Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions

Aquatic fireflies are important indicators of the quality of freshwater environments and key models for research on insect adaptation to freshwater environments. For these investigations, gene expression analyses using quantitative real-time PCR are heavily dependent on reliable reference genes. In...

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Autores principales: Yang, Xiao-Jie, Zheng, Hai-Long, Liu, Ying-Yang, Li, Hong-Wei, Jiang, Yu-Hang, Lin, Lian-Bing, Deng, Xian-Yu, Zhang, Qi-Lin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573347/
https://www.ncbi.nlm.nih.gov/pubmed/33123022
http://dx.doi.org/10.3389/fphys.2020.555233
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author Yang, Xiao-Jie
Zheng, Hai-Long
Liu, Ying-Yang
Li, Hong-Wei
Jiang, Yu-Hang
Lin, Lian-Bing
Deng, Xian-Yu
Zhang, Qi-Lin
author_facet Yang, Xiao-Jie
Zheng, Hai-Long
Liu, Ying-Yang
Li, Hong-Wei
Jiang, Yu-Hang
Lin, Lian-Bing
Deng, Xian-Yu
Zhang, Qi-Lin
author_sort Yang, Xiao-Jie
collection PubMed
description Aquatic fireflies are important indicators of the quality of freshwater environments and key models for research on insect adaptation to freshwater environments. For these investigations, gene expression analyses using quantitative real-time PCR are heavily dependent on reliable reference genes. In this study, based on a transcriptome assembly and annotation for the aquatic firefly Aquatica leii at the adult and larval stages, 10 candidate reference genes (α-tubulin, β-tubulin, β-actin, EF1A, SDHA, UBQ, GST, GAPDH, RPS31, and RPL13A) were identified for analyses of expression stability. Quantitative real-time PCR analyses for each candidate reference genes in A. leii was conducted for four developmental stages, four adult tissue types, two adult sexes, and two ecological stressors [adults exposed to five temperatures and larvae exposed to four concentrations of benzo(a)pyrene]. Results were evaluated by three independent algorithms (geNorm, NormFinder, and BestKeeper) and one comparative algorithm (RefFinder). The expression stability of candidate reference genes in A. leii differed under various conditions. Reference genes with the most stable expressions levels in different tissues, temperatures, sexes, developmental stages, and concentrations of benzo(a)pyrene were α-tubulin, GST, β-actin, β-tubulin, and α-tubulin, respectively. Furthermore, the optimal normalization factors (NFs) for the quantification of the expression levels of target genes by quantitative real-time PCR analyses of A. leii were identified for each experimental group. In particular, NF = 2 for different tissues (α-tubulin + β-tubulin), different sexes (β-actin + EF1A), and larvae exposed to different concentrations of benzo(a)pyrene (α-tubulin + EF1A); NF = 3 for developmental stages (GST + GAPDH + SDHA) and adults exposed to different temperatures (β-tubulin + EFA + GST). In addition, we surveyed the expression profiles of two target genes (CYP3A and CSP8) in larvae exposed to different concentrations of benzo(a)pyrene and in different adult tissues. The results further validated the reliability of the reference genes. The optimal reference genes for various experimental conditions identified in these analyses provide a useful tool for ecological studies of aquatic fireflies.
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spelling pubmed-75733472020-10-28 Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions Yang, Xiao-Jie Zheng, Hai-Long Liu, Ying-Yang Li, Hong-Wei Jiang, Yu-Hang Lin, Lian-Bing Deng, Xian-Yu Zhang, Qi-Lin Front Physiol Physiology Aquatic fireflies are important indicators of the quality of freshwater environments and key models for research on insect adaptation to freshwater environments. For these investigations, gene expression analyses using quantitative real-time PCR are heavily dependent on reliable reference genes. In this study, based on a transcriptome assembly and annotation for the aquatic firefly Aquatica leii at the adult and larval stages, 10 candidate reference genes (α-tubulin, β-tubulin, β-actin, EF1A, SDHA, UBQ, GST, GAPDH, RPS31, and RPL13A) were identified for analyses of expression stability. Quantitative real-time PCR analyses for each candidate reference genes in A. leii was conducted for four developmental stages, four adult tissue types, two adult sexes, and two ecological stressors [adults exposed to five temperatures and larvae exposed to four concentrations of benzo(a)pyrene]. Results were evaluated by three independent algorithms (geNorm, NormFinder, and BestKeeper) and one comparative algorithm (RefFinder). The expression stability of candidate reference genes in A. leii differed under various conditions. Reference genes with the most stable expressions levels in different tissues, temperatures, sexes, developmental stages, and concentrations of benzo(a)pyrene were α-tubulin, GST, β-actin, β-tubulin, and α-tubulin, respectively. Furthermore, the optimal normalization factors (NFs) for the quantification of the expression levels of target genes by quantitative real-time PCR analyses of A. leii were identified for each experimental group. In particular, NF = 2 for different tissues (α-tubulin + β-tubulin), different sexes (β-actin + EF1A), and larvae exposed to different concentrations of benzo(a)pyrene (α-tubulin + EF1A); NF = 3 for developmental stages (GST + GAPDH + SDHA) and adults exposed to different temperatures (β-tubulin + EFA + GST). In addition, we surveyed the expression profiles of two target genes (CYP3A and CSP8) in larvae exposed to different concentrations of benzo(a)pyrene and in different adult tissues. The results further validated the reliability of the reference genes. The optimal reference genes for various experimental conditions identified in these analyses provide a useful tool for ecological studies of aquatic fireflies. Frontiers Media S.A. 2020-10-06 /pmc/articles/PMC7573347/ /pubmed/33123022 http://dx.doi.org/10.3389/fphys.2020.555233 Text en Copyright © 2020 Yang, Zheng, Liu, Li, Jiang, Lin, Deng and Zhang. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Yang, Xiao-Jie
Zheng, Hai-Long
Liu, Ying-Yang
Li, Hong-Wei
Jiang, Yu-Hang
Lin, Lian-Bing
Deng, Xian-Yu
Zhang, Qi-Lin
Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title_full Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title_fullStr Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title_full_unstemmed Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title_short Selection of Reference Genes for Quantitative Real-Time PCR in Aquatica leii (Coleoptera: Lampyridae) Under Five Different Experimental Conditions
title_sort selection of reference genes for quantitative real-time pcr in aquatica leii (coleoptera: lampyridae) under five different experimental conditions
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573347/
https://www.ncbi.nlm.nih.gov/pubmed/33123022
http://dx.doi.org/10.3389/fphys.2020.555233
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