Function Analysis Of G α Q In The20-hydroxyecdysone Signaling Pathway In Helicorverpa Armigera

Background, scientific questions and significance of the researchInsects undergo development to grow up to be pupae generation by generarion. Molting and metamorphosis are two necessary processes during life cycle. They are regulated mainly by two kinds of hormones,20-hydroxyecdsone (20E) and juvenile hormone (JH).20E can antagonize JH to regulate molting and metamorphosis coordinately. At the prime stage,20E has a relative high titer and could trigger molting; while during the final phase, the titer of20E is elevated again and metamorphosis is started.20E is widely known as a steroid hormone and has a classical genomic pathway to tranduce siganaling, that is,20E binds to its nuclear receptors followed by modulation of gene transcription. However, an increasing number of study found that some kinds of steroid hormones have nongenomic pathways such as estrogen and brassinolide. Both even have membrane receptors. G protein coupled receptors, GPCRs act as membrane receptors to play pivotal roles in numerous signaling pathways, which transmit the first signaling outside the cell to the second signaling, thus initiating cascade reactions downstream. Heterotrimer G proteins couple to GPCRs and consist of the subunits of a,(3and y. Gaq belongs to the subtype of Ga, our research mainly focus on the reaction mode of participating in the20E signaling pathway and the influence on molting and metamorphosis. We should attach importance to the study in two aspects. It could reveal the mechanism of molting and metamorphsis of insects to understand the nongenomic pathways of steroid hormones deeper in theory; it could provide target genes for controlling the pests in application.Results and conclusions The Helicoverpa armigera, a Lepidoptera insect, is a holometabolic insect which has four stages of development, egg, larva, pupa and adult. We use the cotton bollworms as a model to study the functions of Gaq in the molting and metamorphosis. We use RNAi in larva and HaEpi cell line (Helicoverpa epidermis cell line), analysis of PKC phosphoprylation and calcium assay to reveal that Gaq participates in the20E sinaling at the pre-genomic stage. The results are as follows1.20E via Gaq regulates gene expression and metamorphosisGaq is visibly upregulated during metamorphosis and could be induced by20E. RNAi in the larva shows that Gaq is essential for20E-promoted metamorphosis and knockdown of Gaq delays the metamorphosis even death. A series of genes in the20E pathway are suppressed when Gaq is knocked down in larva and in the HaEpi cell line.2.20E via Gaq induces Gaq to translocate toward the cell membraneImmunocytochemistry is performed to find that Gaq is mainly distributed in the cytoplasm without hormone induction. However,20E induces Gaq to move toward the cell membrane. GPCR inhibitor blocks the membrane trafficking.20E regulates Gaq PKC phosphorylation, which determines the Gaq membrane traffic.3.20E via Gaq regulates calcium increase in HaEpi cells20E increases cytosolic Ca2+, which peaks in a short time. Gaq is essential for the20E-induced Ca2+release and influx in the cells. However, when Gaq is knocked down by dsGaq transfection in the cells,20E induces neither the release of intracellular Ca2+nor the influx of extracellular Ca2+. The data suggest that Gaq could regulate the20E-induced Ca2+release and influx in the cells.4.20E via Gaq regulates CDK10and USP1phosphorylation20E via Gaq regulates CDK10and USP1PKC phosphorylation. After knockdown of Gaq by infecting dsGaq in the cells, CDK10and USP1are not PKC-phosphorylated by20E induction.20E via Gaq regulates the phosphorylation of Gaq, thus determining gene transcription in the20E genomic pathway to result in the molting and metamorphosis.Innovation and importance of the researchThis work explored the role of Gaq in the20E signaling pathway, which suggested that Gaq participates in20E signaling at the pre-genomic stage by regulating the increase in intracellular Ca2+and phosphorylation of transcription complex proteins. This finding provided experimental evidence to screening the receptors of20E on the cell membrane, thereby to control the pests in application.