小麦种质N9436抗白粉病的特异基因表达谱分析

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小麦抗性种质N9436的抑制性消减杂交文库。从文库中随机挑取140个阳性克隆, 测序结果表明, 冗余重复序列占32.86%, 其中谷胱甘肽转移酶表达频率最高, 其次为参与能量代谢的二磷酸核酮糖大小亚基。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的核酸和蛋白质数据库进行同源性比对和功能分析。BlastX比对结果表明, 有49条EST与已知功能蛋白同源性较高, 主要涉及抗病与防御(包括生物及非生物胁迫)、能量代谢、细胞结构、蛋白质合成及加工、转运及信号转导等过程的相关蛋白。BlastN比对NCBI的非冗余核酸数据库, 其中69条序列与EST数据库中的Unigene具有较高的同源性, 20条与EST数据库中的同源性较高, 另外5条序列找不到同源序列。通过对核酸和蛋白质同源性的比较和功能分析发现, 比对结果一致的序列有33条, 涉及白粉病抗性的相关蛋白22个, 其中与抗病信号传导相关的蛋白6个, 过敏性坏死反应(HR)体系表达蛋白2个, 系统获得性抗性(SAR)体系病程相关蛋白4个, SAR体系诱导防卫蛋白10个。

Expression of Special Genes Resistant to Powdery Mildew (Blumeria graminis f. sp. tritici) in Wheat Germplasm N9436

Powdery mildew, caused by Blumeria graminis f. sp. tritici (syn. Erysiphe 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. Their sizes ranged from 200 to 1 000 bp with an average of 238 bp. Among the 140 clones, 32.86% showed redundant and repeated sequences by sequence analysis. The most frequent se-quence was glutathione transferase and followed by ribulose-1,5-bisphosphate carboxylase/oxygenase small/large subunit. After screening repeat and redundant sequences, 94 ESTs were acquired. Nucleic acid and protein homology search were performed using the BLAST (Basic Local Alignment Search Tool) program with the default settings at NCBI website (http://www.ncbi. nlm.nih.gov). BlastX results in nr-protein database revealed that 49 ESTs were highly homologous with known proteins involved in disease resistance and defenses, energy metabolism, cell structure, protein synthesis and processing, transport and signal trans-duction. BlastNr results showed that 69 and 20 ESTs had high identities with known Unigene and function-unknown ESTs, re-spectively, and 5 ESTs matched none in the nr-database. Compared with BlastX and BlastNr analysis, 33 ESTs were both in the nucleic acid and protein databases including 22 ESTs associated with powdery mildew resistance, out of which 6 for signal trans- duction, 2 for hypersensitive necrosis reaction (HR) system, and 14 for systemic acquired resistance (SAR) system, respectively.