TcLr24小麦NBS-LRR同源基因的克隆与分析

目前已克隆的小麦抗叶锈病基因多数含有NBS类保守结构域,为克隆高抗叶锈病Lr24基因及其抗病相关基因,根据NBS保守结构域设计引物,对小麦抗叶锈病近等基因系TcLr24cDNA进行扩增,得到534bp的抗病基因同源序列。对该序列进行同源性检索并验证,获得1 408bp的电子延伸序列。以此通过RACE技术分别获取2 797bp和3 466bp的cDNA与gDNA全长产物,其翻译的蛋白质序列含有NBS保守区的P-loop、Kinase 2a、Kinase 3a、HD和LRR保守结构,与大麦等单子叶植物NBS类蛋白序列同源性较高,生物信息学分析表明其为稳定的亲水性蛋白,分子质量104.28ku,理论等电点6.15。半定量RT-PCR表明该片段所在基因序列为组成型表达。

Isolation and Characterization of NBS-LRR Homologue from TcLr24

Conserved domain of NBS is mostly contained in the cloned wheat leaf rust resistance (Lr) genes. In order to clone high resistant gene Lr24 and its related genes, NBS like conserved primers were designed and 534 bp target fragment was isolated from cDNA of TcLr24,1 408 bp electronic extended sequence was acquired and verified. Then full-length products with 2 797 bp and 3 466 bp were isolated from cDNA and gDNA of TcLr24, respectively. It showed high homology to some NBS type resistance protein like Hordeum vulgare. Conserved domain of P-loop,Kinase 2a,Kinase 3a, and HD of NBS, and LRR domain were contained in this sequence. Bioinformatics analysis showed it was a hydrophilic protein with good stability, and with molecular weight 104.28 ku and theoretical isoelectric point 6.15. It implied the constitute expression model of this gene by semi-quantitative RT-PCR. The results provide basis for further study on cloning the leaf rust resistance genes in wheat.