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作物学报 ›› 2010, Vol. 36 ›› Issue (3): 401-409.doi: 10.3724/SP.J.1006.2010.00401

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用cDNA-AFLP技术分析小麦成株抗条锈性差异基因表达特征

张岗1,**，董艳玲1,**，夏宁1，张毅1，王晓杰1，屈志鹏1，李依民1，黄丽丽1，康振生1,2,*   

  1. 1西北农林科技大学植物保护学院, 陕西杨凌 712100; 2西北农林科技大学 / 陕西省农业分子生物学重点实验室, 陕西杨凌 712100
  • 收稿日期:2009-10-16 修回日期:2009-12-08 出版日期:2010-03-12 网络出版日期:2009-12-21
  • 通讯作者: 康振生, E-mail: kangzs@nwsuaf.edu.cn
  • 基金资助:

    本研究由国家自然科学基金重点项目（30930064），现代农业产业技术体系技术专项资金，高等学校学科创新引智计划项目（B07049）和国家“十一五”科技支撑计划项目（2006BAD08A05）资助。

cDNA-AFLP Analysis Reveals Differential Gene Expression in Wheat Adult-Plant Resistance to Stripe Rust

ZHANG Gang1,**,DONG Yan-Ling1,**,XIA Ning1,ZHANG Yi1,WANG Xiao-Jie1,QU Zhi-Peng1,LI Yi-Min1,HUANG Li-Li1,KANG Zhen-Sheng1,2,*   

  1. 1 College of Plant Protection, Northwest A&F University, Yangling 712100, China; 2 Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture / Northwest A&F University, Yangling 712100, China
  • Received:2009-10-16 Revised:2009-12-08 Published:2010-03-12 Published online:2009-12-21
  • Contact: KANG Zhen-Sheng, E-mail: kangzs@nwsuaf.edu.cn

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被引次数

31

摘要：

采用cDNA-AFLP技术，对成株抗条锈小麦品种兴资9104在成株期受条锈菌生理小种CY32侵染后5 d内9个时间点的基因表达谱进行了分析。共筛选64对引物，产生32 320个转录本(TDF)；用37对引物检测到2 201个(6.81%)差异TDF，其中926个TDF诱导表达，1 275个下调表达。经大规模克隆、测序分析，最终获得330个差异TDF，聚类分析得到259个EST (unigenes)，命名为aTaPST1至aTaPST259 (GenBank注册号：FL645754~FL646011和FL646262)。经BLASTX比对和功能分类分析，其中96条EST(37.07%)未找到同源性匹配，68条(26.25%)与未知功能蛋白同源性较高；其余95条ESTs主要涉及能量(11.20%)、基础代谢(4.63%)、转录调控(3.86%)、抗病与防御(3.86%)、蛋白质运输和储存(3.09%)、蛋白质合成和细胞生长(各2.32%)、以及信号转导(1.54%)等。选取抗病与防御、转录调控及信号转导类等相关的6个差异基因，qRT-PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。小麦成株抗条锈性分子机制涉及植物多方面生理生化反应，包括抗病与防御、转录调控、蛋白质代谢、信号转导、以及非生物胁迫等多种途径相关基因的协同控制。

关键词: 小麦, 条锈菌, 抗病性, 基因表达, cDNA-AFLP, qRT-PCR

Abstract:

Wheat (Triticum aestivum L.) stripe rust is one of the most devastating diseases of wheat throughout the world. Adult plant resistance (APR) to stripe rust in wheat conferring durable resistance, thus, plays a pivotal role in the control of the disease. In the present study, to elucidate molecular mechanism of wheat APR to stripe rust, we conducted extensive transcription profiling of adult-plant wheat cultivar Xingzi 9104 infected by Puccinia striiformis Westend f. sp. tritici Erikss. pathotype CY32 using cDNA-AFLP technique. We analyzed transcription profiling of the incompatible reaction across nine sampling time points within five days after inoculation. Of the total 32,320 transcript derived fragments (TDFs) obtained using cDNA-AFLP with 64 primer pairs, 2201 (6.81%) displayed altered expression patterns after inoculation, of which 926 showed up-regulated and 1275 down-regulated. Three hundred and thirty differentially expressed TDFs produced reliable sequences after cloning and sequencing, of which 259 expressed sequence tags (ESTs) of unigenes were obtained after assembling, designated from aTaPST1 to aTaPST259, deposited in GenBank with accessions numbers from FL645754 to FL646011 and FL646262. BLASTX analyses and functional annotations were then performed and the results revealed that the 95 ESTs had predicted gene products mainly implicated in energy (11.20%), metabolism (4.63%), transcription (3.86%), disease/defense (3.86%), protein destination and storage (3.09%), protein synthesis and cell growth (each accounted for 2.32%), and signal transduction (1.54% of the sequenced total 259 ESTs). Six differential genes related to disease/defense, transcription, and signal transduction were chosen for further qRT-PCR expression patterns, which confirmed the cDNA-AFLP profiles. Our results indicated that wheat APR to stripe rust involved in multifaceted biochemical and physiological reactions, including concerted regulation of the genes involved in different pathways like disease/defense, transcription, protein metabolism, signal transduction, as well as abiotic stresses. These results provide information for further elucidation of molecular mechanism of wheat APR to stripe rust.

Key words: Wheat, Stripe rust fungus, Adult-plant resistance, Gene expression, cDNA-AFLP, qRT-PCR


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