Screening and Validation of Stable Reference Genes for qRT-PCR Analysis in Epicauta gorhami (Coleoptera: Meloidae)
by
,
Guofeng Yang
1,†, 
Xuetao Yu
1,†,
Yan Zhang
1,
Jinhua Luo
2,
Xiaofei Li
1,
Li Zhu
1,
Huanhuan Zhang
3,
Lin Jin
4
,
Gang Wu
1,
Xiaohong Yan
1,* and
Chenhui Shen
1,* 1
Key Laboratory of Agricultural Genetically Modified Organisms Traceability, Oil Crops Research Institute of Chinese Academy of Agricultural Science/Supervision and Test Center (Wuhan) for Plant Ecological Environment Safety, Ministry of Agriculture and Rural Affairs, Wuhan 430062, China
2
Enshi Tujia and Miao Autonomous Prefecture Academy of Agricultural Sciences, Enshi 445000, China
3
Institute of Vegetable, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850032, China
4
Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Insects 2024, 15(12), 942; https://doi.org/10.3390/insects15120942
Submission received: 26 October 2024
/
Revised: 22 November 2024
/
Accepted: 28 November 2024
/
Published: 29 November 2024
(This article belongs to the Special Issue Research on Insect Molecular Biology)
Simple Summary
The reverse transcription-quantitative polymerase chain reaction (qRT-PCR) is often employed to examine the gene expressions under diverse treatments. Screening optimal reference genes are necessary for obtaining reliable expression results of qRT-PCR. Here, the stability of 10 selected reference genes in Epicauta gorhami was assessed under three conditions (adult ages, tissues/organs and temperatures). These findings displayed that the best suitable reference genes were as follows: SOD and RPS18 for different adult ages and various temperatures and RPS18 and RPS28 for adult tissues. The relative expression patterns of uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (EgUAP) in diverse adult tissues was employed to verify the results. Our study will lay a vital basis for future functional gene expressions in E. gorhami.
Abstract
Epicauta gorhami is a hypermetamorphic insect that mainly forage soybeans during the adult stage. However, the lack of appropriate references hinders our studying of the gene function in E. gorhami. In this study, referring to five computational tools (Ct value, geNorm, NormFinder, BestKeeper and RefFinder), the stability of 10 housekeeping genes (GAPDH, ACT, RPL4, RPL27, α-TUB, RPS18, EF1α, RPS28, RPL13 and SOD) was assessed by qRT-PCR under three different conditions (adult ages, tissues/organs and temperatures). The findings suggested that SOD and RPS18 were the most ideal references for examine gene transcripts among diverse adult ages and at various temperatures; a pair of RPS18 and RPS28 was the most reliable genes to assess gene expressions in diverse adult tissues. Finally, the relative expression levels of EgUAP were computed after normalization RPS18 and RPS28 with across diverse adult tissues. As expected, EgUAP expression was abundant in the foregut, trachea and antenna and scarce in the midgut, hindgut and epidermis. These findings will lay a solid foundation for analyzing the gene expression of E. gorhami.
Keywords:
Epicauta gorhami; Glycine max; reference genes; qRT-PCR; RefFinder
