Identification And Functional Analysis Of Genes Encoding Peptidoglycan Recognition Proteins In Bactrocera Dorsalis(Hendel)

Innate immune system is the main mechanism for insects to against pathogen.The innate immune system of insects includes humoral and cellular immunity and the humoral immunity produces antibacterial peptide,lectins,lysozyme and protease inhibitors,the pattern of cellular immunity is cell phagocytosis and coating.The main single pathways are Toll and Imd pathway in humoral immunity in insects.Toll signaling pathway is mainly involved in the immunization process to against Gram-positive bacteria,while Imd is mainly involved in the immune process for Gram-negative bacteria.Peptidoglycan recognition proteins(PGRPs)are very important in these two pathways and they are the front line of self-defense against infectious non-self.There are a lot of PGRPs with multiple functions in insects.The studies about PGRPs will help us to understand the innate immune system in insect and to carry out more scientific measures in pest control.As one of the most important pests in fruits and vegetables,little research focus on the PGRPs of Bactrocera dorsalis.In this study,the PGRPs are identified and analyzed from the databases.We detected the expression of PGRPs in different developmental stages of B.dorsalis by quantitative real time PCR(qRT-PCR).Combing the micro-injection technology,the change of PGRPs expression upon pathogen injection and the expression of PGRP-LC upon dsRNA injection and the survival rate after the Escherichia coli were detected.The result of this study will be helpful to comprehensively understand the PGRPs in B.dorsalis and lay the foundation for the further study of PGRPs.The main results are as follows: 1 The identification of PGRPs of B.dorsalis and the sequence analysisTwelve genes encoding 22 proteins were identified from the databases in NCBI and transcriptomes.Five of these genes had been idenfied with alternative splicing,in which PGRP-LA/LB/SC2 with 2,PGRP-LC with 4,PGRP-LD with 3 isoforms,respectively.The isoforms of PGRP-LC were more complex than others.In the phylogenetic,we found that the PGRPs of B.dorsalis were close to the Bactrocera oleae and Ceratitis capitata,followed by Drosophila melanogaster.In the function domain analysis of protein sequences,the conserved amino acid residues were found and amide enzyme activity of PGRP-LB,-SB1/2,-SC2,-SC2 b,-NSB and-SC were identified.All PGRPs,except PGRP-SA,PGRP-SCb and PGRP-NSC2,had Arg amino acid residue,which may be involved in the recognition of DAP-PGN(Pepridoglycan of Gram-negative bacteria).2 The expressions of PGRPs in different developmental stages of B.dorsalisThe differential expressions of PGRPs in different developmental stages showned that there were different functions in the growth process of B.dorsalis.The expressions of PGRP-LA/LB/LC were significant up-regulated in metamorphosis,but the expressions of PGRP-LE,-SB1 and PGRP-SC2/S2 b were mainly expressed in the adult and larval stages.The results of PGRP-SC2/S2 b indicated that the function of regulating the microorganism in mid-gut be similar to Drosophila.PGRP-SA was expressed in all stages,while PGRP-SD mainly in adult,however,the expression of PGRP-SCa has on regular in developmental stages.3 The inducible expression of PGRPs upon pathogens infection in B.drosalisThe inducible expression of PGRPs were ananlyzed upon E.coli,PGN-EB(Pepridoglycan from E.coli O111:B4)and PGN-SA(Pepridoglycan from Staphylococcus aureus)injection with qRT-PCR and micro-injection technology.The expressions of PGRP-LAa,PGRP-LB,PGRP-SC2 and PGRP-SC2 b were down-regulated in different time points when challenged by E.coli and PGN-EB.Combining the results of phylogenetic,we infered that these genes may be the negative regulator for Imd pathway.The expression pattern of PGRP-LE were up-regulated after induction sugggesting an important role in Imd pathway to sense Gram-negative bacteria in B.dorsalis.PGRP-SB1,PGRP-SB2,PGRP-SA and PGRP-SD were up-regulated upon E.coli and PGN-SA injection suggesting their roles in immune for Gram-negative bacteria and Gram-positive bacteria.4 Functional analysis of PGRP-LC in B.drosalisThe four isoforms were mainly expressed in mid-gut and fat body with q RT-PCR.After fed with antibiotics,the expressions of PGRP-LCa/b/c were extremely significantly changed,in which PGRP-LCa was down-regulated and PGRP-LCb/c were up-regulated.However,the expression of PGRP-LCd did not changed.The expressions of the four isoforms were significantly up-regulated upon the E.coli and PGN-EB infection,respectively.PGRP-LCd had the strongest response in the four isoforms.In Beauveria bassiana injection,the expressions of PGRP-LCc/d were down-regulated;while in PGN-SA injection,the expression of PGRP-LCb/d were down-regulated.Different functions of each isoforms in insect immune system had also been proved in B.dorsalis.PGRP-LCa was more widely in immune system and PGRP-LCd was more specific for immunization.The survival rates of B.dorsalis were down-regulated became after PGRP-LC were down-regulated by RNAi,suggested B.dorsalis were more sensitive to E.coli,especially PGRP-LCa/c/d down-regulated in the same time.