Identification And Expression Analysis Of Bmtol In Silkworm,Bombyx Mori

Silkworm(Bombyx mori)is a model organism of Lepidoptera species,is also an important economic insect,has a long history in our country domestication.Meanwhile,silkworm belongs to the metamorphosis insects,the whole life development history of silkworm include egg,larva,pupa and adult 4 stages,regulation of growth and development is mainly affected by juvenile hormone(Juvenile hormone,JH)and 20-hydroxyecdysone(20-hydroxyecdysone,20E)from larvae to adult.The juvenile hormone binding protein(juvenile hormone binding,JHBP)is the carrier of JH transport and play a functional in vivo,and it is a general term of the Takeout/JHBP proteins family.The proteins family contain a conserved JHBP structure domain,the two Cys residues of N terminal form a two disulfide that connect the N terminal and the first bending of alpha 1 helix,this part is considered to be the core part of the family proteins to bind ligand.The research of JHBP will is beneficial to the mechanism of the growth and development of silkworm and the development of silk industry.takeout(to)gene was first found in Drosophila melanogaster,and the homologous genes of to gene was also found in other insects,and the genes family is unique in insects.Several genes have been identified in silkworm,and found that the structure is similar to that of other insects,but few research have been studied on its function.In this study,we screened a new takeout like(tol)gene in silkworm by bioinformatics methods from SilkDB and NCBI datas base,The Bmtol gene was identified by bioinformatics analysis,gene cloning,prokaryotic expression,Western Blot and hormone treatment etc methods,and the distribution and function of the Bmtol gene is researching in silkworm,the aim of this study was to provide theoretical support for the further research on the role of JHBP in the growth,reproduction and metamorphosis of silkworm.The main research contents and results are as follows:(1)Cloning and bioinformatics analysis of Bmtol in silkwormThe cDNA of Dazao head tissues at 3 day 5th instarl as template,the Bmtol gene was successfully cloned by PCR.The bioinformatics analysis result show that the ORF of the Bmtol gene is 759 bp,including 6 exons and 5 introns,encoding 252 amino acid,its the theoretical molecular weight is 27.72 KD,and isoelectric point is 6.16.The protein contains a signal peptide of 23 amino acid,and the cell location prediction found that the protein may be a secreted protein,the hydropathicity prediction results show that is a hydrophilic protein.(2)Prokaryotic expression,protein purification and antibody preparation of Bmtol gene of silkwormThe protein sequence of the Bmtol gene encoding contains a signal peptide in N terminal,in order to successfully express the protein,we choice a region with no signal peptide to constructed the prokaryotic expression vector,the pET28(a)is expression vector,the expression strain is E.coli(DE3)BL21 strain.The recombinant protein was expressed in 28?,the concentration of IPTG was 0.6 mM and the induction time was 8 h as the best condition of prokaryotic expression.The recombinant protein was purified by Ni-NTA chromatography,and it was successfully purified.The polyclonal antibody was prepared by the recombinant protein,and the titer of the antibody was detected by ELISA,The titer of antibody was more than 512000.(3)The expression analysis and functional exploration of Bmtol geneBased on the Bmtol gene expression characteristics were analyzed by RT-PCR and qPCR in the tissues,that expression of the gene are higher at the head in silkworm,secondly are the epidermis and testis,but the expression is very low or not in other tissues,may also be related to the regulation of individual development and reproductive development,the main mechanism of action need further research.The expression characteristics of each period was found higher expression mainly in silkworm immediately after ecdysis by RT-PCR,and expression level of Bmtol gene was lower from at 3 days 5th instar to 7 days pupa,began to increase after the moth,Bmtol gene except control growth and development in larvae,also may be involved in other regulation of moth.Using the Western Blot to analyze the expression of BmTOL at the protein level in different tissues of silkworm,and it was found that the results were similar to those detected at the nucleic acid level.Based on the previous experimental results,expression of Bmtol gene in the head found that the experimental group and the control group difference is not obvious for hormone treatment on silkworms,but its changes has no big difference at different time after treatment.This gene may be associated with a lower expression in 5 instar.Analysis of localization and qualitative in the head tissues of silkworm by immunohistochemical,the results showed that there were no obvious changes in the head tissues.