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本文是在先期研究工作的基础上,利用从柑桔与枳属间杂种的基因组DNA所获得的类受体激酶(RLK)的候选抗病基因序列,重新设计引物,对柑桔抗溃疡病材料和感病材料开展以PCR扩增为基础的对比分析,其中一对引物‘19h16/DdeI’的扩增产物经限制性内切酶DdeI酶切,揭示了抗性材料(IchangPape-da,Meiwakumquat,MaruhikumquatandNagamikumquat)和感病材料(FlyingDragon,ValenciaorangeandPalestinesweetlime)之间的多态性差异,同时,在C.Ichangensis的自交F1代和Palestinesweetlime×IchangPapeda的杂交F1代群体中也见明显的多态性差异;对这些F1代个体进行溃疡病病原[Xanthomonasax-onopodispv.Citri(Xac)]接种作抗病性表型鉴定,结果表明,该分子标记‘19h16/DdeI’与柑桔溃疡病抗性密切相关;经纯化测序的结果进一步证明,抗性材料PCR扩增产物具有完全一致的序列和DdeI酶切位点,但感病材料缺少这一DdeI酶切位点;染色体步行(primerwalking)在BAC文库的对应单克隆中获得了一个完整的抗病基因序列,与以前获得的2个抗病基因序列‘17o6RLKP’和‘26m19RLKP’相比,‘19h16RLKP’也具有Xa21抗病基因蛋白的所有特征,包括含有一个信号肽、同样数目的亮氨酸重复序列、跨膜域和激酶域等。基于该序列的开放读码框,发展了特异性更强可靠性更高的另一分子标记,在今后的研究工作中具有较大的应用潜力。
Based on the previous research work, this paper reuses the candidate resistance gene sequences of RLK derived from the genomic DNA of the interspecific hybrid between Citrus and Citrus aurantium, And susceptible materials were compared based on PCR amplification. The amplified product of a pair of primers ’19h16 / DdeI’ was digested with restriction enzyme DdeI to reveal that the resistant material (IchangPape-da, Meiwakumquat, MaruhikumquatandNagamikumquat) and the susceptible material (FlyingDragon, Valenciaorange andPalestinesweetlime). At the same time, significant differences in polymorphism were also observed in the F1 population of selfing F1 hybrids of C.Ichangensis and of Palestinesweetlime × IchangPapeda. The results showed that the molecular marker ’19h16 / DdeI’ was closely related to the resistance of citrus canker. The purified F1 progenies of these F1 plants were susceptible to citrus canker disease. Sequencing results further proved that PCR products of resistant material had exactly the same sequence and DdeI cleavage site, but the susceptible material lacks this DdeI restriction site; chromosome walking (prime rwalking) obtained a complete resistance gene sequence in the corresponding monoclonal of BAC library. Compared with the two resistance gene sequences’ 17o6RLKP ’and ’26m19RLKP’ obtained previously, ’19h16RLKP’ also has the Xa21 resistance gene protein All the features, including a signal peptide, the same number of leucine repeats, transmembrane and kinase domains. Based on the open reading frame of the sequence, another molecular marker with higher specificity and reliability is developed, which has great potential in future research.