Analysis Of Expression Profiles And Metabolome Differences Between Diapause Pupae And Non-pause Pupae In Bactrocera Minax

The Chinese fruit fly,Bactrocera minax(Diptera: Tephritidae),is an oligophagous pest of citrus fruits.Diapause,a physiological phenomenon in which insects and their related arthropods stop developing at specific development stages,plays an important role in coordinating the relationship between organisms and the environment.In view of the complexity of the diapause mechanism,it is necessary to analyze the differences between the diapause pupae(D)and the non-diapause pupae(ND)of B.minax.In this study,Chinese fruit fly is the research object.Based on the differences between D pupae and ND pupae,we used transcriptome and metabolome technology to analyze the pupae of Chinese fruit fly in different stages,and also studied the molecular characteristics and functions of TTLL3 B gene,so as to enrich the further understanding of diapause mechanism of Chinese fruit fly.The main findings of this dissertation are as follows:1 Analysis of metabolic in D pupae and ND pupae of Chinese fruit flyIn this paper,NMR technique was used to studied the metabolic profile differences between D pupae and ND pupae of Bactrocera minax on the same day and at different stages,and the metabolites with significant difference in different comparisons,as well as their potential effects were also explored and analyzed.The results showed that the metabolic profiles of D pupae were significantly different among the three stages,namely,1 d,15 d and 30 d.It also showed that the metabolic profiles of ND pupae with20 E injection were not of significant difference between 15 d and 30 d,but both of them were of significant difference with that of 1 d after injection.At the three stages,D1-ND1 、 D15-ND15 and D30-ND30,the metabolic profiles of D pupae were all significantly different from those of ND pupae.By calculating the VIP value,it was confirmed that the 21 metabolites contributed the most to the differences between the metabolome groups.Among them,citric acid,glucose,maltose,proline,trehalose,glutamic acid,asparagine,and N-acetylglutamic acid had the greatest number of showing significant difference,and the VIP value was much higher than other metabolites.Among the metabolites with the most significant differences,citrate,glucose,maltose,proline,trehalose,glutamate,and asparagine can directly or indirectly enter the tricarboxylic acid(TCA)cycle,indicating that the energy metabolism and substance transformation had significant changes during diapause.In addition,thesignificant differences of trehalose,proline,N-acetylglutamate,and inositol contribute to the regulation of freezing tolerance during diapause.2 Analysis of transcriptome in D pupae and ND pupae of Chinese fruit flyTranscriptome sequencing was used to sequence the D pupae and ND pupae at different stages,and a data size of 15 G was obtained.The number of the sequences was108564,the length 356974766 bp,and the N50 reached 3868 bp.All the sequences were submitted to NCBI nr,GO,KEGG,eggNOG,Swissprot databases for prediction.Finally,85,494,21,693,6,828,77,828,58,878 were respectively annotated in nr,GO,KEGG,eggNOG,Swissprot database,meanwhile,4,536 in nr,GO,KEGG,eggNOG,Swissprot database.In the NR annotation,it was found in species homology analysis that the Bactrocera has the highest degree of sequence homology,accounting for76.94%.And the similarity of 40.14% sequence surpassed 60%,in which that of 8.29%reached95%.A total of 21,693 sequences in the GO database were successfully annotated into 54 secondary classifications,with the largest number of cellular process,binding,and catalytic activity.A total of 6,828 sequences in the KEGG database were successfully annotated into 33 pathways,With the largest number of signaling transduction,followed by translation.A total of 77,828 sequences in eggNOG were successfully annotated into 25 secondary classifications,with the greatest number of general function prediction,signal transduction mechanism and post-translational modification / protein conversion / associated protein.Differential analysis of gene expression by using DESeq showed that D15-ND15 obtained the most differentially expressed genes,and a total of 2,485 genes had significant changes,with an increase of621 and a decrease of 1,864.It is shown that D30-ND30 has the smallest differentially expressed genes,with only 19 genes in total,and all of them were up-regulated.3 Analysis of differential gene expression between D pupae and ND pupae of Chinese fruit flyBased on the transcriptome data,the expression patterns of genes highly related to diapause were analyzed by QRT PCR and semi quantitative PCR,and 7 genes were selected.The 7 genes were cyp6a9,fru,kspi,lyz1,MSta,ttll3 b and UC2.The results of QRT PCR and semi quantitative PCR showed that there were significant differences between D pupae and ND pupae.Among them,genes of cyp6a9,fru,MSta,ttll3 b andUC2 were positively correlated with diapause,while kspi and lyz1 were negatively correlated with diapause.4 Cloning and sequence analysis of TTLL3 B gene of Chinese fruit flyThe full-length TTLL3 B gene cDNA was cloned by RACE technology and named as TTLL3B-1,TTLL3B-2,and TTLL3B-3.The cDNA of the three genes contained open reading frame of 2097 bp,2034bp and 2121 bp,encoding 699,678 and 707 amino acid residues.The predicted molecular of TTLL3B-1,TTLL3B-2,and TTLL3B-3 were80.90 kDa,78.56 kDa and 81.70 kDa,and the isoelectric point are 9.44,9.36 and 9.44 respectively.Phylogenetic analysis showed that TTLL3 B gene and TTLL3 B of all Diptera were grouped into one.qRT-PCR was applied to analyze the expression levels of TTLL3 B in pupa and adult tissues of Chinese fruit fly.The results showed that TTLL3 B was expressed during the whole pupa period,and the expression levels of TTLL3B-2 and TTLL3B-3 were similar.With the deepening of diapause,its expression level increased gradually.The highest expression level was from the 45 th day to the105 th day,and it decreased gradually at the end of the diapause.In different tissues,the expression of TTLL3B-2 was found highest in fat body,followed by midgut.The expression of TTLL3B-3 was found in the highest level in cuticula,followed by fat body and midgut.