The Study Of Apolipoprotein D Gene,Cloning And Functional Analysis In Silkworm,Bombyx Mori

Apolipoprotein D is a glyprotein, which is mainly exists in the High density lipoprotein in the blood and widely distributed in different tissues. Apolipoprotein D as a lipid, cholesterol transporter, and it also involved in the repair of central and peripheral nervous system, innate immune system, resistance to oxidative stress, enhanced locomotor behavior and may act a role in neurodegeneration disease. Apolipoprotein D is widespread in different species, the structure of ApoD shows that it has an eight-stranded antiparallel β-barrel with a ligand-binding pocket is relatively well conserved, which could combined with hydrophobic small molecules. With ligand binding, ApoD could transport to the target organ and involved in the regulation of the development of the organization and other physiological functions. There is a deep research on apolipoprotein D in human, mice and Drosophila, but few studies have been reported in the silkworm.The silkworm, Bombyx mori, as an important economic insect of Lepidoptera, has been widely concerned. The development of silkworm is accompanied with great changes of morphology and function. With proteomics techniques, we identified two apolipoprotein D gene from the late pupae in silkworm, which contain lipocalin domain, the entry in SilkDB is BGIBMGA002703,BGIBMGA002704.The study is to identify high level expression protein in late pupae and investigate the tissue localization, expression characteristic, potential function and the regulation mechanism through gene clone, bioinformatics analysis, prokaryotic expression, antibody production, western blot, immunohistochemistry, Quantitative-PCR. The main findings as follows: 1. Identification and bioinformatics analysis of Apolipoprotein D gene fromBombyx moriAfter 2-DE electrophoresis analysis on molting fluid and blood of Bombyx mori, we identified two gene, the entry in SilkDB is BGIBMGA002703, BGIBMGA002704. The gene located on chromosome fifth, the nscaf2529 from 4191766 to 4194594（+strand） and nscaf2529 from 4202485 to 4205359（+strand）. Gene microarray and RT-PCR shows ApoDⅠ is mainly expressed in head and testis, ApoDⅡ is mainly expressed in head, middle and posterior silk gland.The structure of gene shows it has 4 exons and 3 introns, ApoDⅠ contains a lipocalin（PF00061） domain, ApoDⅡ contains a lipocalin₂（PF08212） domain. The similarity between two amino acid sequence alignments is 24.65%. From sequence analysis, we found they all have signal peptide. 3D structure prediction shows they can form an eight-stranded antiparallel β-barrel with a ligand-binding pocket. Phylogenetic analysis indicated that ApoDⅠ gene is similar to Hyphantriacunea and ApoDⅡ gene is similar to Nevada termite. We also used Multiple sequence alignment in SilkDB to identified 9 lipocalin super family member, cluster analysis of microarray data shows, it can clustered into three groups, one is mainly expressed in midgut, another one is mainly expressed in silk gland, the third one is widely expressed in gonad, malpighian tube, fat body, head and Hemolymph. 2. The Expression, Purification, and antibody preparation of Apolipoprotein D inBombyx moriPrimers were designed by sequence analysis, we using pupae 2days cDNA as a template for PCR amplification. After gel recovery and enzyme digestion, we received the fragment and inserted it into the p28 vector. After sequencing, The plasmid was transformed into Transetta（DE3） expression strain and an insoluble form of ApoDⅠ and ApoDⅡ recombinant protein were obtained under the final concentration of 0.1 mM IPTG, at 37℃. The recombinant protein of ApoDⅡ was purified by Ni-NTA affinity chromatography and rubber cutting combined with electroelution. Polyclonal antibody was raised against with rabbit, the result of Elisa shows that the titer of polyclonal antibody is subsequent for experiment. In order to obtain soluble protein, we constructed two plasmid pET-SUMO-ApoD Ⅰ and pET-SUMO-ApoD Ⅱ, after sequencing, we transformed plasmid into Transetta（DE3） expression strain and we obtain soluble recombinant protein. After enzyme digestion and purification, we obtained the ApoDⅡ soluble protein for the next experiment. 3. The expression patterns analysis and tissue localization of ApoDⅠand ApoDⅡThe result of RT-PCR shows that tissues from fifth larval 3days silkworm, ApoDⅠ gene is mainly expressed in head and testis, ApoDⅡ gene is mainly expressed in head and silk gland. We also used RT-PCR and Q-PCR to analyze the expression of developmental stages, found that ApoDⅠ is highly expressed on pupae 3rd day, and ApoDⅡ is higher expressed in early stage of pupa and a high amount of expression in the stage of moth. Western blot shows that the protein of ApoDⅡ has been found in head, fatbody, gonad, middle silk gland and poster silk gland in fifth 3rd days silkworm, the result of different stage hemolymph from pupae shows ApoDⅡ is highly expressed during the early stage of fifth larval, wandering stage and late stage of pupae. The result of head and ovary in pupae stage indicted that ApoDⅡ is highly expressed in head on pupae 6 day and in the ovary ApoDⅡ is highly expressed on late pupae stage and moth 1st day. Immunohistochemistry shows that we detected the signal in fifth larval 3rd day testis and ovary, wandering stage brain and eyeball from moth 1st day. 4. Preliminary investigation the expression regulation of Apolipoprotein D gene inresponse to Oxidative stress and Bacterial challengeWe selected UV and H2O2 as an external pressure signal, Escherichia coli and Serratia marcescens as a strain to challenge silkworm. The result of QPCR indicted that ApoDⅠand ApoDⅡ gene is up-regulated in response to stress signal, and in bacterial treatment, ApoDⅠand ApoDⅡ gene is also up-regulated in response to bacterial challenge. For further explanation of the regulated mode, we have analysized the 5` upstream promoter 1500 bp sequence fragment, Combining with the literature analysis, we predicted and identified some cis-elements that related to DNA damage and immune recognition. We also identified some BRC transcriptional factor binding site in the promoter religion, we hypothesized that ApoDⅡ gene may be related with ecdysone regulation, then we used the 20 E to treated BmE cell, after 6 hours, we found that ApoDⅡ gene is up-regulated in response to ecdysone.