Gene Cloning Of Glycine Oxidase From Aspergillus Oryzae A-F04and Construction Of The Recombinant Engineering Strain

In this paper, the early screening Aspergillus oryzae strain in lab was used as theexperimental strain. The glyphosate degradation rates of different Aspergillus oryzaestrains were measured by the High Performance Liquid Chromatography (HPLC)method. Compared with the National Centre for Biotechnology information (NCBI),Glycine oxidase was identified as glyphasate degradation enzyme of Aspergillusoryzae strain through looking up the relevant literature. The DNA from differentAspergillus oryzae strains made the gene sequence of glycine oxidase obtained byPCR. And the sequence can be analyzed by suitable software. The Aspergillus strainof having the strongest degradation ability was used as follow-up experiments strain.The gene sequence of glycine oxidase was obtained through extracting RNA andmaking RT-PCR. Finally, the engineering strain, having the stronger ability ofsecreting enzyme, was gotten via importing the above sequence to the genome ofPichia pastoris by means of genetic engineering technology.The main research cotent of this article was summarized as follows:1. The glyphosate degradation rates of different Aspergillus oryzae strains weremeasured by the High Performance Liquid Chromatography (HPLC) method. Thestrain, the highest degradation rate12.5%, was Aspergillus oryzae A-F04.2. The different glycine oxcidase sequence from different Aspergillus oryzaestrains were received get through extracting DNA and amplification by PCR. Thegenetic mutational situation would be known by comparing with each other.3. Compared with the normal group of having no glyphosate, there are somemany different phenomenones in the culture medium of adding glyphosate. Thenumber of Aspergillus oryzae hypha was less and the size was big in the latter culturemedium, while the number of Aspergillus oryzae hypha was more and the size wassmall in the former culture medium. This means glyphosate has certain toxicity toAspergillus oryzae strain. The results were that the strain of having the higher degradation ability can grow rapidly and the strain of having no ability to utilizingglyphosate would be weed out4. The gene sequence of glycine oxidase was obtained through extracting DNA.The PCR produce, obtained from cutting rubber recycling and purification, and theexpression vector pPlC9K, from Pichia pastoris strain, were both cutting by twoenzymes, which are restriction endonuclease EcoR Iå'ŒNot I. The cutting productswere linked by ligase and the connecting product was imported to the competent cellof E.coli DH5α. Finally, the restructuring plasmid pPIC9K-AFGO was gaineddepending on the screening of the ampicillin resistance and the identification of PCRtechnology.5. The linearized restructuring plasmid pPIC9K-AFGO was guided into thegenome of Pichia pastoris GS115and the engineering strain GS115-AFGO, havingsecreting glycine oxidase ability, was obtained by screening.