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作物学报 ›› 2008, Vol. 34 ›› Issue (12): 2121-2125.doi: 10.3724/SP.J.1006.2008.02121

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦抗白粉病SSH-cDNA文库中差异基因的表达模式

吴金华1,2;胡银岗1;王新茹3;张宏1;王长有1;王秋英1;吉万全1,*   

  1. 1 西北农林科技大学农学院 / 陕西省农业分子生物学重点实验室 / 国家小麦改良中心杨凌分中心，陕西杨凌712100；2 河北农业大学农学院，河北保定071001；3 陕西省农药管理检定所，陕西西安710003
  • 收稿日期:2008-03-07 修回日期:2008-06-05 出版日期:2008-12-12 网络出版日期:2008-09-06
  • 通讯作者: 吉万全
  • 作者简介:吴金华(1978-),女,山西朔州人,博士,主要从事小麦抗病育种及其分子生物学研究.
  • 基金资助:

    国家重点基础研究发展计划(973计划)前期项目(2006CB708208);国家科技支撑计划项目(2006BAD01A02)

Expression Pattern of Special Genes Resistant to Powdery Mildew (Blumeria graminis f. sp. tritici) in SSH-cDNA Library of Wheat

WU Jin-Hua1,HU Yin-Gang1,WANG Xin-Ru2,ZHANG Hong1,WANG Chang-You1,WANG Qiu-Ying1,JI Wan-Quan1*   

  1. 1 College of Agronomy, Northwest A＆F University / Yangling Branch of China Wheat Improvement Center / Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, Shaanxi; 2 College of Agronomy, Agricultural University of Hebei, Baoding 071001, Hebei; 3 Institute for the Control of Agrochemicals of Shaanxi Province, Xi’an 710003, Shaanxi, China
  • Received:2008-03-07 Revised:2008-06-05 Published:2008-12-12 Published online:2008-09-06
  • Contact: JI Wan-Quan

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被引次数

20

摘要：

为了解白粉菌诱导下抗白粉病小麦的抗病机制，构建了白粉菌接种初期的抑制性消减杂交cDNA(SSH-cDNA)文库。从中随机挑取140个阳性克隆进行测序，去除冗余序列和重复序列后，得到94条EST，利用NCBI的BLAST在线序列比对工具对GenBank的蛋白质数据库进行同源性比对及功能分类。结果表明，49个EST与已知功能蛋白同源性较高，主要涉及初级代谢(2%)、能量代谢(24%)、细胞结构(2%)、转录(2%)、蛋白质合成加工与储藏(16%)、转运(4%)、信号转导(4%)和抗病与防御(30%)。经文库比较，筛选出与病程相关蛋白基因同源的EST(Z25-1)和与谷胱甘肽硫转移酶同源的EST(Z440-1)，GenBank登录号为EX567369和EX567360。以其EST序列为依据设计引物，通过RT-PCR分析它们在白粉菌诱导下的表达模式，结果该2基因表达存在明显差异。其中，Z25-1在未接种白粉菌时具有一定的表达量，白粉菌侵染后表达量开始上升，侵染72 h时表达量最高，然后下降；Z440-1在未接种时也具有一定的表达量，但在接种初期表达微弱，甚至不表达，24 h后表达开始上升，到72 h时达最高，然后下降。本研究表明，病程相关蛋白和谷胱甘肽硫转移酶属于诱导型表达基因，且参与白粉病抗病反应，在白粉菌诱导72 h时表达量最高。

关键词: 小麦, 白粉病, 抑制性消减杂交(SSH), 反转录聚合酶链式反应(RT-PCR)

Abstract:

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important fungal diseases of common wheat (Triticum aestivum L.) worldwide and causes severe yield losses. Wheat germplasm N9436, developed by our research group, is a resistant material to powdery mildew. In the present study, a suppression subtraction hybridization (SSH) cDNA library was constructed with cDNA from N9436 leaf inoculated by Blumeria graminis as the tester and cDNA from N9436 healthy leaf as the driver. A total of 140 positive clones were randomly chosen from the SSH-cDNA library and were amplified with sp6 and t7 primers to examine the insert size, which ranged from 200 to 1 000 bp with an average of 238 bp. After screening repeat and redundancy sequences, 94 expressed sequence tags (ESTs) were acquired. Protein homology search in nr-protein database revealed that 49 ESTs were highly homologous with known proteins functioning in primary metabolism (2%), energy metabolism (24%), cell structure (2%), transcription (2%), protein synthesis and processing (16%), transport (4%), signal transduction (4%), and disease resistance and defenses (30%). Compared with the EST sequences among the SSH-cDNA library, 2 ESTs (GenBank accession number: EX567369 and EX567360) were highly similar to pathogenesis-related protein (Z25-1) and glutathione-S-transferase (Z440-1), respectively. On the basis of the EST sequences, 2 pairs of primers were designed and used to detect the expression differences of the 2 genes when inoculating with powdery mildew by Reverse Transcriptase PCR (RT-PCR). Z25-1 and Z440-1 were both expressed under inoculation. Their expression amounts were the largest in 72 h after inoculation with powdery mildew. But their expression trends were different. At early stage of inoculation (24 h after inoculation), the expression amount of Z440-1 was very a little, then increased in 72 h, and finally decreased in 96 h. The expression amount of Z25-1 increased from 24 h to 72 h after inoculation and decreased after 96 h. It suggests that pathogenesis-related protein and glutathione-S-transferase belong to inducible expression genes and are involved in the resistance to powdery mildew.

Key words: Wheat, Powdery mildew, Suppression subtraction hybridization (SSH), Reverse transcriptase PCR

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