山东滨海两种大型植物(大叶藻与芦苇)内生菌的多样性研究
李海林
学位类型硕士
导师龚骏 ; 范德朋
2016-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词内生细菌 大叶藻 天鹅湖 季节 群落结构 滨海湿地 芦苇 内生真菌 盐度胁迫
摘要植物与内生菌在长期的进化过程中形成了互利共生的关系。对陆生植物的研究表明内生菌(细菌、古菌及真菌)的多样性很高,许多内生菌在植物的生长及抵御环境胁迫中起着非常重要的生态学作用。尽管如此,对沿海典型生态系统及滨海盐渍化土壤中大型植物内生微生物多样性的研究还很少。海草床是海岸带生物多样性及碳汇的重要生境,芦苇在黄河三角洲区域具有巨大的生物量。本工作以海草床及盐沼中的两种典型植物为对象研究内生菌多样性,希望为海岸带植物的生态适应及微生物资源研究提供基础数据。
在山东威海天鹅湖于夏、秋、冬季采取大叶藻根、叶组织样品,利用16S rRNA基因克隆文库和高通量测序等技术解析大叶藻内生细菌和内生古菌的群落组成、结构及其在季节和组织间的变化情况。对4个细菌文库和4个古菌文库,29个古菌样品和30个细样品的高通量测序与分析表明:古菌克隆文库共鉴定出3个已知门类(广古菌门、泉古菌门、奇古菌门)和1个未知古菌类群。系统发育分析表明,7个进化枝未确定分类,其中6个属于广古菌门,推测可能代表新的古菌类群。多样性分析显示大叶藻叶中古菌多样性高于根中,冬季多样性高于夏秋季,Chao1指数受季节显著性影响。非度量多维尺度分析表明内生古菌群落结构在不同组织间的差异极显著,季节同样显著影响内生古菌群落结构,ANOSIM分析表明秋、冬2季群落结构差异显著,而夏秋、夏冬季间群落结构无显著性差异。细菌克隆文库和高通量数据均显示变形菌门相对丰度最高,其次是厚壁菌门;根中内生细菌多样性高于叶中,夏季多样性最高,其次为秋季。Chao1、丰富度、香农和辛普森指数均表现了显著的季节差异,但组织间差异不显著。非度量多维分析表明季节变化能够显著性影响内生细菌的群落结构,ANOSIM分析显示冬季样品细菌群落结构与夏、秋季均有极显著性差异。通过比较不同季节间的差异发现,冬季内生细菌群落结构与夏、秋季均呈极显著性差异,夏、秋两季内生细菌群落结构差异不显著。
从黄河三角洲滨海湿地1350个芦苇组织切片中分离得到318株内生真菌,通过对rDNA ITS的分型、测序及系统进化分析,研究了该地区芦苇植株根、茎、叶中可培养内生真菌的种类组成及在不同盐度、不同组织中的分布情况。根据序列相似性(以98%为阈值),共获得12个真菌分类操作单元(OTUs)。在门级分类水平上,子囊菌门为绝对优势菌群, 在各组织和站位中均有分布。芦苇根组织中分离得到的内生真菌OTU数相对较多,叶组织和茎组织中分离到的OTU数相同,且叶中的OTU在根中均存在。潮上区(低盐区)内生真菌OTU数最多,高潮区次之。根中可培养内生真菌的丰富度和多样性指数最高,叶中多样性最低;潮上区丰富度最高,中潮区多样性最高。尽管不同盐度梯度及不同芦苇组织中都有其特异的菌株,但总体看,土壤盐度及不同组织对可培养内生真菌的种类组成的影响并不显著。链格孢属是所有样品共有的优势菌。
其他摘要Endophytes play an important ecological role in promoting growth and alleviating environmental stress of host plants, and form a symbiotic relationship with their hosts. It has been known that terrestrial plants usually have a high diversity of endophytic microbes. However, little is known about the diversity of coastal and marine macrophytes. In this study, we collected Zostera marina from the Swan Lake in Weihai city, Shandong province in summer, autumn and winter of 2015. The seasonal and among-tissue variations of community composition and structure of endophytic archaea and endophytic bacteria were investigated using clone library and high throughput sequencing (HTS) and prokaryotic 16S rRNA gene as molecular markers. Four endophytic bacterial and archaeal clone libraries were obtained, and 29 and 30 samples were subjected to HTS. From the 4 clone libraries of endophytic archaea, we identified 3 archaeal phyla (Euryarchaeota, Crenarchaeota and Thaumarchaeota) and an unclassified group. The phylogenetic tree shows that there are 7 clusters that could not be classified, of which 6 clusters belonged to Euryarchaeota. Archaeal diversity in leaves and in winter was higher than these in roots and in summer and autumn, respectively. Chao1 of archaeal phylotype in winter was significantly different from those in summer and autumn, but no difference was found between the summer and the autumn. NMDS showed that the archaeal community structure was significant different between root and leaf and between seasons. ANOSIM showed archaeal community structure was significant differenct between autumn and winter. Proteobacteria had the highest proportion based on both clone library and HTS data, followed by Firmicutes. The HTS showed bacterial diversity was higher in roots than in leaves, and the summer had the richest diversity among all seasons studied. All of alpha diversity estimators (Chao1, richness, Shannon and Simpson) showed significant differences among seasons (P < 0.05). NMDS analysis showed that seasonality of endophytic bacterial community, with the wintertime community significantly different compared to the summer and autumn (P = 0.001), but the latter two were statistically similar.
We also investigated the diversity and community composition of endophytic fungi isolated from the common reed in wetlands of the Yellow River Delta. A total of 318 endophytic fungal isolates were obtained from 1350 tissue (leaf, stem, and root) fragments and from three tidal zones with different soil salinity gradients. rDNA ITS regions of these isolates were PCR amplified, sequenced, and phylogenetically analyzed. All the sequences were divided into 12 operational taxonomic units (OTUs) at the 98% similarity threshold. Ascomycota was found to be the most abundant flora at the level of the phylum and distributed in all tissues and sites. There were generally richer OTUs in roots than in leaves and stems, and leaves and stems shared the same OTU numbers. All the OTUs retrieved from leaves appeared in roots. The supra tide (low salinity zone) had the most OTUs among the three sampling sites, followed by high tide. Furthermore, the roots had the highest richness and diversity, whereas the leaves had the lowest; supra tide held the highest richness, and the middle tide had the highest diversity. Although unique OTUs could be found in different tissues and tidal zones of different salinity, the communities of culturable endophytic fungi were not substantially different among tissues or tidal zones (ANOSIM, P > 0.05). Alternaria sp. was the shared dominant taxa among all samples.
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/13827
专题中国科学院烟台海岸带研究所知识产出_学位论文
作者单位中国科学院烟台海岸带研究所
第一作者单位中国科学院烟台海岸带研究所
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李海林. 山东滨海两种大型植物(大叶藻与芦苇)内生菌的多样性研究[D]. 北京. 中国科学院大学,2016.
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