基于转录组测序的姬松茸镉胁迫下内参基因筛选

Screening of reference genes under cadmium stress in Agaricus brasiliensis based on transcriptome sequencing

摘要:

[目的] 筛选不同实验条件下，尤其是镉胁迫下的姬松茸内参基因，为研究姬松茸镉富集相关基因功能研究奠定基础。[方法] 根据不同浓度Cd（0、2、5 mg/L）胁迫下菌丝中转录组表达量数据，筛选出18个候选基因，设计了30对引物；根据引物扩增的特异性、扩增效率初步筛选出来自不同基因的5对引物；运用qRT-PCR检测了5个基因在不同浓度Cd胁迫下的菌丝样品、原基、菌柄和菌盖中的C_t值，利用geNorm、NormFinder和BestKeeper对这些基因的表达稳定性进行评价。[结果] SGT2和STK是菌丝阶段Cd胁迫下最稳定的2个候选内参基因，GAPDH是不同组织中最稳定的内参基因，SGT2、STK和GAPDH是所有实验条件下最稳定的3个内参基因。[结论] 在姬松茸镉胁迫条件下，本研究筛选出的SGT2和STK比常用的内参基因表现出更强的稳定性。

Abstract:

[Objective] The aim of this study was to screen the reference genes of Agaricus brasiliensis under different experimental conditions, especially under cadmium (Cd) stress, and to lay the foundation for studying the functional studies of cadmium enrichment-related genes in A. brasiliensis.[Methods] We screened 18 candidate genes and designed 30 primer pairs based on the transcriptomes of mycelia under the stress of different concentrations of Cd (0, 2, 5 mg/L). According to the specificity and amplification efficiency of primers, 5 pairs of primers from different genes were selected. The Ct values of mycelia, primordium, stipe, and pileus of 5 genes under different concentrations of cadmium stress were detected by QRT-PCR, and the expression stability of these genes was evaluated by geNorm, NormFinder, and BestKeeper. [Results] The results showed that Small glutamine-rich tetratricopeptide repeat-containing protein 2 (SGT2) and Serine/threonine-protein kinase (STK) were the two most stable candidate reference genes under Cd stress in the mycelial stage, GAPDH was the most stable reference gene in different tissues and SGT2, STK and GAPDH were the three most stable reference genes under all experimental conditions. [Conclusion] Under Cd stress conditions in A. brasiliensis, SGT2 and STK screened in this study were more stable compared to the commonly used reference genes.