Background: The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris.
Results: Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three diﬀerent algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long time, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable housekeeping genes for cold treatments in short and long time, respectively. Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as housekeeping genes leads to biased results.
Conclusions: Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.