Selection and validation of endogenous reference genes for RT-qPCR normalization in different stresses and tissues of the tiger milk mushroom, <i>Pleurotus tuber-regium</i>

Abstract

Pleurotus tuber-regium is a sclerotium-producing mushroom with pharmacological and nutritional value. Many researches applied molecular biological strategies that allow analyzing numerous genes expressed in response to different growth stresses or tissues. In order to capture accurate and reliable results in gene expression studies, it is necessary to select appropriate internal reference genes. They must have relatively uniform expression in the majority of tested samples, thereby reducing the impact of the sample itself on the RT-qPCR results. The selection and validation of 14 candidate reference genes, including α-tub, β-tub, γ-tub, GAPDH, Tif-5a, Tef-1α, ATPase, acyl-CoA, U2ribo, S/G, Wlp, VtpAsE, E3upl, and Sdh, were carried out for gene expression analysis in P. tuber-regium mycelia in response to different temperatures, drought levels, and salinity shifts, fruitbody, and sclerotium. Four statistical algorithms, NormFinder, geNorm, BestKeeper, and RefFinder, were recruited to evaluate the transcription stability of candidate reference genes. The RT-qPCR expression stability analysis indicated that the E3upl and Tif-5a were the most stable expressed genes among all experimental samples, so the combination of these two reference genes is suitable for the analysis of gene expression patterns in P. tuber-regium.

Development of reference genes for RT-qPCR analysis of gene expression in Pleurotus pulmonarius for biotechnological applications

“…As such, this gene also adheres to the guidelines for ideal reference genes, with conservation in the target DNA sequence, species specificity, constant copy number, and without allelic variation 36 . Previously, the GAPDH gene was reported to be unsuitable for normalizing expression in the basidiomycete P. tuber-regium , according to data generated using the geNorm algorithm 37 . Here, the GAPDH gene was also shown to be unsuitable for normalizing gene expression in P. pulmonarius cultivated on jatropha cake, as was the ACTIN gene.…”

“…Castanera and colleagues 19 reported that the constitutive genes ACTIN , GAPDH1 , and β-TUB , according to results generated using Genorm and NormFinder, were the least stable for normalizing gene expression in P. ostreatus cultivated on lignocellulosic residue. Clearly, the use of constitutive genes as reference genes without validation of their stability can lead to errors in gene expression analyses 37 , 46 . Given the previous reports across the three Pleurotus species, the constitutive gene GAPDH does not appear to be adequate for normalizing the expression of genes in basidiomycetes of the genus Pleurotus .…”

“…The genetic stability of reference genes has previously been investigated in different species of the genus Pleurotus , including Pleurotus ostreatus 19 , 33 – 35 ; Pleurotus eryngii 36 , and Pleurotus tuber-regium 37 . Given the potential in P. pulmonarius for both the degradation of phorbol esters and concomitant application in biorefinery models using jatropha cake as carbon source, analysis of fungal gene expression is warranted.…”

Genome and Comparative Transcriptome Dissection Provide Insights Into Molecular Mechanisms of Sclerotium Formation in Culinary-Medicinal Mushroom Pleurotus tuber-regium

“…The reactions were executed in QuantStudio TM 6 Flex System (Thermo Fisher Scientific, United States) using the following parameters: 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s, and 60 • C for 1 min, and the melt curve for each product was detected. Three biological and three technical replicates were performed for each sample using the combination of E3upl and Tif-5a as the internal reference genes (Sun et al, 2021). The log 2 Fold Change of sclerotia relative to mycelia was calculated using the 2 − Ct method.…”