白叶枯病菌胁迫下云南普通野生稻SSH文库的构建及抗病相关基因分析

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.

Construction of SSH Library and Resistance Gene Analyses of Yunnan Oryza rufipogon Griff. under Xanthomonas oryzae pv. Oryzae Stress

A cDNA-SSH library was established by suppression subtractive hybridization （SSH） using leaves of Yunnan Oryza rufipogon Griff. treated by Xanthomonas oryzae pv. oryzae. All the positive clones were sequenced, 494 availableexpressed sequence tags （ESTs） were obtained by cluster analysis. The results of BlastN showed that 417 ESTs hadhomologous sequences in GenBank database. The BlastX results indicated that 104 ESTs had higher similarity withthe unknown proteins and putative proteins, and 341 ESTs had significant protein homologies while 49 ESTs had noprotein homologies. The study suggested that a large group of Xanthomonas oryzae pv. oryzae of stress-inducedgenes were found in cDNA library, which are involved in many metabolism pathways such as energy metabolism,protein metabolism, nucleic acid metabolism, defense and resistance response, signal transduction, photosynthesisand transmembrane transport. We studied the mRNA expression of several ESTs which may be bacterial blightresistance genes in control and Xanthomonas oryzae pv. oryzae stress of Yunnan Oryza rufipogon Griff. bysemi-quantitative RT-PCR. The results showed that the expression of the clone No. OR7, OR68 and OR826 ESTwere induced by Xanthomonas oryzae pv. oryzae stress, and were up-regulated. EST OR826 that had no proteinhomology in protein database may belong to a class of new bacterial blight resistance genes. The mRNA expressionof OR143 could be detected in the leaf of control and Xanthomonas oryzae pv. oryzae stress. Its expression wasgradually strengthened 48 hours after Xanthomonas oryzae pv. oryzae stress. We suggested these genes participatedin resistance defense response in Yunnan Oryza rufipogon Griff. directly. This research provided theoretical basis fordiscovery of new bacterial blight resistance genes, and laid the foundation for further studies of the molecularmechanisms of bacterial blight resistance in Yunnan Oryza rufipogon Griff.