Study On Preparation Of Protoplast And Construction Of PyrG Disruption Strains In Aspergillus Oryzae

Aspergillus oryzae is an important industrial brewing strain and an ideal host for heterologous protein expression strain. It has been received increasing attention that using genetic engineering techniques to construct Aspergillus oryzae genetic transformation system, such as the study of genetically engineered strains and efficient heterologous protein expression systems. Formation and regeneration conditions of protoplasts for industrial strains of A.oryzae3.951and the model strain of A. oryzae RIB40were optimized. The protoplast yield and regeneration rate were greatly improved. The targeting vector pPGT was transformed into the protoplasts of A.oryzae3.951use a PEG-CaCl2way and the pyrG-strains were isolated by resistance to5-FOA. The main contents and finding were eapounded as follows:1. The optmized Conditions of preparing protoplast from A. oryzae3.951and A. oryzae RIB40Formation and regeneration conditions of protoplasts for A.oryzae3.951and A.oryzae RIB40were optimized, the effect of material, cell age, osmotic stabilizer, enzyme proportion, enzyme acting time and acting temperature on the formation and regeneration of protoplasts from A. oryzae3.951and A. oryzae RIB40were studied. The optimized conditions of A. oryzae3.951was:mycelial as material and its age was14h,0.8M NaCl as osmotic stabilizer, the enzyme solution including1.0%(w/v) cellulase,1.0%(w/v) lysing enzyme and0.1%(w/v) snailase, the enzyme acting time was3h and enzyme acting temperture was35℃. Similarly, the optimized conditions oiA.oryzae RIB40were the same as A.oryzae3.951but the better age of its mycelial was12h. Under optimized conditions, A.oryzae3.951released6.43×107protoplast/g and the regeneration rate was23.5%, A.oryzae RIB40released4.24×107protoplast/g and the regeneration rate was22.41%. 2. Constructed of pyrG-strains based on the auxotrophic resistance in A.oryzae3.951The targeting vector pPGT was transferred into A.oryzae3.951use PEG-mediated protoplast transformation method on the base of high protoplast yield. About1000pyrG-strains were isolated by resistance to5-FOA. About600transformations got stable resistance after passaged to the seven generation. Phenotypic validation results showed that all transformants were able to grow normal on the SM and MM medium. PCR validation results showed that only8transformants showing the expected bands(1.5kb) with the original article(2.0kb). Further southern blot analiysis of the8transformants show that only two transformations (A138and A168) occurred a homologous recombination with the targeting vector. There was421bp delection in the middle of pyrG gene, the pyrG gene was succefully inactivated in A.oyrzae3.951.