The Molecular Identification Of Helicoverpa Assulta Sex Peptide Receptor And The Effect Of Helicoverpa Armigera Mating On Antimicrobial Peptides Expression

Seminal proteins and antimicrobial peptides(AMPs) are important life substanceswhich participate in insect reproduction and immunity. The previous studies showedthat insect males transfer seminal proteins to females during copulation, thusinfluence reproduction behaviours and immune responses of females. Obviously,ascertaining the molecular mechanism of seminal proteins roles has theoretical andpractical significance for comprehensivly understanding the mating regulatory lawson females reproduction behaviors and immunocompetence changes, and developingmolecule interference technologys which impact pests reproductive process andimmunocompetence. Therefore, this study selected Helicoverpa related species H.assulta(Guenée)and H. armigera(Hübner) as experimental material, and made theresearch separately on clone and expression of H. assulta sex peptide receptor geneand expression of seven insect antimicrobial peptide genes in abdoimen of H.armigera female under diffierent mating state, subsequently, discussed the influencerule of mating on sex peptide receptor gene and antimicrobial peptide genesexpression level. The main results are as following:(1) A cDNA sequence of2048bp encoding an sex peptide receptor protein frompheromone gland of Helicoverpa assulta was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR). Sequences analysis result showed open readingframe (ORF) of this gene was1275bp in size, encoding424amino acid residues.Thisreceptor predicted molecular weight (MW) was48.6kD and isoeletric point was9.25.Amino acid sequences analysis showed that this protein contained seven putative transmembrane domains which are characterisistic of G-protein coupledreceptor(GPCR) and shared more80%amino acid identity with other Lepidopteramoth homologs. This gene, named as HassSPR, had been deposited in the GenBankdatabase with accession number AFH53182.1.(2) The tissular and temporal expression of HassSPR transcript was measured byusing real time quantitative PCR. The results revealed that HassSPR could be detectedin pheromone gland, bursa copulatrix, ovary, brain, thorax ganglion and musule of2day old H. assulta female. Among them, HassSPR was richest in brain. And thatHassSPR in adult female pheromone gland could be detected with a lowertranscription level on day1before emergence, reached expression peak on day3afteremergence and was subsequently down-regulated.(3)The effect of mating on expression of HassSPR transcript in differenttissue(including pheromone gland, bursa copulatrix, brain and ovary) was studied byusing real time quantitative PCR.The results showed that after mating, HassSPR geneexpression level of female brain and pheromone gland was significantly up-regulation,while that of bursa copulatrix and ovary was significantly down-regulation.(4) The effect of mating on seven AMP genes transcription in H. armigerafemale was studied by using real time quantitative PCR.The results showed thatspecific AMP genes responded differentially to mating, mating inhibited Cecropin3gene transcription, but induced CecropinD, Moricin, Defensin, Lebocin, Attacin andGloverin gene transcription,While this effect was only limited in12h after mating.