| The Chinese citrus fly,Bactrocera minax(Enderlein)is a devastating pest of citrus plants.It enters the obligatory diapause in overwintering pupae and arrest the physiological development to resist harsh environmental conditions and ensure the reproduction of the population.Trehalose is the blood sugar of insects and plays an important role in insect growth and diapause.This dissertation conducts in-depth research on metabolic profiles through pupal stage of B.minax,and the molecular characteristics and functions of three genes of trehalose-6-phosphophosphatase(TPPB,TPPC1,TPPC2).This research provides a theoretical basis for clarifying the molecular mechanism of diapause.The main findings are as follows: 1 Analysis of metabolic through pupal stage of B.minaxThe pupae at five developmental stages,pre-,early-,middle-,late-,and post-diapause,were collected for evaluating the variation of metabolomics profiles by NMR spectroscopy.Principal component analysis(PCA)and least squares discriminant analysis(PLS-DA)showed that the difference in metabolic profile was consistent with that of expression profile.They both indicated that the differences among the early-,middle-and late-diapause were marginal,while that between pre-diapause and post-diapause is remarkable.Nine metabolites proline,trehalose,N-acetylglutamate,succinic acid,glutamic acid,alanine,glycerophosphoryl choline,glutamine and 2-oxoglutaric acid,were found to contribute significantly to variations among metabolomic profiles by calculating the VIP value of each metabolite.Of 9 metabolites,proline and trehalose had much higher VIP values than other,and their concentration was maintained at a high level throughout the diapause stage.Proline and trehalose act as cryoprotectants during the winter,protecting individuals from cold damage.2 Patial and temporal expression patterns of BmTPPB,BmTPPC1 and BmTPPC2The expressions of BmTPPB,BmTPPC1 and BmTPPC2 were evaluated by qPCR and droplet digital PCR.The results showed that BmTPPB,BmTPPC1,and BmTPPC2 were expressed lower in the pupal stage than did in the larval and adult stages.In addition,the expression patterns of BmTPPB,BmTPPC1,and BmTPPC2 were slightly different during the whole developmental stage.BmTPPB is highly expressed in the epidermis,BmTPPC1 is mainly expressed in the malpighian tube,and the expression level in other tissues is close to zero,and BmTPPC2 is the highest in the fat body.3 TPPB,TPPC1,TPPC2 protein biological information analysisSequence analysis of TPPB,TPPC1 and TPPC2 proteins showed that it has a conserved region of the HAD superfamily: motif I-DXXX(T/V);motif II-(S/T)GX;motif III-K,Motif IV-(G/S)(D/S)XXX(D/N).The motif I sequence has a DxD characteristic,a highly conserved threonine or serine amino acid of Motif II,and Motif III is centered on a conserved lysine,which occurs around the N-terminal portion of the α-helix region,and Motif II and III contribute to the stability of the hydrolysis intermediate.Motif IV is characterized by a conserved acidic residue.The acidic residue of Motif IV typically exhibits one of three characteristics: DD,GDxxxD or GDxxxxD(where x is any amino acid).The marker for Bactrocera minax TPP Motif IV is represented by the GDxxxD acidic residue.4 Functional analysis of BmTPPB,BmTPPC1 and BmTPPC2 based on RNAi2.0 μg ds-BmTPPB,ds-BmTPPC1,ds-BmTPPC2 and mixed dsRNA were injected into the Chinese citrus fruit fly third-instar larvae.The qPCR was used to detect the silencing efficiency after injection.The results showed that the target gene silencing effect was obvious after 48 hours of injection,and there was no mutual interference and off-target effect.At the same time,the interference of BmTPP genes also led to a decrease in TPP enzyme activity and trehalose content,and a decrease in the expression of genes involved in the chitin synthesis pathway.5 Functional expression of recombinant enzymesRecombinant proteins of BmTPPB,BmTPPC1 and BmTPPC2 were expressed by Pichia pastoris expression system,respectively.The analysis of recombinant protein activity showed that TPPB had the strongest enzyme activity at 37.5°C and pH7.8,TPPC1 had the strongest enzyme activity at 27.5°C and pH7.4,while TPPC2 had the strongest enzyme activity at 35°C and pH7.4.The recombinant protein TPPC1 had higher affinity for substrates,and the enzymatic reaction rate of TPPC2 was the fastest.In summary,this dissertation analyzes the metabolic profiles of different diapause periods,this complements previous gene expression profiling studies.In addition,the sequence characteristics and spatiotemporal expression patterns of BmTPPB,BmTPPC1 and BmTPPC2 were comprehensively analyzed,and the functions of TPP gene were analyzed from the two layers of gene silencing and gene overexpression using RNAi and eukaryotic expression.The above results provide an important basis for excavating the important role of trehalose-6 phosphate phosphatase in the metabolism of insect trehalose and providing a data base for further understanding of the molecular mechanism of stagnation of Bactrocera minax. |
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