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作物学报 ›› 2011, Vol. 37 ›› Issue (10): 1763-1770.doi: 10.3724/SP.J.1006.2011.01763

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

黑胫病菌诱导的烟草SSH文库构建及其分析

苏振刚1，杨爱国1，孙玉合1，罗成刚1，刘贯山1，周佳2，李元元1，杨帆1，赵百英1，王元英1,*   

  1. 1 中国农业科学院烟草研究所 / 烟草遗传改良与生物技术重点开放实验室，山东青岛 266101；2 云南瑞升烟草技术(集团)有限公司, 云南昆明 650106
  • 收稿日期:2011-03-15 修回日期:2011-06-25 出版日期:2011-10-12 网络出版日期:2011-07-28
  • 通讯作者: 王元英, E-mail: wyytob@126.com, Tel: 0532-88703857
  • 基金资助:

    本研究由国家烟草专卖局科技项目(110201002002, 110200902044)和山东省烟草专卖局科技项目(200837)资助。

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

SU Zhen-Gang1,YANG Ai-Guo1,SUN Yu-He1,LUO Cheng-Gang1,LIU Guan-Shan1,ZHOU Jia2,LI Yuan-Yuan1,YANG Fan1,ZHAO Bai-Ying1,WANG Yuan-Ying1,*   

  1. 1 Tobacco Research Institute of Chinese Academy of Agricultural Sciences / Key Laboratory of Tobacco Genetic Improvement and Biotechnology, Qingdao 266101, China; 2 Yunnan Reacend Tobacco Technology (Group) Co. Ltd, Kunming 650106, China
  • Received:2011-03-15 Revised:2011-06-25 Published:2011-10-12 Published online:2011-07-28
  • Contact: 王元英, E-mail: wyytob@126.com, Tel: 0532-88703857

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1338

被引次数

12

摘要： 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16 d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。

关键词: 烟草, 黑胫病病菌, 抑制差减杂交, 反向Northern斑点杂交, 非亲和互作

Abstract: 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 thatPR1b 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.

Key words: Tobacco, Phytophthora parasitica var. nicotianae, Suppression subtractive hybridization (SSH), Reverse Northern dot-blot, Incompatible interaction

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