Establishment Of Transformation Systems For Filamentous Fungi

Fusarium oxysporum Schl is an important pathogenic fungus of the vascular tissue disease of many plants, and can cause blight. Functional genome, especially pathogenic functional genome of F. oxysporum is of theoretically and practically significant for the probe of the relationship between F. oxysporum and its host. Establishing muntants through DNA insertion is an important method in studying gene function.Thermomyces lanuginosus Tsiklinsky is a thermophilic fungus with higher growth temperature, and is shown to produce thermostable protease, glucomylase, xylanase, chitinase, andα-galactosidase. The genes of glucomylase and chitinase have been cloned and expressed. However, it is not very clear about the mechanism how thermophilic fungi recept and transfer signal. Through genetic transformation, exterior plasmids have been inserted into genome randomly to establish the mutant of this fungus. It is meaningful to do some research on the thermophilic mechanism.Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation systems of F. oxysporum and T. lanuginosus, conditions for the protoplasts isolation and regenration of the mycelia of F. oxysporum and T. lanuginosus were examined, including the effects of the hyphal age, enzymes and their concentration, time and temperature of digestion, osmotic stabilizers on the protoplast preparation and the effect of osmotic stabilizers on the protoplast regeneration. The results indicated that optimum conditions for preparing protoplasts of F. oxysporum were: the condia cultured in liquid PDB medium for 18 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 10mg/mL Lywallzyme used to digest the hyphae for 4 hours at 30℃. And 0.7mol/L sucrose was the osmotic stabilizer for the regeneration of protoplasts. Optimum conditions for preparing protoplasts of T. lanuginosus were: the condia cultured in liquid PDB medium for 28 hours, 0.7mol/L NaCl used as the osmotic stabilizer, 15mg/mL Lywallzyme used to digest the hyphae for 4 hours at 30℃. And 0.7mol/L sucrose was the osmotic stabilizer for the regeneration of protoplasts.To study the pathogenicity mechanism, we used the restriction enzyme-mediated integration approach to transform F. oxysporum. We have transformed protoplasts with vector pUCATPH which has hygromycin B-resistant gene, and obtained over 200 transformants. It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification. Most of the transformants were fully stable after five rounds successive culture and 20 phenotypic mutants were gained by REMI. But no transformant was got after transforming T. lanuginosus.The plant expression vector pROK2 was ligated to hygromycin B resistance gene cassette (PtrpC-hph-TtrpC) with the fungus promoter from plasmid pUCATPH. Thus, the vector pROK2-PtrpC-hph-TtrpC was constructed. The vector was identified with the enzymes digestion and PCR, and then transferred into Agrobacterium LBA4404. By using this transformation system, F. oxysporum was successfully transformed. PCR analysis showed that the hph gene was integrated into the genome. But no transformant was got after transforming T. lanuginosus.By using the system of Agrobacterium tumefacines-mediated transformation, we successfully transformed F. oxysporum and obtained T-DNA insertion mutants. Under an optimum condition (F.oxysporum spores: 106/mL; A.tumefacines D600=0.25; cetosyringone: 300μg/mL, co-cultivation time: 48h), transformation efficiency reached 6~15 transformants per 106 spores. More than 400 transformants have been obtained and most of them were quite stable after five rounds successive culture. PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division. The transformation system is the basis for studying on pathogenicity mechanism and functional gene of the fungi.To get the transformants of F. oxysporum and T. lanuginosus, two systems, REMI and ATMT, were used for transformation of the two fungi. As a result, the two systems were successful in transforming F. oxysporum, and phenotypic mutants of F. oxysporum were gained. But both of the systems were failed in tranforming T. lanuginosus. According to the description above, this paper made an analysis on aspects of marker genes, Agrobacterium strains, vector, and promoter of marker genes and so on.