Study On The Gene Expression Profiles Along With Pual Development And Trehalose Metabolism-relative Genes In Bactrocera Minax

Bactrocera minax(Enderlein)is a devastating oligophagous pest of citrus.Diapause is a physiological phenomenon of insects into a state of arrested development under adverse environments such as high temperature,low temperature,drought,and food shortage.Trehalose is the major blood sugar in insects,which plays a crucial role in insect development and diapause.In this research,genes encoding trehalose-6-phosphate synthase(TPS),trehalose-6-phosphatase(TPP)and trehalase(Tre)were identified and characterized.The purpose of the research provided with a theoretical basis for further clarifying the molecular mechanism of diapause.The main results are as follows:1.Respiratory rate trajectory reveals occurrence of diapause in pupae of Chinese citrus flyThe respiratory rate of Bactrocera minax was measured throughout the pupal stage,and the respiratory rate trajectory demonstrated the occurrence of diapause in pupae.To our knowledge,this is the first direct evidence of diapause in B.minax pupae.In addition,respiratory rate trajectory of 20-hydroxyecdysone(20E)treated pupae showed that 20 E application averted the occurrence of diapause.2.Transcriptomic profiles of Bactrocera minax,along with pupal development provide insight into diapause programThe pupae at five developmental stages,pre-,early-,middle-,late-,and post-diapause,were collected for evaluating the variation of transcriptomic profiles by the next-generation sequencing technology.A total of 4,808 genes were significantly regulated in ten pairwise comparisons,representing the major shift in metabolism,signal transduction,endocrine system,and digestive system.The gene expression profiles were validated by qRT-PCR analysis.Moreover,the samples collected within maintenance of diapause(early-,middle-,and late-diapause)only showed marginal variation with each other in transcriptomic profiles.3.Molecular cloning and sequence analysis of trehalose metabolism-relative genes from Bactrocera minaxIn this study,we cloned and characterized five full length c DNA sequences of trehalose metabolism-relative genes,BmTPS,BmTPPB,BmTPPC-1,BmTPPC-2 and BmTre-1,respectively,in Bactrocera minax.The cDNA of BmTPS contains an open reading frame(ORF)of 2,445 bp,encoding 814 amino acid residues.The cDNA of BmTPPB,BmTPPC-1,and BmTPPC-2 contain open reading frame(ORF)of 825 bp,822 bp,and 822 bp,encoding 274,273,and 273 amino acid residues,respectively.The cDNA of BmTre-1 contains an open reading frame(ORF)of 1,665 bp,encoding 554 amino acid residues.The predicted molecular of BmTPS,BmTPPB,BmTPPC-1,BmTPPC-2,and BmTre-1 are 92.05 kDa,31.27 kDa,30.42 kDa,30.85 kDa and 63.78 kDa,the isoelectric point are 6.05,7.10,5.63,5.24 and 5.15,respectively.Phylogenetic analysis was performed by MEGA 5.05 with the neighbor-joining method,all species from the same order were clustered together.4.Gene expression,enzymatic activities,trehalose content and glucose content related to trehalose metabolism during diapause of Bactrocera minaxThe relative expression levels of BmTPS,BmTPP,and BmTre were determined by qPCR.The results showed that the expression patterns of BmTPS and Bm Tre-1 with the opposite trend,the expression level of BmTre-1 was lower after pupariation,while the expression level of BmTPS was higher after pupariation.Besides,the relative expression levels of BmTPPB,BmTPPC-1,and BmTPPC-2 have subtle differences among each other.And then BmTPS,BmTPP,and BmTre enzyme activities were measured in the whole pupal stage.The results showed that the activity of BmTPP followed a trend very similar to that of BmTPS.The concentration of trehalose maintained a high level during the whole pupal stage in Bactrocera minax,and the concentration of trehalose fluctuated over a small range.Interestingly,the level of glucose followed an opposing trend compared to the trehalose content in pupal stage.The activities of trehalose-6-phosphate synthase and trehalose-6-phosphatase were significantly up-regulated 30 days after injection of 20 E compared with control group.Moreover,trehalose and glucose contents were significantly decreased 30 days after injection of 20 E.5.Establishment of RNA interference system in Bactrocera minaxIn order to establish the appropriate system of RNA interference,we injected dsRNA into the chinese citrus fruit fly third-instar larvae,the injection of 2.0 or 2.5 ?g dsBmTPS caused high RNAi efficiency 12,24,and 48 h after injection,while only that of 2.0 ?g dsBmTPS was the most effective and maintain the RNAi efficiency for 72 h.Injection of double-stranded RNA into third-instar successfully silenced the transcription of BmTPS in B.minax,and thereby decreased the activity of TPS and trehalose content.Additionally,silencing of BmTPS inhibited the expression of three key genes in the chitin biosynthesis pathway and exhibited 52% death and abnormal phenotypes.Moreover,the 20-hydroxyecdysone(20E)induced the expression of BmTPS and three genes in the chitin biosynthesis pathway.