Institute of Grain Storage and Logistics, National Engineering Research Center of Grain Storage and Logistics, Academy of National Food and Strategic Reserves Administration, Beijing, China.
School of Culinary and Food Sciences, Zhengzhou Tourism College, Zhengzhou, China.
J Econ Entomol. 2024 Apr 12;117(2):629-637. doi: 10.1093/jee/toae004.
Rhyzopertha dominica is a serious stored grain insect pest around the world. Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely used experimental method in molecular biology for detecting the expression of target genes. As appropriate reference genes are essential for normalizing gene expression, the selection of suitable reference genes is the basis of RT-qPCR experiments. In this study, the expression profiles of 7 candidate reference genes of rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA were analyzed under 4 different experimental conditions. The expression stability of candidate genes was evaluated using the ΔCt, GeNorm, BestKeeper, NormFinder, and RefFinder methods. The results revealed that different reference genes were suitable for various experiments. Specifically, rps3 and rps6 were appropriate for the developmental stages and all samples: 18S rRNA and rps13 for temperature-related experiments, actin and rps6 for sex-related experiments, and rps6 and gadph for starvation stress. Our results lay essential groundwork for the normalization of RT-qPCR analyses and contribute to genomic and gene functional research of R. dominica.
赤拟谷盗是一种世界性的严重仓储害虫。实时荧光定量聚合酶链反应(RT-qPCR)是分子生物学中广泛使用的一种实验方法,用于检测靶基因的表达。由于合适的参考基因对于标准化基因表达至关重要,因此选择合适的参考基因是 RT-qPCR 实验的基础。在这项研究中,分析了 rps3、rps6、rps13、肌动蛋白、GAPDH、微管蛋白和 18S rRNA 这 7 个候选参考基因在 4 种不同实验条件下的表达谱。使用 ΔCt、GeNorm、BestKeeper、NormFinder 和 RefFinder 方法评估候选基因的表达稳定性。结果表明,不同的参考基因适用于不同的实验。具体来说,rps3 和 rps6 适合于发育阶段和所有样本:18S rRNA 和 rps13 适用于温度相关实验,肌动蛋白和 rps6 适用于性别相关实验,rps6 和 GAPDH 适用于饥饿应激。我们的研究结果为 RT-qPCR 分析的归一化奠定了重要基础,有助于赤拟谷盗的基因组和基因功能研究。