扩展青霉菌实时定量 PCR 内参基因挖掘与应用

实时定量聚合酶链式反应(polymerase chain reaction, PCR)技术因其高效且便捷的特点在基因表达检测中被广泛应用。实时定量PCR结果数据处理策略之一是使用内参基因进行基因表达数据的标准化。文章基于扩展青霉菌孢子不同生长阶段的RNA-seq数据，挖掘和注释了694个稳定表达的候选内参基因；随机挑选出Knr4、Isy1、Spt5、Nucb、Hp1、Hp2、Whth、Hp3、Gph3、Pwi、Taf4、Hp4、Asy、GTPase在内的14个基因，以4、25℃条件下PDB培养基生长6、12、24、36 h的扩展青霉菌为材料，进行实时定量PCR实验。实时定量PCR数据基于geNorm和NormFinder2种软件的综合分析，表明Isy1、Spt5、Nucb、Hp1适合作为内参基因。以Isy1和Spt5分别作为内参基因，检测Gh30基因在4、25℃条件下扩展青霉菌生长发育过程中的基因表达，显示出一致的基因表达水平，进一步验证了挖掘得到的内参基因的可靠性。该研究为扩展青霉菌的分子生物学研究提供了优良的内参基因，同时提供了一种高效的内参基因的挖掘和鉴定方法。

Based on efficient and convenient characterization, the quantitative real-time polymerase chain reaction (qRT-PCR) technology is widely applied in gene expression analyses. Currently, one important strategy of result data processing is expression data normalization based on internal reference genes. In this study, 694 stably expressed genes were identified in the RNA-seq data from the spores of Penicillium expansum in different growth stages. Through qRT-PCR experiments, the gene expression levels of 14 randomly selected genes, including Knr4, Isy1, Spt5, Nucb, Hp1, Hp2, Whth, Hp3, Gph3, Pwi, Taf4, Hp4, Asy and GTPase, were determined in P. expansum after 6, 12, 24, and 36 hours cultivation in PDB medium at 4 °C and 25 °C, respectively. Analyzed through geNorm and NormFinder, Isy1, Spt5, Nucb and Hp1 were proved to be suitable as internal reference genes. Using Isy1 and Spt5 as internal reference genes, the expression profiles of Gh30 gene were verified in P. expansum of different growth stages at 4 °C and 25 °C, respectively, showing consistent expression profiles. The results could provide well internal reference genes for molecular studies on P. $ \expansum $, and an efficient method for the exploitation and verification of internal reference genes.