研究报告

条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析

展开
  • 1. 中国农业科学院植物保护研究所, 北京100193 2. 中国农业大学农学与生物技术学院, 北京100193 3. 中国农业科学院农业资源与农业区划研究所, 北京 100081

收稿日期: 2011-01-24

  修回日期: 2011-04-27

  网络出版日期: 2011-09-25

基金资助

国家重点基础研究发展规划(973计划)项目(编号:G2000016202)资助

Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by Puccinia striiformis Westend. f. sp. tritici

Expand
  • 1.Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China 2. College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China 3. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received date: 2011-01-24

  Revised date: 2011-04-27

  Online published: 2011-09-25

摘要

为分析条锈菌诱导下的小麦抗病与感病近等基因系之间差异表达的基因, 以接种小麦条锈菌CY26小种的抗病近等基因系Yr4/6×Taichung 29幼苗叶片cDNA作为实验方, 接种CY26的感病亲本Taichung 29幼苗叶片cDNA为驱动方, 利用抑制消减杂交(SSH)技术构建了一个包含1 300余克隆的消减文库。对文库中600个克隆进行了反向Northern点杂交筛选, 对获得的阳性克隆进一步进行了Northern杂交验证, 获得显著差异的克隆12个。经测序和BlastX分析, 其中6个差异表达序列的推测产物分别为亮氨酸重复序列蛋白、过氧化氢酶、硫氧还蛋白、RNA结合蛋白、抗坏血酸过氧化物酶和热激蛋白。除亮氨酸重复序列为信号传导类蛋白外、其他几个均为抗病防御类蛋白。

本文引用格式

舒伟,陈晓红,牛永春 . 条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析[J]. 遗传, 2011 , 33(9) : 1011 -1016 . DOI: 10.3724/SP.J.1005.2011.01011

Abstract

To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

参考文献

[1] 李振岐, 曾士迈. 中国小麦锈病. 北京: 中国农业出版社, 2002.
[2] 吴立人, 牛永春. 我国小麦条锈病持续控制的策略. 中国农业科学, 2000, 33(5): 46-54.
[3] Chen XM. Epidemiology and control of stripe rust (Puccinia striiformis f. sp. tritici) on wheat. Can J Plant Pathol, 2005, 27(3): 314-337.
[4] 徐世昌, 吴立人, 万安民, 王凤乐, 赵文生, 牛永春. 以Taichung 29为背景的小麦抗条锈病近等基因系转育进展. 植物保护, 2004, 30(2): 19-22.
[5] Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J. Acquired resistance in Arabidopsis. Plant Cell, 1992, 4(6): 645-656.
[6] Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JD. Molecular genetics of plant disease resistance. Science, 1995, 268(5211): 661-667.
[7] Diatchenko L, Lau YF, Campbell AP, Chenchik A, MoqadamF, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93(12): 6025-6030.
[8] Guo P, Bai G, Carver B, Li R, Bernardo A, Baum M. Transcriptional analysis between two wheat near-isogenic lines contrasting in aluminum tolerance under aluminum stress. Mol Genet Genomics, 2007, 277(1): 1-12.
[9] 骆蒙, 孔秀英, 刘越, 周荣华, 贾继增. 小麦抗病基因表达谱中的文库构建与筛选方法研究. 遗传学报, 2002, 29(9): 814-819.
[10] 郭新红, 姜孝成, 潘晓玲, 戴玉池, 姜维明, 陈良碧. 用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段. 植物生理学报, 2001, 27(5): 401-406.
[11] 于秀梅, 喻修道, 屈志鹏, 韩青梅, 郭军, 黄丽丽, 康振生. 条锈菌诱导的小麦抑制差减杂交文库构建及其表达序列标签研究. 植物病理学报, 2007, 37(1): 50-55.
[12] 朱龙付, 涂礼莉, 张献龙, 聂以春, 郭小平, 夏启中. 黄萎病菌诱导的海岛棉抗病反应的SSH文库构建及分析. 遗传学报, 2005, 32(5): 528-532.
[13] 萨姆布鲁克J, 拉塞尔DW. 分子克隆实验指南 (第三版) (黄培堂等译). 北京: 科学出版社, 2002.
[14] Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W, van Staveren M, Stiekema W, Drost L, Ridley P, Hudson SA, Chalwatzis N. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature, 1998, 391(6666): 485-488.
[15] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet, 2000, 25(1): 25-29.
[16] 吴金华, 胡银岗, 王新茹, 张 宏, 王长有, 王秋英, 吉万全. 小麦抗白粉病SSH-cDNA文库中差异基因的表达模式. 作物学报, 2008, 34(12): 2121-2125.
[17] Duan YH, Guo J, Ding K, Wang SJ, Zhang H, Dai XW, Chen YY, Govers F, Huang LL, Kang ZS. Characterization of a wheat HSP70 gene and its expression in response to stripe rust infection and abiotic stresses. Mol Biol Rep, 2011, 38(1): 301-307.
[18] Rebrikov DV, Britanova OV, Gurskaya NG, Lukyanov KA, Tarabykin VS, Lukyanov SA. Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res, 2000, 28(20): E90.
[19] Ji W, Wright MB, Cai L, Flament A, Lindpaintner K. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics, 2002, 3: 12.
[20] Matin GB, Bogdanove AJ, Sessa G. Understanding the functions of plant disease resistance proteins. Annu Rev Plant Biol, 2003, 54: 23-61.
[21] Dilbirligi M, Erayman M, Sandhu D, Sidhu D, Gill KS. Identification of wheat chromosomal regions containing expressed resistance genes. Genetics, 2004, 166(1): 461-481.
[22] Catherine F, Silvia T, Nils S, Albar L, Nublat A, Keller B. Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome. Proc Natl Acad Sci USA, 2003, 100(25): 15253-15258.
[23] Krattinger SG, Lagudah ES, Spielmeyer W, Singh RP, Huerta-Espino J, McFadden H, Bossolini E, Selter LL, Keller B. A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science, 200
文章导航

/