DNA Transformation On Fusarium Oxysporum And The Analysis Of Some Phenotypes Of These Transformants

Fusarium oxysporum causes plant vacicular wilt diseases worldwide and is one of the most economically important phytopathogens. A better understanding of the molecular basis involved in the pathogenesis of the fungus would be invaluable in obtaining targets for fungicide fungicide development and therefore control of the disease. Obviously, the identification of genes required for pathogenicity is a key step in achieving this goal. REMI(restriction enzyme-mediated integration), a widely applied gene tagging technique, is an efficient way to identify genes required for pathogenicity in some filamentous fungi. Recently ATMT (Agrobacterium tumefaciens mediated transformation) has also shown to be potentially useful for the transformation of filamentous fungi.In this paper, REMI transformation technique of Fusarium oxysporum f. sp. cucumerinum was established and some transfomation parmeters were optimized. The results showed REMI tranformation efficiency of the fungus was significantly increased by the improvement of protoplast preparation and regeneration. Meanwhile an optimized protocol of the protoplast preparation and regeneration of Fusarium oxysporum was developed by analyzing key factors which could influence protoplast release and vigor, such as fungal cultures, cell wall degradation enzymes, digesting conditions and regeneration media. Normally we could isolate 3ml protoplasts of Fusarium oxysporum with 5.0 X 107~1.44X 108 cell/ml by digesting fungal culture in osmotic buffer containing 12mg/ml lywallzyme and 4mg/ml Snailase for 4-5h. In REMI transformation of Fusarium oxysporum, we also found that plasmids and restriction enzymes used in the transformation can effect on the transformation efficiency. The results showed that pCB1004's REMI transformation efficiency is higher than pCB1003's and the BamH I's REMI transformation efficiency is higher than Hind Ill's. REMI transformation efficiency was 19.12~22.72 times higher than normal plasmid transformation without restriction enzyme.Based on REMI transformation techniques, we obtained 123 REMI transformants of Fusarium oxysporum f. sp. cucumerinum ATCC16416. We had analysed some phenotypes of these transformants, such as growth, conidiation and pathogenicity. Thirteen transfonnants were reduced in conidiation by 1-4.5 times that of the wild strain. Twelve transformants were 1.35-3.59 times more in conidiation. Ten transfonnants grew slower on PDA than wild strain. Meanwhile, the results of pathogenicity assay showed two of the transformants were of reduced virulence.In this study, a binary vector ATMT1 was also constructed based on pCAMBIA1300. The key point in this construction was 35S (CaMV35S) promoter replaced by trpC promoter to make it suitable for the transformation of filamentous fungi.