Cloning,Localization And Functional Identification Of Ionotropic Receptors From Helicoverpa Armigera

Insects mainly rely on sensitive olfactory systems to sense various chemicals in the environment.These chemical signals are further processed and integrated to trigger specific behavioral responses,such as the location of host plants and the selection of oviposition sites.In the process of olfactory recognition,a variety of olfactory proteins are involved.With the technological development of transcriptome and genomic sequencing,plenty of chemosensory genes have been identified from Helicoverpa armigera.At present,the research on olfactory proteins of H.armigera were focused on odorant receptors and odorant binding proteins.Nonetheless,the function of ionotropic receptors,which were identified as another class of olfactory receptor,has not been investigated.The sequences of six ionotropic receptor genes were cloned and their structure were analyzed based on the antennal transcriptome of H.armigera.The structures of HarmIR8 a and HarmIR25 a also were analyzed and then the localizations of the two IRs were performed on the antenna of H.armigera.The function of neurons in the antennal coeloconic sensilla was recorded by single sensilla recording.Finally,the function of six IRs in vitro were studied.These researches provide molecular basis for insight into the mechanism of chemical recognition in H.armigera and developing new targets for behavior regulation.The main results are shown as follows:1.The full length sequences of six ionotropic receptor genes were cloned by PCR and RACE technique,including HarmIR8 a,HarmIR25a,HarmIR21 a,Harm IR41 a,HarmIR76b and HarmIR64 a.2.Bioinformatics methods were used to analyze the amino acid sequences,transmembrane domains and functional domains.The results of amino acid sequence alignments showed that the similarity of HarmIR8 a and HarmIR25 a with other insects were relatively high and were relatively conserved,while the results of amino acid sequences alignment showed that HarmIR21 a,HarmIR41a,HarmIR76 b and HarmIR64 a were relatively low conserved with other insects.The structural analysis of HarmIR8 a and HarmIR25 a proteins revealed that they contained the amino acid terminal domain(ATD),transmembrane domains(M1,M2 and M3),the ion channel(P),the ligand bingding domain(S1 and S2)and the intracellular C terminus.3.The relative expression levels of Harm IR8 a,HarmIR25a,Harm IR76 b,HarmIR21a,Harm IR41 a and HarmIR64 a in the eight tissues of male and female adults of H.armigera were detected by real-time fluorescent quantitative PCR assay.The results showed that HarmIR8 a was expressed in the head and antenna of the female and male adults and was highly expressed in the antennae of the female and male.The expression level of HarmIR25 a was highest in the female and male antennae and was lower in other tissues.HarmIR76 b was highly expressed in the proboscis except in the antennae.HarmIR64 a was expressed in all tissues especially with higher expression level in the antennae.HarmIR21 a and HarmIR41 a were highly expressed in the antennae.4.The localization of HarmIR8 a and HarmIR25 a in antennal sensilla of H.armigera were preliminarily confirmed by in situ hybridization.The results showed that two ionotropic receptors were expressed mainly under the antennal coeloconic sensilla,and the hybridization signals were not detected in other sensilla.5.The function of neurons in the antennal coeloconic sensilla of H.armigera was identified by single sensilla recording.The results showed that the neurons were activated by acids,ammonia,amines,aldehydes and esters.But there are no electrophysiological responses to two terpenes,(R)-(+)-Limonene and α-Pinene.At the same time,the neurons of coeloconic sensilla was divided into four functional subtypes,including Type A,Type B,Type C and Type D.The neurons in different coeloconic sensilla were functionally divergent.6.The in vitro function of six ionotropic receptors in different combinations were preliminarily studied by Xenopus oocytes heterologous expression combined with two-electrode voltage clamp recording system.The results showed that none of the test odors activated electrophysiological responses of the selected combination of IRs.