New reference genes in Trichoderma reesei

Abstract

Trichoderma reesei is a highly significant producer of various enzymes used in industry and agriculture. Due to its relatively high secretion efficiency, it holds potential for a wide range of applications. However, the complexity of its gene expression mechanisms and the unpredictable behavior of newly developed genetically modified strains have made it a subject of extensive research. A particularly important method for gene expression analysis is the RT-qPCR, which requires stably expressed reference genes to ensure reliable results. The absence of properly evaluated reference genes, assessed using modern techniques, poses a significant challenge in qPCR-based research on Trichoderma reesei. In this study, potential new reference genes for gene expression analysis via RT-qPCR were evaluated against the currently used reference genes, act1 and sar1. Candidate genes were identified in silico by analyzing publicly available RNA-Seq datasets. Five potential reference genes with promising expression stability were validated by RT-qPCR analysis of biological samples collected under a wide range of experimental conditions. Both in silico and RT-qPCR analysis ranked the novel gene, bzip, which appears to play a role in histone methylation, as the most stable in terms of expression. Additionally, all five novel genes exhibited higher expression stability compared to act1 and sar1. As a result of these findings, we propose using the gene pair bzip/traff instead of act1/sar1 for RT-qPCR analysis in Trichoderma reesei.