Zarivi Osvaldo, Cesare Patrizia, Ragnelli Anna Maria, Aimola Pierpaolo, Leonardi Marco, Bonfigli Antonella, Colafarina Sabrina, Poma Anna Maria, Miranda Michele, Pacioni Giovanni
Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
Department of Life, Health and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.
Phytochemistry. 2015 Aug;116:78-86. doi: 10.1016/j.phytochem.2015.02.024. Epub 2015 Mar 13.
The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable genes for fruiting body developmental stages.
共生真菌黑孢块菌（皮埃蒙特黑松露）属于子囊菌门，与树木和灌木的根系形成互利共生关系。这种松露在全球市场上具有很高的价值，在南北半球的许多国家都有种植。黑孢块菌基因组的公布为研究人员提供了独特的机会，使他们能够更多地了解这种真菌的生物学特性。实时定量PCR（qRT-PCR）是一种确定样本间转录基因表达水平差异的定量技术。为了促进基因表达研究并获得更准确的qRT-PCR数据，需要相对于稳定的管家基因进行标准化。这些管家基因在给定的实验条件下必须表现出稳定的表达，qRT-PCR结果才会准确。不幸的是，目前尚无关于黑孢块菌发育过程中所使用的管家基因稳定性的研究。在本研究中，我们对黑孢块菌子实体的发育阶段进行了形态学和显微镜分类，并研究了12个候选参考基因（18S rRNA；5.8S rRNA；延伸因子1-α；延伸因子1-β；α-微管蛋白；60S核糖体蛋白L29；β-微管蛋白；40S核糖体蛋白S1；40S核糖体蛋白S3；葡萄糖-6-磷酸脱氢酶；β-肌动蛋白；泛素结合酶）的表达水平。为了评估这些基因作为内参的适用性，我们使用了五种基于软件的方法和一个基于网络的综合工具（RefFinder）来分析和排名测试基因。我们在此证明，18S rRNA基因在黑孢块菌发育过程中表现出最稳定的表达，并且一组三个基因，即18S rRNA、延伸因子1-α和40S核糖体蛋白S3，最适合对所有分析发育阶段的qRT-PCR数据进行标准化；相反，18S rRNA、葡萄糖-6-磷酸脱氢酶和延伸因子1-α是子实体发育阶段最适合的基因。
