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利用已克隆的抗病基因(resistance-gene,R基因)与病程相关蛋白基因?-1,3-葡聚糖酶基因(PR2)的保守序列设计简并引物,从海岛棉品种海7124基因组中成功分离了79条核苷酸结合位点(NBS,nucleotidebindingsite)类R基因类似物(RGAs,resistancegeneanalogs),21条丝氨酸/苏氨酸蛋白激酶(STK,Serine/Threoninekinase)类RGAs和11条防卫基因类似物(DGAs,defensegeneanalogs).将NBS类RGAs与棉花中已经克隆的该类RGAs进行序列分析,鉴定了1个新的亚族.具有连续ORF的48条NBS类RGAs和20条STK类RGAs分别可以划分为TIR-NBS与non-TIR-NBS两个亚类和A与B两个亚类.DGA中有4条具有连续的ORF.对这些具有连续ORF的序列进行表达分析发现,6条NBS类与1条STK类RGA受黄萎病菌诱导表达,1条DGA经黄萎病菌诱导后表达量明显提高.随机选取的4条TIR类与4条non-TIR类NBS类RGAs中有3条呈现2~3条杂交带,其余的都有5~10条或以上的杂交带.可见,NBS类RGAs大多数以多基因家族的形式存在.另外,有3条non-TIR类NBS类RGAs出现了一样的杂交带型,可能这3条RGAs在基因组中成簇分布.
Degenerate primers were designed based on the conserved sequence of cloned resistance-gene (R gene) and disease-related protein gene -1,3-glucanase gene (PR2) A total of 79 RGAs (resistance gene analogs) from NBS (nucleotide binding site), 21 RGAs (Serine / Threoninekinase) and 11 defense genes (DGAs, defensegenealogs) .A novel subfamily of N-type RGAs and RGAs that have been cloned in cotton was identified, and 48 NBS RGAs and 20 STK RGAs with continuous ORF Which can be divided into two subtypes TIR-NBS and non-TIR-NBS and two subtypes A and B. There are four consecutive ORFs in the DGA.Analysis of these sequences with consecutive ORFs revealed that six NBS And one STK RGA was induced by Verticillium dahliae, and the expression of one DGA was significantly increased after Verticillium dahliae infection.Among three randomly selected TIRs and four non-TIR NBS RGAs, three 2 to 3 hybridization bands, and the rest have 5 to 10 or more hybrid bands.It can be seen that most of NBS RGAs Form of the gene family. In addition, there are three non-TIR-based NBS class RGAs appeared with the same type of hybridization, which may be distributed in the three clusters RGAs genome.