Preliminary Study Of Peptidoglycan Recognition Protein BmPGRP-L4 In Silkworms

Bombyx mori is an important economic insect.It is easily infected during the process of mulberry planting and silkworms raising,which has an impact on the silk industry.Innate immunity is the only protective barrier for insects,and the molecular mechanism of innate immunity of silkworm is the key to the prevention and control of silkworm disease.As an important immune factor in innate immunity,peptidoglycan recognition protein plays a key role in identifying pathogens,activating the Toll pathway and IMD pathway,and regulating the immune response in insects.In this study,in order to clarify the role of the peptidoglycan recognition protein Bm PGRP-L4 in the innate immune response of the silkworms,molecular biology techniques were used to preliminarily explore the function of Bm PGRP-L4.In this study,the complete ORF of Bm PGRP-L4 gene was cloned using RT-PCR and TA cloning technology.Gene sequence and encoded amino acid sequence of Bm PGRP-L4 gene were analyzed by bioinformatics.The expression profile of Bm PGRP-L4 gene were detected by Real-time quantitative PCR.Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus were fed and injected to 3^rdday of 5^thinstar silkworm larvae.The relative expression level of Bm PGRP-L4 in different tissues at different time points post treatment was detected by Real-time quantitative PCR.Using RNA interference?RNAi?technology,the double-stranded RNA was injected to 5^thinstar silkworm larvae,and the relative expression level of Bm PGRP-L4 gene in different tissues at different time points were detected by Real-time quantitative PCR.The Bm PGRP-L4 expression vector was constructed,Bm PGRP-L4 protein was expressed,the expression time was optimized,and the bacterial components of Bm PGRP-L4 protein was explored.In this study,Bm PGRP-L4 gene sequence from silkworm larvae was obtained.Its full ORF is 915 bp in length,encoding 304 amino acids which contains a transmembrane domain,a PGRP functional domain,and an amidase-2 functional domain without signal peptide.The amino acid sequence of Bm PGRP-L4 has the highest identities with Bm PGRP-L2,and the two Bm PGRPs have the closest genetic relationship with the amino acid sequence of Ms PGRP-SC2 of Manduca sexta.Bm PGRP-L4 lacks four residues?Y,H,T,C?of five Zn²⁺dependent binding sites,without amidase activity.The expression profile showed that Bm PGRP-L4 was expressed in the epidermis,fat body and midgut at different developmental stages,and the expression level in silk glands was extremely low,of which was mainly expressed in high abundance in the midgut of 5^thinstar larva.After feeding of E.coli and S.aureus,Bm PGRP-L4 gene was up-regulated significantly in the midgut and epidermis,with significant up-regulation at 6 h,12 h and 24 h.In the fat body,the expression of Bm PGRP-L4gene was up-regulated at 24 h.In silk glands,the expression of Bm PGRP-L4 gene was up-regulated at 12 h.After injection of E.coli and S.aureus,the expression level of Bm PGRP-L4 gene was slightly up-regulated 24 h after S.aureus injection in the epidermis.The expression of Bm PGRP-L4 gene was up-regulated when fat body were injected with E.coli for 6 h.In the midgut and silk gland,the relative expression level of Bm PGRP-L4 gene was up-regulated 24 h after E.coli injection.The results of RNAi showed that in the epidermis RNAi effect appeared 48 h;no RNAi effect in the fat body;and the RNAi effect in the midgut was the best,in which the gene suppression expression appeared at three time points of 12 h,24 h and 48 h.Through the prokaryotic expression system of E.coli,Bm PGRP-L4 protein could be induced expressed by IPTG,and the protein expression level increased with the increase of time in the four time points of 1 h,2 h,4 h,and 8 h,and mainly expressed in cell pellets.In summary,Bm PGRP-L4 gene was expressed in different tissues at different ages,and it could be induced to up-regulate the expression at different time points in different tissues after bacterial feeding and injection infection.It is speculated that Bm PGRP-L4 gene may be involved in the immune response of silkworm larvae to recognize bacteria.After RNAi,the expression level of Bm PGRP-L4 gene was suppressed.Bm PGRP-L4 protein could be induced in prokaryotic system and expressed in cell pellets.The above results laid a data foundation for the further study of Bm PGRP-L4 receptor protein involved in immune response and other functions in silkworms.