| The insect innate immune pathway functions via immune recognition,immune cascade,and immune effectors.As an important special economic insect,the silkworm has formed a relatively complete set of innate immune mechanism in the long co-evolution with microorganisms.Activation of innate immunity by microorganisms is initiated by pathogen-associated molecular patterns（PAMPs）,including peptidoglycan（PGN）,lipopolysaccharide（LPS）,mannan,andβ-1,3-glucan,which are bacterial and fungal cell wall and membrane components.After being recognized by pattern recognition receptors（PRRs）,PAMPs activate the immune cascade pathway and release immune effectors.There are numerous proteins in the hemolymph,nutrient-storage proteins and protease inhibitors are very abundant.Protease inhibitors have been reported to have antifungal effects in silkworms,and Kazal-type protease inhibitors（KPIs）have been reported to be antimicrobial in bees,jellyfish,crayfish and other species.In 2019,our team used proteomics to analysis the upregulation of protein components in the hemolymph of silkworms after induction by microorganisms,and found that Bm SPI65 was significantly upregulated after induction by E.coli and S.aureus.So does Bm SPI65,a KPI of silkworm,also have antimicrobial function?What is the molecular mechanism involved in the immune defense of the silkworm?At present,there is no report on the involvement of silkworm KPIs in immune defense.This study is expected to elucidate the biological function of Bm SPI65 and its molecular mechanism involved in the immune defense of silkworm.We studied the structural activity,expression and distribution characteristics,protease inhibitory activity,antifungal activity,and fungal binding characteristics of Bm SPI65.The main results obtained are as follows:1.Structural Activity and Physicochemical Properties of Bm SPI65Using bioinformatics analysis to analyze the sequence of Bm SPI65,it was found that Bm SPI65 is composed of three Kazal domains.Each domain contains 6 conserved cysteine residues,which are responsible for intramolecular disulfide bridge formation.The predicted signal peptide of Bm SPI65 is the 19 amino acid N-terminal sequence,indicating that Bm SPI65 might be extracellularly secreted.Based on the ORF sequence of Bm SPI65,specific amplification primers were designed for PCR amplification,and the expression vector p ET28a-Bm SPI65 was constructed and transferred into the expression strain BL（DE3）.The small amount expression test results showed that Bm SPI65 detected in the supernatant of BL21 cells after incubation at 16°C for 20 h.After the recombinant protein was subjected to nickel column affinity chromatography and gel filtration chromatography,a high-purity recombinant protein Bm SPI65 was obtained,and antibodies were prepared.The results of non-reducing electrophoresis showed that the recombinant protein Bm SPI65 formed a multimer,and Bm SPI65tended to exist as a multimer in the silkworm serum.The secondary structure of the recombinant protein Bm SPI65 was analyzed by circular dichroism spectroscopy.The results showed that Bm SPI65 mainly had anα-helical structure and was thermally stable.Using q PCR technology to analyze the expression characteristics of Bm SPI65,the results showed that Bm SPI65 was highly expressed on the day 5 and day 7 of fifth instar larvea,and specifically expressed in hemocytes.2.Bm SPI65 immune function researchUsing q PCR and Western blot to detect the expression characteristics of Bm SPI65after microbial induction,the results showed that Bm SPI65 was significantly upregulated in hemocytes and fat bodies after infection with E.coli,Staphylococcus aureus,Saccharomyces cerevisiae,Beauveria bassiana.By studying the antifungal activity of Bm SPI65,we found that it significantly inhibited the growth of three fungal spores of Candida albicans,Saccharomyces cerevisiae and Beauveria bassiana.The three-dimensional structure of homology modeling showed that the Bm SPI65 P1residues are Arg28,Tyr79,and Arg136.Analysis of Bm SPI65 inhibitory activity against seven proteases revealed that Bm SPI65 has significant inhibitory activity against trypsin,chymotrypsin,pronase,and proteinase K.However,inhibitory activity against subtilisin,thrombin,and papain was not detected.3.The molecular mechanism of Bm SPI65 in the silkworm immunityBy incubating Bm SPI65 with B.bassiana spores,we found that Bm SPI65 binds B.bassiana spores to aggregate them together.However,PI staining results showed that Bm SPI65 could not cause the death of B.bassiana.To identify the Bm SPI65 PAMP binding spectrum,binding assays were performed using two fungal polysaccharides,β-D-glucan and mannan.The results showed that Bm SPI65 binds to PGN and LPS,which are bacterial membrane components.The results also showed that Bm SPI65exhibited high,dose-dependent affinity forβ-D-glucan,but could not bind with mannan.Further,we found that Bm SPI65 binds to fungi by bindingβ-D-glucan on the fungal cell wall and promotes fungal spore aggregation,but Bm SPI65 does not bind mannan.Generally,N-linked glycosylation in insects contains abundant mannose.The specific PAMPs recognition mechanism of Bm SPI65 is a self-protection mechanism,which could avoid causing glycoprotein aggregation in silkworms.In order to investigate whether Bm SPI65 participates in the cellular immunity of silkworm,we cross-linked Bm SPI65 with agarose beads and incubated in hemocytes to simulate the binding of Bm SPI65 to PAMPs.The results showed that Bm SPI65 can promote the encapsulation and melanization of hemocytes.Pre-incubating Bm SPI65-coated beads with anti-Bm SPI65 rabbit antibody effectively blocked hemocyte encapsulation.In order to study the function of Bm SPI65 in cellular immunity,we added Bm SPI65 and fungal spores to hemocytes in vitro.The results indicate that Bm SPI65 recognizes and binds to fungi,promotes hemocyte aggregation and encapsulation,and helps hemocytes to phagocytose fungi. |
