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以云南普通野生稻为材料,利用抑制差减杂交技术(SSH),构建了白叶枯病菌胁迫的云南普通野生稻特异表达基因的差减文库.通过对文库所有阳性单克隆进行测序,聚类分析后共获得494条高质量的表达序列标签(EST).经过BlastN分析,有417条与已知功能的序列有较高同源性;经BlastX分析,有104条EST与未知功能蛋白或假定蛋白有较高相似性,49条EST未能找到同源匹配,341条EST与已知功能蛋白有较高同源性.初步分析发现,这些基因主要涉及能量代谢、蛋白质代谢、核酸代谢、防御与抗逆应答反应、信号转导、光合作用及膜运输等代谢过程.使用半定量RT-PCR研究了7个可能与白叶枯病抗性相关的EST序列在云南普通野生稻对照和白叶枯病菌处理的叶片中的表达情况,并获得这些基因的表达谱.结果发现,克隆编号为OR7,OR68和OR826的EST受白叶枯病菌胁迫诱导上调表达,其中OR826 EST在蛋白数据库中无同源序列,可能是一类新的白叶枯病抗性基因,而组成型表达的OR143 EST在对照和接菌处理的叶片中均能检测到其mRNA的表达,但其表达量在白叶枯病菌胁迫48 h后逐渐增强,推测这些基因直接参与了云南普通野生稻抗病防御反应.本研究为从云南普通野生稻中发掘和克隆新的白叶枯病抗性基因提供了理论依据,为进一步研究云南普通野生稻抗白叶枯病的分子机制奠定了基础.
A subtractive library of common Oryza rufipogon (Oryza sativa) wild-type (Oryza sativa) differential genes was constructed by using SSH in Yunnan common wild rice (Oryza sativa L.) as a material.All the positive clones were sequenced and clustered After analysis, a total of 494 high quality ESTs were obtained.After BlastN analysis, 417 ESTs had high homology with known function sequences. According to BlastX analysis, there were 104 ESTs with unknown or hypothetical proteins 49 ESTs could not find homologous matches and 341 ESTs had high homology with known functional proteins.A preliminary analysis showed that these genes mainly involved in energy metabolism, protein metabolism, nucleic acid metabolism, defense and anti-retrograde responses Reaction, signal transduction, photosynthesis and membrane transport, etc. Seven semi-quantitative RT-PCR studies of EST sequences that may be related to bacterial leaf blight resistance were conducted in Yunnan wild rice (Oryza sativa) The results showed that ESTs with clone numbers OR7, OR68 and OR826 were up-regulated and induced by the bacterial leaf blight, and OR826 EST expressed in the protein number There was no homologous sequence in the library, which may be a new type of bacterial leaf blight resistance gene. However, constitutively expressed OR143 EST could detect its mRNA expression in both control and inoculated leaves, but its expression The results showed that these genes were directly involved in the defense response of common wild rice (Oryza rufipogon Griff) in Yunnan wild rice (Oryza sativa) The theoretical basis for the further study on the molecular mechanism of the common wild rice resistance to bacterial blight in Yunnan laid the foundation.