Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses
Selection and validation of reliable reference genes for Tolypocladium guangdongense gene expression analysis under differentially developmental stages and temperature stresses
复制标题
差异发育阶段和温度胁迫下广东毛叶霉基因表达分析可靠内参基因的选择和验证
DOI：
10.1016/j.gene.2020.144380
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发表时间：
2020-04-15
期刊：
影响因子：
3.5
通讯作者：
Li, Taihui
中科院分区：
文献类型：
--
作者：
Wang, Gangzheng;Cheng, Huijiao;Li, Taihui
广东弯颈霉（Tolypocladium guangdongense），原名广东虫草（Cordyceps guangdongensis），是冬虫夏草属（Cordyceps s.l.）在过去的12年里，我们研究过的物种。它有可能被用于健康和制药行业的许多应用，因为根据以前的动物研究，它已显示出其高营养和药用价值。qRT-PCR（quantitative reverse transcription polymerase chain reaction，定量逆转录聚合酶链反应）广泛用于分析功能基因在差异实验条件下的表达模式和分子机制。用于标准化的参考基因的表达稳定性决定了qRT-PCR结果的可靠性，表明在基因表达分析之前选择和验证参考基因的重要性。本研究采用geNorm、Normalization和BestKeeper三种统计算法对19个候选参考基因（CRG）在T.广东话。在不同发育阶段（菌丝体、原基、幼嫩子实体和成熟子实体）、不同碳源、冷热胁迫等条件下进行了研究。结果表明，在所有实验样品中，组蛋白H4和微管蛋白β链2（β-tub 2）分别是最稳定和最不稳定的基因。此外，对单个数据集的分析显示出参考基因的不同稳定性和表达谱。液泡蛋白分选基因VPS是在不同发育阶段和温度胁迫下表达最稳定的基因，而H4是在不同碳源下表达最稳定的基因。因此，可以提出VPS和H4是在不同实验条件下用于基因表达标准化的优选参考基因。本研究的结果将使T.利用最佳参考基因进行qRT-PCR分析。
Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (beta-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.