Construction And Analysis Of Tobacco SSH Library Induced By Race 0 Of Phytophthora Parasitica Var. Nicotianae

The lost caused by Phytophthora parasitica var. nicotianae is one of the most important diseases of tobacco production output and quality in China, with the characteristics of wide distribution, transmission diversity and highly destructive. Study on the cultural characteristics under different conditions of Phytophthora Nicotianae var. nicotianae and the process of tobacco resisting pathogen infection both can grasp the pathogenic of fungi and reveal the mechanism of host plant resistance. It is good for rational use of anti-fungal disease of tobacco material and significant to sustainable agricultural development of modern tobacco agriculture. In order to elucidate the tobacco resistant Phytophthora parasitica var. nicotianae mechanism at the molecular level, a cDNA-SSH library was established by suppression subtractive hybridization using roots and stems of Gexin 3 and pathogen-induced genes were verified by Reverse Northern dot-blot. The main results included in the research were as follows:1. The results showed that race 0 of Phytophthora parasitica var. nicotianae was more like oatmeal medium. In dark conditions, oatmeal medium was suitable for mycelium growth and production of sporangium. 0.1% KNO3 solution soaking mycelium contribute to sporangium production and 1% glucose solution can promote sporangia to release zoospores, and it can also prolong time of zoospore's activities.2. Based on the experimental period, mycelium cultivated for 21 days was more appropriate for induced, many sporangiums were producted after 72 h cultivation by 26℃, sudden drop of 12℃for 0.5 h soaked by 0.1% KNO3 + 1% glucose solution. Bacteria in the artificial creation of appropriate conditions of sporangia produced compound inducing agents can better induce the production of sporangia, in order to gain optimal concentration of zoospore, the method is simple and fast, saving time and effort.3. A cDNA-SSH library was established by suppression subtractive hybridization using roots and stems were sampled at 12 h, 1 d, 3 d, 6 d, 10 d and 16 d after inoculation of Gexin 3 and 960 positive clones were picked out. The size of gene fragments of the library were concentrated in 500 bp-1 100 bp, among them, 240 positive clones were verified by Reverse Northern dot-blot and ESTs were obtained by cluster analyses of the ESTs sequencing.4. 57 differentially expressed EST sequences were screened out, and 33 high quality non-redundant ESTs were obtained by cluster analyses of the ESTs sequencing. Initially identified with differentially expressed genes related to types and abundances between tobacco and Phytophthora parasitica var. nicotianae. Indicated that PR1b protein, cysteine proteinase, senescence-associated protein, glycine gene, etc involved in disease resistance of tobacco, play an importent role in the process of the incompatible interaction between tobacco and Phytophthora parasitica var. nicotianae and EF1-α,α-tubulin, aquaporin, peroxisomal membrane protein signaling processes involved in disease resistance.5. Selection of elongation factor 1-α(A1-62), cytochrome P450 (B1-12), pathogenesis-related protein (A2-91), age-related protein (B2-72) such as the cloning of full-length candidate genes. The experiments based on the findings of functional genes, cloned for further separation of black shank resistance gene to provide a basis for the integrated control of black shank and lay the foundation for breeding of resistant varieties.