Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants

Authors

Chunxiao Yang, University of KentuckyFollow
Evan L. Preisser, University of Rhode Island
Hongjun Zhang, University of Kentucky
Yong Liu, Hunan Academy of Agricultural Sciences, China
Liangying Dai, Hunan Agricultural University, China
Huipeng Pan, University of KentuckyFollow
Xuguo Zhou, University of KentuckyFollow

Abstract

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔC_t method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Document Type

Article

Publication Date

11-8-2016

Notes/Citation Information

Published in Frontiers in Plant Science, v. 7, article 1672, p. 1-10.

Copyright © 2016 Yang, Preisser, Zhang, Liu, Dai, Pan and Zhou.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Digital Object Identifier (DOI)

https://doi.org/10.3389/fpls.2016.01672

Funding Information

This research was supported by a grant from USDA BRAG grant (3048108827), the National Natural Science Foundation of China (31501642), and a Special Fund for Agroscience Research in the Public Interest (201303028).

Related Content

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.01672/full#supplementary-material

Repository Citation

Yang, Chunxiao; Preisser, Evan L.; Zhang, Hongjun; Liu, Yong; Dai, Liangying; Pan, Huipeng; and Zhou, Xuguo, "Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants" (2016). Entomology Faculty Publications. 107.
https://uknowledge.uky.edu/entomology_facpub/107