【作者】 阎冰；

【导师】 喻子牛； 洪葵；

【作者基本信息】 华中农业大学 ， 微生物学， 2007， 博士

【摘要】 红树林是生长在热带、亚热带海岸潮间带，以红树植物为主体的木本植物群落，是生物多样性高度浓缩的海岸生态关键区。对于红树林生态系统的微生物是海洋类群还是陆地类群和多样性是否丰富等仍存争议。本研究通过直接提取我国红树林保护区的土壤总DNA为模板，以细菌和古菌的16S rDNA通用引物PCR扩增红树林土壤细菌和古菌群落的16S rRNA基因，构建16S rDNA克隆文库，经rDNA扩增限制性片段长度多态性分析(ARDRA)、DNA序列测定和系统发育分析等，对海南东寨港国家级自然保护区红树林土壤细菌和古菌多样性进行了研究。本研究结果表明，红树林土壤细菌包括了变形细菌门(Proteobacteria)、绿屈挠菌门(Chloroflexi)、酸杆菌门(Acidobacteria)、拟杆菌门(Bacteroidetes)、脱铁杆菌门(Deferribacteres)、浮霉菌门(Planctomycetales)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)、疣微菌门(Verrucomicrobia)、硝化螺旋菌门(Nitrospira)、梭杆菌门(Fusobacteria)、蓝细菌门(Cyanobacteria)、Candidate division OP11、Candidate division WS3、Candidate division OP8、Candidate division TM6等16个类群，其中变形细菌和绿屈挠菌是优势类群。古菌包括了嗜泉古菌界(Crenarchaeota)和广域古菌界(Euryarchaeota)，其中嗜泉古菌占优势。嗜泉古菌包含了Marine Benthic Group A、Marine Benthic Group B、Marine Benthic Group C、Marine GroupⅠ和2个尚不能确定分类地位的分枝MSA-cluster-6和MSA-cluster-78等6个类群，其中Marine Benthic Group C占优势；广域古菌包含了Marine Benthic Group D、Marine Benthic Group E、Marine GroupⅣ、Rice ClusterⅠ、Rice ClusterⅢ、Methanogen(MA)和Anaerobic methane-oxidizing archaeon(ANME)等7个类群，其中Marine Benthic Group D占优势。多样性指数(H′)和物种丰富度指数(Schaol)的计算结果表明，细菌的多样性程度比古菌高。不同土壤深度微生物群落结构和多样性程度有明显差异。在土壤深度0～30 cm范围内，随着深度增加：细菌多样性程度降低，a-变形细菌、拟杆菌、放线菌和Candidate division OP11主要分布在表层，d-变形细菌、γ-变形细菌、酸杆菌和脱铁杆菌在中层以及绿屈挠菌在深层的比例大；古菌多样性程度先上升后降低，嗜泉古菌所占的比例增加，广域古菌所占的比例减少。以16S rDNA同源性≥97％为同一个种的标准进行评估，红树林土壤中至少有76.3％的细菌和古菌是还未发现的种，97.7％的种尚未获得纯培养。通过与不同生态环境中的文献研究结果相比较，红树林土壤微生物多样性高于海洋沉积物，与陆地土壤相当。GenBank数据库中与红树林土壤细菌和古菌同源性最高的相同的种51.5％来源于海洋，30.1％来源于陆海过渡带，18.4％来源于陆地和淡水，说明红树林土壤微生物群落是以海洋微生物为主的海洋、陆地微生物的混合体。此外，红树林土壤中以硫还原菌为主的分布也表明了海洋沉积物特征。根据最新的微生物生物地理地图的概念，我们将红树林土壤定位于海洋——潮间带——红树林。不同红树林植被状况下土壤微生物群落结构和多样性程度差异显著。外来种无瓣海桑林土壤细菌和古菌的多样性显著低于秋茄林，甚至明显低于相邻无红树林裸滩沉积物；秋茄林土壤微生物群落结构和无红树林裸滩沉积物更相似。结合样地的地理位置综合分析，推断无瓣海桑的种植导致林下土壤微生物群落结构改变和多样性降低。更多还原

