定量实时PCR(qRT-PCR)被广泛接受为定量分析功能基因表达的精确和方便的方法。数据标准化强烈依赖于稳定的参考基因。豆虫,Riptortuspedestris(半翅目:Alydidae),是豆科作物的重要害虫,广泛分布于东南亚。在这项研究中,共有16个候选内参基因(RPL32、RPS23、SDHA、UBQ,UCCR,GST,TATA-box,HSP70,GAPDH,RPL7A,SOD,RPS3,肌动蛋白,α-微管蛋白,AK,和EF1)是在R.pedestris中精心选择的,在各种条件下评估它们的表达水平,包括不同的发育阶段,不同的组织,温度处理,成年年龄,蜕皮时间,和交配状态。在此之后,使用四种算法(ΔCt,GeNorm,NormFinder,和最佳守护者)。最终,综合排名是使用在线工具RefFinder确定的。我们的结果表明,在R.pedestris中进行qRT-PCR分析的参考基因取决于特定的实验条件。RPL7A和EF1是发育阶段的最佳参考基因。此外,α-微管蛋白和EF1在各种成体组织中表现出最稳定的表达。RPL32和RPL7A在成年年龄表现出最稳定的表达。对于若虫来说,RPL32和SOD显示最稳固的表达。对于温度条件,RPS23和RPL7A被鉴定为最适合监测基因表达。最后,我们验证了在整个发育阶段评估气味结合蛋白37(RpedOBP37)和细胞色素P4506a2(RpedCYP6)表达水平的可行性,组织,和温度条件。这些发现是对R.pedestris的qRT-PCR分析研究的重要补充,作为未来研究R.pedestris以及其他生物体中稳定参考基因的基础。
Quantitative real-time PCR (qRT-PCR) is widely accepted as a precise and convenient method for quantitatively analyzing the expression of functional genes. The data
normalization strongly depends upon stable reference genes. The bean bug, Riptortus pedestris (Hemiptera: Alydidae), is a significant pest of leguminous crops and broadly distributed across Southeast Asia. In this study, a total of 16 candidate reference genes (RPL32, RPS23, SDHA, UBQ, UCCR, GST, TATA-box, HSP70, GAPDH, RPL7A, SOD, RPS3, Actin, α-tubulin, AK, and EF1) were carefully chosen in R. pedestris, and their expression levels were assessed across various conditions, including different developmental stages, diverse tissues, temperature treatments, adult age, molting time, and mating status. Following this, the stability of these reference genes was evaluated using four algorithms (ΔCt, GeNorm, NormFinder, and BestKeeper). Ultimately, the comprehensive rankings were determined using the online tool RefFinder. Our results demonstrate that the reference gene for qRT-PCR analysis in R. pedestris is contingent upon the specific experimental conditions. RPL7A and EF1 are optimal reference genes for developmental stages. Furthermore, α-tubulin and EF1 exhibit the most stable expression across various adult tissues. RPL32 and RPL7A exhibit the most stable expression for adult age. For nymph age, RPL32 and SOD display the most stable expression. For temperature conditions, RPS23 and RPL7A were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 (RpedOBP37) and cytochrome P450 6a2 (RpedCYP6) throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on R. pedestris, serving as a fundamental groundwork for future investigations on stable reference genes in R. pedestris as well as other organisms.