Wang Liuyang, Liu Qingyu, Guo Pei, Gao Zhanlin, Chen Dan, Zhang Tao, Ning Jun
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences, Integrated Pest Management Center of Hebei Province, Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture, Baoding 071000, China.
Insects. 2023 Dec 18;14(12):960. doi: 10.3390/insects14120960.
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, (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 (, , , , , , -, , , , , , , α-, , and ) were carefully chosen in , 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 is contingent upon the specific experimental conditions. and are optimal reference genes for developmental stages. Furthermore, α- and exhibit the most stable expression across various adult tissues. and exhibit the most stable expression for adult age. For nymph age, and display the most stable expression. For temperature conditions, and were identified as the most suitable for monitoring gene expression. Lastly, we verified the practicability of evaluating expression levels of odorant-binding protein 37 () and cytochrome P450 6a2 () throughout developmental stages, tissues, and temperature conditions. These findings are a significant addition to the qRT-PCR analysis studies on , serving as a fundamental groundwork for future investigations on stable reference genes in as well as other organisms.
定量实时聚合酶链反应（qRT-PCR）作为一种精确且便捷的功能基因表达定量分析方法被广泛接受。数据归一化强烈依赖于稳定的内参基因。豆蝽（半翅目：缘蝽科）是豆科作物的一种重要害虫，广泛分布于东南亚。在本研究中，在豆蝽中精心挑选了总共16个候选内参基因（、、、、、、-、、、、、、、α-、、和），并在包括不同发育阶段、不同组织、温度处理、成虫年龄、蜕皮时间和交配状态等各种条件下评估它们的表达水平。在此之后，使用四种算法（ΔCt、GeNorm、NormFinder和BestKeeper）评估这些内参基因的稳定性。最终，使用在线工具RefFinder确定综合排名。我们的结果表明，豆蝽中用于qRT-PCR分析的内参基因取决于特定的实验条件。和是发育阶段的最佳内参基因。此外，α-和在各种成虫组织中表现出最稳定的表达。和在成虫年龄方面表现出最稳定的表达。对于若虫年龄，和表现出最稳定的表达。对于温度条件，和被确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、组织和温度条件下评估气味结合蛋白37（）和细胞色素P450 6a2（）表达水平的实用性。这些发现是豆蝽qRT-PCR分析研究的重要补充，为未来豆蝽以及其他生物中稳定内参基因的研究奠定了基础。
