Molecular Characterization, Expression Of 4 Neuropeptieds From Clostera Anastomosis And Discussion On Mechanisms Of Allatostains

Insect neuropeptides regulate some key development procedure such as growth, development, metamorphism and production, as neuro hormones, neuro transmitters or neruo regulators. Molecular cloning and characterazing the genes of insect neruopepties and research on their expression and function would provid more theory avidences to discover the mechnism of neuropeptids and to develop new insectcides based on neuropedtides. In this paper, three cDNAs encoding allatostatin (AS), allatotropin (AT), diapause hormone (DH) and pheromone biosynthesis activation neuropeptide (PBAN) were cloned respectively, and DNAs encoding Cloan-AT and Cloan-DH-PBAN were also cloned respectively. The tissue specific expression of Cloan-AS, Cloan-AT and Cloan-DH-PBAN mRNAs were analysised by RT-PCR. A semi-quantitative RT-PCR technique was developed to detect the expression of Cloan-AS, Cloan-AT and Cloan-DH-PBAN mRNAs, and a prokaryotic expression vector pQEAT was constructed to express Cloan-AT in Escherichia coli. Main findings of the paper were as fellows:The cDNA encoding Cloan-AS was 580bps in full length, in which 372 bps encoding 124 amino acids was CDS. The 5' UTR was from 1 to 111, and the 3' UTR was from 484 to 580. The most likely cleavage site of predicted signal peptide was between position 26 and 27 (IHA-AP). The similarity of Cloan-AS to Spofr-AS, Samcy-AS, Pseun-AS and Drome-AS were 83%, 77%, 83% and 42%, respectively.The cDNA encoding Cloan-AT was 790 bps in full length, in which 399 bps encoding 133 amino acids was CDS. The 5' UTR was from 1 to 83, and the 3' UTR was from 483 to 790. The most likely cleavage site of predicted signal peptide was between position 22 and 23 (AVA-AP). The similarity of Cloan-AT to Spofr-AT, Helar-AT, Pseun-AT, Mytse-AT, Manse-AT, Samcy-AT and Bommo-AT were 92.5%, 91.9%, 90.4%, 89.7%, 85.8%, 84.4% and 80.6%, respectively.The cDNA encoding Cloan-DH-PBAN was 796bps in full length, in which 588 bps encoding 196 amino acids was CDS. The 5' UTR was from 1 to 22, and the 3' UTR was from 611 to 796. The most likely cleavage site of predicted signal peptide was between position 20 and 21 (VEA-TN). The cDNA of Cloan-DH-PBAN encoded 1 DH gene, 1 PBAN gene and other 3 SGNPs. The similarity of Cloan-DH to Spoex-DH, Spoli-DH, Agrip-DH, Helar-DH, Samcy-DH, Manse-DH, Helze-DH,Helas-DH, Antpe-DH, Helvi-DH, Bomma-DH, Bommo-DH and Adosp-DH were 86.2%, 81.2%, 93.1%, 79.3%, 79.3%, 79.3%, 75.9%, 75.9%, 79.3%, 75.9%, 75.9%, 75.9% and 37.9%, respectively. The similarity of Cloan-PBAN to Spoex-PBAN, Spoli-PBAN, Agrip-PBAN, Helar-PBAN, Samcy-PBAN, Manse-PBAN, Helze-PBAN,Helas-PBAN, Antpe-PBAN, Helvi-PBAN, Bommo-PBAN, Mambr-PBAN, Adosp-PBAN and Lymdi-PBAN were 90.9%, 90.9%, 87.9%, 93.9%, 87.9%, 85.7%, 93.9%, 93.9%, 87.9%, 91.2%, 90.9%, 90.9%, 63.9% and 87.9%, respectively.The DNA sequence encoding Cloan-AT was 814 bps in length, including 2 exons and 1 intron, and the DNA sequence encoding Cloan-DH-PBAN was 3694 bps in length, including 6 exons and 5 introns.Cloan-AS, Cloan-AT and Cloan-DH-PBAN mRNAs were detected by RT-PCR in the head and gut of C. anastomosis and was not detected in the integument. The result of a semi-quantitative RT-PCR showed that all these three mRNAs were expressed higher in the head of adult than in pupae and larvae.In order to express Cloan-AT in Escherichia coli fused to His, a prokaryotic expression vector, pQEAT, was reconstructed based on the plasmid pQE30. But after transformed into JM109, there was no production was induced by IPTG.Based on previous studies, the mechanism of AS was disscused. Most insect allatostatins inhabit the biosynthesis of JH by corpora allata (CA), which may function at the first step of pathway of biosynthesis of JH and take cAMP as second messenger. But some allatostatins of type A have no effect on CA of source insects for there may be another mechenism of inhabiting JH synthesis in these insects, including different allatostatins and second messenger. Several allatostatins of type A from the same insect are origined from a single proprecursor, which maybe function via a complex mix of differing affinity interactions with receptors in the CA. Allatostains of type A maybe inhabit vitellogenesis in orthopteroid insects through inhabition of biosynthesis and/or uptake of vitelloginin, and this function maybe correct to inhabition of biosythesis of JH by CA. Allatostatins put myoinhabitory effects on foregut, midgut and hindgut with results of inhabition of food taking, growth and more rate of mortality. Furthermore, Allatostatins inhabit contraction of musles in heart, dorsal vessel and oviducts. The myoinhabitory role of allatostatins may be mediated by a Gi-protein, which may be different from the receptor coupling with AS to inhabit the activity of CA.