Identification Of Allatotropin Receptor And The Developmental Expression Of The Related Neuropeptide Receptors In Bombyx Mori

Neuropeptides and their G protein coupled receptors (GPCRs) play key rolesin regulation of growth, development, reproduction, nervous transduction,metabolism, behavior and other aspect of insect life, which were regarded as atarget model to develop novel agents for pest control. Allatotropin is an importantneuropeptide in Bombyx mori, which functions include the stimulation of juvenilehormone biosynthesis, cardioacceleratory functions, and inhibition of active iontransport. Thus, the allatotropin receptor gene can be used as target gene to controlinsect growth. In the present research, the full length of cDNA of the geneenconding allatotropin G protein coupled receptor in Bombyx mori (Bom-ATR)was cloned and idengtified. The developmental expression of16neuropeptidereceptor genes (including allatotropin receptor) were demonstrated.The open reading frame (ORF) of Bom-ATR was1254bp in length, whichencoding416amino acids. The secondary structure of Bom-ATR was predicted asseven trans-membrane proteins, which in accord with the typical characteristics ofGPCR family members. The predicted3-D structure of Bom-ATR contained18helixes in total and7of which wasα-helix,in consistent with the7trans-membranedomains. Phylogenetic analysis showed that Bom-ATR was in close to that ofManduca sexta, followed by BNG5, Aedes aegypti, Nasonia and Triboliumcastaneum, which was in coincidence with the traditional relationship. TheBom-ATR ORF was inserted into pcDNA5-FRT vector, and the recombinantplasmid was obtained (pcDNA5-FRT-Bom-ATR-ORF). The recombinant DNA wastrandfected into HEK cell line; and then was used to interaction with Bom-AT.The results showed that the responsive curve was in a mode of dose-dependent, andthe EC50value of AT-ATR was2.16nM, which demonstrated that the Bom-ATR wecloned was the G-protein coupled receptor of Bom-AT.The expression levels of Bom-ATR mRNA in Brain-Suboesophageal ganglioncomplex (Br-SG) from1st in5th instar to2nd adult were quatified by Real-TimePCR. Bom-ATR mRNA in5th larval instar was higher than those in pupal stage andadult. The expression level of Bom-ATR mRNA maintained high level from1st to5th day in5th larval instar, with a highest level on2nd day, which may be relatedto the synthesis of juvenile hormone. The developmental expression profiles of other15G-protein coupledneuropeptide receptor genes in Br-SG were investigated. The expression profileswere different to each others. Within the investgated genes, the receptors of sNPF1and other six neuropeptides expressed high level in early5th larval instar, and thesNPF2receptor, Tachykinin2receptor, CAPA1receptor and Sulfakinin receptorshowed high level during wandering pupal stage. During the adult stage, onlyTachykinin2receptor and neuropeptide receptor A2gene kept a relative high level,and the other receptor genes showed a lower level.