Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses

Gangzheng Wang; Huijiao Cheng; Min Li; Chenghua Zhang; Wangqiu Deng; Taihui Li

doi:10.1016/j.gene.2020.144380

Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses

Gene. 2020 Apr 15:734:144380. doi: 10.1016/j.gene.2020.144380. Epub 2020 Jan 21.

Authors

Gangzheng Wang¹, Huijiao Cheng², Min Li³, Chenghua Zhang⁴, Wangqiu Deng⁵, Taihui Li⁶

Affiliations

¹ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China.
² Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China; South China Agricultural University, Guangzhou 510642, China.
³ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China; College of Agriculture and Animal Husbandry, Tibet University, Nyingchi 860000, Tibet, China.
⁴ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China. Electronic address: zhangch@gdim.cn.
⁵ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China. Electronic address: dengwq@gdim.cn.
⁶ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, China. Electronic address: lith@gdim.cn.

PMID: 31978511
DOI: 10.1016/j.gene.2020.144380

Abstract

Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (β-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.

Keywords: Cold and heat stress; Cordyceps guangdongensis; Differential development stages; H4; Normalization; VPS.

Publication types

Validation Study

MeSH terms

Algorithms
Cordyceps / genetics*
Cordyceps / growth & development
Gene Expression Profiling
Genes, Fungal*
Histones / genetics
Reference Standards
Stress, Physiological / genetics
Temperature
Tubulin / genetics

Substances

Histones
Tubulin