小麦成株抗条锈性抑制差减杂交文库构建及表达序列标签分析  

Construction of Suppression Subtractive Hybridization cDNA Library of Wheat Adult Plant Resistance to Stripe Rust and Analysis of Its Expressed Sequence Tags

作　　者：黄雪玲[1] 喻修道[1] 屈志鹏[1] 王晓杰[1] 韩青梅[2] 黄丽丽[1] 康振生[1] 

机构地区：[1]西北农林科技大学植物保护学院,杨凌712100 [2]陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2007年第6期976-981,共6页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)前期专项(No.2006CB708208);教育部重大科技培育项目(No.2004-295);教育部长江学者和创新团队发展计划项目(No.IRT0558);国家自然科学基金项目(No.30671350);高等学校学科创新引智计划项目(No.B07049)资助。

摘　　要：以成株期抗条锈小麦(Triticum aestivum)品种兴资9104为材料,依照CLONTECH公司PCR-SelectTMcDNA Subtraction Kit方法构建了小麦成株期条锈菌(Puccinia striiformis)诱导的抑制差减杂交(suppression subtractive hybridization,SSH)cDNA文库。建库质量分析表明,接头连接效率高,插入片段平均300bp,文库质量较好。对文库中随机挑取的96个阳性克隆进行测序,用CAP3软件聚类拼接,得到unigenes41个。与GenBank已知序列进行Blastx分析表明,所得基因主要涉及植物的信号传导、转录调控、能量与代谢、蛋白质合成与代谢、膜转运、细胞生长分化等。利用RT-PCR对3条基因进行分析,明确其在抗病过程中的表达模式。A suppression subtractive hybridization （SSH） cDNA library was constructed with an incompatible-combination between wheat （Triticum aestivum） cultivar Xingzi 9104 and Puccinia striiformis f. sp. tritici at adult plant stage by PCR-SelectTM cDNA Subtraction Kit （CLONTECH）. Detection and analysis of the library construction showed that the technique was efficient in the adaptors ligation, and the average insert fragment size was 300 bp. Plasmids were extracted from 96 positive clones randomly selected from the library and then sequenced and assembled using CAP3 software and 41unigenes were obtained. ESTs similarity analysis was finished by comparing sequences with BLASTx software in non- redundance database of GenBank. The results showed that they related to many biological processes including signal transduction, transcription regulation, protein synthesis and metabolism, membrane transport and cell growth and division. Three sequences were selected to testify their expression profile at different time after inoculating with P. striiformis race CY32 by reverse transcription PCR（RT-PCR）.

关 键 词：小麦 条锈菌 抑制差减杂交 表达序列标签(EST) 成株抗性 

分 类 号：S188[农业科学—农业基础科学]