蜜环菌(Armillaria mellea)内参基因的筛选

Selection of reference genes for real-time quantitative PCR of Armillaria mellea

摘要:

【背景】蜜环菌属(Armillaria)是一类营腐生或寄生生活的药食两用型真菌，研究其功能基因表达具有重要意义。【目的】筛选并获得蜜环菌(Armillaria mellea)最稳定的内参基因。【方法】以蜜环菌(A. mellea) 541为研究对象，以马铃薯琼脂糖培养基培养的菌丝和菌索为对照组，以添加还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate，NADPH)氧化酶抑制剂氯化二亚苯基碘鎓(diphenyleneiodonium chloride，DPI)培养的菌丝和菌索为抑制剂组，以添加木屑培养的菌丝和菌索为诱导剂组，利用RT-qPCR技术和BestKeeper程序系统评估候选内参基因ACT-1、α-TUB、β-TUB 1、γ-TUB、UBQ、EF-1γ、18S rRNA biogenesis protein基因(18S rRNA BP)和GAPDH的表达量稳定性。【结果】内参基因EF-1γ在对照组、DPI抑制剂组和木屑诱导剂组中的表达量稳定性最好。【结论】EF-1γ为蜜环菌(A. mellea)的最佳内参基因，为蜜环菌属真菌功能基因表达研究提供了参考。

Abstract:

[Background] Armillaria are medicinal and edible fungi having a parasitic or saprophytic phase, and it is vital to study the differentially expressed genes in these fungi. However, there are few reports on the internal reference genes of them. [Objective] To screen out the optimal internal reference genes for Armillaria mellea 541 under different experimental conditions. [Methods] In this study, ACT-1, α-TUB, β-TUB 1, γ-TUB, UBQ, EF-1γ, 18S rRNA BP, and GAPDH were used as candidate reference genes of A. mellea 541. The A. mellea 541 hypha (AH) and rhizomorph (AR) cultured on potato dextrose agar (PDA) medium were used as the control group, and those cultured on the PDA medium supplemented with diphenyleneiodonium chloride and sawdust as inhibitor group and inducer group, respectively. The expression levels of the eight genes were evaluated by RT-qPCR and BestKeeper. [Results] EF-1γ was selected as the optimal reference gene for A. mellea 541 because of its stable expression under different experimental conditions. [Conclusion] This study provides a reliable reference gene for analyzing the gene expression in Armillaria spp..