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

• Published in: Scientific reports Vol. 13; no. 1; p. 12296
• Main Authors: Gomes, Taísa Godoy, de Assis Fonseca, Fernando Campos, Alves, Gabriel Sergio Costa, de Siqueira, Félix Gonçalves, Miller, Robert Neil Gerard
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 29-07-2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/2017/2003; 631/326/193; 631/61/168; Actin; Agaricales; Animal Feed; Animals; Biorefineries; Biotechnology; Combined treatment; Detoxification; Enzymes; Esters; Feeds; Gene expression; Humanities and Social Sciences; Inactivation; Industrial applications; Jatropha; Jatropha curcas; multidisciplinary; Oilseeds; Phorbol esters; Pleurotus - genetics; Pleurotus pulmonarius; Reverse Transcription; Science; Science (multidisciplinary); Stability analysis
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-39115-4

Jatropha curcas
is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary.
Pleurotus pulmonarius
is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes
β-TUB
,
ACTIN
,
GAPDH
,
PHOS
,
EF1α
,
TRPHO
,
LAC
,
MNP3
,
MYP
and
VP
were evaluated following growth of
P. pulmonarius
on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified
PHOS
,
EF1α
and
MNP3
as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the
CHU_2040
gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for
P. pulmonarius
will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.