Identification and validation of reference genes for RT-qPCR normalization in Komagataella phaffii at different growth stages

Abstract:

[Background] The methylotrophic yeast Komagataella phaffii (K. phaffii) or Pichia pastoris has received extensive attention as a cell factory to produce recombinant proteins and build biosynthetic pathways. Real-time quantitative PCR (RT-qPCR) is a rapid and efficient technique for detecting gene expression levels in the study of the K. phaffii expression system. However, normalization is required to ensure the reliability of the results obtained. [Objective] To screen and verify the most stable reference genes in K. phaffii at different growth stages for accurate normalization of RT-qPCR. [Methods] Sixteen candidate reference genes (rps8b, rpl35a, rpl10, eif5a, rpl19a, por1, rpl23b, 0887, tif1, ole1, rpl14b, gs, sun, sdh2, trx1, and ccp1) were preliminarily screened by transcriptome data analysis. After the C _t values of the candidate reference genes were detected by RT-qPCR, their expression stability was analyzed by applying qBASE geNorm combined with NormFinder. [Results] The optimal number of reference genes by geNorm analysis was 2, and the most stable genes were rpl19a and tif1. The NormFinder analysis demonstrated that the most stable gene was tif1. In addition, we validated the candidate reference genes using fdh encoding formate dehydrogenase and afdh encoding the bifunctional alcohol/formaldehyde dehydrogenase. [Conclusion] Two reference genes, tif1 and rpl19a, were required for accurate normalization of RT-qPCR at different growth stages of K. phaffii. This study provided a reliable basis for the analysis of expression quantification of related functional genes and enriched the reference genes in RT-qPCR analysis, facilitating the study of gene expression regulation and application at different growth stages of K. phaffii.

Key words:Komagataella phaffii;reference gene;RT-qPCR;geNorm;NormFinder