大豆过氧化物酶基因的克隆及其在毕赤酵母中的表达-生物化学与分子生物学专业论文.docx

ABSTRACT Soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily(EC). SBP is very stable at high temperature, extremes of pH, and in organic solvent.At the same time, it is highly reactive towards both organic and inorganic substrates,SBP has a wide range of of particular interest for engineering purposes .SBP was found to be a very effective of biocatalyst and biosensor which was used in wastewater treatment and phenolic resin synthesis. We choose Pichia pastoris expression system to express SBP. the system not only can make the express SBP glycosyl and fold for natural activity conformation, and can be induced in methanol under high level, the secretion of SBP expression. This makes the in vitro directed evolution and SBP follow-up of purification process becomes more simple and feasible. Each part introduces experimental are as follows:: The sbp gene were cloned and analyzed from soybean root.In this process ,we firstly obtained the cDNA of total mRNA using RT-PCR technology ,and then obtained the sbp gene using designed primer by PCR technology. The agarose gel electrophoresis of DNA fragments, and the size 1kb expected results. In the construction of the vector expression of reorganization, the design of experiments for two sticky end directional cloning, sbp gene and the pPICZ?-A vector were digested by restriction endonuclease then linked, got the recombinants expression vector pPICZ?-A-sbp,which were transformed into E.coli by the method of CaCl2. Positive recombinants were selected and sequenced. The results showed that the cloning DNA sequence from soybean root was homologous by 92% to the sbp(U51191(GmEpa1) DNA sequence reported by Huabang Chen. The sbp gene was transformed successfully into Pichia pastoris GS115, using Pichia pastoris espression system (from invitrogen Corporation). The sbp gene was expressed successfully in Pichia pastoris GS115 and got the bioactive of SBP. Pichia pastoris espression system can be induced in