Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca 2+ and Na + on L. edodes lentinan content were investigated. Metal ion concentrations and induction times were optimized according to mycelial biomass, and intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and total polysaccharide (TPS) content. The activities and gene expression of phospho-glucose isomerase (PGI), phosphoglucomutase (PGM), and UDP-glcpyrophosphorylase (UGP) were also measured. Ca 2+ and Na + concentration and induction time affected biomass, IPS, and EPS concentrations. Na + increased EPS, IPS and TPS, while Ca 2+ increased biomass, IPS, and TPS. During fermentation, mycelial biomass varied greatly under Ca 2+ induction, while IPS, EPS and TPS varied greatly under Na + induction. PGM and UGP activities increased in the presence of Na + , while PGI increased with Ca 2+ . Compared to control samples, pgi and pgm expression under Na + was greater at days 45 and 60, respectively, while under Ca 2+ , ugp expression was greater at day 45. IPS content correlated significantly with enzyme activity, while EPS correlated with PGM activity. Our data contributes to better understanding how Na + and Ca 2+ affect mycelial growth and secondary metabolite production, and of polysaccharide biosynthesis mechanisms of L. edodes.

IntroductionHypsizygus marmoreus is an industrial mushroom that is widely cultivated in East Asia. Its long postripening stage before fruiting severely limits its industrialized production.MethodsFive different mycelial ripening times (30, 50, 70, 90, and 100 d) were chosen and primordia (30P, 50P, 70P, 90P, and 110P) were collected for comparative transcriptomic analyses. The corresponding substrates (30F, 50F, 70F, 90F, and 110F) were used for nutrient content and enzyme activity determination.ResultsIn pairwise comparisons between 110P and other primordia, a total of 1,194, 977, 773, and 697 differentially expressed genes (DEGs) were identified in 30P_110P, 50P_110P, 70P_110P, and 90P_110P, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG) functional enrichment analyses revealed that the DEGs were mainly associated with amino acid metabolism, and lipid and carbohydrate metabolism pathways. Tyrosine, tryptophan, phenylalanine and histidine metabolism were enriched in all groups. Among the main carbon nutrients, the contents of cellulose and hemicellulose were high, and the lignin content decreased with the extension of the ripening time. Laccase had the highest activity, and acid protease activity decreased with the extension of the ripening time.DiscussionThe highly enrichment for amino acid metabolic pathways in primordia reveals that these pathways are essential for fruiting body formation in H. marmoreus, and these results will provide a basis for the optimization of its cultivation.