Transformation Of The Green Fluorescent Protein Gene Into Fusarium Oxysporum F.sp.cubense And Cloning Its Beta-Glucosidase Gene

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense(Foc), was one of the most serious fungal diseases in banana, and reported to be one of the major limiting factors for banana production worldwide. Especially, Foc race 4 caused great threat to banana production. To date, few effective and economically safe methods to protect banana from Fusarium wilt disease had been developed. It is important for us to find the pathogenic mechanism and genetic characteristic to research biochemical and molecular survey of Fusarium wilt of banana.Transform Foc isolate with the gfp gene, was made a useful tool to study Foc infection. It helps to further study pathogenic mechanisms and biocontrol mechanisms for banana fusarium wilt. Beta-glucosidase genes coloning for the Foc isolates is the foundation to study the gene function. These major results are as follows:1. Fusarium strains collected from banana wilt disease in Zhangzhou, Fujian Province, by single spore isolation, molecular characteristics and pathogenicity tests, were 21 Foc Race 4 strains.2. The PEG-mediated transformation system of Foc was established. Preparation of Fusarium protoplast is the key step during the transformation. Many factors affect the quality and quatity of the protoplast including the appropriate enzymes, mycelium age, the digestion temperature and time of the cell wall. This transformation was used in 0.8 mol·L-1 of NaCI dissolved collapse enzyme (Drislase) and dissolved wall enzymes (Lysing Enzyme); culturing 10-14 h of tender mycelium; digest temperature in 28℃then shaking at 70 r·min-1 at for 2-3 h.3. Strain Foc-3076 was transformed with gfp gene by protoplast method. The morphology of mycelium, conidia, chlamydospores of labeled-gfp strains were the same as the parent strain's, and all transformants appeared green under ultraviolet fluorescence. PCR amplification showed that gfp gene has been transferred into strains Foc-3076. And the transformants resistance ability to hygromycin B increased greatly than the original strain. Transformants were quite stable and remained strong green fluorescence after six successive subcultures. The morphology, pH value and growth rate of the gfp-tagged transformants are the same as the wild type strain. The pathogenicity of the wild type strain and the gfp-tagged strain was over 90 percent.4 Using laser scanning confocal microscope, colonization and infection were visualized:the colonization sites on the root surface by spore germination or mycelium, then along the extension of host cells within the gap, and spread upward through the plant vascular tissue growth to the top of pseudo stem.5 Degenerated primers were designed by the conserved domains of nucleotide sequence, which came from beta-glucosidase genes of known fungus on GenBank. After extracting RNA from strain Foc-3076, beta-glucosidase gene cDNA was amplified with the degenerated primers. And we obtain a cDNA clone of beta-glucosidase with 1473 bp nucleic acids from strain Foc-3076 by 3'RACE PCR amplified.