RESUMO
Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. ß-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.
Assuntos
Quimotripsina/genética , Genes Essenciais , Genes de Insetos , Gossypium/parasitologia , Lepidópteros/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Actinas/genética , Actinas/metabolismo , Animais , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de RNAr , Gliceraldeído-3-Fosfato Desidrogenase (NADP+)(Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (NADP+)(Fosforiladora)/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Ribonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (1518 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.
Assuntos
Inativação Gênica , Mariposas/genética , RNA de Cadeia Dupla/administração & dosagem , Animais , Quimotripsina/genética , Quimotripsina/metabolismo , Dieta , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Reação em Cadeia da PolimeraseRESUMO
Helicoverpa armigera (the cotton bollworm) is a significant agricultural pest endemic to Afro-Eurasia and Oceania. Gene suppression via RNA interference (RNAi) presents a potential avenue for management of the pest, which is highly resistant to traditional insecticide sprays. This article reviews current understanding on the fate of ingested double-stranded RNA in H. armigera. Existing in vivo studies on diet-delivered RNAi and their effects are summarized and followed by a discussion on the factors and hurdles affecting the efficacy of diet-delivered RNAi in H. armigera.