Preliminary Study On The Functions Of BmPGRP-S1 And BmPGRP-S4 In Bombyx Mori

In the long-term evolution process,insects form a complete immune system,the innate immune system,to protect against microbial infections.The innate immunity of insects mainly relies on pattern recognition receptors to identify pathogen-associated molecular patterns of pathogens,and these pathogen-related molecular patterns exist only in microorganisms and are not present in hosts,such as lipopolysaccharide,peptidoglycan,lipoteichoic acid,etc.Among them,peptidoglycan recognition protein as an important pattern recognition receptor can recognize peptidoglycan,which is the main component of the cell wall of pathogenic microorganisms.So far,the study of peptidoglycan recognition proteins is relatively thorough in fruit flies,but there are few studies on peptidoglycan recognition proteins in silkworm.In this paper,we mainly studied the peptidoglycan recognition proteins of silkworm,namely BmPGRP-S1 and BmPGRP-S4.The results are as follows:1.Bioinformatics was used to analyze BmPGRP-S1 and BmPGRP-S4.The sequence numbers of these two genes were identified as NM-001043371 and XM-004928822,respectively.They encode 196 and 199 amino acids respectively,and all have signal peptides and a conserved PGRP domain,without transmembrane domain.Sequence alignment and phylogenetic tree analysis showed that the two genes were highly conserved in insects.The gene transcription analysis of BmPGRP-S1 and BmPGRP-S4 in different developmental stages and tissues or organs of silkworm showed that BmPGRP-S1 was mainly expressed in 5th instar larvae and fat bodies,while BmPGRP-S4 was mainly expressed in early larvae and hemolymph of silkworm.2.HE staining technique was used to study the midgut tissue of silkworm infected with bacteria.It was found that with the prolongation of bacterial infection time,intestinal tissues gradually developed pathological changes,which showed the enlargement and gradual exfoliation of cell nuclei,the rupture of midgut tissues,until the midgut tissues gathered into clusters,and no complete cell structure appeared,which eventually led to the death of silkworm.Immunohistochemical experiments showed that BmPGRP-S1 protein was expressed in the cytoplasm and evenly distributed throughout the intestinal cavity under bacterial infection.3.Quantitative Real-time PCR was used to study the changes of gene at transcriptional level.It was found that the expression of immune related genes(Imd and Toll pathway related genes and antimicrobial peptide genes)and BmPGRP-S1 and BmPGRP-S4 genes were induced by bacterial infection.BmPGRP-S1 participates in the innate immune process of silkworm,and the expression of BmPGRP-S1 is different in different tissues induced by bacteria.At the same time,for the same tissue,the expression of BmPGRP-S1 is different due to the sensitivity of different microorganisms.BmPGRP-S4 is mainly responsive to bacterial infection in the hemolymph,but when the silkworm is infected with E.coli,there is no significant change in BmPGRP-S4 expression compared with other bacteria.4.Five kinds of antimicrobial peptide gene promoters were constructed on PGL3 vector by double luciferase reporter gene method,and the activity of promoters was detected by measuring luciferase activity.It was found that BmPGRP-S1 and BmPGRP-S4 proteins could activate the promoter of antimicrobial peptide,which indicated that BmPGRP-S1 and BmPGRP-S4 proteins could activate the expression of antimicrobial peptide gene.5.Using RNA interference technology,the expression of BmPGRP-S1 gene was successfully knocked down in silkworm and midgut tissues,and the expression of BmPGRP-S4 gene was successfully knocked down in hemolymph.The results show the expression of CecA,Glv1,Leb3 and Mor decreased significantly after RNA interference of BmPGRP-S1 gene in silkworm body and midgut tissues,while the expression of Dredd and FADD genes involved in Imd pathway decreased,but had no effect on the expression level of Myd88 and Tube genes involved in the Toll pathway.It is speculated that BmPGRP-S1 protein participates in the activation of Imd pathway in silkworm innate immunity,and produces antimicrobial peptides by activating Imd pathway,thus killing the invading bacteria.After RNA interference of BmPGRP-S4 gene in silkworm haemolymph tissue,the expression of CecA,Leb3 and Morantimicrobial peptide genes increased significantly,while the expression of FADD gene in Imd pathway increased,but the expression of Pelle and Tube genes in Toll pathway was not affected.It is speculated that BmPGRP-S4 protein may inhibit the Imd pathway and inhibit the expression of antimicrobial peptides by inhibiting the activation of the Imd pathway,thus preventing the over-activation of the Imd pathway to damage the host.