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利用cDNA末端快速扩增技术对小麦抗叶锈病近等基因系TcLr35中所获得的抗病基因同源片段S2A25′端和3′末端序列进行扩增,并根据拼接序列设计特异性引物,进行全长基因的扩增,获得了1个通读的NBS类抗病同源基因S2A2cDNA序列,该序列全长为3 476 bp,编码866个氨基酸序列。经BLASTp比较,该片段含有NB-ARC保守结构域和多个LRR结构域,与已知植物抗病基因I2C1-、L6、RPS2等相应区域相一致。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TcLr35小麦中成功获得了抗病同源基因,这为最终克隆小麦抗叶锈病基因Lr35奠定了基础。
The rapid amplification of cDNA ends was used to amplify the S2A25 ’and 3’ end sequences of the resistance gene homologous fragment TcLr35 in wheat leaf rust resistance and to design specific primers according to the splicing sequence The sequence of S2A2 cDNA of NBS resistance gene was obtained through the amplification of the long gene. The full length of this gene was 3 476 bp, which encoded a 866 amino acid sequence. Compared with BLASTp, the fragment contains NB-ARC conserved domain and multiple LRR domains, which are consistent with corresponding regions of known plant resistance genes, such as I2C1-, L6, RPS2. Semi-quantitative RT-PCR analysis showed that the gene was low-abundance constitutively expressed in wheat leaves. This study successfully obtained the disease-resistant homologous genes in TcLr35 wheat, which laid the foundation for the cloning of wheat leaf rust resistance gene Lr35.