Key Laboratory of Agricultural Genetics and Breeding of Shanghai, National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungal Resources and Utilization (South), Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China.
Hongzhen Agricultural Science and Technology Co. Ltd., Jinghong, China.
PLoS One. 2023 Sep 27;18(9):e0288982. doi: 10.1371/journal.pone.0288982. eCollection 2023.
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.
美味牛肝菌（Boedijn）是一种具有吸引力的可食用蘑菇，被认为是唯一一种在体外实现人工栽培的牛肝菌。基因表达分析已广泛应用于食用菌研究，对于阐明涉及复杂生物过程的基因功能非常重要。选择合适的参考基因对于确保可靠的 RT-qPCR 基因表达分析结果至关重要。在我们的研究中，根据 9 个发育阶段的 3 个转录组中的表达稳定性，从 25 个传统管家基因中选择了总共 12 个候选对照基因。然后，在不同条件和发育阶段下，使用 geNorm、NormFinder 和 RefFinder 进一步评估这些基因。结果表明，MSF1 结构域蛋白（MSF1）、突触融合蛋白（SYB）、丝裂原活化蛋白激酶基因（MAPK）、TATA 结合蛋白 1（TBP1）和 SPRY 结构域蛋白（SPRY）是所有样品处理中最稳定的参考基因，而伸长因子 1-α（EF1）、肌动蛋白和泛素结合酶（UBCE）是最不稳定的表达基因。根据转录组结果选择了基因 SYB，并将其鉴定为 P. portentosus 中的一个新的参考基因。这是对该真菌中参考基因鉴定的首次详细研究，可能为选择基因和定量基因表达提供新的见解。
