Cloning,Expression And Functional Analysis Of GRP-LE Gene In Musca Domestica

Invertebrate innate immunity?Innate immunity?system is initiated by pattern recognition receptors?PRRs?in the body to identify pathogen-associated molecular patterns?PAMPs?on the surface of invading pathogenic microorganisms.Peptidoglycan recognition proteins?PGRPs?are a family of proteins used to recognize peptidoglycan?PGN?in the innate immune system of animals and play an important role in activating the immune pathway.In this study,we took Musca domestica as the research object,cloned the MdPGRP-LE gene,studied a systematic bioinformatics analysis of the PGRP family of Musca domestica.In addition,the function of MdPGRP-LE in the innate immunity of Musca domestica was studied at the molecular level.The findings are as follows:1.We screened 26 MdPGRP genes,including 8 PGRP-L and 18 PGRP-S.The analysis results showed that most of PGRP-S had signal peptides,except for MdPGRP-LB,there were no signal peptides in other PGRP-L gene members.In addition,MdPGRP was distributed on different scaffolds in the housefly genome and showed an uneven distribution.PGRP-L generally has more exons than PGRP-S.2.Analyzing the amino acid sequence of the MdPGRP protein family,it was found that there were differences in the physicochemical properties and motif composition of the family members.In addition,sequence alignment analysis showed that PGRP-LE of Musca domestica had high similarity to the amino acid sequence of PGRPs found in Drosophila melanogaster and Bombyx mori and T7 lysozyme.Bioinformatics predicts that it contains five key sites for amidase activity,but the key residue Cys?cysteine?is mutated to Ser?serine?.3.Using qPCR detection technology,MdPGRP-LE was found to have the highest expression in the intestine of the housefly and the second instar stage of larvae.After bacterial stimulation,the transcription level of MdPGRP-LE gene showed a trend of first up-regulation and then down-regulation.4.After 30 minutes of feeding GFP expressing host bacteria?pGFP-ECO?to the larvae of the MdPGRP-LE gene interference experiment,it was found that the fluorescence intensity decreased significantly with time.5.After knocking down the MdPGRP-LE gene using RNAi technology,the expression levels of transcription factor?Dorsal?and antimicrobial peptide?Muscin?were up-regulated and the expression levels of transcription factor?Relish?and other antimicrobial peptides?Attacin,Diptericin,Cecropin?were down-regulated.After the MdPGRP-LE gene was knocked down and stimulated by E.coli,the expression levels of 2 transcription factors?Dorsal,Relish?and 4 antibacterial peptides?Attacin,Diptericin,Cecropin,Muscin?were down-regulated,and the expression of Relish and 4 antibacterial peptides was significantly down-regulated;while the expression of 2 transcription factors and 3 antibacterial peptides?Attacin,Diptericin,Cecropin?were down-regulated by S.aureus stimulation,except for the antimicrobial peptide?Muscin?gene.6.MdPGRP-LE interference can significantly reduce the phenoloxidase activity in Musca domestica larvae.7.After feeding bacteria?E.coli and S.aureus mixed 1:1?to knock-down MdPGRP-LE expression,the survival rate of the MdPGRP-LE interference group was significantly lower than that of the control group?GFP interference group?,but the pupation time and weight of the larvae did not change significantly compared with the control group.8.In vitro recombinant protein rMdPGRP-LE was obtained through prokaryotic expression,and the activity of rMdPGRP-LE binding bacteria was detected by Western blot.The results showed that rMdPGRP-LE could be combined with Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Serratia marcescens and Salmonella typhimurium,respectively.ELISA test found:rMdPGRP-LE is combined with two peptidoglycans?PGN-EK,PGN-SA?,lipopolysaccharide?LPS?,galactose?Galactose?,mannose?Mannose?and glucan?Glucan?in a dose-dependent manner,which has strong binding activity to Galactose and Mannose,and relatively weak binding to Glucan.9.In the presence of Ca²⁺,rMdPGRP-LE caused agglutination of Escherichia coli,Staphylococcus aureus,Bacillus subtilis,Serratia marcescens and Salmonella typhimurium,respectively.10.In the presence of Zn²⁺,rMdPGRP-LE has no amidase activity on peptidoglycan?PGN-EK,PGN-SA?.11.In the presence of Zn²⁺,rMdPGRP-LE has antibacterial activity against both E.coli and S.aureus.