The Research On Molecular Mechanism Of Sex Pheromone Biosynthesis Regulated By Calcineurin In Bombyx Mori And Helicoverpa Armigera

Lepidoptera moth sex pheromone,produced and released by pheromone gland?PG?of the female moth,is the key factor to attract conspecific males for mating and reproduction.Pheromone Biosynthesis Activating Neuropeptide?PBAN?was found to be a neuropeptide that regulate sex pheromone biosynthesis and release.PBAN action is species-dependent in lepidoptera moths.The research regarding signal transduction mechanism regulated by PBAN is vital for understanding insect sex pheromone biosynthesis and the evolution of insect species.As a key enzyme in PBAN signal transduction,calcineurin?CaN?plays an important role in sex pheromone biosynthesis of Bombyx mori and Helicoverpa armigera,respectively.However,the down-stream targets regulated by CaN and corresponding regulatory mechanism still keep elusive.In the present study,the molecular characterics,down-stream targets and regulatory mechanism of CaN were studied by using molecular biology,biological chemistry and so on.The main results and conclusions are as follows: 1.Analysis of protein expression levels of B.mori fatty acyl reductase gene?BmpgFAR?in the PG of B.mori.The full-length of the cDNA sequence of BmpgFAR is 2152bp?The GenBank accession number: NM₀01043502?,encoding 460 amino residues,and predicted protein relative molecular weight is 52325.3 Da.The analysis of BmpgFAR protein expression at different developmental stages in B.mori PGs by using western blot revealed that BmpgFAR protein begins to express at 48 h before emergence?-48 h?,gradually increases to the peak at 24 h after eclosion?24 h?,subsequently declines slowly,which is similar to the expression pattern of transcription studied previously,both abundantly are expressed in the critical period of pheromone synthesis.These results further suggested that BmpgFAR plays an important role in the biosynthesis of sex pheromone in B.mori.2.The regulatory role of CaN on BmpgFAR in the PG of B.moriThe regulatory role of CaN on BmpgFAR were studied by using co-immunoprecipitation and western blot.The results revealed that phosphorylation level of BmpgFAR significantly decreased following the stimulation of PBAN.CaN inhibitor significantly counteracted PBAN induced dephosphorylation of BmpgFAR.These results strongly manifested that CaN activates BmpgFAR by dephosphorylation responding to PBAN challenges,thus mediating sex pheromone biosynthesis in silkworm.3.Molecular characteristics of CaN in the PG of H.armigeraThe cDNA sequence of H.armigera CaN was obtained by transcriptome sequencing.H.armigera CaN has two subunits,the catalytic subunit A and structural subunit B.Sequence analysis manifested that the open reading frame?ORF?of cDNA sequence of CaN catalytic subunit A?CaNA?is 1488 bp,encoding 495 amino residues,and predicted protein relative molecular weight is 55946.8 Da.CaNA shared 61.41%-98.99% amino acid identity with other insects;the ORF of cDNA sequence of CaN structural subunit B?CaNB?is 513 bp,encoding 170 amino residues,and predicted protein relative molecular weight is 19356.9 Da,CaNB shared 91.76%-100% the amino acid identity with other insects.Developmental expression analysis showed The expression pattern of CaNA transcription was age-dependent,started to express at 48 h before emergence?-48 h?,gradually increased to the peak at 24 h after eclosion?24 h?until 72 h after eclosion?72 h?,then began to decline,which agreed with the critical period of sex pheromone biosynthesis in H.armigera.RNAi-mediated knockdown of CaNA and CaN inhibitor treatment significantly lead to reduction of sex pheromone production,suggesting that CaN plays an essential role in regulating the sex pheromone biosynthesis in H.armigera.4.Molecular characteristics of acetyl coenzyme A carboxylase?ACC?gene in the PG of H.armigeraThe cDNA sequence of H.armigera ACC was obtained by transcriptome sequencing,Sequence analysis manifested that the ORF of ACC cDNA sequence is 7092 bp,encoding 2363 amino residues,and predicted protein relative molecular weight is 265444.5Da,ACC shared 58%-95% amino acid sequence identity with other insects.Developmental expression analysis showed that expression pattern of ACC transcription was age-dependent,which started to express at 48 h before emergence,gradually increased to the peak at 24 h after eclosion until 48 h,then began to decline,which abundantly expressed in the critical period of pheromone synthesis.RNAi-mediated knockdown of ACC and ACC specific inhibitor treatment could siginificantly result in reduction of sex pheromone production.All these results show ACC plays an essential role in the biosynthesis of sex pheromone in H.armigera.5.The regulatory role of CaN on ACC in the PG of H.armigeraGC/MS results manifested that sex pheromone production significantly increased following PBAN stimulation from 0 to 60 min,during which ACC activity was significantly increased.However,CaN specific inhibitor suppressed the increase of ACC activity induced by PBAN.PBAN-dependent phosphoproteomics releaved that CaN and ACC were regulated by PBAN through phosphorylation/dephosphorylation,it is that CaN was regulated by phosphorylation and ACC was regulated by dephosphorylation.Subsequent CaN-dependent phosphoproteomics revealed deficiency of CaN significantly increased phosphorylation level of ACC.All these results strongly suggested that during the sex pheromone synthesis in H.armigera,CaN activates ACC by dephosphorylation responding to the stimulation of PBAN and thus mediating sex pheromone biosynthesis.