A Study On Breeding Of Griseofulvin Producing Strain (Penicillium Urticae Bainier) By Genome Shuffling

Genome shuffling is a new breeding technology which builds on the protoplast fusion technology. Penicillium urticae Bainier, the griseofulvin high production strain, asits incubation period is long, increased the production cost and prolonged the production period. Accordingly, this research plans means to obtain shorter fermentation period or higher titer Penicilliumg urticae Bainier strain by genome shuffling. The main results of this research are as follows:(1) This research has explored the highly effective protoplast preparation and regeneration method of Penicillium urticae Bainier: the optimum osmotic pressure stabilizer is 0.6 mol/L NaCl, the optimum age of the strain is 20 hours cultivated in the temperature28, P.urticae Bainier hypha pretreated 0.5 hours by DTT, enzymed 3.5 hours by 0.7% cellulase +0.7% snailase +0.5% lysonzyme in the temperature 28, and separated on the double-decked high infiltrate PDA culture medium plate. The results show that the Protoplast yield reaches 2.09×10~6/ml, and the regeneration rate of protoplast reaches21.2%;(2) Through the research of mutagenesis conditions, has chosen the optimal mutafacient scheme, the original strain mutagenized by the complex mutagen of UV andNTG, using 254 nm wavelength UV mutated 150 seconds, and then 1mg / ml NTG solution mutated 35 minutes.Among the mutated strains, obtained 3 shape-different mutant and high-vitality effective strains by the antimicrobial screening experiment, has achieved the desired result basically;(3) The mutant strains and original strains fused 5 minutes by 30% PEG4000 in 30°C,after two rounds of recursive-protoplast fusion, we have obtained three high fermentation vitality strains,theirs titer increased by about 15%. Analysis by RAPD indicated that the existence of genetic differences between the mutant or shuffled strains and the original strain, achieved the breeding purpose on the whole.