Molecular Indentification Of Related Genes Involved In Bombyx Mori Sex Pheromone Biosynthesis

Sex pheromone is the key factor that insects release to external to attractthe opposite sex and quickly seek partner for successful mating. The biosynthesis andrelease of sex pheromone are in fact regulated by a neuropeptide, named pheromonebiosynthesis activating neuropeptide (PBAN). It is well-known that the molecularmechanism of PBAN is to firstly bind with its receptor, which expresses at the sexpheromone gland cytomembrane, then go through a series of signal transduction andfinally result in sex pheromone biosynthesis and release. Sex pheromones are derivedfrom acetyl-CoA through fatty acid synthesis, desaturation, and chain-shorteningreactions followed by the reductive modification of the carbonyl carbon. Themechanism of sex pheromone biosynthesis is species-dependent. Currently, theresearches regarding to sex pheromone biosynthesis mainly focus on Bombyx moriand Helicoverpa zea. In the present study, B. mori is used as the model to studyPBAN action. In B. mori, the binding of PBAN and PBANR activates a series ofcritical genes involved in the biosynthesis of bombykol, that is, accelerates both thelipolysis of bombykol precursor and the reduction of fatty acyl group to generate thefinal bombykol products.The main research contents are summarized as follows:Molecular identification of pheromone gland specific PBANR gene in B. mori;The effects of mating on the changes of related genes from B. mori pheromonegland by digital gene expression (DGE) method;Effects of JH deficiency and overexpression on bombykol production;Effect of Met1gene knockout mediated by RNAi on bombykol production;The temporal expression profile of pheromone gland specific pancreaticlipase-like gene (BmPLLG) in B. mori;Effect of BmPLLG gene knockout mediated by RNAi on bombykol production;Polyclonal antibody preparation and Western blot detection of BmPLLG protein atdifferent development stages of B. mori.The main results are as follows: 1. Molecular identification of pheromone gland PBANR gene in B. moriSequence analysis indicates that the full-length cDNA sequence of PBANR geneis2780bp and contains a complete open reading frame of1242bp that encodesdeduced polypeptides of413amino acids with45.9KD molecular weight and9.28predicted isoelectric point. The temporal expression profile analysis shows that thePBANR transcript steady increase from96h before eclosion to48h after eclosion inage-dependent pattern. Real-time PCR analysis indicates that decapitated treatmentobviously inhibited PBANR expression at different development stages. Methoprenetreatments in vitro obviously inhibited the transcript level of PBANR from-72h to-48h after decapitating, but had no effect at other stages. Mating also distinctlysuppressed the expression of PBANR at different development stages.2. Effect of mating on sex pheromone biosynthesis of B. moriThe effects of mating on the expression profiles of related genes in sexpheromone gland were determined by DGE method. The results manifested that aserial of genes were up/down-regulated by mating, especially, sex pheromonebiosynthesis related genes significantly were down-regulated. Interestingly JH signalgenes were up-regulated after mating, similar results were found regarding to someimmune related genes. Therefore, we further studied the effect of JH on the B. morisex pheromone biosynthesis after mating. Effect of methoprene treatment in vivo onbombykol production was studied by GC/MS approach. The results manifested thatmethoprene treatments on decapitated and normally developmental femalessignificantly inhibited bombykol production. Effect of Met1and Met2, involving inJH signal-transduction, on bombykol production was further studied. Real-Time PCRanalysis shows that it is Met1, not Met2, takes part in JH signal transduction inpheromone gland. The further gene knockout of Met1induced by RNAi leads to theobviously reduction of bombykol product.3. Molecular identification of sex pheromone gland BmPLLG gene in B. moriThe expression levels of all genes from sex pheromone gland at different stages(-72h,0h,72h) were studied by using DGE. Seven specific lipase genes of sexpheromone gland in B. mori were screened out from DGE results. The molecularcharacteristics of a pancreatic lipase-like gene, designated as BmPLLG, wasidentified. Sequence analysis indicated that BmPLLG gene belongs to the pancreas lipasefamily member, the complete open reading frame of BmPLLG sequence is1086bp insize and encodes deduced polypeptide of361amino acids with40.3KD predictedmolecular weight and8.77predicted isoelectric point, the identity of BmPLLG aminoacid sequence with other insects homologs is relative low, only16-49%. Temporalexpression analysis showed that the expression of BmPLLG gene was age-dependentpattern at different development stages in B. mori. The analysis of BmPLLG protein atthe different development stages by using corresponding antibody found that thepattern of protein expression similar to the transcriptional expression patterns andboth highly expressed at the critical period of sex pheromone biosynthesis.Spatio expression analysis revealed that BmPLLG gene was present in alldetective tissues, but lower expressed in eggs. Further gene knockout of BmPLLGgene induced by RNAi obviously inhibited bombykol production. All those resultsshow that BmPLLG gene plays an essential role in the biosynthesis of bombykol.