|
Tobacco black shank (Phytophthora parasitica var. Nicotianae) the harm caused by tobacco production is one of the most important diseases, which hosts a wide range of bacteria, transmission diversification, highly destructive, seriously endangering the production of tobacco production and quality. Phytophthora culture characteristics to carry out research and tobacco against Phytophthora infection process of research, both can better grasp the pathogenic Phytophthora law, but also can reveal non-host plants immune affinity interaction mechanism for tobacco anti-fungal disease rational use of materials and modern tobacco agriculture sustainable development is of great significance. In this study, SSH technology to build anti-tobacco black shank cDNA library, and use reverse Northern hybridization screening blackleg pathogen-induced differentially expressed genes, to explore the mechanism of resistance of tobacco black shank disease has laid a theoretical foundation. The main results of this paper are as follows: 1. Tobacco black shank No. 0 races on a selective medium, preference for oats medium; dark conditions conducive to mycelial growth, sporangium number ones in dark conditions ; 0.1% KNO 3 solution induced mycelium can hasten sporangia produce; 1% glucose solution helps sporangia release zoospores or extending their active time. 2 best suitable bacteria induced cultured 21 d of age of mycelium, the amount of 0.1% KNO 3 1% glucose solution at 26 ℃ dark induced 72 h, and then transferred to 14 ℃ culture 0.5 h, can get a lot of zoospores. In the artificial creation of suitable nicotianae sporangium conditions, the compound can be better inducer induced sporangia production, so as to obtain the suitable concentration of zoospores, the method is simple and efficient, saving time and effort. 3 In this study, the 3rd of innovative materials, respectively, after pathogen inoculation 12 h, 1 d, 3 d, 6 d, 10 d and 16 d sampling by suppression subtractive hybridization cDNA library obtained 960 positive cloning. Library focused on gene fragment size 500 bp-1 100 bp between the use of reverse Northern blot hybridization technique which 240 positive clones were screened by hybridization, differentially expressed ESTs were picked for analysis. 4 out of 57 screened significantly differentially expressed genes, EST clustering and redundant to go after the total of 33 non-repeating sequence, the resulting gene sequence alignment analysis, preliminary clear and black shank of tobacco during the interaction Differentially expressed genes in the type and abundance. PR1b tobacco pathogenesis-related proteins presumably, cysteine ??protein, senescence-associated protein, glycine genes involved in the disease process of tobacco, tobacco and black shank in non-affinity interactions may play an important role in the process; EF1-α, α-tubulin, the water channel protein, peroxisomal membrane proteins involved in disease resistance signaling processes. 5 pick elongation factor 1-α (A1-62), cytochrome P450 (B1-12), pathogenesis-related protein (A2-91), senescence-associated protein (B2-72), etc., as a candidate gene for full-length clone. Obtained in this study based on the findings of functional genes, in order to further isolate and clone blackleg resistance gene to provide a basis for the integrated control of blackleg and lay the foundation for breeding resistant varieties.
|