Selection and validation of internal control genes for quantitative real-time RT‒qPCR normalization of Phlebopus portentosus gene expression under different conditions

Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions...

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Published in:PloS one 2023-09, Vol.18 (9), p.e0288982-e0288982
Main Authors: Hu, Chen-Menghui, Zhou, Chen-Li, Wan, Jia-Ning, Guo, Ting, Ji, Guang-Yan, Luo, Shun-Zhen, Ji, Kai-Ping, Cao, Yang, Tan, Qi, Bao, Da-Peng, Yang, Rui-Heng
Format: Article
Language:English
Subjects:
Actin
Analysis
Biological activity
Biology and Life Sciences
Dehydrogenases
Design
Developmental stages
Elongation
Evaluation
Fungi
Gene expression
Genes
Genetic aspects
Kinases
MAP kinase
Muscle proteins
Mushrooms
Physical Sciences
Polymerase chain reaction
Protein binding
Protein kinases
Proteins
Research and Analysis Methods
RNA polymerase
Synaptobrevin
TATA-binding protein
Transcriptomes
Ubiquitin-conjugating enzyme
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Summary:Phlebopus portentosus (Berk. and Broome) Boedijn is an attractive edible mushroom and is considered the only bolete for which artificial cultivation in vitro has been achieved. Gene expression analysis has become widely used in research on edible fungi and is important for elucidating the functions of genes involved in complex biological processes. Selecting appropriate reference genes is crucial to ensuring reliable RT-qPCR gene expression analysis results. In our study, a total of 12 candidate control genes were selected from 25 traditional housekeeping genes based on their expression stability in 9 transcriptomes of 3 developmental stages. These genes were further evaluated using geNorm, NormFinder, and RefFinder under different conditions and developmental stages. The results revealed that MSF1 domain-containing protein (MSF1), synaptobrevin (SYB), mitogen-activated protein kinase genes (MAPK), TATA-binding protein 1 (TBP1), and SPRY domain protein (SPRY) were the most stable reference genes in all sample treatments, while elongation factor 1-alpha (EF1), actin and ubiquitin-conjugating enzyme (UBCE) were the most unstably expressed. The gene SYB was selected based on the transcriptome results and was identified as a novel reference gene in P. portentosus. This is the first detailed study on the identification of reference genes in this fungus and may provide new insights into selecting genes and quantifying gene expression.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0288982