光处理下粘虫头部组织定量PCR分析中内参基因的筛选

Identification of reference genes in the heads of Mythimna separata with real-time quantitative PCR under different light sources

【目的】筛选不同光处理下粘虫Mythimna separata稳定表达的内参基因,为基因定量研究提供基础。【方法】以粘虫头部组织为材料,选取18s rRNA、EF-1α、β-actin、GAPDH和AK 5种候选内参基因进行实时荧光定量PCR(qRT-PCR)分析,然后通过ΔCt法,BestKeeper、GeNorm、Normfinder和RefFinder软件对候选内参基因的稳定性进行分析;利用GeNorm软件进行基因配对差异分析,以判断内参基因的最适组合。【结果】5种候选内参基因的Ct值都处于15-28之间。4种软件对5个候选基因稳定性的分析结果存在一定差异。综合分析各种软件的分析结果,推荐粘虫成虫不同光处理条件下采用AK和GAPDH作为内参基因。【结论】根据特定试验体系选择合适的内参基因对于qRT-PCR定量结果的准确定和可靠性具有重要意义。本研究对后续粘虫在不同光处理条件下目标基因的准确定量具有重要意义。

[Objectives] To select the most stable reference genes for real-time quantitative PCR(qRT-PCR) in Mythimna separate. [Methods] We assessed mRNA expression of candidate reference genes in the heads of M. separata under different light sources. Five candidate genes, including 18s rRNA, EF-1α, β-actin, GAPDH, and AK, were selected for qRT-PCR analysis. The expression stabilities of these genes were evaluated with the Ct method, BestKeeper, GeNorm, Normfinder and RefFinder. Pairwise variation(V) of candidate reference genes were calculated by geNorm. [Results] The Ct values of candidate genes were between 15 and 28. The four software packages differed somewhat in estimating gene stability, however, overall, AK and GAPDH were the best reference genes for M. separata adults under different light conditions. [Conclusion] It is important to select suitable reference genes for specific conditions to ensure the accurate quantification target genes in qRT-PCR. This study has important practical value for the accurate quantification of target genes of M. separata adults exposed to different light conditions.