Study On Immune Functions Of C-type Lectin CTL10 In Silkworm,Bombyx Mori

Insect C-type lectins（CTLs）belong to a large family of pattern recognition receptorswith a conserved carbohydrate recognition domain（CRD）.CTLs usually recognize different glycosyls by the Ca²⁺-dependent mechanism and play important roles in regulating the humoral and cellular immune responses.Previous studies have shown that Bm CTL10 is an immulectin containing two carbohydrate recognition domains（CRDs）in the silkworm,Bombyx mori.CTLs can bind various pathogenic microorganisms and pathogen-associated molecular patterns（PAMPs）.However,the functions of Bm CTL10 as an opsonin to enhance hemocyte-mediated cellular immune responses and its other functions in innate immune responses have not been reported.Therefore,this study focuses on Bm CTL10-mediated immune responses in silkworms.Firstly,gene cloning,sequence and phylogenetic tree analysis of Bm CTL10 were conducted.The expression patterns of Bm CTL10 in silkworm tissues and different developmental stages,and the expression patterns induced by pathogenic microorganisms and 20-hydroxyecdysone（20E）were analyzed.The agglutination of microorganisms by Bm CTL10 and the binding of Bm CTL10 to bacteria,fungi,PAMPs and hemocytes were determined.The regulatory effect of Bm CTL10 on the expression of immune genes in hemocytes was analyzed by RT-q PCR.Finally,the interaction between Bm CTL10 and silkworm integrinβsubunits was analyzed by yeast two-hybrid experiment.The main results are as follows:1.Analysis of sequence and phylogenetic tree:BmCTL10 gene has a 972 bp open reading frame,which encodes a 323 aa immunolectin containing two carbohydrate recognition domains（CRDs）.Phylogenetic analysis of amino acid sequences showed that Bm CTL10 had a distant evolutionary relationship with homologous immulectins in B.mori.2.Expression patterns in different tissues,developmental stages and induced expression patterns:The q PCR analysis showed that Bm CTL10 was mainly expressed in second to fifth instar larvae,wandering stage larvae,prepupa,and adults.In fifth instar larvae,Bm CTL10 was predominantly expressed in the fat body and epidermis.The expression of Bm CTL10 was significantly downregulated in second instar larvae immersed in Beauveria bassiana.BmCTL10 was upregulated by E.coli and S.aureus in the epidermis of the fifth instar larvae.Bm CTL10 was upregulated in hemocytes and epidermis by B.bassiana conidia.In the fat body,20-hydroxyecdysone upregulated Bm CTL10 at 6 h and downregulated at 12 h.3.The immune functions of recombinant protein（rBmCTL10）in vitro.Agglutination experiments showed that the agglutination of Escherichia coli,Staphylococcus aureus,Bacillus subtilis and Serratia marcescens by r Bm CTL10 was enhanced with Ca²⁺.While the agglutination was weakened by EDTA.r Bm CTL10 can bind to silkworm hemocytes and various pathogen-associated molecular patterns to enhance hemocyte-mediated nodulation,phagocytosis and encapsulation.4.Regulation of immune genes by BmCTL10:Injection of r BmCTL10 caused significant upregulation of most antimicrobial peptide（AMP）genes and Bm NOS1 genes in larval hemocytes.However,Bm Def,Bm PPO1 and Bm PPO2 were significantly downregulated.5.Interaction between BmCTL10 and integrin:Yeast two-hybrid demonstrated that integrinβ3 andβ4 subunits can interact with Bm CTL10.Further analysis showed that only CRD2 can interact withβ3,while both CRD1 and CRD2 can interact with theβ4.Taken together,this study demonstrates that BmCTL10 has multiple functions in the innate immune responses of silkworm,and two integrinβsubunits are the potential receptors.The relevant research results laid a foundation for in-depth study on the role of Bm CTL10 in the immune defense system of silkworm,and provided new ideas for the prevention and control of silkworm disease,as well as ideas for the development of biological control measures.