【作者】 张敏；

【导师】 沈德龙；

【作者基本信息】 中国农业科学院 ， 微生物学， 2008， 硕士

【摘要】 本文以甘草为研究材料,运用传统的分离培养方法并结合非培养方法PCR-DGGE技术,对采自内蒙古鄂尔多斯地区的野生及栽培甘草的不同部位内生细菌的多样性进行了研究,比较了野生及栽培甘草不同部位内生细菌群落的多样性差异,并获得了一批内生细菌菌株资源。对野生及栽培甘草根、茎、叶组织中的内生细菌进行分离和计数,发现野生及栽培甘草根、茎、叶部位的内生细菌菌落数分布在3.10×102～1.37×10~6 (单位:cfu/g鲜重)上,总体来说,野生甘草内生细菌菌落数大于栽培甘草,内生细菌在甘草根、茎、叶部位的菌落数和种群数量均有差异,野生及栽培甘草均表现出根和叶部的内生细菌种类比茎部丰富。应用分子方法ERIC-PCR方法对分离内生细菌进行菌株水平的多样性检测,共得到内生细菌121株。对其中82株进行16S rDNA片段测序分析,结果表明这些内生细菌分别与Genbank中α、β、γ-Proteobacteria、Firmicutes、Actinobacteria五类细菌中的19个已知属相似性达到97-100%。其中γ-Proteobacteria和Firmicutes最多,分别占45.78%和42.17%,剩余的12.05%为其余三类。内生细菌的主要优势种群为芽孢杆菌属(Bacillus sp.)、假单胞菌属(Pseudomonas sp.)、泛菌属( Pantoea sp.)和沙雷氏菌属(Serratia sp.)。对同一批甘草样品进表面灭菌后,以CTAB法提取植物基因组DNA,用引物799f-1492r扩增细菌16S rDNA片段,应用变性梯度凝胶电泳(DGGE)技术对内生细菌菌群多样行进行分析。DGGE指纹图谱显示野生及栽培甘草不同生长部位的优势内生细菌较为相似,个别部位有其特有的优势菌。对其中的1-9号亮度较高的条带回收并克隆测序,显示他们与Genbank中γ-Proteobacteria类细菌中的Enterobacter sp.、Pantoea sp.、Klebsiella oxytoca、Serratia sp.相似性达到97%-100%。采用变性梯度凝胶电泳(DGGE)方法对可培养内生细菌及非培养内生细进行比对分析,结果显示可培养细菌的DGGE条带未能与基因组内生细菌中的条带产生很好的对应,可培养内生细菌样品条带分布在凝胶中较高浓度梯度区域,而非培养内生细菌条带的范围则分布在较低浓度梯度区域。在野生和栽培的根、茎、叶六组样品中,仅有栽培茎(CS)中的分离菌株CS1b可以与其非培养内生细菌中的的9号条带(Pantoea agglomerans)很好的对应。对可培养内生细菌进行16S rDNA片段测序后,发现在Genbank中与菌株WS10b相匹配的菌株多为非培养细菌。应用生理生化、APi 20E、Biolog细菌鉴定系统、16S rDNA全序列测定等多种方法对其进一步鉴定,初步确定其属于肠杆菌科的泛菌属,对其是否是新种的确定还需进一步研究。更多还原

【Abstract】 In this study, To completely know the diversity of endophytic bacteria from Glycyrrhiza uralensis plants—a important Chinese traditional medicine, PCR-DGGE and traditional culture-dependent methods were combined to examine the microbial community of endophytic bacteria from wild and cultivated Glycyrrhiza uralensis plants which collected from Erdos Innermongolia province.The isolation of endophytic bacteria from the roots,stems and leaves of wild and cultivated Glycyrrhiza uralensis plants indicated that Glycyrrhiza uralensis plant has plenty of endophytic bacterium in density and population. The endophytic bacterium density were between 3.10×102～1.37×10~6 (cfu/g fresh weight)and it is higher in wild Glycyrrhiza uralensis than in cultivated Glycyrrhiza uralensis. There were differences in endophytic bacterium density and population between roots,stems and leaves of Glycyrrhiza uralensis. the isolate result show that more endophytic bacterium in roots and leaves than in stems. 121 strains of endophytic bacteria identified by ERIC-PCR . Partial sequence analysis of 16S rDNA gene of 82 strains indicated that these strains were in a high similarity with 19 known genus which belong toα,β,γ-Proteobacteria,Firmicutes and Actinobacteria .γ-Proteobacteria was the first dominant group (45.78% of the total), then the Firmicutes (42.17% of the total), and 12.05% of the total was another three groups. The dominant genus were Bacillus sp.,Pseudomonas sp.,Pantoea sp. and Serratia sp..Using CTAB method to extract the plant genome DNA and amplifying endophytic bacteria 16S rDNA directly from Glycyrrhiza uralensis tissues by using bacterial 16S rDNA primers 799f-1492r, then using PCR-DGGE to examine the microbial community. The DGGE profile show that the species of dominant endophytic bacteria in different tissues were similar, but some tissues still has its own dominant endophytic bacteria. Sequence analysis of DGGE bands indicated that they are in high similarity with Enterobacter sp.,Pantoea sp.,Klebsiella oxytoca,Serratia sp. which belong toγ-Proteobacteria.The integrated DGGE profiles of endophytic bacteria explored with culture-dependent and unculture- dependent approaches indicated that the bands of bacteria DNA from culture-dependent approache were in higher denaturing gradientgel area than the bacteria DNA from unculture-dependent approache, the bands of them couldn’t matching well, and only one bacteria species from culture-dependent approache exhibited band in relevant DGGE fingerprints of the endophytic bacteria from unculture-dependent approache.The partial sequence analysis of 16S rDNA gene of isolated strain WS10b show that it was similar with uncultured endophytic bacteria from Genbank. The result of characterazation,APi 20E bacterial identify system,Biolog bacterial identify system and whole 16S rDNA sequence show that it belonged to Pantoea sp.. It still need time to validate weather it was a new srain or not.更多还原