| Insects are the most abundant animal group on the earth.The hemolymph in their body flows in the space between various tissuess and forms an open circulatory system to play the roles of material transportation,nutrient metabolism and immune defense.The silkworm is one of the important economic insects.After thousands of years of domestication,it has also become an important model insect.The hemolymph of silkworm contains a lot of proteins,such as 30 K protein,hemolymph proteases,protease inhibitors,and nutrient storage proteins.Among them,serine protease inhibitors(SPIs),as important regulatory factors,play an important role in the growth,development and immune process of silkworm.At present,most research reports on serine protease inhibitors are the Serpin family,and there are few studies on the function of other families such as Kunitz,Kazal and the mucus family.In our previous study,80 serine protease inhibitors wre identified from the silkworm genome.SPIs of silkworm have a wide range of distribution characteristics.For example,Bm SPI38 and Bm SPI39 are expressed in silk glands and can resist fungal infection,while Bmserpin5 and Bmserpin32 are distributed in hemolymph and participate in the regulation of melanization in humoral innmunity of silkworm.In previous studies,many SPIs were found in the hemolymph of different silkworm strains,several of them belonged to chymotrypsin inhibitor(CI),which was named CI-1～CI-13 according to their electrophoretic mobility.At present,there are relatively few studies on CIs in the silkworm.Although there are many reports of CIs natural isolation and purification,the understanding of their functions are still very limited.Based on previous studies in our laboratory,we found that CI-13 is a Kunitz type SPI,which is widely distributed in various silkworm strains and has a high abundance in hemolymph.SPIs have a wide range of regulatory roles in organisms.However,the function of silkworm CI-13 is still unknown so far.Therefore,it is necessary to further study the function of the Kunitz type SPIs,CI-13,which is highy expressed in various silkworm strains.In this study,Bombyx mori was used as the research object,and the proteins containing Kunitz domain of silkworm were identified by bioinformatics methods.CI-13 was further studied through gene cloning,protein expression purification,transgenic manipulation,in vitro physiological and biochemical experiments.The main results of this study are as follows:1.Identification and analysis ofproteins containing Kunitz domain in silkwormAccording to Kunitz’s Pfam area number,we used HMMER software to construct a hidden Markov model and searched for Kunitz-containing proteins in silkbase,silk DB3.0,and NCBI databases of silkworms.A total of 18 Kunitz-containing proteins were identified in silkworms.The p H,molecular weight,and tissue distribution characteristics of these proteins are quite different.Among them,CI-13 is highly expressed in this family,which contains a Kunitz domain.It is a secretable protein with a predicted molecular weight of 9.61 k Da.Its P1 active site is phenylalanine,and there are 6 conserved cysteines in Kunitz domian.The simulation of the three-dimensional structure shows that CI-13 is composed of a C-terminal α helix and two anti-parallel βsheets.The phylogenetic tree analysis of Kunitz type CIs in silkworm and other insect species showed that CI-13 has a close relationship with silkworm CI-b1 and the tissue factor pathway inhibition evolutionary relationship in wild silkworm,while the members of other species,such as flies,Spodoptera litura,Helicoverpa armigera,etc.,are divided into different branches due to their distant evolutionary relationship.2.Prokaryotic expression,purification and expression characterization analysis of silkworm CI-13We successfully cloned CI-13 from fat body c DNA of silkworm.The encoding region of CI-13(without the signal peptied)was successfully linked with the p28 expression vector and then transferred it to BL21 transformed cells.After induction,CI-13 was mainly expressed in the form of inclusion bodies at 37°C.We obtained the soluble form of recombinant CI-13 protein through urea denaturation and refolding in a dialysis bag.To obtain the protein,we used nickel column affinity chromatography and gel filtration chromatography to purify the recombinant CI-13 protein.In order to verify the activity of the refolded and purified recombinant CI-13 protein,we performed active gel staining and found that the recombinant CI-13 protein had obvious inhibitory activity on chymotrypsin.In order to analyze the stability of CI-13,the protein was treated with different temperatures and p H,and it was found that it still had inhibitory activity.Morever,the α helix in the secondary structure of CI-13 protein was found to a certain extent after high temperature treatment by circular dichroism(CD)analysis,which further showed that CI-13 protein has good stability.Inhibition activity analysis showed that CI-13 can significantly inhibit chymotrypsin activity,but has a weak inhibitory effect on trypsin activity.In addition,the expression profiles analysis showed that CI-13 was highly expressed in fat body and hemolymph,which was continuously expressed in from the fat body from the third day of the fourth instar to the first day of pupa.CI-13 was mainly synthesized and secreted from the fat body to the hemolymph,and distributed on the surface of hemocytes.These results suggest that CI-13 may play a role in silkworm immune tissues such as fat body and hemolymph.3.Research on the immune function of silkworm CI-13After injecting pathogen-associated molecular patterns(PAMPs)into the silkworm body,it was found that the expression of CI-13 in the fat body was up-regulated,suggesting that it may participate in the silkworm immune process.In order to test whether CI-13 can participate in the regulation of antimicrobial peptide gene expression,we injected CI-13 recombinant protein and induced the immune pathway with Micrococcus luteus,and found that CI-13 can positively regulate the expression of antimicrobial peptides.Further Elisa and Western blot experiments proved that CI-13 protein can bind to pathogens,but cannot directly inhibit the growth of bacteria and fungi as immune effectors.Subsequently,we used CRISPR/Cas9 technology to knock out CI-13.Through quantitative PCR and Western blot detection,it was found that after knocking out CI-13,the expression of antimicrobial peptides in the fat body and hemolymph was significantly reduced.After being challenged with different types of bacteria and fungi,the survival rate of the silkworms in the CI-13 knockout group was lower than that of the control silkworms.The effect of CI-13 on phenol oxidase(PO)activity was detected by enzyme activity test,and it was found that recombinant CI-13 protein can significantly inhibit the PO activity induced by pathogen-related molecular patterns.Similarly,we tested the activity of PO in the hemolymph of the knockout group in the same way,and found that the activity of PO in the hemolymph of the knockout group was significantly higher than that in the control group,which implies that the silkworm CI-13 can inhibit the activity of PO in the hemolymph.To regulate the level of blackening reaction.Based on the above research results,we speculate that Bombyx mori CI-13 can not only be used as an immune recognition molecule to positively regulate the expression of antimicrobial peptides,but also can act as an immune homeostasis regulator by regulating the level of melanization. |
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