Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

Autor: Darron A. Cullen, Gregory A. Sword, Marie Pierre Chapuis, Stephen J. Simpson, Fleur Ponton, Tim Dodgson, Laurence Blondin, Donya Tohidi-Esfahani
Přispěvatelé: Chapuis, Marie Pierre, Centre de Biologie pour la Gestion des Populations (UMR CBGP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Institut de Recherche pour le Développement (IRD [France-Sud])-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), School of Biological Sciences, The University of Sydney, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Department of Entomology, Texas A&M University System, Sixth European Community Framework Programme, ARC Discovery Project grant
Jazyk: angličtina
Rok vydání: 2011
Předmět:
Zdroj: BMC Molecular Biology 7 (12), 1-11. (2011)
BMC Molecular Biology, Vol 12, Iss 1, p 7 (2011)
BMC Molecular Biology
BMC Molecular Biology, BioMed Central, 2011, 12 (7), pp.1-11. ⟨10.1186/1471-2199-12-7⟩
BMC Molecular Biology, 2011, 12 (7), pp.1-11. ⟨10.1186/1471-2199-12-7⟩
ISSN: 1471-2199
DOI: 10.1186/1471-2199-12-7⟩
Popis: Background The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust. Results Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust. Conclusion Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.
Databáze: OpenAIRE
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