Cloning And Identification Of The Neuropeptide Allatotropin G Protein Coupled Receptor Gene In Helicoverpa Armigera

The cotton bollworm, Helicoverpa armigera, which belongs to Lepidoptera Noctuidae, distributes broadly in the world. The cotton bollworm is one of serious pests around the world and often brings great lost in agriculture. Neuropeptide allatotropin (AT) plays key role in regulating the synthesis of juvenile hormone (JH) in coropa allata (CA), and regulation of insect growth, development and reproduction of adults. Insects neuropeptide receptors are mainly G protein coupled receptor (GPCR), which transduct the extracellular signals into the cell and play regulation in insect growth, reproduction, immunity, cell migration, nerve conduction, metabolism and behavior. GPCRs are always the targets genes to develop of green pesticides. Thus, it is very important to clone and identify the allatotropin GPCRs in Helicoverpa armigera, which construct the foundation for further development of green pesticide in control of cotton bollworm.The full-length of cDNA of gene encoding of ATR in H. armigera was cloned using RACE method. The ORF of ATR gene contains 1341 nucleotides and encoding 466 amino acids. The non-coding regions of ATR 3’and 5’terminal are 341 and 236 bp, respectively. The ATR receptor of H. armigera shows the typical character of G-protein couple receptor of 7 transmembrane regions, which is the similar to other Lepidoptera. The phylogenetic data basd on the amino acid sequences of ATRs indicate that ATR of H. armigera and Manduca sexta locate in the same branch with identity of 72.2%.The tissue distributions and the development expression in brain of AT and ATR genes in H. armigera were performed by RT-PCR, The mRNA of Har-AT gene showed highest expression in thymus gland, and next is brain, but no expression in midgut and fat body. The mRNA of ATR expressed in all the detected tissues, and showed highest in brain. The developmental expression pattaern of Har-AT and Har-ATR genes in brain in H. armigera showed some differences, Har-AT express the highest level in 8th day and next at 10th day in pupal stage, but Har-ATR shows the highest level in 2th day and next at 10th day in pupal stage.The recombinant plasmid containing pcDNA5 and the ORF of ATR (pcDNA5-ATR-ORF) was obtained and transfected into CHOW11 cell lines to analysis of the binding activity to AT. Four ATs all can bind to pcDNA5-ATR-ORF and induce significant increasing of intracellular Ca2+, which show the dose-dependent pattern. The EC50 value of binding of Har-AT, Har-ATL-I, Har-ATL-II and Har-ATL-III to ATR were measured as 6.9364×10-5,2.8744×10-7, 1.3061×10-6 and 2.38×10-3 mM, respectively. The above results confirmed that the cloned gene is the G protein coupled receptor of allatotropin in Helicoverpa armigera, and Ca2+ play as second messenger in the Har-ATR signaling pathways; Har-ATL-I showed the highest binging activity to Har-ATR, and next is Har-AT and Har-ATL-II; Har-ATL-III almost can not bind to Har-ATR. The four members also can bind to Har-ATR transfected into HEK293 cell; the order is same to the pattern in CHOWT11 cell, but the binding activities in HEK293 cell are slightly lower than in CHOWT11 cell.The effects of the binding of ATs to Har-ATR in HEK293 cell on intracellular cAMP were detected. After binding, the ATs almost could not induce the response of intracellular cAMP, which demonstrated cAMP is not second messenger, but Ca2+ is the main second messenger to transdustion of signals. During perfirming the above analysis, the pcDNA5 plasmid DNA was also used to bind to ATs, but showed no response.The above results demonstrated that Har-ATL-I, Har-AT and Har-ATL-II were the functional ligands to activity Har-ATR. Three mimetic peptides were designed and synthesized which were followed the five amino acids of C terminal of the corresponding ligands, and named as ATL1, AT and ATL2, respectively. After binding to Har-ATR in CHOWT11 cell, ATL1 induce the slight Ca2+ response with EC50 value of 2.16 ×10-3mM, which is similar to that by Har-ATLIII. AT and ATL2 almost can not induce any responses after binding. Therefore, the design and selection of high activity mimic peptides to Har-ATR still need further works.