Research On The Mutation Breeding Of High Avermectin Producing Strains And The Mutagenic Effect About Genes And Proteins Of Streptomyces Avermitilis

Abamectin which has a high efficiency and low toxicity insecticide drugs andrecommended by the Ministry of Agriculture is a secondary metabolite secreted byStreptomyces avermitilis, and it is the most effective biological parasite-kill drugs byfar., so it has good value and broad market prospects. In this experiment,the S.avermitilis is studied, heavy ion beams provided by the Institute of Modern Physicsof Chinese Academy of Sciences to Irradiation, then screen out the high yieldproperties of strains of S. avermitilis. The gene mutagenic effects of high titers ofmutant strains under the heavy ion beams is analysed in order to looking fordifferences. The protein mutagenic effects of the high titers of mutant strains underthe heavy ion beams is analysed.First of all, the original strain is recoveried, And the energy of the irradiationsource is100Mev/μ Heavy Ion¹²C⁺⁶,0Gy,10Gy,30Gy,40Gy,45Gy,50Gy,70Gyseven dose are set, then avermitilis spores of3900ug/ml titer were irradiated. Thepositive mutation rate experimental results show that the positive mutation rate of45Gy dose up to11.5%and be able to obtain the strains of more titer improve.After observing the morphology of mutant strains,4serotype: paracrinkle type,bread type, hat type, bare type were found, and hat type strains of them produced thestrongest anti-ability. A total of621single-bacteria filter and the fermentation titerdetermination were made in this experiment.44titer improved strains were abtained.147,238and HS had the highest titer which was up to4901ug/mL. Through theoptimization of medium composition which included carbon, nitrogen sources,inorganic salts and trace elements, The optimum medium which contained dextrin11%, soluble starch1%, yeast extract2.5%, soybean meal0.5%, K2HPO4·3H2O0.06%, KCl0.1%, CoCl2·6H2O4mg/Lwas got. At the same time the titer of the hightiter strain HS was increased by8%.28pairs of primer were used to analysis the random amplified polymorphic(RAPD Random Amplified Polymorphic)on the original species, the high titer mutantstrains under the different doses and the DNA of the high titers of strain147, HS. Electrophoresis results showed a large number of different stripes, Analysis of resultsshowed that ion beam processing caused genomic changes of S. avermitilis and had ahigh homology. The gene primers of the original strains and C, D, I, R genes of thehigh titers of strains of147, HS were designed respectively. Amplification productswere abtained by the polymerase chain reaction. And sequencing, comparativeanalysing amplification products showed after a heavy ion beam mutagenesis thenucleic acid of the high titers of mutant strain appeared base mutation, base deletionand base substitution phenomenon. Part of the I gene deletions may have contributedto the improvement of the147, HS titer.The two-dimensional electrophoresis was still the most effective method forseparation of proteins by far. Experiment extracted the soluble protein from the hightiters of strain147, HS and the original strains after45hours of fermentation broth.Then two-dimensional electrophoresis was used to separate proteins. At first,theexperiment optimized the extraction conditions and the electrophoresis procedure.Secondly, selected the appropriate electrophoresis strips. Finally,the well separatedprotein profiles were got. Analysising of the two-dimensional electrophoresis figureof the soluble proteins of AO,147, HS. AO,147, HS, respectively, contain proteins345,418,591, And its molecular weight is mostly for the24KD to67KD. There were22points that the protein concentration of147were three times more than AO and28points were down. There were29points that the protein concentration of HS werethree times more than AO and19points were less than two times. Heavy ionirradiation breeded high titers of S. avermitilis appeared in the early stage offermentation. Because the irradiation effect changed the nucleic acid sequence,whichmakes the protein expression have more differences including the difference ofprotein spots and protein expression differences. The AO protein spots were at least345, Mutations in the efficient price of147and HS was at45hours in thefermentation when the protein is highly expressed, which was more than the originalstrain, This may be one of the reasons of titer improved.