Study On The Method Of Agrobactirium Tumfacience Mediated-Transformation Of Penicillium Griseofulvin And Penicillium Chrysogenum

Penicillium griseofulvin is an important filamentous fungus which can produce antibiotics. Cloning functional genes can promote the study on growth, metabolic pathway and spore formation mechanism of P. griseofulvin. The knowledge can provide theoretical foundation for developing stable, high-efficiency antibiotics. Producing large quantity of mutants by molecular tag method like Agrobactirium tumfacience mediated-transformant (ATMT) is a high efficient method for cloning functional genes. In this study, ATMT method was studied and used to establish mutant libraries of P. griseofulvin as well as Penicillium chrysogenum. The results were as follows:(1) Plasmid and strain constructionsThe plasmid of pulAc43 containg the ble gene was used. After Digest plasmids of pulAc43 and pCAMBIA1302, the target DNA segments were ligated to construct the plasmid of pCAM-ble. Then the pCAM-ble plasmid was transformed successfully into wild-type Agrobacterium tumefaciens strain EHA105.(2) The optimization of ATMT for P. griseofulvinThe factors influencing the transformantion efficiency of ATMT for P. griseofulvin were studied. The results showed that the transformation efficiency was correlated with the 3’5’-Dimethoxy-4-Hydroxy Acetophenone (AS) concentration in induced medium and co-cultivated medium, the induced time of A. tumefaciens, pH of co-culture medium, co-cultivation time, co-cultivation temperature, and the initial concentration of A.tumefaciens and the initial concentration of P. griseofulvin conidia. According to these results, we established optimal conditions for transformation of P. griseofulvin as follows: A. tumefaciens cultured to OD600 0.30~0.5 in induced medium with 200μM AS for 6 hours and then concentrated by 2 to 3 times; mixing the A.tumefaciens ferment broth with same volume P.griseofulvin conidia suspension which concentration was 106 conidia per milliliter; spreading 400μL mixture on the co-cultivation medium (pH5.3, 200μM AS) with a microporous membrane on it; incubating 48h at 25℃. Under these conditions, the transformation efficiency of ATMT for P. griseofulvin was 140~230 mutants per 106 conidia.We constructed a T-DNA insertional mutant library of P.griseofulvin with 200 transformants using the method above mentioned. The quality of the mutant library was also evaluated. All the randomly selected resistant colonies were proved to be stable through mitotic cell division. 100 transformants were screened for spore formation mutants. One mutant was selected from the transformants cultured on PDA medium. Compared with the wild type P. griseofulvin, this mutant, to some degree, showed shorter time for conidia production.(3) The investigation of ATMT for P. chrysogenumThe method of ATMT for P. chrysogenum was also investigated. The transformants of P. chrysogenum were picked up by the method of ATME for P. griseofulvin above mentioned. However, the transformation efficiency, 20~80 mutants per 106 conidia, was much lower than 140~230 mutants per 106 conidia, which is the transformation efficiency of ATMT for P. griseofulvin. Two transformants were screened for spore formation mutants. Compared with the wild type P. chrysogenum, the two mutants showed shorter time for conidia production cultured on PDA medium; and one mutant with lower growth was also selected.We investigate about 100 transformants of P. chrysogenum and found that the ble segments would lost from the genomes of some transformants after several generations, which suggested that the transformants of P. chrysogenum rather than P. griseofulvin is not genetically stable. So it is nessary to research further in order to build the high efficient ATMT method for P. chrysogenum with stability of heredity.