Study On The Expression Characteristics And Its Binding To Mulberry Leaf Metabolites Of JHBP-like Protein In Digestive Tract Of Silkworm,Bombyx Mori

Bombyx mori,derived from the ancient wild silkworm after long-term domestication,is a very important economic insect in China and an important model organism for the study of Lepidoptera.It is a completely metamorphosis insect,which goes through four stages of development,namely egg,larva,pupa and adult,which are completely different in morphology and physiological function.In the silkworm growth process,juvenile hormone(JH)synthesized from the corpora allata and 20-hydroxyecdysone(20E)synthesized from the anterior thymus are two important hormones that regulate silkworm growth and metamorphosis.They acted together to regulate the growth and development process of silkworm.Among them,juvenile hormone can inhibit the appearance of adult characteristics at larval stage,so that the larva can remain in larval stage after molting,while in adult stage,it can control the sexual development,produce sexual attraction,and promote egg maturation.However,Juvenile hormone in silkworm requires the help of Juvenile hormone binding protein(JHBP)in hemolymph to bind to it and transport it to target cells.JHBPs in hemolymph of silkworm,are a kind of specific carrier proteins with a unique JHBP domain,which can specifically bind to JHs and protect them from enzyme degradation and transport them to the site of action.In this study,we found one king of protein molecules with similar structure to silkworm hemolymph JHBP in the digestive tract.Both of them have the same JHBP domain,thus we named this protein BmJHBP-like protein.Previous studies have shown that insect juvenile hormone binding proteins have low sequence similarity at the nucleotide and amino acid levels,but high similarity at the protein structure level.Therefore,we speculated that JHBP-like proteins may have functions similar to those of hemolymph JHBP protein of silkworm.Based on the above questions,this study used bioinformatics methods to identify and analyze BmJHBP-like proteins which have the same JHBP structure domain,verified the expression pattern of BmJHBP-like gene in different tissues and stages of silkworm by quantitative PCR,and through the prokaryotic expression and protein lipid overlay assays to explore the function of BmJHBP-like proteins.The main results are as follows:1.Identification,Cloning and Bioinformatics Analysis of BmJHBP-like GeneFive BmJHBP-like genes,named BmJHBP-L1,BmJHBP-L2,BmJHBP-L3,BmJHBP-L4 and BmJHBP-L5,were identified in the midgut of silkworm by bioinformatics method.Four of the above genes were cloned from N4 strain,except BmJHBP-L5.The amino acid sequence similarity of BmJHBP-like proteins are 40%.The signal peptide prediction results showed that all BmJHBP-like encoded a signal peptide,and the protein structure analysis showed that all of them had the same JHBP domain.By predicting the hydrophobicity and tertiary structure of BmJHBP-like protein,most of the amino acids were hydrophobic amino acids,and the results of predicting the tertiary structure of protein showed obvious similarity with that of silkworm hemolymph JHBP.2.Expression analysis of BmJHBP-like genesThe expression level of BmJHBP-like gene in the midgut,fat body,silk gland,malpighian tube,testis,ovary,epidermis,trachea,head and blood cells of N4 strain silkworm was detected by RT-PCR.The results showed that the expression of five BmJHBP-like genes in different tissues of silkworm showed obvious tissue specificity,with high specificity in midgut,slight expression in epidermis,and almost no expression in other tissues.The expression level of BmJHBP-like gene in midgut of silkworm was detected every 24h from 4th instar to 4th day of 5th instar.The results showed that the expression level of the 5BmJHBP-like genes decreased slightly after the 4th instar,and increased sharply at the 2nd day of the 4th instar,decreased after the 4th instar 2 days,reached the lowest level during the4 th sleep stage,and then increased day by day after the 5th instar,presenting an obvious wave fluctuation.The expression level of BmJHBP-like gene in the midgut of silkworm was detected by RT-PCR after starvation treatment for 24h and 48h at the 5th instar and 3rd day.The results showed that all the other four genes were significantly down-regulated after starvation treatment except for BmJHBP-L4.3.Prokaryotic expression,purification and verification of specific expression of BmJHBP-like proteinBuild BmJHBP-like proteins prokaryotic expression vector,and obtain pCold Ⅰ-BmJHBP-L1 recombinant plasmid,express the purified protein,and synthesis of specific antibody.The expression of BmJHBP-L1 protein in silkworm tissues was detected in midgut,digestive juice,midgut contents and feces of silkworm but not in hemolymph of silkworm usingβ-tubulin as internal reference.4.Verification of binding of silkworm BmJHBP-L1 protein with juvenile hormone and mulberry leaf metabolitesBinding ability of BmJHBP-L1 protein with juvenile hormone and mulberry leaf metabolites was detected by protein lipid overlay assays.The results showed that BmJHBP-L1 protein could bind to juvenile hormone and mulberry leaf metabolites,and the signal intensity was related to the concentration of JH and metabolites.