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

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 (beta-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.