Function Study Of Serine Protease BmSP125Gene In The Cuticle Development Of Silkworm,Bombyx Mori

Serine proteases (SPs) constitute nearly one-third of all the known proteolytic enzymes and maintain normal life for organisms by degradation and synthesis of protein, it can maintenance the vivo environment homeostasis of insects though serine protease inhibitors modulate the bioactivity of target proteins. Serine proteases degradation protein depended on the active center which composed of His, Ser and Asp. According to the integrity of the active site, SPs divided into two class, one is SP which have complete active site and the other is SPH which is have incomplete active site. SPs are divided into several types, including the trypsin, chymotrypsin and elastase, based on the target scissile bond. SPs are usually produced as zymogen, it can be activated when N domain split. SPs play an important role in mammals, including digestion, blood coagulation and complement system. In insects, SPs participate in the digestion and metabolism, but also play an important role in the defense, growth and development. SPs mainly take part in modulating and amplifying exogenous signals in insect immune system and embryo development, and the clip serine proteases are the mainly member. Clip serine proteases are only found in insect so far. The clip SPs can be divided into two classes based on their position in the cascade pathway, terminal proteinases and penultimate proteinases.Our research groups have identified18clip serine proteases in previous study. Based on this, we choose clip serine protease BmSP125gene as the research object. We have completed the sequence analysis and expression pattern analysis. We use the prokaryotic system to obtain recombinant protein and make polyclonal antibody of BmSP125. We use the polyclonal antibody to detection BmSP125protein distribution in the cuticle. In the end, we make yeast eukaryotic expression to obtain activity protein of BmSP125, and analyse its function in the cuticle pigmentation. The main results are as follows: 1. Sequences and expression analysis of BmSP125.Analyzing BmSP125based on the data from NCBI and silkworm genome database. BmSP125is1173bp long, contains4exons and3introns. It encodes390amino acids, the1-19amino acid is signal peptide sequence. After resection of signal peptide of the protein with molecular weight of40.5kDa, PI6.5, activated site is Arg. The analysis of amine acid sequence indicated that BmSP125(Bombyx mori), coagulation factor-like protein2(Hyphantria cunea), hemolymph proteinase17(Manduca sexta) are close to each other, the similarity is respectively71%and68%.Firstly, according to the silkworm microarray data, BmSP125was found expressed most highly in the head and integumentum of3rd fifth instar larvae, and expressed low or no in other tissues. Secondly, the results of RT-PCR of development newly hatched larva to adult showed that the high expression level of BmSP125is after molting, low expression level in molting, expression in the pupal stage gradually increase.2. Prokaryotic expression and polyclonal antibody preparation of BmSP125.The BmSP125gene was cloned into the p28, the inclusionbody protein was expressed in E.coli BL21and purified. Polyclonal antibodies were raised against BmSP125with rabbit. Anti-BmSP125has immune reaction with BmSP125by Western blot, suggesting that antibody and the antigen specific binding.3. The protein synthesis pattern and tissue localization of BmSP125.Using the polyclonal antibody of BmSP125, we analyze the first day of4th, the last day of4th,4instar molting epidermal and the first day to the last day of pupal by Western blot, we found that BmSP125had three synthesis pattern in the epidermis, such as complex, zymogen and mature. Immunofluorescence localization BmSP125in the integument the instar molting of4th and the first day in5instar. The results showed, BmSP125localization in the new cuticle and old cuticle, the result showed that the BmSP125participate in the development of cuticle in silkworm Bombyx mori.4. Activity analysis and function study of BmSP125we use the Pichia pastoris expression to production mature BmSP125.The yeast expression recombinant protein at methanol induction24h,48h,96h in the medium.We purified recombinant protein by ammonium sulfate precipitation and anion exchange column chromatography, and analysis the function of the BmSP125show that it take part in the melanism in integument.