【作者】 邱服斌；

【导师】 宋未；

【作者基本信息】 首都师范大学 ， 植物学， 2007， 博士

【摘要】 本文以人参(Panax ginseng C．A．Mey．)为研究材料，采用构建16S rDNA克隆文库与扩增rDNA限制性分析(ADRDA)的非培养方法，并结合传统的分离培养方法研究了人参根内生细菌的群落多样性。比较了同一类型人参在不同生长时期和不同种植地区的根内生细菌群落多样性差异，并获得了人参根内生细菌的新菌种资源。选用我国人参原产地吉林省抚松县及其种植在北京中国医学科学院药用植物研究所三年生的人参根为材料，经严格表面灭菌，以CTAB法提取植物基因组DNA，采用799f和1492r引物扩增细菌16S rDNA片段，构建了人参根内生细菌16S rDNA克隆文库。药植所样品构建的文库得到有效阳性克隆274个，经ARDRA分型，得到35个操作分类单元(OTU)。经16S rDNA片段测序，有103个克隆属于a-Proteobacteria，占总克隆数的37.59％，归属10个OTUs；140个克隆属于γ-Proteobacteria，占总克隆数的51.09％，归属于13个OTUs，这两个类群属于该克隆文库的优势种群。抚松样品所构建的内生细菌16S rDNA克隆文库中，共得到阳性克隆286个，经ARDRA分型及16S rDNA片段测序比对后发现，有130个克隆归属于47个OTUs。在这些OTUs中有包括10个OTUs在内的43个克隆与未培养(uncultured)细菌具有较高的同源性；37个克隆为CFB(Cytophaga／Flexibacter／Bacteroides)类群，归属于14个OTUs，为该文库优势种群；有19个OTUs(含35个克隆)为Proteobacteria，分属于α-Proteobacteria，β-Proteobacteria和γ-Proteobacteria三个类群，且在这三个类群中的分布也比较均一。此外还发现147个克隆同人参叶绿体16S rDNA具极高的同源性，1个克隆同植物线粒体18S rDNA有较高同源性，还有8个克隆为嵌合序列。在该克隆文库中还发现了3个OTUs共14个克隆的衣原体序列。以不同生长时期、不同种植地点的人参根为材料，应用LB和D(?)berener无氮培养基分别进行了内生细菌的分离。在2005年药植所的样品中，用LB培养基分离到的内生细菌大部分为芽孢杆菌(Bacillus)，其中有2株菌已鉴定为潜在的新种，用D(?)berener无氮培养基分离到的内生细菌则主要是假单胞菌(Pseudomonas sp．)；而在2006年的样品中，用LB培养基和D(?)berener无氮培养基分离到的内生细菌主要为假单胞菌(Pseudomonassp．)，其次是贪噬菌(Variovorax sp．)。在2005年抚松的样品中，从LB培养基上分离到的主要为贪噬菌(Variovorax)、赖氏菌(Leifsonia)和分枝杆菌(Mycobacterium)，在D(?)berener无氮培养基上分离到的主要是贪噬菌(Variovorax)和土壤杆菌(Agrobacterium)。对分自药植所人参根的两株细菌ge10和ge14做了多相分类鉴定，结果表明这两株为芽孢杆菌属中潜在的两个新种；对分自抚松样品中的一株新菌wged11进行了多相分类研究，结果表明该菌株含较高的环己基脂肪酸，为Leifsonia属的一个新种，定名为Leifsonia ginsengi。对分自药植所的7株内生细菌(ge05、ge10、ge12、ge14、ge18、ge21和ge24)做了体外拮抗植物病原真菌的初步研究，结果表明有两株芽孢杆菌属的菌株(ge10和ge14)对引起人参锈腐病的病原菌(Cylindrocarpon destructans)具有较强的拮抗作用，1株类芽孢菌属(Paenibacillus)的菌株(ge21)对引起人参立枯病、人参疫病及水稻稻瘟病的病原菌(Rhizoctonia solani、Phytophthora cactorum、Pyricularia oryzae)均有一定的拮抗作用。本论文研究结果表明，与传统的分离培养方法相比，非培养方法能够更客观地反映植物内生细菌的多样性，但实验易受植物总DNA提取质量、细胞器DNA及PCR反应条件等的影响；从非培养方法的研究结果也证实，生活在不同地理环境下的同一种植物，其内生细菌的群落结构有较大差异，并表明在人参根内存在潜在的新菌种资源。培养方法研究结果表明，假单胞菌和贪噬菌为人参根内生优势种群。用多相分类法已鉴定有三一株细菌wged11、ge10、ge14分别与已报道的Leifsonia及Bacillus在种的水平上有差异。更多还原

