Identification and validation of reference genes for RT-qPCR analysis in Sclerodermus guani (Hymenoptera: Bethylidae).

IF 1.6 3区农林科学 Q2 ENTOMOLOGY Bulletin of Entomological Research Pub Date : 2024-10-01 Epub Date: 2024-10-07 DOI:10.1017/S0007485324000427

Rina Zhao, Xiaomeng Guo, Ling Meng, Baoping Li

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Abstract

Gene expression studies in organisms are often conducted using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and the accuracy of RT-qPCR results relies on the stability of reference genes. We examined ten candidate reference genes in Sclerodermus guani, a parasitoid wasp that is a natural enemy of long-horned beetle pests in forestry, including ACT, EF1α, Hsc70, Hsp70, SRSF7, α-tubulin, RPL7A, 18S, 28S, and SOD1, regarding variable biotic and abiotic factors such as body part, life stage, hormone, diet, and temperature. Data were analysed using four dedicated algorithms (ΔCt, BestKeeper, NormFinder, and geNorm) and one comparative tool (RefFinder). Our results showed that the most stable reference genes were RPL7A and EF1α regarding the body part, SRSF7 and Hsc70 regarding the diet, RPL7A and α-tubulin regarding the hormone, SRSF7 and RPL7A regarding the life stage, and SRSF7 and α-tubulin regarding temperature. To ascertain the applicability of specific reference genes, the expression level of the target gene (ACPase) was estimated regarding the body part using the most stable reference genes, RPL7A and EF1α, and the least stable one, SOD1. The highest expression level of ACPase was observed in the abdomen, and the validity of RPL7A and EF1α was confirmed. This study provides, for the first time, an extensive list of reliable reference genes for molecular biology studies in S. guani.

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Sclerodermus guani (Hymenoptera: Bethylidae) RT-qPCR 分析参考基因的鉴定和验证。

生物体内的基因表达研究通常使用反转录酶-定量聚合酶链反应（RT-qPCR）进行，而 RT-qPCR 结果的准确性取决于参考基因的稳定性。我们研究了林业长角蠹害虫的天敌寄生蜂 Sclerodermus guani 的十个候选参考基因，包括 ACT、EF1α、Hsc70、Hsp70、SRSF7、α-tubulin、RPL7A、18S、28S 和 SOD1，研究涉及身体部位、生命阶段、激素、饮食和温度等可变生物和非生物因素。数据分析采用了四种专用算法（ΔCt、BestKeeper、NormFinder 和 geNorm）和一种比较工具（RefFinder）。结果表明，最稳定的参考基因是与身体部位有关的 RPL7A 和 EF1α、与饮食有关的 SRSF7 和 Hsc70、与激素有关的 RPL7A 和 α-微管蛋白、与生命阶段有关的 SRSF7 和 RPL7A 以及与温度有关的 SRSF7 和 α-微管蛋白。为了确定特定参考基因的适用性，使用最稳定的参考基因 RPL7A 和 EF1α，以及最不稳定的参考基因 SOD1 估算了目标基因（ACPase）在身体部位的表达水平。腹部的 ACPase 表达水平最高，RPL7A 和 EF1α 的有效性也得到了证实。该研究首次为瓜尼蛙的分子生物学研究提供了一份广泛而可靠的参考基因列表。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bulletin of Entomological Research 生物-昆虫学

CiteScore

4.00

自引率

0.00%

发文量

160

审稿时长

6-12 weeks

期刊介绍： Established in 1910, the internationally recognised Bulletin of Entomological Research aims to further global knowledge of entomology through the generalisation of research findings rather than providing more entomological exceptions. The Bulletin publishes high quality and original research papers, ''critiques'' and review articles concerning insects or other arthropods of economic importance in agriculture, forestry, stored products, biological control, medicine, animal health and natural resource management. The scope of papers addresses the biology, ecology, behaviour, physiology and systematics of individuals and populations, with a particular emphasis upon the major current and emerging pests of agriculture, horticulture and forestry, and vectors of human and animal diseases. This includes the interactions between species (plants, hosts for parasites, natural enemies and whole communities), novel methodological developments, including molecular biology, in an applied context. The Bulletin does not publish the results of pesticide testing or traditional taxonomic revisions.