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Effect of diet delivered various concentrations of double-stranded RNA in silencing a midgut and a non-midgut gene of Helicoverpa armigera

Published online by Cambridge University Press:  05 April 2013

R. Asokan ,
G. Sharath Chandra ,
M. Manamohan  and
N.K. Krishna Kumar
R. Asokan*
Affiliation:
Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hesaraghatta Lake (PO), Bengaluru – 560 089, India
G. Sharath Chandra*
Affiliation:
Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hesaraghatta Lake (PO), Bengaluru – 560 089, India
M. Manamohan
Affiliation:
Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hesaraghatta Lake (PO), Bengaluru – 560 089, India
N.K. Krishna Kumar
Affiliation:
National Bureau of Agriculturally Important Insects, H. A. Farm (PO), Bellary Road, Bengaluru – 560 024, India
*
*Authors for correspondence Phone: +91 80 28466420 Fax: +91 80 28466291 E-mail: asokaniihr@gmail.com
Phone: +91 8123564905 E-mail: sharathgsc@gmail.com
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Abstract

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 (15–18 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.

Keywords

Helicoverpa armigeraRNAidsRNAchymotrypsinjhamtRT-qPCR
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 103 , Issue 5 , October 2013 , pp. 555 - 563
Copyright
Copyright © Cambridge University Press 2013 

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