杏鲍菇采后木质化相关基因实时定量PCR中内参基因的选择

Selection of reference gene for quantitative real-time PCR analysis of lignification related genes in postharvest Pleurotus eryngii

【目的】筛选出合适的内参基因,为分析不同贮藏时期杏鲍菇(Pleurotus eryngii)子实体中木质化相关基因的表达提供支持。【方法】以4℃低温贮藏0,3,6,9,12,15和18d的杏鲍菇子实体为材料,采用实时荧光定量PCR(Quantitative real-time PCR,RT-qPCR)技术,分析了β-actin、18SrRNA、β-tubulin、ELF和GAPDH 5个候选内参基因在不同贮藏时期杏鲍菇的表达情况;通过GeNorm和NormFinder 2种软件筛选出最稳定的内参基因;选取最佳内参基因,采用相对表达定量法对木质素合成途径中的关键酶,即苯丙氨酸转氨酶(Phenylalanina ammonia-lyse,PAL)基因的表达进行定量分析。【结果】5个内参基因均能特异扩增,其中GAPDH基因表达丰度较低,不适合用作内参基因;经GeNorm软件分析计算,β-actin、β-tubulin、ELF、18SrRNA表达稳定度平均值M分别为0.527,0.527,0.584,0.875,最适内参基因组合为β-actin、β-tubulin和ELF;由NormFinder软件分析计算,β-actin、β-tubulin、ELF、18SrRNA稳定值S分别为0.221,0.356,0.583,1.092,β-actin稳定性最高;综合分析认为β-actin的表达稳定性最高,为最佳内参基因;以β-actin为内参基因,发现4℃低温贮藏3,6,9,12,15和18d的杏鲍菇子实体中PAL基因表达量分别比新鲜样品(0d)增加了11.3%,10.8%,11.3%,11.5%,11.4%,10.7%。【结论】β-actin可作为杏鲍菇子实体采后贮藏条件下品质变化相关基因表达研究的内参基因。

[Objective] The objective of this experiment was to select suitable reference gene to support analysis of the expression of lignification related genes in Pleurotus eryngii at different storage periods. [Method] In this study,quantitative real-time PCR （RT-qPCR） was used to analyze the mRNA expression of five candidate reference genes,βactin, 18S rRNA,βtubulin ,ELF and GAPDH, in Pleurotus eryngii stored at 4 ℃ for different periods （0,3,6,9,12,15 and 18 d）. The most stable genes were selected by GeNorm and NormFinder. Then, with the relative quantification method, the expression of Phenylalanina ammonia-lyse （PAL）,a key enzyme in lignin biosynthesis pathway,was valuated. [Result] All of the five reference genes could amplify specially. GAPDH was unsuitable because of its less abundant expression. Mean stabilities of βactin ,βtubulin, ELF, and 18S rRNA were 0. 527,0. 527,0. 584, and 0. 875, analyzed by GeNorm,respectively. The optimum reference genes were a combination of β-actin ,βtubulin and ELF. Stability values of β-actin,βtubulin, ELF, and 18S rRNA analyzed by NormFinder were 0. 221,0. 356, 0. 583 ,and 1. 092 ,and β-actin was the most stable one. Comprehensive analysis showed that βactin with the highest stability was the most suitable gene. Then using β-actin as the reference gene, relative changes of PAL expression in Pleurotus eryngii at different storage periods （3,6,9,12,15 and 18 d） increased significantly （11.3% ,10.8%,11.3% ,11.5%,11.4%,and 10.7%,respectively） compared to fresh sample （0 d）. [Conclusion] β-actin was the most suitable reference gene to normalize mRNA levels of quality related genes in Pleurotus eryngii.