【Abstract】 The community diversity of endophytic bacteria (CDEB) in Panax ginseng root was investigated by using culture-independent and culture-dependent approaches. The differences of CDEB for one congeneric ginseng root sampled from two regions and different growth stages were compared with constructions of 16S rDNA clone library and amplified DNA restriction analysis (ARDRA). Some novel bacterial species inhabiting the interior of ginseng roots were isolated and identified.Congeneric ginseng roots that have grown for 3 years were sampled from Fusong County in Jilin Province (FS), the original habitat, and from Institute of Medicinal Plant Department (IMPD) of Chinese Academy of Medical Sciences which lies in Beijing. Samples were strictly surface-sterilized followed by CTAB DNA Extraction Protocol to obtain their genome DNA respectively. To construct 16S rDNA clone libraries, primers 799f and 1492r were selected to amplify 16S rDNA segments. Thirty five OTUs, including 274 positive clones, were assigned according to their band pattern similarity in IMPD clone library. The representatives of each OTU were sequenced. The results showed that γ-Proteobacteria was the most dominant group (13 OTUs, 51.09% of the total clones), followed by α-Proteobacteria (10 OTUs, 37.59% of the total clones). A total of 286 positive clones were obtained in FS clone library, in which 130 clones were assigned to 47 OTUs according to ARDRA patterns. 10 (43 clones) out of 47 OTUs had most identity with uncultured bacteria, and 14 OTUs (37 clones) had identity with CFB (Flexibacter/Cytophaga/Bacteroides). CFB group was the most dominant group in the known species. Proteobacteria (19 OTUs, 35 clones) were only accounted for 26.9% and classified into α-, β-, and γ proteobacteria. In FS libray, 147 clones were highly homologous to chloroplast small sub-unit 16S rDNA and 1 clone was homologous to mitochondrion small sub-unit 18S rDNA. 8 chimeric DNA sequences were checked in this library and 3 OTUs (14clones) belonged to chlamydiae.Congeneric ginseng samples from different regions, different growth stages, and different culture media were used to isolate endophytic bacteria from ginseng roots. Most of Bacillus were isolated from IMPD samples on LB medium in 2005. Four of them had potential as new bacteria species. However, plenty of pseudomonas were isolated from free-nitrogen Doberener medium from the same samples. Pseudomonas spp. and Variovorax spp. as the dominantspecies were isolated on both LB medium and free-nitrogen Doberener medium from IMPD samples in 2006. Variovorax, Leifsonia and Mycobacterium from FS samples were isolated on LB medium and Variovorax spp. and Agrobacterium spp. were isolated on free-nitrogen Doberener medium in 2005.Two strains (strains ge10 and ge14) isolated on LB medium from IMPD samples were studied according to a polyphasic approach. The results showed that they were novel and different species subject to the genus Bacillus. One strain (stain wged11) isolated on free-nitrogen Doberener medium from FS samples was also explored according to a polyphasic approach. The data indicated that wged11 containing the unusual ω-Cyclohexylundecanoic acid was a novel species of the genus Leifsonia.In this study the antagonistic activities of 7 endophytic bacterial strains (ge05、 ge10、 ge12、 ge14、 ge18、 ge21and ge24) from IMPD on PDA media were assayed. The results showed that two Bacillus strains (ge10 and ge14) were antagonistic to Cylindrocarpon destructans. Paenibacillus strain (ge21) was antagonistic to Rhizoctonia solani, Phytophthora cactorum and Pyricularia oryzae.Three endophytic bacterial strains (wged11, ge10, ge14) from ginseng roots had been identified as potential novel specieses.更多还原