Establishment Of Transformation Systems And Mutant Bank For Fusarium Oxysporum F.sp. Niveum

Fusarium oxysporum . sp. niveum (WTF) is the pathogenic fungus of the vascular tissue disease of many plants, which is of world-wide distribution and of economic importance.Functional genomic, especially pathogenic functional genomic of WTF is of theoretically and practically significant for the probe of the relationship between the host and WTF, breeding and selection of host resistant varieties, and creating new control strategies to WTF diseases. In this thesis, a high efficient and stable transformation systems of WTF based on Agrobacterium tumefaciens-mediated transformation (ATMT) was established, and 1884 T-DNA insertion mutants(TF) were obtained by using this system.1. The key conditions, factors and parameters of the transformation system of WTF were screened and optimized. The results showed as followings: among the culture media tested, the most suitable medium for WTF spore formation was PDA, then the CA medium; the spore production of WTF under continuous light was higher than the one under dark. Hygromycin B could inhibit the growthof WTF, but the effect of inhibition activities was different from culture medium to culture medium. On MM , the growth of WTF mycelia and spores were well inhibited at 50g/ml of hygromycin B. On PDA, however, WTF still grew well even at 50g/ml Hygromycin B. Therefore, 75g/ml of Hygromycin B in PDA culture medium was used in the mutant screening in this transformation systems. Among the A. tumefaciens isolates tested, the transformation efficiency of WTF with isolate AGL-1 was significantly higher than the isolate LBA4404 and EHA105; A. tumefaciens incubated for 5-6h in broth IM with 400Mmol/L of AS before co-cultivation resulted in highest transformation efficiency; A. tumefaciens (OD660 0.15-0.2) and WTF spore (1 106 conidia per ml) solution were mixed by equal volume, which was then plated onto a 60mm diameter nitrocellulose filter(0. 45Mm pore) on the surface of IM solid medium, 150 per filter, at 26 for 48h, and this procedure resulted in good transformation.2. The transformation experiment on WTF was carried out by using the optimized conditions and parameters described above, afterwards the results were testified. The results showed as the followings: For WTF conidia, 320-500 of Hygromycin B resistant transformants were obtained on average; with a pair of primers (H1, H2) designed by Hygromycin Phosphotransferase gene in plasmid pBHt2, a fragment of 819bp was separately amplified by PCR from all clones randomly selected from TF, confirming the Hygromycin gene had been really inserted into the TF genome; The transformants still maintained mitotically stable, maintaining their hygromycin Bresistance after 6 subcuture on PDA medium that absent Hygromycin B, showing that Hygromycin B resistance of the mutants obtained was stable.3. Large scale transformation on WTF was carried out. The colony characteristics and color, spore production and morphology between the TF and WTF were compared. A total of 1884 TF was obtained. Among the 867 TF randomly chosen, 3. 45% of all mutants, that was 26 number of mutants were morphology different from WTF. Among the morphology mutants, the colony characteristics, colony color and spore sterilization mutants accounted for 0. 239 3.0% 0.92% of the all mutants obtained respectively; Among these 867 TF, 4 TF completely lost virulences to watermelon seedlings, 1 TF showed much weak virulence than WTF. It showed that the Hygromycin gene had inserted into the pathogenic-related genes of these 5 TF. The 5 TF were named as FOCN2192 FOCN2195 FOCN3280 FOCN3181 and FOCN3179 respectively.