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黑胫病是危害严重的世界性烟草主要病害之一,探究烟草对黑胫病的抗病机制,可以为该病的综合防治提供理论依据。本研究以烟草黑胫病抗性品种革新3号为材料,利用黑胫病0号生理小种诱导其水平抗性,分别在接种后0.5、1、3、6、10和16d取样,通过抑制差减杂交技术(SSH)构建cDNA文库,获得了960个阳性克隆。利用反向Northern斑点杂交技术对其中240个阳性克隆进行杂交筛选,筛选出57个表达差异明显的基因,序列拼接后获得33条高质量EST,测序及其比对分析表明,革新3号对烟草黑胫病抗性相关基因主要涉及抗病防卫、光合作用、信号传导和能量代谢等方面。进一步基因功能分析显示,病程相关PR1b蛋白、半胱氨酸蛋白、延伸因子1-α、α-微管蛋白、细胞色素P450、精胺合成酶、水通道蛋白、过氧化物酶体膜蛋白、甘氨酸延伸因子等可能参与了烟草与黑胫病菌非亲和互作过程。
Black shank is one of the most serious diseases in the world. Studying the mechanism of tobacco resistance to black shank disease can provide a theoretical basis for the comprehensive prevention and treatment of the disease. In this study, a new tobacco black shank cultivar, Gexin 3, was used as a material to induce horizontal resistance of black shank 0 race. The samples were collected at 0.5, 1, 3, 6, 10 and 16 days after inoculation, Subtractive hybridization (SSH) was used to construct a cDNA library, and 960 positive clones were obtained. A total of 240 positive clones were screened by reverse Northern dot blot hybridization. Fifty-seven genes with significant differences were screened and 33 high-quality ESTs were obtained after splicing. Sequencing and comparison showed that GenBank 3 Black shank resistance related genes are mainly involved in disease resistance and defense, photosynthesis, signal transduction and energy metabolism. Further analysis of gene function showed that the duration of disease associated PR1b protein, cysteine protein, elongation factor 1-α, α-tubulin, cytochrome P450, spermine synthase, aquaporin, peroxisome membrane protein, Glycine elongation factor may be involved in the non-affinity interaction between tobacco and Phytophthora parasitica.