黑胫病菌诱导的烟草SSH文库构建及其分析

Construction and Analysis of Tobacco SSH Library Induced by Phytophthora parasitica var. nicotianae

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 of tobacco is one of the major disease which harmful to tobacco in the world, the incompatible interaction between tobacco and Phytophthora parasitica var. nicotianae was studied to provide more information for integrated control. A cDNA-SSH library was established by suppression subtractive hybridization using roots and stems sampled at 0.5, 1, 3, 6, 10, and 16 d after inoculation of Gexin 3, a horizontal resistant cultivar to race 0 of Phytophthora parasitica var. nicotianae, and 960 positive clones were picked out. Among them, 240 positive clones were verified by Reverse Northern dot-blot. A total of 57 differentially expressed EST sequences were screened out, and 33 high quality non-redundant ESTs were obtained by cluster analyses of the ESTs sequencing. The results of BlastN showed that the resistant genes of tobacco mainly related to disease defense, photosynthesis, signal transduction, and energy metabolism. Further analysis of gene function indicated that PR1b protein, cysteine proteinase, EF1-α, α-tubulin, cytochrome P450, putative spermine synthase, aquaporin, peroxisomal membrane protein, glycine for the elongation factor-1α may be involved in the process of the incompatible interaction between tobacco and Phytophthora parasitica var. nicotianae.