Analysis of reference genes stability and histidine kinase expression under cold stress in Cordyceps militaris

PLoS One. 2020 Aug 12;15(8):e0236898. doi: 10.1371/journal.pone.0236898. eCollection 2020.

Abstract

The development of fungal fruiting bodies from a hyphal thallus 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. 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 periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, 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 RGs leads to biased results. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cold-Shock Response / genetics*
  • Cordyceps / enzymology
  • Cordyceps / genetics*
  • Cordyceps / physiology*
  • Gene Expression Profiling / standards*
  • Histidine Kinase / genetics*
  • Reference Standards

Substances

  • Histidine Kinase

Grants and funding

This work was supported by the National Natural Science Foundation of China (NO. 31772374 and 31900027), the Natural Science Foundation of Hunan Province (NO. 2020JJ5972), the Scientific Research Fund of Hunan Provincial Education Department, China (NO. 17K106 and 18B167), the National Key R & D Programs of Intergovernmental International Science and Technology Cooperation (NO. 2017YFE0108100), the Project of International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province (NO. 2018WK4008), and Aid Program for Science and Technology Innovative Research Team in Higher Educational Instituions of Hunan Province. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.