《植物生理学报》 2010, 46(5): 453-458

大白菜霜霉菌诱导抑制性消减杂交cDNA 文库的构建和分析

唐永洽^1,2, 于拴仓², 朱月林¹, 张凤兰^2,*, 余阳俊², 赵岫云², 张德双²

1 南京农业大学园艺学院, 南京210095; 2 北京市农林科学院蔬菜研究中心, 北京100097

通信作者：张凤兰；E-mail: zhangfenglan@nercv.org；Tel: 010-51503 038

摘　要：

以抗霜霉病大白菜双单倍体系(DH) ‘T12-19’ 为材料, 构建了霜霉病诱导表达的正向抑制性消减文库, 并利用反向 Northern 斑点杂交技术对 768 个阳性克隆进行了筛选, 共获得 57 个病原菌诱导上调表达的克隆。测序后得到 55 条通读表 达序列标签(ESTs), 对这些 ESTs 序列进行聚类和拼接分析, 共获得 50 个 unigenes。Blast 分析表明, 37 个 unigenes 与已知基 因高度同源, 占全部非重复序列的 67.3%。对已知功能基因按 MIPS 的分类方法进行功能分类, 发现这些基因的功能主要 涉及物质与能量代谢、转录调控、蛋白质合成与代谢、膜及转运、信号转导、抗病防御等。为了验证文库筛选结果的可 靠性, 采用实时荧光定量 PCR 技术分析了其中 2 个克隆 BFCH10 和 BFIA7 的表达谱。结果表明, 这 2 个克隆在接种病菌 6 h 后明显上调表达, 与反向 Northern 斑点杂交结果基本一致。

关键词：大白菜; 霜霉病; 抑制性消减文库; 实时荧光定量PCR

收稿：2010-01-27   修定：2010-04-28

资助：国家科技支撑项目(2009BADB8B00-03)、大宗蔬菜产业技术体系建设专项资金、北京市自然科学基金(6102011)和北京市科技计划项目(Z09090501040902)

Construction and Analysis of Suppression Subtractive Hybridization cDNA Library in Chinese Cabbage (Brassica rapa ssp. pekinensis) Leaves Induced by Peronospora parasitica

TANG Yong-Qia^1,2, YU Shuan-Cang², ZHU Yue-Lin¹, ZHANG Feng-Lan^2,*, YU Yang-Jun², ZHAO Xiu-Yun², ZHANG DeShuang²

¹College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China; ²Biejing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

Corresponding author: ZHANG Feng-Lan; E-mail: zhangfenglan@nercv.org; Tel: 010-51503 038

Abstract:

A forward suppression subtractive hybridization (SSH) library was constructed from leaves of Chinese cabbage (Brassica rapa ssp. pekinensis) double haploid (DH) line ‘T12-19’ resistant to downy mildewwhich was induced by Peronospora parasitica. By reverse Northern blot analysis, 57 up-regulated expressed sequence tags (ESTs) were identified from 768 clones of the SSH library and were sequenced to generate 55 high-quality ESTs. These sequences clustered into 50 unigenes. Blast analyses showed that 37 unigenes shared high identity with genes of known functions involved in energy metabolism, transcription regulation, protein synthesis and fating, membrane transportation, signal transduction, defense, etc. To verify the screening efficiency, the expression profiling of two clones, BFCH10 and BFIA7, was analyzed by real-time PCR. The significant upregulation was observed in leaves of 6 h post-inoculation for these two clones. These results matched the study by reverse Northern blot analysis.

Key words: Brassica rapa ssp. pekinensis; downy mildew; suppression subtractive hybridization (SSH); realtime PCR