Cloning And Identification Of The PBAN Receptor Gene, And The Expression Of The Genes Related To The Pheromone Biosynthesis In Antheraea Pernyi

The oak silkworm (Antheraea pernyi) is an important economic insect, which origin from China. Because of easy rearing and large body size, A. pernyi has been extensively used as a model animal to analyse the photoperiodic time measurement, regulation of the neuroendocrine and molecular genetics. Pheromone biosynthesis activating neuropeptide (PBAN) is a member of FXPRLamide neuropeptide family, which plays key role in mating and breeding of insects by regulation of the synthesis and release of sex pheromone. PBAN receptor is a kind of G-protein coupled receptors, which can transmit extracellular signal to regulate cell behavior after binding to the corresponding ligands. In the present degree thesis, the full length of cDNA of PBAN receptor gene of A. pernyi was cloned and sequenced. By analysis of the intracellular Ca2+level, the PBAN receptor gene was identifed. Some mimetic peptides whch showed high binding activity to PBAN receptor were obtained. The distributions and the developmental expression in pheromone gland of PBAN, PBAN receptor and the genes related the pheromone biosynthesis was performed in A. pernyi.Using RACE method, the full-length of the cDNA of the PBAN receptor gene in A. pernyi was obtained. The ORF of PBAN gene contains 933 nucleotides and encoding 311 amino acids. The non-coding regions of PBANR 3'and 5'terminal are 330 and 171bp, respectively. The PBAN receptor of A. pernyi shows the typical character of G-protein couple receptor of 7 transmembrane regions, which is the same as other Lepidoptera. The phylogenetic tree basd on the sequences of PBANR indicates that PBANR of A. pernyi locate in the same branch with Bombyx mori and Manduca sexta. The PBANR of A. pernyi showed the identity of 83.9%,83.6%,83.6% and 73.1% with M. sexta, B. mori, Helicoverpa zea and Plutella xylostella, respectively.The recombinant plasmid in PCDNA5 contained the ORF PBAN receptor was obtained and transfected to CHO cell to analysis of the binding activity to PBAN. The binding of PBAN receptor to PBAN induced the significant intracellular Ca2+response, and the response showed the dose-dependent pattan. The value of EC50 was 1.95nM, which comfirmed the gene we cloned was PBAN receptor. The PBAN receptor of A. pernyi showed corss binding activity the other four neuropeptides of FXPRL family, the highest is Anpy-SGNP, and the next is Anpp-SGNP, Anpa-SGNPand AnpDH. The above corss activities to other four member imply that the C-terminal hexapeptide of PBAN play key role in binding to PBAN receptor. The mimetic peptide containg the nine amide residues of PBAN showed similar binding activity of that of full PBAN. The deletion in the C-terminal hexapeptide (FSPRLa) revealed that the P, R and L residue play higher influences in binding to PBAN receptor. In addition, the amidation to the mimetic peptides play key role in binding to PBAN receptor.The expression of the mRNA of PBAN receptor gene was highest in the pheromone gland, and the next is fat body of A. pernyi. The mRNA of PBAN receptor gene in pheromone gland increased significantly with the female moth eclosion. The distribution and the developmental expression in pheromone gland of PBAN and DHR were different to the pattern of PBAN receptor of A. pernyi.Based the data of transcriptome of single pupae of A. pernyi, the sequences of 15 genes related to the pheromone biosynthesis were obtained. Two genes showed the highest expression in pheromone gland, including Acetyl CoA Carboxylase, elongation of very longchain fatty acids. The other thirteen genes express highly in midgut or fat body, including fatty acid synthase, desaturase, Aldehyde oxidase, Aldehyde dehydrogenase, Acyl-CoA oxidase, enoyl-CoA hydratase,3-Hydroxyacyl-CoA dehydrogenase, Alcohol dehydrogenase, Acetyltransferase, Lipase, Esterase, Acyl-CoA dehydrogenase, and acyl-CoA binding protein gene. Different to the 3-Hydroxyacyl-CoA dehydrogenase gene, the mRNA of other 14 genes change significantly with moth eclosion. The mRNA of eight genes in the pheromone gland of 2 days after eclosion are higher that those in the gland of 2 days before eclosion, including Acyl-CoA dehydrogenase gene, Acyl-CoA oxidase gene, etc. The other six genes show higher expression level in the pheromone gland of 2 days before eclosion than those in the gland of 2 days after eclosion, including Fatty acid synthase gene, Desaturases gene, etc.