【作者】 张华；

【导师】 刘凤权； 胡白石；

【作者基本信息】 南京农业大学 ， 植物病理学， 2004， 硕士

【摘要】 水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)和细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola)，是黄单胞菌属水稻黄单胞菌的不同致病变种。水稻白叶枯病是一个世界性病害，以亚洲稻区发生为重，水稻细菌性条斑病的危害仅次于水稻白叶枯病，主要发生于我国南部地区，是国内重要的检疫性病害。这两种病害同时也被许多国家列为检疫性有害生物，限制从这两种病害的疫区进口水稻种子及稻米。因此，发展一种灵敏专化、准确便捷的同时可从水稻上检测这两种细菌的检疫技术无论对国内植物检疫还是对保证我国水稻种子及稻米的出口均有重要意义。 本研究分别选用16S rDNA、ITS、23S rDNA序列的三对通用引物Srl(5’-AGAGTTTGATCATGGCTCAG-3’)和Sr2(5’-ACGGTTACCTTGTTACGACTT-3’)、L1(5’-AGTCGTAACAAGGTAGCCGT-3’)和L2(5’-GTGCCAAGGCATCCACC-3’)、C1(5’-ATTTCCGAATGGGGIAACCC-3’)和C3(5’-TGTCTCACGACGTTITAAACCCAGCTC-3’)对水稻白叶枯病菌、条斑病菌和野油菜黄单胞菌的三个菌株进行扩增，所有菌株均分别得到了一条分子量约为1.6kb、600bp、2.3kb的扩增产物。对参试的3个菌株的扩增产物进行克隆和测序，将这些序列与数据库中已经报道的其它16S rDNA、ITS、23S rDNA序列进行同源性比较，结合已经发表的这两种病原细菌的基因序列，在此基础上分别设计并合成了白叶枯病菌的专化性引物OSF1-OSR1和条斑病菌的专化性引物XoocF-XoocR。并且用这两对专化性引物分别对67个白叶枯病菌菌株、31个条斑病菌菌株、20个其它参试菌株进行PCR扩增，证明这两对专化性引物都具有很强的专化性。同时研制了免疫吸附PCR技术可以使检测纯菌的灵敏度比标准PCR技术提高约10倍。 本研究通过将这两对专化性引物配对以及不断优化PCR反应条件，成功建立了双重PCR技术，同时对水稻白叶枯病菌和细菌性条斑病菌实施快速精确的检测。该双重PCR对单一的白叶枯病菌能够扩增出一条特异性条带，大小约1500bp；对单一的细菌性条斑病菌能够扩增出一条特异性条带，大小约338bp；对白叶枯和条斑的混合菌能够扩增出两条大小显著区别的特异性条带，分别为1500bp和338bp；而对其它参试菌不能扩增出任何条带。由于该双重PCR是直接对菌液进行扩增，所以只需常规PCR操作即可，简便快速;并且所用材料普通，只需一般PCR的试剂即可，成本低;而且不需要特别的技术。因此该双重PCR技术非常适合应用于日常的植物检疫工作。关键词:水稻白叶枯病菌;水稻细菌性条斑病菌;序列分析;专化性引物;双重PCR技术;检测更多还原

【Abstract】 Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola are important pathogens which cause rice disease. Bacterial blight and bacterial leaf streak of rice are quarantine pathogens in some countries in the world. It is very important to detect the bacteria pathogens rapidly and specifically for plant quarantine.Universal PCR primers Sr1 (5’ -AGAGTTTGATCATGGCTCAG-3’ ) and Sr2 ( 5 ’ -ACGGTTACCTTGTTACGACTT-3 ’ ) , L1 ( 5 ’ AGTCGTAACAAGGTAGCCGT-3 ’ ) and L2 ( 5 ’ -GTGCCAAGGCATCCACC-3 ’ ) . C1 ( 5 ’ -ATTTCCGAATGGGGIAACCC-3 ’ ) and C3 ( 5 ’ -TGTCTCACGACGTTITAAACCCAGCTC-3’ ) that target the 16S rDNA, ITS, 23S rDNA region were utilized in the assay. A similar fragment about 1.6kb, 600bp or 2.3kb was amplified from each of 3 tested strains by PCR. The PCR products were cloned and sequenced. We compared these sequence alignments with the homology array from the database on the internet and combined the published gene sequences of two bacteria pathogens , and then two pairs of specific primers were designed and synthesized. Primers were used to amplify the specific region from 67 strains of Xanthomonas oryzae pv. oryzae , 31 strains of Xanthomonas oryzae pv. oryzicola and 20 other tested bacteria strains and proved the specificity of this two pairs of primers was high. We developped the immuno-capture PCR method, by which could enhanced 10 times of sensitivity of detection pure culture.We setted up succeedly multiplex-PCR and optimize its reactive condition, by which we could detect Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola rapidly and specifically. A specific fragment about 1500bp was amplified from strains of Xanthomonas oryzae pv. oryzae by this multiplex -PCR; a specific fragment with 338bp was amplified from strains of Xanthomonas oryzae pv. oryzicola; two specific fragments with 1500bp and 338bp were amplified from the mixture of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola; but not other tested bacteria strains. It issimple and convenient and rapid because this multiplex -PCR amplify directly from pure culture; and its cost is low. So this multiplex -PCR technique is adapt to routine plant quarantine in port.更多还原