The Physiological Functions Of Tachykinin Signaling System In Tribolium Castaneum

Coleoptera is the biggest order in insects with a wide variety of species and complex and diverse,infesting agricultural and forestry economic crops.Tribolium castaneum,a model insect and an important stored products insect pest,has strong fertility and short interval between generations.Neuropeptides are important signaling molecules for cell communication,and are involved in the regulation and coordination of physiological processes in invertebrates.They were proved to regulate a variety of physiological functions via activating their receptors in insects.However,their receptors belong to the G-protein coupled receptors?GPCRs?,which are important signaling molecules for cell communication.Interfering with normal GPCRs function by blocking or over stimulating the signal transduction of these neuropeptides signaling systems can cause disruption of the important physiological and biological process in insects,which provide ideal target for the development of novel insect control agents.So,the tachykinin signaling system,one of the neuropeptides signaling systems,involves many important physiological functions in insects,including olfactory perception,lipid metabolism and aggression and so on.In the present study,the precursor gene sequences of tachykinin?TcTK?precursor and its receptor?tachykinin receptor,TcTKR?were screened based on the genomic data of the T.castaneum.Furthermore,their cDNA sequences were cloned by RT-PCR.The sequences and structures analysis of TcTK precursor and its receptor were reported.Moreover,to confirm the reconstructed plasmid pcDNA3.1?+?-TcTKR is active,a calcium reporter assay was performed with its endogenous ligands in Chinese hamster ovary?CHO?cells.Besides,the temporal and spatial distributions of TcTK and TcTKR transcripts were investigated by real-time quantitative PCR?RT-qPCR?.As indicated by the RT-qPCR,TcTK-expressing and TcTKR-expressing in the central nervous system of T.castaneum were investigated using immunohistochemistry.Finally,by observing the phenotypes of the behavioral traits such as mating and oviposition after RNA interference?RNAi?,we revealed the important physiological functions of tachykinin signaling system in the T.castaneum adults.In a word,the results of this study will not only enrich our finding out the physiological functions of the tachykinin signal system for coleoptera insects,but also provide certain theoretical basis especially for the research on coleoptera insect pest control agents represented by T.castaneum.1 Molecular cloning and heterologous expression of TcTK and TcTKRGene sequences of TcTK and TcTKR were screened based on T.castaneum genome dataset,and the cDNA sequences were verified by RT-PCR.Sequence analysis showed that the TcTK precursor gene has an ORF of 795 nucleotides encoded264 amino acid residues.Seven T.castaneum TK mature peptides were predicted and all of them?TcTK1-7?possess the general C-terminal motif of FxGxRamide.Besides,phylogeneticanalysis showed that TcTK precursor shared highest homology with the Blattella germanica.Moreover,the ORF of TcTKR contained 1287 nucleotides,encoded 428 amino acid residues.It is the typical GPCR with seven transmembrane domains.Multiple sequence alignment indicated that TcTKR shares highly conserved transmembrane domains with Drosophila melanogaster and Bombyx mori.In the phylogenetic tree,TcTKR was clustered with bettles such as Aethina tumida,Anoplophora glabripennis and Leptinotarsa decemlineata.TcTKR was successfully expressed using CHO heterologous expression system in vitro.A cell based intracellular calcium mobility assay method was applied to determine the ligand-receptor binding.The results showed that the endogenous ligand TcTK5 could activate TcTKR in a concentration-dependent manner.The 50%effective concentration(EC₅₀)value reached nanomolar level,which was 36.98 nM.As shown in the in vitro assay,TcTKR we cloned is functionally validated.2 The temporal,spatial expression patterns and subcellular localization in the central nervous systemThe RT-qPCR was used to analyze the temporal and spatial expression patterns of TcTK and TcTKR in T.castaneum.The results showed that TcTK and TcTKR were stably and highly expressed at all the tested developmental stages except the egg stage.Moreover,for the different tissues in adult,both TcTK and TcTKR were superlatively expressed in CNS.However,in addition to CNS,TcTK was also slightly expressed in the midguts,and TcTKR was also slightly expressed in the hindguts.There was no expressing in other tissues.At the protein level,subcellular localization of TcTK and TcTKR was identified by immunohistochemistry in the central nervous system of T.castaneum.By using a rabbit antibody against L.migratoria TK peptide,we found that only a pair of neurons was stained by TK antibody in tritocerebral neuron area.However,no neurons were found to be stained in the nerve cord.On the contrary,we found a total of 32 pairs of neurons expressing TcTKR in the central nervous system of adults.Among them ten pairs of neurons in brain and twenty two pairs of neurons in ganglions except for the first ganglion.3 RNAi and phenotypic analysisThe expression of TcTK and TcTKR were silenced by dsRNA injection mediated RNA interference.The results indicated both TcTK and TcTKR could be effectively silenced.Compared with control groups,the mating and oviposition behavior of the adult cannot be significantly affected and also have no effect of testes and ovary development after silencing of TcTK.Meanwhile,silencing of TcTKR could not cause reduced mating times in adults,but could cause significantly reduced spawning number.Besides,the ovary development was also serious influenced.In conclusion,the TcTKR has an important physiological function in regulating ovary development of T.castaneum female adults.In conclusion,this dissertation deeply studies the function of TK and TKR in the coleopteran model insect T.castaneum,and contributes to the improvement of the function of the Tachykinin signaling system in the whole insecta.What's more,it provides a theoretical basis for the development of coleopteran harmful insects with GPCR as a target agent for new insecticides.