Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Abstract

Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.T he basidiomycete Pleurotus ostreatus is an efficient producer of extracellular enzymes, such as laccases (Lacs; EC 1.10.3.2) and manganese peroxidases (MnPs; EC 1.11.1.13), which are notable for their application in multiple industrial and biotechnological processes (1, 2). It has a special relevance in agroindustry due to its worldwide cultivation for mushroom production, and it has also been studied for its nutritional and medicinal value (3). Currently, two genomic sequences of P. ostreatus are publicly available, and it has become a very popular model organism for the study of fungal genetics and comparative genomics (4-6). Additionally, many studies on the production of ligninolytic enzymes, with a focus on culture optimization for enzyme production, have recently been published. Most of these enzymes are encoded by genes organized in gene families, which are occasionally arranged into clusters as a result of gene duplications.Despite the information uncovered by sequencing efforts, substantial work on the functional characterization of gene family members remains to be performed. In this sense, transcriptomic analyses are powerful tools for providing an understanding of gene regulation and the presumptive function of genes. Despite the high productivity and low cost of transcriptome sequencing (RNA-seq), reverse transcription (RT) followed by real-time PCR (RT-quantitative PCR [RT-qPCR]) is likely the best option for analyzing the expression patterns of certain genes under multiple conditions. Additionally, it is the most reliable technique for validating RNA-seq and microarray data because of its specificity, reproducibility, and capacity...

Laccase Production and Differential Transcription of Laccase Genes in Cerrena sp. in Response to Metal Ions, Aromatic Compounds, and Nutrients

“…We selected the appropriate reference genes for laccase gene transcription profiling. Housekeeping genes are commonly used as normalization references in relative quantification qPCR experiments, although they may show expression variability with different experimental factors (Kozera and Rapacz, 2013 ; Castanera et al, 2015 ). For example, GAPDH is one of the most widely used reference genes, but it was unstable with various carbon/nitrogen sources in this study.…”

Systematic Analysis of the Pleurotus ostreatus Laccase Gene (PoLac) Family and Functional Characterization of PoLac2 Involved in the Degradation of Cotton-Straw Lignin

“…Each reaction was performed in triplicate. PCR amplification conditions were as follows: 50 °C for 2 min and 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 20 s. The relative expression levels of target genes were calculated using the 2 −ΔΔCT method [ 53 ], with the sar gene used as an internal control [ 54 ].…”

Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation

“…The procedure for reverse transcription quantitative PCR experiments was adapted from (Castanera et al 2015 ). RT-qPCRs were performed in a StepOnePlus® (Applied Biosystems), using SYBR green dye to detect product amplification.…”