【作者】 孙磊；

【导师】 宋未；

【作者基本信息】 首都师范大学 ， 植物学， 2006， 博士

【摘要】 本文对非培养方法在水稻内生细菌和根结合细菌研究中的应用进行了探索。主要采用了构建16S rRNA基因克隆文库、DGGE两种非培养方法并结合传统的细菌分离培养方法，对水稻(Oryza sativa L.)内生细菌和根结合细菌群落多样性及群落动态变化进行了研究。 建立了直接从水稻根组织扩增内生细菌16S rDNA的方法。选择了扩增细菌16S rDNA的引物(799f-1492r)，该对引物可以通过PCR的方法直接从水稻根总DNA中扩增出内生细菌的16S rDNA片段，而不扩增水稻叶绿体DNA。水稻线粒体DNA可通过扩增产物中片段大小差异与细菌的16S rDNA分开。通过对水稻内生细菌16S rRNA基因克隆文库中阳性克隆的序列测定证实该对引物完全适用于非培养的分子生物学方法对水稻内生细菌的研究。该项研究结果尚未见报道。 对分蘖期水稻(Oryza sativa L. japonica 90-3)的内生细菌16S rRNA基因克隆文库中192个阳性克隆的16S rDNA片段进行ARDRA分型，根据ARDRA分型结果将该克隆文库分为52个OTUs；16S rDNA序列系统发育分析揭示水稻根内生细菌中包含α-、β-、γ-、δ-、ε-五个亚类的Proteobacteria，其中γ-Proteobacteria为克隆文库中最优势的细菌类群，占克隆总数的25％。除此之外水稻根内生细菌中还含有低GC的革兰氏阳性细菌及Cytophaga／Flexibacter／Bacteroides(CFB)类细菌。文库中28.64％的克隆与尚未培养的细菌具有较高的序列相似性。该文库中还有6个克隆属于古菌，这是首次通过非培养方法研究证实水稻内生菌群落中包含有古菌。这些结果均说明水稻内生菌具有丰富的种群多样性。 采用构建细菌16S rDNA克隆文库并结合分离培养方法，研究了水稻根结合细菌群落结构。ARDRA分型结果显示，克隆文库包含43个OTUs，分离培养物包含17个OTUs。两种研究方法的结果均说明γ-Proteobacteria为水稻根结合细菌群落中的优势类群。气单胞菌属为克隆文库中的最优势菌群；与16S rDNA克隆文库比较，分离培养物中的最优势菌群则为假单胞菌属。 采用PCR-DGGE技术研究了在水稻生长的不同年份及不同生长时期两个品种的水稻(Oryza sativa L. japonica 90-3，Oryza sativa L. japonica 90-12)根内生细菌和根结合细菌的群落多样性及其动态变化。DGGE图谱中主要条带的序列分析结果显示，水稻内生细菌和根结合细菌群落中均有α-、β-、γ-、δ-四个亚类的Proteobacteria及低GC的革兰氏更多还原

【Abstract】 The rice endophytic bacteria and root-associated bacteria were explored with culture-independent approaches. The diversity and population dynamics of endophytic bacteria and root-associated bacteria of two rice cultivars (Oryza saliva L. japonica 90-3 and Oryza sativa L. japonica 90-12) grown in the same paddy field were studied by using both 16S rDNA cloning, sequencing and denaturing gradient gel electrophoresis (DGGE) as well as traditional cultivation technique.The method of amplifying endophytic bacterial 16S rDNA from rice roots directly was established. A pair of bacterial 16S rDNA primers (799f-1492r) was selected to amplify bacterial sequences directly from rice root tissues by PCR for exclusion of chloroplast DNA. The mitochondrial sequence from rice was separated from the PCR-amplified bacterial 16S rDNA sequence by the different size of fractionations. Sequence analysis of clones in the 16S rDNA library of rice endophytic bacteria indicated that this pair of primers was available for the study of endophytic bacteria communities associated with rice plant. To our knowledge, this is the only one report of the primers used for culture-independent approaches on the analysis of endophytic bacterial communities associated with rice plants.Among the 196 positive clones in the 16S rDNA library of endophytic bacteria, 52 OTUs were formed according to the similarity of the banding patterns obtained by amplified DNA restriction analysis (ARDRA). The sequence analysis revealed a broad phylogenetic spectrum of bacteria, including α 、 β、 γ、 δ and ε subgroup of the Proteobacteria, low G+C gram-positive bacteria, and some microbes belonging to the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the library. The most dominant group was y - Proteobacteria (25% of the total). 28.64% of the total clones showed the highest similarity to uncultured bacteria. To our knowledge, it is the first report that archaea was identified as endophytic bacteria associated with rice by the culture-independent approach. It is suggested that a abundant diversity of endophytic bacterial community occurred within rice roots.The structure of bacterial community associated with rice roots was studied with both construction of 16S rDNA clone library and culture-dependent approache. ARDRA patterns showed 43 OTUs in the 16S rDNA clone library as well as 17 OTUs in the bacterial isolates. Y更多还原