Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, (Hemiptera: Alydidae)

Overview

Journal Insects

Specialty Biology

Date 2023 Dec 22

PMID 38132633

Authors

Liuyang Wang

Qingyu Liu

Pei Guo

Zhanlin Gao

Dan Chen

Tao Zhang

Jun Ning

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Characterization of the ligand-binding properties of odorant-binding protein 38 from when interacting with soybean volatiles.

Guo J, Liu P, Zhang X, An J, Li Y, Zhang T Front Physiol. 2025; 15():1475489.

PMID: 39835200 PMC: 11743672. DOI: 10.3389/fphys.2024.1475489.

Screening and Validation of Stable Reference Genes for qRT-PCR Analysis in (Coleoptera: Meloidae).

Yang G, Yu X, Zhang Y, Luo J, Li X, Zhu L Insects. 2025; 15(12.

PMID: 39769544 PMC: 11678893. DOI: 10.3390/insects15120942.

Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in under Diverse Biological Conditions and Environmental Stresses.

Gong Z, Zhang J, Chen Q, Li H, Zhang Z, Duan Y Insects. 2024; 15(9).

PMID: 39336629 PMC: 11432719. DOI: 10.3390/insects15090661.

Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer (Lepidoptera: Pyralidae).

Wang Z, Shang X, Wei J, Tian X, Liu Y, Zhang G Insects. 2024; 15(8).

PMID: 39194799 PMC: 11354500. DOI: 10.3390/insects15080594.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in (Lepidoptera: Noctuidae).

Wang L, Yang C, Liu Q, Zhang X, Mei X, Zhang T Insects. 2024; 15(3).

PMID: 38535380 PMC: 10970824. DOI: 10.3390/insects15030185.

References

Zhang H, Wang Y, Wang Z, Ding W, Xu K, Li L . Modelling the current and future potential distribution of the bean bug Riptortus pedestris with increasingly serious damage to soybean. Pest Manag Sci. 2022; 78(10):4340-4352. DOI: 10.1002/ps.7053. View

Li W, Gao Y, Hu Y, Chen J, Zhang J, Shi S . Field Cage Assessment of Feeding Damage by on Soybeans in China. Insects. 2021; 12(3). PMC: 8002668. DOI: 10.3390/insects12030255. View

Lu J, Yang C, Zhang Y, Pan H . Selection of Reference Genes for the Normalization of RT-qPCR Data in Gene Expression Studies in Insects: A Systematic Review. Front Physiol. 2018; 9:1560. PMC: 6232608. DOI: 10.3389/fphys.2018.01560. View

Fu S, Duan Y, Wang S, Ren Y, Bu W . Comparative Transcriptomic Analysis of (Hemiptera: Alydidae) to Characterize Wing Formation across All Developmental Stages. Insects. 2021; 12(3). PMC: 7999114. DOI: 10.3390/insects12030226. View

Han S, Qin Q, Wang D, Zhou Y, He Y . Selection and Evaluation of Reference Genes for qRT-PCR in (Lepidoptera: Noctuidae). Insects. 2021; 12(10). PMC: 8538597. DOI: 10.3390/insects12100902. View

Mitra T, Bilic I, Hess M, Liebhart D . The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models. Vet Res. 2016; 47(1):105. PMC: 5073923. DOI: 10.1186/s13567-016-0388-z. View

Ferguson B, Nam H, Hopkins R, Morrison R . Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes. PLoS One. 2010; 5(12):e15208. PMC: 3001470. DOI: 10.1371/journal.pone.0015208. View

Kozera B, Rapacz M . Reference genes in real-time PCR. J Appl Genet. 2013; 54(4):391-406. PMC: 3825189. DOI: 10.1007/s13353-013-0173-x. View

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

10.

Thellin O, Zorzi W, Lakaye B, de Borman B, Coumans B, Hennen G . Housekeeping genes as internal standards: use and limits. J Biotechnol. 2000; 75(2-3):291-5. DOI: 10.1016/s0168-1656(99)00163-7. View

11.

Zhang H, Chen J, Lin J, Lin J, Wu Z . Odorant-binding proteins and chemosensory proteins potentially involved in host plant recognition in the Asian citrus psyllid, Diaphorina citri. Pest Manag Sci. 2020; 76(8):2609-2618. DOI: 10.1002/ps.5799. View

12.

Kong D, Shi D, Wang C, Zhai R, Lyu L, He Y . Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in (Hemiptera: Cimicidae). Insects. 2022; 13(9). PMC: 9502763. DOI: 10.3390/insects13090784. View

13.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

14.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

15.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

16.

Shen C, Peng L, Zhang Y, Zeng H, Yu H, Jin L . Reference Genes for Expression Analyses by qRT-PCR in (Lepidoptera: Gelechiidae). Insects. 2022; 13(2). PMC: 8879603. DOI: 10.3390/insects13020140. View

17.

Xie J, Liu T, Khashaveh A, Yi C, Liu X, Zhang Y . Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions. Front Physiol. 2021; 12:669510. PMC: 8165390. DOI: 10.3389/fphys.2021.669510. View

18.

Arya S, Jain G, Upadhyay S, Sarita , Singh H, Dixit S . Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions. Sci Rep. 2017; 7(1):13520. PMC: 5648885. DOI: 10.1038/s41598-017-13925-9. View

19.

Liu Y, Zhou J, Qiu Z, Hu P, Chen X, Yang Z . Identification and Validation of Reference Genes for Expression Analysis Using RT-qPCR in Fisher and La Salle (Hymenoptera: Eulophidae). Insects. 2023; 14(5). PMC: 10231091. DOI: 10.3390/insects14050456. View

20.

Liu P, Guo J, Wei H, Feng L, Gao Z, Zhang T . Genome-wide identification of candidate chemosensory receptors in the bean bug (Hemiptera: Alydidae) and the functional verification of its odorant receptor co-receptor (Orco) in recognizing aggregation pheromone. Front Physiol. 2023; 14:1224009. PMC: 10375722. DOI: 10.3389/fphys.2023.1224009. View