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.pdfVIP

农业生物技术学报 Journal ofAgricultural Biotechnology 2007，1 5 (6)：976～98 1 · 研究论文· 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 黄雪玲 ，喻修道 ，屈志鹏 ，王晓杰 ，韩青梅z，黄丽丽 ，康振生--z (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) 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．SelectTM cDNA 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300 bp，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unigenes 41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。 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 中图分类号：S188 文献标识码：A 文章编号：1006．1304(2007)06．0976．06 Construction of Suppression Subtractive Hybridization cDNA Library of Wheat Adult Plant Resistance to Stripe Rust and Analysis of Its Expressed Sequence Tags HUANG Xue—ling ，YU Xiu—dao ，Qu Zhi—peng ，WANG Xiao-jie ， HAN Qing—mei2,HUANG Li—li ．KANG Zhen—sheng r』．College ofHantProtection，NorthwestAgricultureandForesuy University，Yangling 712100，China；2．ShaanxiKeyLaboratory of MolecularBiologyforAgriculture,Yangling，712100，China) Ab血act A suppression subtractive hybridization(SSH)cDNA library was constructed with an incompatible’combination between wheat(Trificum aestivum)cultivar Xingzi 9104 and Puccinia striiformis￡sp．tritici at adult plant stage by PCR—SelectTM cDNA Subtraction Kit(CLONTECH)．Detection and analysis ofthe 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