Evaluation of endogenous reference genes in Bactrocera cucurbitae by qPCR under different conditions

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.瓜实蝇（Bactrocera cucurbitae，melon flies）是中国南方地区的重要入侵害虫。为筛选适用于四类不同处理条件（温度、虫态、日龄及性别）下瓜实蝇组织样本比较分析的稳定内参基因（reference gene），本研究采用实时荧光定量PCR（real-time fluorescent quantitative PCR）对12个候选内参基因在不同处理条件下的表达水平进行了分析。通过geNorm、NormFinder、BestKeeper及RefFinder软件进行综合分析后，结果显示稳定性最优的内参基因为RPL60，稳定性最差的内参基因为actin-5。本研究选取热休克蛋白基因（HSP-90）对该结果进行验证，所得结论与之一致。以RPL60作为内参基因时，HSP-90的相对表达量随处理时间延长基本保持稳定；而以actin-5作为内参基因时，HSP-90的表达量则随处理时间延长发生显著变化。本研究结果可为瓜实蝇的基因表达相关后续研究提供参考依据。