糙皮侧耳热胁迫背景下RT-qPCR内参基因的筛选

The Screening of Reference Genes in RT-qPCR under Heat Stress of Pleurotus ostreatus

通过文献报道和对糙皮侧耳基因组进行搜索,得到14个候选基因。以40℃热胁迫不同时间(0,1,2,4,8,12 h)的菌丝作为材料,通过反转录得到的候选基因片段,进行实时荧光定量PCR筛选,使用geNorm、NormFinder、BestKeeper 3种软件对结果进行分析,筛选得到稳定的内参基因β肌动蛋白(β-actin)、假定蛋白(sar1)。此外,选取2种热应激反应基因Hsp9、Hsp90作为靶基因,分别以筛选得到的稳定、不稳定基因以及多个内参基因作为内参进行分析,结果显示选用多个内参基因时能够缩小不同基因作为内参导致的结果差异。

In this study,14 candidate genes were obtained through reviewing literatures and searching the genome of Pleurotus ostreatus.P.ostreatus mycelium treated with heat stress at 40℃for different times(0,1,2,4,8,12 h)as experimental materials.Through software analysis(geNorm,NormFinder,BestKeeper),we obtained the stability of individual candidate gene.The most stable internal reference genes wereβ-actin and sar1.In addition,Hsp9 and Hsp90 were selected as target genes.The results showed that the relative expression difference between the stable gene and unstable gene could be reduced by multiple genes as the internal reference gene with the most stable and unstable reference genes,and multiple reference genes for RT-qPCR verification.