摘要
以结球甘蓝高抗TuMV自交不亲和系 84 0 75为材料 ,构建了cDNA文库。根据抗病基因保守序列 (NBS LRR)设计一对简并引物 ,以 84 0 75的基因组DNA和cDNA为模板 ,进行PCR扩增 ,分别扩增出一条 5 13bp片段 ,对扩增片段进行了克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针 (命名Bor1,Bor2 ) ,对构建的cDNA文库进行筛选 ,得到一个阳性克隆 (命名TuR2 )。测序及序列分析表明 ,该基因总长为 76 2bp ,编码 2 2 6个氨基酸 ,包含 6 81bp的开放阅读框 ,与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针 ,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明 ,TuR2可能以单拷贝形式存在 ,其表达是组成型的 ,且无组织特异性 ;
A cDNA library was constructed from cabbage 84075 which resists TuMV. The degenerate primers was designed with the disease resistance gene conservative domain (NBS LRR). The fragments of 513 bp were amplified by RT PCR and genomic DNA PCR from resistant material 84075, then cloned and sequenced. Two recombinants which are highly homologous with the resistance genes cloned in other plants were used as probes, (named as Bor1, Bor2), the cDNA library was screened. A positive clone was obtained, named as TuR2, whose length was 762 bp, which encodes 226 amino acids, contains a long 681 bp open reading frame (ORF), and has different homology score of amino acid compared with the cloned resistance disease genes of other plants. TuR2 was used as probe. Southern blotting hybridization with genomic DNA shows that TuR2 is probably present in single copy; No. rthern blotting hybridization with RNA shows that the gene expression is constitutive and not differential in every part of the resistance plant 84075, the result of separating detection in F 2 population shows that TuR2 gene is probably related to resistance to TuMV in cabbage.
基金
教育部中青年骨干教师项目资助~~
