Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

Raman Bansal; Priyanka Mittapelly; Yuting Chen; Praveen Mamidala; Chaoyang Zhao; Andy Michel

doi:10.1371/journal.pone.0152730

Quantitative RT-PCR Gene Evaluation and RNA Interference in the Brown Marmorated Stink Bug

PLoS One. 2016 May 4;11(5):e0152730. doi: 10.1371/journal.pone.0152730. eCollection 2016.

Authors

Raman Bansal¹, Priyanka Mittapelly¹, Yuting Chen¹, Praveen Mamidala², Chaoyang Zhao¹, Andy Michel¹

Affiliations

¹ Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, 44691, United States of America.
² Department of Biotechnology, University College of Science, Telangana University, Dichpally, Nizamabad, Telangana, 503322, India.

Abstract

The brown marmorated stink bug (Halyomorpha halys) has emerged as one of the most important invasive insect pests in the United States. Functional genomics in H. halys remains unexplored as molecular resources in this insect have recently been developed. To facilitate functional genomics research, we evaluated ten common insect housekeeping genes (RPS26, EF1A, FAU, UBE4A, ARL2, ARP8, GUS, TBP, TIF6 and RPL9) for their stability across various treatments in H. halys. Our treatments included two biotic factors (tissues and developmental stages) and two stress treatments (RNAi injection and starvation). Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). The qRT-PCR results indicated ARP8 and UBE4A exhibit the most stable expression across tissues and developmental stages, ARL2 and FAU for dsRNA treatment and TBP and UBE4A for starvation treatment. Following the dsRNA treatment, all genes except GUS showed relatively stable expression. To demonstrate the utility of validated reference genes in accurate gene expression analysis and to explore gene silencing in H. halys, we performed RNAi by administering dsRNA of target gene (catalase) through microinjection. A successful RNAi response with over 90% reduction in expression of target gene was observed.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Gene Expression Profiling
Genes, Insect
Heteroptera / genetics*
RNA Interference*
Reverse Transcriptase Polymerase Chain Reaction

Grants and funding

Funding for this work was provided by the Ohio Soybean Council, and by state and federal funds appropriated to The Ohio State University, Ohio Agricultural Research and Development Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.