The Research On Genetic Breeding Of P.Chrysogenum

P.chrysogenum belongs to the filamentous fungus,a kind of mold that widely exists in nature。People began the study of P·chrysogenum from the sixties of last century, The physical mutagenesis and chemical mutagenesis methods were developed for P. chrysogenum breeding to improve the yield of penicillin, The high-yield strain was achieved by doing a lot of screening work to get the high-yield strains.In the past irrational strategy is commonly used for breeding of P.chryso genum,including random strategy and semi-rational strategy, The strain breeding work shifting from rational strategy to irrational strategy because of a deeper understanding to metabolic engineering of the fungi and understanding the metabolic pathways of penicillin and the control genes. It is difficult to knock-in gene by homologous recombination in filomentous fungi because the non-homologous recombination jointing(NHRJ) repair is stronger than the homologous recombination repair. So it is of significance to knockout the ku70 gene, encoding a key enzyme at the NHRJ system to get a no-scar strain for P. chrysogenum breeding. The results are as followed.1)In this paper,the cefG(encoding DAC acetyltransferase, DAC-AT), cefD1(encoding isopenicillin N-acetyl-COA synthetase),cefD2(encoding isopenicillin N-acetyl-COA isomerase), cefEF(encoding deacetylation oxygen cephalosporin C synthase-hydroxylase) gene were cloned from cephalosporium acremonium by RT-PCR method and there size were 1158 bp, 1830 bp, 1152 bp, 999 bp.The HSV-Tk(herpes simplex virus thymidine kinase; HSV-TK) gene were synthesized using templateless PCR technology.The first step is to link the oligonucleotide chains together by TPCR,and the second step is to connect the small fragment DNA together by FPCR,and the third step is to insert the large fragement into a clone vector and sequence.2)The upstream and downstream homologous fragment of ku70 gene and the intergenic fragment between pcbAB gene and pcbC gene(PpcbAB-PcbC) from P.chrysogenum were cloned by PCR and size is 1586 bp, 1000 bp, 1015 bp respectively; The 608 bp TrpCTer gene was amplified from Aspergillus nidulans by PCR method, The 720 bp green fluorenscence protein gene(gfp) and 712 bp bleomycin(ble) gene were amplified from the corresponding plasmid.The upstream and downstream homolo gous fragment of ku70 gene, PpcbAB-PcbC, TrpCTer, GFP, and ble gene were assembled together in yeast by homologous recombination.The target gene’s size is 5641 bp pKOku70.3)The factors influencing protoplast prepation and its regeneration were studied. The results showed that the most suitable preparation age of P.chrysogenum was 48 h, the cellulase and snailase volume ratio is 3:2 and the hydrolysis time is 4h.The pKOku70 plasmid was transformed into P.chrysogenum mediated by PEG-CaCl2 and integrated into the genome of P.chrysogenum.The results showed that the transformants contain the ble and gfp gene identified by PCR and the mycelia of P. chrysogenum showed green fluorescence under UV excitation.4)The mutants of P. chrysogenum was also obtained by plasma mutation breeding. It was easy to obtain mutants under the following conditions:spore concentration was 105/mL,the distance of mutation platform was 2 mm and the mutation time is 150 s.