Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress

Abstract

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying

“…In later studies, the expression levels of the housekeeping genes were found unstable under abiotic and biotic stress [26,30], suggesting the importance of reliable reference genes for qRT-PCR analysis. The stability of reference genes was evaluated under different development stages, nutrient conditions, and abiotic stresses in G. lucidum , V. volvacea, L. edodes , and Pleurotus ostreatus [21,24,26,31]. In the present study, we focused on the reference genes for shiitake mushroom in the early stage of the hot-air drying process.…”

“…Several studies have been performed on the selection and evaluation of stable reference genes under different conditions, such as different development stages and nutrient conditions as well as abiotic stresses in Morchella [19], Ganoderma lucidum [20,21,22], Volvariella volvacea [23,24], and L. edodes [25,26]. Nevertheless, the information about the reference genes used for qRT-PCR normalization of the hot-air-dried L. deodes is rather limited.…”

Effect of the heme oxygenase gene on mycelial growth and polysaccharide synthesis in Ganoderma lucidum

“…The hyphae cultured in CYM for 7 days were used to extract RNA, and the extraction method of the total RNA has been described previously [25]. We used quantitative real‐time PCR (qRT‐PCR) to detect messenger RNA expression of the HMX1 gene and other testing genes in the WT, Sicontrol, and HMX1 ‐silenced strains, as previously described [26]. We used the 18S rRNA housekeeping gene to normalize the expression of other target genes, and the primers used are listed in Table 1.…”