小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

Cloning and Characterization of a NBS Resistance Gene Homology cDNA Sequence from Wheat

根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19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目的基因奠定了基础。

Resistance gene homology sequence from wheat was isolated by using homology-based method.A pair of degenerated primer was designed according to the nucleotide binding site conserved domains of the cloned plant disease resistance（R） genes.RT-PCR and RACE were used to obtain the full length sequence of the disease resistance homology gene in the near isogenic lines TcLr19.One open-reading NBS class of resistance gene analogs（RGAs） named S11A11 was obtained,which was 2923bp in length and encoded 878 amino acids.Bioinformatics analysis showed the deduced amino acids of S11A11 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats（LRR） domains.The phylogenetic tree analysis indicated a considerable identity of the protein encoded by S11A11 with that of wheat leaf rust resistance gene Lr1,but a lower similarity with Lr10.The S11A11 gene appeared not to be induced by Puccinia triticina and was a constitutive gene with low abundance in wheat leaf tissue by semi-quantitative RT-PCR.The resistance homology sequence was successfully obtained in TcLr19,which provides the shortcut for cloning of wheat leaf rust resistance gene.