小麦抗白粉病SSH-cDNA文库中差异基因的表达模式  

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

作　　者：吴金华[1,2] 胡银岗[2] 王新茹 张宏[2] 王长有[2] 王秋英[2] 吉万全[2] 

机构地区：[1]河北农业大学农学院,河北保定071001 [2]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 [3]陕西省农药管理检定所,陕西西安710003

出　　处：《作物学报》2008年第12期2121-2125,共5页Acta Agronomica Sinica

基　　金：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)

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72h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,24h后表达开始上升,到72h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72h时表达量最高。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, two ESTs （GenBank accession numbers： 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, two pairs of primers were designed and used to detect the expression differences of the two 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 incr

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) 

分 类 号：S435.121[农业科学—农业昆虫与害虫防治]