摘要：用根据抗病基因保守区设计的一对简并性引物 ,从小麦 簇毛麦易位系 6VS 6ALcDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点 (Nucleotidebindingsite,NBS)结构特点的DNA片段克隆N7。从小麦 簇毛麦易位系6VS 6AL基因组TAC(Transformation competentartificialchromosome,TAC)文库的 2 2块 96孔板提取所有 2 112个克隆池(每个池含约 10 0 0个克隆 )的质粒 ,再根据N7的核苷酸序列设计一对特异引物 ,用克隆池PCR(pooledPCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针 ,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。

摘要：Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5'-end sequence of an RGA N5 was obtained by 5'-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2.