title: Selection of Reference Genes for RT-qPCR Analysis inCoccinella septempunctatato Assess Un-intended Effects of RNAi Transgenic Plants.
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 ladybeetleCoccinella 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 ofC. septempunctata v-ATPase Agene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms,geNorm, Normfinder, BestKeeper, the ΔCtmethod, andRefFinder, 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 inC. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.
authors.affiliation: College of Plant Protection, Hunan Agricultural UniversityHunan, China; Institute of Plant Protection, Hunan Academy of Agricultural SciencesHunan, China; Department of Entomology, University of Kentucky, LexingtonKY, USA.
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Biological Sciences, University of Rhode Island, Kingston RI, USA.
authors.forename: Evan L
authors.initials: EL
authors.lastname: Preisser
authors.affiliation: Department of Entomology, University of Kentucky, LexingtonKY, USA; Institute for the Control of Agrochemicals, Ministry of AgricultureBeijing, China.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Institute of Plant Protection, Hunan Academy of Agricultural Sciences Hunan, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Plant Protection, Hunan Agricultural University Hunan, China.
authors.forename: Liangying
authors.initials: L
authors.lastname: Dai
authors.affiliation: Department of Entomology, University of Kentucky, LexingtonKY, USA; Department of Entomology, South China Agricultural University, Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong ProvinceGuangzhou, China.
authors.forename: Huipeng
authors.initials: H
authors.lastname: Pan
authors.affiliation: Department of Entomology, University of Kentucky, Lexington KY, USA.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Zhou
keywords: Coccinella septempunctata; RNAi transgenic plants; RT-qPCR; environmental risk assessment; plant incorporated protectant; reference gene