addition of ethanol and sodium butyrate on Day 3, followed by cultivation for an additional period of 2 days. At the end of the cultivation period (5 days), the liquid phase was collected for product analysis. Cells were collected for the determination of gene expression levels and then used in bioconversion using resting cells for another period of 2 days. The liquid phase was measured to determine the output of the target products and the expression levels of the key genes related to the biosynthesis of these compounds. Consequently, co-culture with Alternaria MG1 and addition of Tu-chung bark in the medium efficiently increased Pin, PMG, and PDG production yield in the biosynthesis systems using potato dextrose broth medium and resting cells of Phomopsis sp. XP-8. The key genes related to the biosynthesis of these compounds were significantly upregulated. However, in the majority of cases, the addition of ethanol and sodium butyrate, and ultrasound treatment decreased the production yield of Pin, PMG, and PDG. The change in production yield was not consistently accompanied by a change in gene expression.

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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)

Cited by 11 publications

References 60 publications

Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

Genomic sequencing, genome-scale metabolic network reconstruction, and in silico flux analysis of the grape endophytic fungus Alternaria sp. MG1

Strategies to enhance the production of pinoresinol and its glucosides by endophytic fungus (Phomopsis sp. XP-8) isolated from Tu-chung bark

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