| Fusarium wilt of banana caused by Fusarium oxysporum f. sp. Cubense (Foe) is a devastating disease in banana production. Among4races of Foe, the race4(Focr4) harms most greatly banana planting due to its wild host range, and the absence of banana varieties with high resistance to the disease. Up to now the pathogenic mechanism of Foc4is still little known. The Focr4genome has been sequenced and the mutant library of Focr4has been initially built as well. Based on these previous works, several insertion mutant strains whose pathogenicity have been lost or seriously weakened are selected from the insertion mutant library by the pathogenicity identifications in this thesis. The insertion sites of the mutant strains are obtained by DW-ACP technique and further located to the complete genome sequences of Focr4. The full DNA sequence of the gene where the insertion site located is obtained and the non-induced expression of this gene in the wild strain are confirmed by RT-PCR. Additionally, in this thesis the knockout and complementation of the pathogenicity-related gene are both performed and the knockout mutant strain△Focr4-1701as well as the complementary mutant strain△Focr4-1701-cp-1is obtained. Taking the wild strain Focr4-193-6, the T-DNA inserted mutant strain Focr4-1701, the gene-knockout mutant strain (△Focr4-1701) and the complementary mutant strain (△Focr4-1701-cp-1) as research materials, relationships between the pathogenicity and the mutant gene due to the T-DNA inserted are completely studied by the biology phenotype observation. The research achievements of this thesis include three following aspects:(1) The DNA sequence of the gene marked by insertion site is obtained for the mutant strain Focr4-1701whose pathogenicity has extremely reduced. According to the online comparison analysis, the author predicts that the ORF of the gene is composed of nine exons and separated by eight introns. This gene is further judged as a helicase-regulated gene.(2) The Knockout vector labelled by GFP (green fluorescent protein) and Hyg is constructed for the above-mentioned gene. Sequentially, the designated gene-knockout is performed for the wild strain Focr4-193-6. Southern hybridization is performed using partial sequence of GFP as the probe and the knockout mutant△Focr4-1701is verified as a mutant with a single knockout site. Pathogenicity detection results show that the pathogenicity of the knockout mutant strain△Focr4-1701is significantly diminished, compared with the wild strain Focr4-193-6. The biology phenotypes of△Focr4-1701coincide with those of the T-DNA inserted mutant strain Focr4-1701. The pathogenicity of the reverse mutation strain △Focr4-1701-cp-1is as same as the wild type Focr4-193-6. The study reveals that the T-DNA lost gene of F. oxysporum is probably the pathogenicity-related gene.(3) The biology phenotypes of the T-DNA inserted mutant strain Focr4-1701, gene-knockout mutant strain AFocr4-1701, the complementary mutant strain△Focr4-1701-cp-1and the wild strain Focr4-193-6are compared. The results show that no obvious disease symptoms are observed on the leaves of the banana plants inoculated by the T-DNA inserted mutant strain Focr4-1701and the gene-knockout mutant strain△Focr4-1701. Inversely, the leaves of the banana plants inoculated by Focr4-193-6and△Focr4-1701-cp-1both show obvious disease symptoms. Focr4-1701and△Focr4-1701both can not penetrate the cellophane to grow. The spore germination rate, the spore yield and the toxin-producing ability of Focr4-1701and△Focr4-1701are significantly less than those of the other two types of strains. The activities of pectin methyl-galacturonase (PMG) and pectin methyl transeliminase (PMTE) produced by Focr4-1701and△Focr4-1701are also less than those of AFocr4-1701-cp-1and Focr4-193-6. These phenotype differences indicate that the remarkable pathogenicity attenuation of Focr4-1701may be caused by the reduction of the infection ability and toxin-producing ability of the inactivated strain obtained by T-DNA insertion. |
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