Identification and Evaluation of Reference Genes for Quantitative PCR Normalization in Alligator Weed Flea Beetle (Coleoptera: Chrysomelidae)
Open Access
- 1 September 2021
- journal article
- research article
- Published by Oxford University Press (OUP) in Journal of Insect Science
- Vol. 21 (5)
- https://doi.org/10.1093/jisesa/ieab067
Abstract
Stably expressed reference genes are critical internal standards for the quantification of gene transcription levels using quantitative real-time PCR. Housekeeping genes are commonly used as reference genes but their expressions were variable depending on experimental conditions in many insect species studied. Here we report the identification and evaluation of 10 housekeeping genes in alligator weed flea beetle, Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae), a biocontrol agent of alligator weed. The 10 housekeeping genes are: beta-actin (Actin), ribosomal protein L13A (PRL13a), succinate dehydrogenase complex subunit A (SDHA), ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), glyceraldehyde phosphate dehydrogenase (GAPDH), TATA-box-binding protein (TBP), ribosomal protein L32 (RPL32), tubulin alpha-1 chain (TUBULIN), and elongation factor-1 alpha (ELF). Five programs, geNorm, NormFinder, BestKeeper, ΔCt method, and RefFinder, were used to evaluate the expression stability of the 10 genes among various A. hygrophila body parts and with different nutrient types (starvation, diet types). The expression stability analysis showed that RPS32 and RPL13a were reliable reference genes for the study of gene transcription in different body parts; Actin and RPL13a were optimal reference genes for different nutrient types. The selections of reference genes were validated using a CarE gene (GeneBank No: KX353552). The results of this study provide useful bases for studies of gene expression in various aspects relating to A. hygrophila.Keywords
Funding Information
- State Key Laboratory of Sustainable Dryland Agriculture
- College of Plant Protection
- Shanxi Agricultural University (202003-4)
- National Natural Science Foundation of China (31301723, 31570436)
- Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi of China (2017143)
- Key Research and Development Project of Shanxi province of China (201803D221004-7)
This publication has 41 references indexed in Scilit:
- Sequential loss of genetic variation in flea beetleAgasicles hygrophila(Coleoptera: Chrysomelidae) following introduction into ChinaInsect Science, 2013
- Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)PLOS ONE, 2013
- Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)International Journal of Biological Sciences, 2013
- Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locustBMC Molecular Biology, 2011
- Quantification of mRNA using real-time RT-PCRNature Protocols, 2006
- 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 SetsCancer Research, 2004
- Genetic uniformity of Alternanthera philoxeroides in South ChinaWeed Research, 2003
- The effect of temperature on the development and survival ofAgasicles hygrophilaSelman & Vogt (Coleoptera: Chrysomelidae), a biological control agent for alligator weed (Alternanthera philoxeroides)New Zealand Journal of Zoology, 1999
- Assignment of the L32 ribosomal protein gene (RPL32) to human chromosome 3q13.3→q21 by in situ hybridizationCytogenetic and Genome Research, 1997
- Expression of protease genes in the gastric mucosa during agingExperimental Gerontology, 1993