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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

PDF

1379

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

[1]Zhu X-C(朱贤朝), Wang Y-T(王彦亭), Wang Z-F(王智发). China Tobacco Diseases (中国烟草病害). Beijing: China Agriculture Press, 2002. pp 21–37 (in Chinese)
[2]Hou M-S(侯明生), Huang J-B(黄俊斌). Agricultural Plant Pathology (农业植物病理学). Beijing: Science Press, 2011. pp 321–334 (in Chinese)
[3]Shang Z-Q(尚志强). Recent research advance on pathogen and disease occurrence of tobacco black shank and its integrated control. Rev China Agric Sci Technol (中国农业科技导报), 2007, 9(2): 73–76 (in Chinese with English abstract)
[4]Wang D-L(王德龙), Ye W-W(叶武威), Wang J-J(王俊娟), Song L-Y(宋丽艳), Pan W-L(樊伟丽), Cui Y-P(崔宇鹏). Construction of SSH Library and its analyses of cotton drought associated genes under drought stress. Acta Agron Sin (作物学报), 2010, 36(12): 2035–2044 (in Chinese with English abstract)
[5]Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E D, Siebert P D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025–6030
[6]Botha A M, Lacock L, van Niekerk C, Matsioloko M T, du Preez F B, Eduard Venter S L, Kunert K J, Cullis C A. Is photosynthetic transcriptional regulation in Triticum aestivum L. cv. ‘TugelaDN’ a contributing factor for tolerance to Diuraphis noxia (Homoptera: Aphididae)? Plant Cell Rep, 2006, 25: 41–54
[7]Li Y-J(李艳军), Zhang X-Y(张新宇), Yang H-N(杨会娜), Tian X-H(田新惠), Sun J(孙杰). Isolation of specifically expressed genes in brown colored cotton by suppression subtractive hybridization. Mol Plant Breed, 2009, 7(1): 194–198 (in Chinese with English abstract)
[8]Peng Q(彭琦), Hu Y(胡燕), Du P-F(杜培粉), Xie Q-X(谢青轩), Ruan Y(阮颖), Liu C-L(刘春林). Construction of SSH library with different stages of seeds development in Brassica napus L. Acta Agron Sin (作物学报). 2009, 35(9): 1576–1583 (in Chinese with English abstract)
[9]Tian Z D, Liu J, Xie C H. Isolation of resistance related-genes to Phytophthora infestans with suppression subtractive hybridization in the R-gene-free potato. Acta Genet Sin, 2003, 30: 597–605
[10]McIntyre J L, Taylor G S, Sereening tobacco seedling for resistance to Phytophthora parasitica var. nicotianae. Phytopathol, 1976, 66: 70–73
[11]Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature, 1998, 391: 485–488
[12]Ji W, Wright M B, Cai L, Flament A, Lindpaintner K. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics, 2002, 3: 12
[13]Wang X(王雪), Duan Y-X(段玉玺), Chen L-J(陈立杰), Wang Y-Y(王媛媛). Correlation analysis of amino acids components in soybean cultivars root exudates and resistance to soybean cyst nematode. J Shenyang Agric Univ (沈阳农业大学学报), 2008 39: 677–681 (in Chinese with English abstract)
[14]Liu S-P(刘素萍), Wang R-X(王汝贤), Zhang R(张荣), Yang Z-W(杨之为). Effects of sugar and amino acid in root exudation of different resistant cotton cultivars on cotton fusarium wilt pathoge. Acta Univ Agric Boreali-occidents (西北农业大学学报), 1998, 26(6): 30–35 (in Chinese with English abstract)
[15]Pan K(潘凯), Wu F-Z(吴凤芝). Correlation analysis of amino acids components in cucumber root exudates and fusarium wilt resistance. Acta Ecol Sin (生态学报), 2005, 27(5): 1945–1950 (in Chinese with English abstract)
[16]Burger C, Rondet S, Benveniste P, Schaller H. Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14α-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L. J Exp Bot, 2003, 54: 1675–1683
[17]Hutv?gner G, Barta E, B?nfalvi Z. Isolation and sequence analysis of a cDNA and a related gene for cytochrome P450 proteins from Solanum chacoense. Gene, 1997, 188: 247–252
[18]Irmler S, Schröder G, St-Pierre B, Crouch N P, Hotze M, Schmidt J, Strack D, Matern U, Schröder  J. Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J, 2000, 24 : 797–804
[19]Takemoto D, Hayashi M, Doke N, Takemoto D, Hayashi M, Doke N, Nishimura M, Kawakita K. Molecular cloning of a defense-response-related cytochrome P450 gene from tobacco. Plant Cell Physiol, 1999, 40: 1232–1242
[20]Chang J S, Seok H, Kwon T K, Min D S, Ahn B H, Lee Y H, Suh J W, Kim J W, Iwashita S, Omori A, Ichinose S, Numata O, Seo J K, Oh Y S, Suh P G. Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. J Biol Chem, 2002, 277: 19697–19702
[21]Bassell G J, Powers C M, Taneja K L, Singer R H. Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts. J Cell Biol, 1994,126:863–876
[22]Bunai F, Ando K, Ueno H, Numata O. Tetrahymena eukaryotic translation elongation factor 1A(eEF1A) bundles filamentous actin through dimer formation. J Biochem (Tokyo), 2006, 140: 393–399
[23]Murray J W, Edmonds BT, Liu G, Condeelis J. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filamentends. J Cell Biol, 1996, 135: 1309–1321
[24]Duttaroy A, Bourbeau D, Wang X L, Wang E. Apoptosis rate can be accelerated or decelerated by overexpression or reduction of the level of elongation factor-1 alpha. Exp Cell Res, 1998, 238: 168–176
[25]Chen E, Proestou G, Bourbeau D, Wang E. Rapid up-regulation of peptide elongation factor EF-1 alpha protein levels is an immediate early event during oxidative stress-induced apoptosis. Exp Cell Res, 2000, 259: 140–148
[26]Münch-G S, Neuhaus J M, Boller T, Kemmerling B, Kogel K H. Expression of beta-1,3-glucanase and chitinase in healthy, stem-rust-affected and elicitor-treated near-isogenic wheat lines showing Sr5-or Sr24-specified race-specific rust resistance. Planta, 1997, 201: 235–244
[27]Ebner C, Hoffmann-Sommergruber K, Breiteneder H. Plant food allergens homologous to pathogenesis-related proteins. Allergy, 2001, 56: 43–44
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