草菇不同菌株RT-qPCR参考基因筛选

Selection of optimal RT-qPCR reference genes for examining different strains of Volvariella volvacea

适合的参考基因是应用实时荧光定量PCR(RT-qPCR)技术进行基因表达分析的前提。本研究以7个食用菌常用参考基因(β-TUB 1、GPD、ACTB、Ras、α-TUB、β-TUB 2和SPRYp)为候选基因,利用RT-qPCR检测其在草菇常用生产菌株(CPS)V844、V5、V971和V844继代退化菌株(SDS)T8、T12、T16、T20中的表达;用Genorm、NormFinder和BestKeeper3种软件分析候选基因的表达稳定性,并结合几何平均数法筛选出最佳参考基因。结果表明,SPRYp、α-TUB和β-TUB2基因适用于草菇常用生产菌株检测,SPRYp、GPD和α-TUB基因适用于草菇继代退化检测,SPRYp基因适用于两种条件的检测。两两差异分析表明,草菇常用生产菌株的最佳参考基因组合为SPRYp、α-TUB和β-TUB 2,继代退化菌株的最佳参考基因组合为SPRYp和GPD,两种条件混合菌株的最佳参考基因组合为SPRYp和α-TUB。

A suitable reference gene is the prerequisite for gene expression analysis by real-time quantitative PCR(RT-qPCR).In this study,seven commonly used reference genes of edible fungi(β-TUB 1,GPD,ACTB,Ras,α-TUB,β-TUB 2,SPRYp)were selected as candidate genes,and RT-qPCR was used to examine their expression in the commonly used production strains(CPS)V844,V5 and V971 and V844 subsequent degraded strains(SDS)T8,T12,T16 and T20 of V.volvacea.Three kinds of software(Genorm,NormFinder,BestKeeper)were used to analyze the expression stability of candidate genes,and finally the best reference gene was screened by geometric mean method.The results suggest that SPRYp andα-TUB andβ-TUB 2 genes are suitable for the detection of commonly used production strains of V.volvacea,SPRYp,GPD andα-TUB genes are suitable for the detection of subculture degradation,while SPRYp gene is suitable for both the detection.The pairwise difference analysis showed that the best reference gene combinations for detection of commonly used production strains of V.volvacea were SPRYp,α-TUB andβ-TUB 2,and the best reference gene combination for detection of subsequent degraded strains were SPRYp and GPD.The best reference gene combination for mixed detection of the strains is SPRYp andα-TUB.