Functional Study On The Silkworm Class B Scavenger Receptor BmSRB3 In Cellular Lipid Transport And Immune Response

There are a variety of pattern recognition receptors(PRRs)in insects.Scavenger receptors(SRs),as a class of PRRs with diverse structures and biological functions,are involved in the regulation of insect immune defense,lipid transport,pheromone perception and other physiological processes are of great significance to the growth and development of insects.The silkworm(Bombyx mori)is an important economic insect of Lepidoptera and one of the important model organisms in the field of entomology.Bombyx mori scavenger receptors are divided into A,B and C classes,of which the B class scavenger receptor has the most members and the most diverse functions.Studies have shown that class B scavenger receptors play important functions in the silkworm humoral immunity and lipid transport.Bm SRB3(Cameo1)is the only tandem gene of Cameo2 on the C gene of the yellow cocoon,which interacts with the carotenoid transporter CBP on cells,but they are not involved in the transport of lutein and β-carotene.As a member of the silkworm class B scavenger receptor,although Bm SRB3 is not involved in the transport of carotenoids in mulberry leaves,is it involved in the transport of other lipids? Could Bm SRB3 function as a pattern recognition receptor in innate immunity? Based on this,this study took the silkworm class B scavenger receptor Bm SRB3 as the research object,combined with bioinformatics analysis,molecular biology and physiological and biochemical techniques in vitro and in vivo,and started from lipid transport and immune defense.The function of Bm SRB3 was explored,and the main findings obtained are as follows:1.Bioinformatics and expression analysis of Bm SRB3The sequence and structure of Bm SRB3 were analyzed by the method of bioinformatics.The Bm SRB3 protein consists of 495 amino acids and is a secondary transmembrane protein.Outer loop CD36 domain.Evolutionary analysis showed that among lepidopteran insects,silkworm Bm SRB3 and moths clustered into one clade,and butterflies clustered in different clade due to their distant evolutionary relationship.The prokaryotic expression vector p ET28a-r SRB3 was constructed,the soluble r SRB3 was obtained by gradient renaturation and nickel column affinity chromatography,and its polyclonal antibody was prepared.The p SL1180-Op IE2-Bm SRB3 cell overexpression vector was constructed,and the subcellular localization showed that Bm SRB3 was localized on the cell membrane.The spatiotemporal expression profiling analysis showed that Bm SRB3 had the highest expression level in the midgut of the silkworm,followed by blood cells and fat body,and its expression level was significantly higher in the midgut feeding period of larvae than in the dormant period.These results suggest that Bm SRB3 may function in the silkworm midgut and blood cells.2.The role of Bm SRB3 involved in the transport of lipids and vitamins in the midgutTo investigate whether Bm SRB3 is involved in the transport of nutrients in mulberry leaves in the midgut,the effect of starvation treatment on the expression of Bm SRB3 was first analyzed.The results showed that the expression of Bm SRB3 was significantly decreased in the midgut and significantly increased in the fat body after starvation treatment.We speculate that Bm SRB3 may be involved in nutrient absorption and mobilization in the midgut and fat body.Subsequently,immunofluorescence localization analysis showed that Bm SRB3 was mainly localized on the inner surface of the intestinal lumen in the anterior part of the midgut,and was located on the brush border and the tonoplast inside the goblet cells in the posterior part of the midgut,suggesting that Bm SRB3 may be located in the midgut.Involved in the transport of nutrients.Finally,in order to explore whether Bm SRB3 can transport mulberry leaf lipids,this study carried out the transport experiment of Bm SRB3 at the cellular level.We used lipid vitamin A,vitamin D2 and vitamin E as transport targets to prepare the corresponding medium,and CCK-8 assay showed that the prepared lipid vitamin medium had no cytotoxicity.After overexpressing Bm SRB3 at the cellular level,extracts from Bm E cells were detected by GC-MS.The results showed that cells could absorb vitamin A and vitamin D2,and Bm SRB3 could promote the absorption of vitamin D2 by cells.Concentration gradient and time gradient incubation results further indicated that the absorption of vitamin D2 by cells was concentration-dependent.3.Research on the involvement of Bm SRB3 in blood cell immune functionIn order to study the role of silkworm Bm SRB3 in immune defense,the response of Bm SRB3 to pathogens was firstly analyzed.The results of pathogenic bacteria induction experiments showed that the expression of Bm SRB3 in blood cells was significantly upregulated at each time point,suggesting that it may be involved in immune defense in blood cells.Secondly,a variety of pathogenic microorganisms and their pathogenassociated molecular patterns(PAMPs)were incubated with recombinant r SRB3 protein respectively.Western blot and ELISA results showed that r SRB3 can interact with a variety of bacteria and fungi and their corresponding PAMPs directly combined.Subsequently,we stimulated the Bm SRB3-overexpressing cells with LPS to induce an immune response.The results showed that the expression levels of various antimicrobial peptide genes in cells overexpressing Bm SRB3 did not change significantly,indicating that Bm SRB3 may not be involved in regulating the expression of antimicrobial peptides in humoral immunity.The results of bacterial coagulation experiments showed that r SRB3 recombinant protein had obvious ability to coagulate bacteria,which may be involved in the nodular role in cellular immunity.In addition,the results of in vitro blood cell encapsulation and melanization experiments showed that the r SRB3 recombinant protein significantly promoted the encapsulation and melanization of blood cells,and the Bm SRB3 antibody could effectively block the cyst and melanization.Finally,the bacterial clearance experiments at the individual level of silkworms showed that Bm SRB3 could significantly promote the clearance of Escherichia coli,Micrococcus luteus and Staphylococcus aureus in silkworms.These results suggest that Bm SRB3 is involved in the immune defense process of silkworm by mediating the nodulation and encapsulation responses of blood cells.Based on the above findings,we speculate that Bombyx mori Bm SRB3 may be a receptor with dual functions,which is involved in the transport of lipid nutrient vitamin D2 in the midgut and in the process of cellular immune response in blood cells.