Functional Analysis Of BdNub In IMD Immunity And Intestinal Mocrobiota Homeostasis In Bactrocera Dorsalis

The innate immune system of insects can distinguish various pathogens that invade the insect body and quickly response.Insects’ humoral immunity mainly include Imd and Toll signaling pathways,while the delicate immune system still requires a variety of regulatory factors coordinated to achieve immune balance.Insect gut microbes have multiple functions to the host,which can provide nutrients and resist to pathogenic bacteria.In this study,the horticultural pest Batrocera dorsalis was selected as the candidate,database bioinformatics analysis found that there are two transcript variants of gene BdNub.Sequence analysis,functional domain prediction,the expression patterns of different tissues and periods of BdNub were studied.Molecular biological methods RNAi and q PCR were used to study the function of BdNub gene in the systemic immunity and intestinal immunity of B.dorsalis,and its role in the regulation of intestinal microbes.The main results are as follows:1.Sequence analysis and expression pattern study of BdNubThrough NCBI database analysis,we found that the BdNub gene has two alternative splicing modes,resulting in two transcript variants.The BdNub X1 transcript variant contains 5 exons,and the BdNub X2 transcript variant contains 4 exons.The two variants have a great overlap,and there are also some different fragments.The phylogeny showed that the BdNub is most closely related to the Bactrocera latifrons(pepper fruit fly).And the Pit-Oct-Unc(POU)and Homeodomain(HOX)domains of the BdNub are highly similar to those in Drosophila melanogaster.Functional domain prediction analysis showed that the isoforms of BdNub X1 and BdNub X2 have the same structural domain at the C-terminus of the protein,indicating that the different transcriptional isoforms have the same ability to bind to the target gene.There are some differences in the N-terminal sequence of the protein.This part of the results suggests that BdNub gene has a transcriptional regulatory function in B.dorsalis.qPCR was used to study the expression pattern of BdNub gene transcription levels,the results show that BdNub X1 and BdNub X2 are mainly expressed in the last pupal stage and the newly emerged adults among different developmental stages.In the expression pattern of different tissues of the adult,BdNub X1 had the highest expression in midgut.It was highly expressed in foregut and testis,while BdNub X2 was highly expressed in foregut and midgut.The high expression of BdNub gene in gut tissues suggested that BdNub has an important function in the intestinal immunity of B.dorsalis.2.Study on the function of BdNub X1 and BdNub X2 in the immunity of B.dorsalisAfter systemic infection of Gram-negative bacteria Escherichia coli in adults,the expression levels of Imd signaling pathway effect genes antimicrobial peptides Dpt,Cec,Att A,Att B,and Att C were significantly up-regulated,while BdNub X1 and BdNub X2 did not appear any immune response in the systemic infection 1-24 h post infection.The relative expression levels of antimicrobial peptide genes Dpt,Cec,Att A,Att B,and Att C were significantly up-regulated after intestinal 6h and 9h after infection of the gram-negative bacterium P.rettgeri.BdNub X1 was significantly up-regulated after 6h of intestinal infection with P.rettgeri,but did not appear to be significantly up-regulated in other time periods.BdNub X2 was significantly up-regulated at 6h and 9h of intestinal infection,and it was up-regulated by 1.61-1.69 folds compared with the control group.Through the RNAi of BdNub X1 and BdNub X2,the function of genes in immunity was analysed.The results showed that the expression level of antimicrobial peptide genes after the system infection of the test insects after RNAi has no significant difference with the control group after system infection,indicating that BdNub X1 and BdNub X2 does not participate in the regulation of the antimicrobial peptides of the Imd pathway of B.dorsalis.After intestinal infection of the test insects after RNAi,the expression levels of antimicrobial peptide genes Dpt,Cec,Att A,Att B and Att C in the BdNub X1 group were significantly down-regulated after intestinal infection 6 hours with those in the control group after intestinal infection.The expression levels of antimicrobial peptide genes Dpt,Cec and Att C in the BdNub X2 group 6h after intestinal infection were significantly up-regulated by 1.98-2.59 folds compared with those in the control group after intestinal infection.This part of the results indicates that BdNub X1 regulates AMPs in B.dorsalis intestinal immunity as transcriptional activator,and BdNub X2 regulates antimicrobial peptides in B.dorsalis intestinal immunity as transcriptional repressor.Both RNAi of BdNub X1 and BdNub X2 made B.dorsalis more sensitive to P.rettgeri intestinal infection,showing a significant reduction in survival rate.3.Study on regulation of BdNub X1 and BdNub X2 on intestinal microbial community structureThrough RNAi of BdNub X1 and BdNub X2,the role of BdNub gene in the regulation of intestinal microbes of B.dorsalis was studied.The results showed that 5d after BdNub X1 RNAi,the total intestinal bacteria was significantly up-regulated by 1.39 folds,the abundance of Enterobacteriaceae did not change significantly,while the abundance of Pseudomonas and Salmonella were significantly up-regulated by 1.95 times and 3.42 folds.3d and 5d after RNAi of BdNub X2,the total intestinal bacteria were significantly down-regulated by 43.6%-59.9%,and the abundance of Enterobacteriaceae was significantly down-regulated by 45.7%-46.3%,Salmonella was significantly up-regulated.This part of the results suggested that the knockdown of the two transcriptional isoforms of the BdNub gene caused changes in the structure of the intestinal microbial community,indicating that the BdNub gene was involved in the regulation of the homeostasis of the gut microbial community.