牛樟芝不同发育阶段菌丝体实时荧光定量PCR中内参基因的筛选

Reference Genes Selection for Quantitative Real-time PCR in Antrodia cinnamomea Mycelium of Different Development Stages

实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)技术具有较高的敏感性、准确性和特异性,被广泛应用于基因表达分析。在基因表达分析中,选择合适的内参基因是衡量样品表达量和准确性的重要前提和保障。本研究以液体发酵7、21、35 d的牛樟芝菌丝体为样本,采用qRT-PCR技术检测牛樟芝菌丝体内Actin、TPK、Floxuridine、CAP-Gly、α-Amylase、Phosphatase、HA2-helicase、MAGT1等8个候选内参基因在3组样本中的表达水平,采用geNorm、NormFinder、BestKeeper3个软件对其分别进行稳定性比较和评价。结果表明,geNorm软件计算得出Floxuridine、HA2-helicase、TPK、CAP-Gly在牛樟芝菌丝体中具有更高的稳定性;NormFinder软件计算得出TPK、CAP-Gly、Floxuridine、HA2-helicase在牛樟芝菌丝体中具有较高的稳定性;BestKeeper软件计算得出HA2-helicase的表达水平最好,其次是TPK和MAGT1。综合分析结果认为TPK和HA2-helicase比其他内参基因更稳定,更适合作为牛樟芝菌丝体研究的内参基因。可见,通过牛樟芝菌丝体转录组数据来筛选和挖掘稳定表达的内参基因具有可靠性、高效性和可行性,为牛樟芝菌丝体基因表达分析提供了可靠的内参基因。

Quantitative Real-time PCR(qRT-PCR) technology is widely used in the analysis of gene expression because of its high sensit-ivity, high accuracy and high specificity. Therefore, it is important to select appropriate reference genes to measure the expression and accuracy of the sample. In this study, the expressions of 18 candidate internal reference genes of Actin, TPK, Floxuridine, CAP-Gly, α-Amylase, Phosphatase, HA2-helicase and MAGT1 were detected by qRT-PCR with A. cinnamomea Mycelium under 7, 21 and 35 d liquid fermentation time. The stability of the internal reference genes were analyzed using softwares of geNorm, NormFinder and BestKeeper. Floxuridine/HA2-helicase, TPK, CAP-Gly genes were found more stable with geNorm. TPK, CAP-Gly, Floxuridine, HA2-helicase were found more stable with NormFinder. HA2-helicase was found to be best expressed with BestKeeper, followed by TPK and MAGT1. The comprehensive analysis showed that TPK and HA2-helicase had the best stability and were more suitable as the internal reference genes for A. camphorata. The results of this study would have a practical guiding role in the analysis of the selection of internal reference genes in the gene expression of A. cinnamomea by qRT-PCR.