小麦NBS-LRR类抗病基因同源序列的分离与鉴定  

Isolation and Characterization of NBS-LRR Resistance Gene Homology Sequences from Wheat

作　　者：王海燕[1] 杨文香[1] 刘大群[1] 

机构地区：[1]河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心,保定071001

出　　处：《中国农业科学》2006年第8期1558-1564,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30170602);河北农业大学博士基金;国家"973"前期项目(2005CCA01600)

摘　　要：目的拟利用同源序列法分离小麦抗病基因同源片段。方法根据已克隆植物抗病(R)基因NBS-LRR保守区段设计两对引物,采用RT-PCR方法对小麦抗叶锈病近等基因系材料TcLr35的RNA进行扩增。结果获得了3个通读的小麦抗病基因NBS-LRR类抗病基因同源片段PS13-1、PS13-2和S2A2,长度分别为239bp、289bp和539bp,编码78、84和177个氨基酸。核苷酸比较分析表明,PS13-1和PS13-2与已克隆小麦抗白粉病基因PM3b同源性为91%;S2A2与大麦一个来源于mRNA的同源片段同源性为91%;经BLASTp比较,3个片段均含有NB-ARC保守结构域,与已知抗病基因I2C-1,L6,RPS2等相应区域相一致,具有抗病基因NBS特征结构域(P环、激酶2a等)。Northern杂交分析表明,3个同源片段在小麦叶片中为低丰度组成型表达。结论本研究在TcLr35小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病基因奠定了基础。[ Objective ] In this study, resistance gene homology fragments from wheat were isolated using homology-based method. [Method]Two pairs of primers were designed according to the amino acid conserved regions NBS-LRR of the cloned plant disease resistance genes, and the reverse transcription-polymerase chain reaction （RT-PCR） was used. [Result] Three open-reading NBS resistance gene analogues （RGAs） have been obtained. They were named as PS 13-1, PSI 3-2, and S2A2 respectively from the RNA of TcLr35 carrying the Lr35 gene conferring resistance against wheat leaf rust by a reverse transcription-polymerase chain reaction （RT-PCR）. The nucleotide sequences of the three RGAs were 239bp, 289bp and 539bp encoding 78, 84 and 177 amino acids respectively. Homology research showed that the nucleotides of P S 13-1 and PS 13-2 were 91% identical to wheat powdery mildew resistance gene PM3b, and the nucleotide of S2A2 was 91% identical to the sequence from the mRNA of barley. BLASTp analysis indicated that three RGAs contained the conserved motifs of NB-ARC （such as P-loop, kinase2, kinase3a and transmembrance domain）. Northern hybridization showed that the RGAs expression was not induced by Puccinia recondite inoculation suggesting that the gene be a constitutive gene with low abundance in wheat. [ Conclusion ] In this study, three resistance homology sequence were obtained in TcLr35, which provide the shortcut for the cloning of wheat leaf rust resistance genes.

关 键 词：小麦抗叶锈病基因 NBS-LRR 同源序列 

分 类 号：S512.1[农业科学—作物学]