【Abstract】 Mangrove forests occur in the inter-tidal zones along the tropical and subtropicalcoast with the majority of Rhizophoraceae tree. Mangrove ecosystem are ecologicallycritical areas where biodiversity and valuable biological resources are highly concentrated.There exist arguments on whether "microorganism in mangrove soil are marine type orterrestrial type?" and "microorganism in mangrove soil are really rich?"In this research, total DNA were extracted directly from the soil of a mangrovereserve zone in Hainan, China, and using as template for polymerase chain reaction (PCR)amplification, which employ specific primer for Bacteria and Archaea 16S rRNA genesequences. The 16S rRNA gene libraries were then constructed. Biodiversity wereanalyzed by amplitied rDNA restriction analysis (ARDRA), DNA sequencing andphylogenetic analysis.From the results, there are sixteen phyla in Bacteria domain: Proteobacteria,Chloroflexi, Acidobacteria, Bacteroidetes, Deferribacteres, Planctomycetales,Actinobacteria, Firmicutes, Verrucomicrobia, Nitrospira, Fusobacteria, Cyanobacteria,Candidate division OP11, Candidate division WS3, Candidate division OP8 andCandidate division TM6 with the predominant phyla of Proteobacteria and Chloroflexi.In Archaea domain, it was found Crenarchaeota and Euryarchaeota kingdom andpredominate by Crenarchaeota. There are six divisions in Crenarchaeota kingdom:Marine Benthic Group A, Marine Benthic Group B, Marine Benthic Group C, MarineGroupⅠand two other groups of MSA-cluster-6 and MSA-cluster-78, which do notbelong to any already known archaea divisions. Marine Benthic Group C dominated inCrenarchaeota. There are seven divisions in Euryarchaeota kingdom: Marine BenthicGroup D、Marine Benthic Group E、Marine GroupⅣ、Rice ClusterⅠ、Rice ClusterⅢ、Methanogen (MA) and Anaerobic methane-oxidizing archaeon (ANME), dominate byMarine Benthic Group D. The results of diversity Shannon-Wiener index (H’) andspecies richness (Schaol) calculated indicated that Bacteria are richer in diversity thanArchaea in mangrove soil. There are distinct differences in soil microbial community structure and diversityamong different depth. Bacterial diversity reduced with the depth increase among thedepth of 0～30 cm. a-Proteobacteria, Bacteroidetes, Actinobacteria and Candidatedivision OP11 mainly distribute in upper layer, d- andγ- Proteobacteria, Acidobacteriaand Deferribacteres are mainly distribute in middle and deep layer. Chloroflexi are mainlydistributed in upper and deep layer. With the depth increase, archaeal diversity firstincreased and then reduced, the proportion of Crenarchaeota increased andEuryarchaeota reduced.Employ a standard of using16S rDNA similarity≥97% as the same species, there areat least 76.3% of bacteria and archaea species undiscovered and 97.7% of species havenot obtained pure culture in mangrove soil. Comparing with similar studies in theliteratures indicated that microbial diversity in mangrove soil is richer than marinesediment and similar to the terrestrial soil.Comparing in the GenBank database, the most closest species to mangrove soilbacteria and archaea, 51.5% were from marine origin,. 30.1% were from connect zone ofmarine and continent origin, and 18.4% were from continent and freshwater. Mangrovesoil microbial communities appear to be a mixture of organisms found in a variety ofenvironments with the majority being of marine origin. The predominated sulphatereduction bacteria also show the characteristic of marine sediment. According to the mostrecent concept of "Microbial biogeography map", we put mangrove soil inmarine—inter-tidal—mangrove on the map.There are distinct differences in mangrove soil microbial community structure anddiversity between different vegetation. Soil microbial diversity under the imported specieSonneratia apetala is much lower than Kandelia candel, even lower than naked tidal flatsediment near mangrove. According to the sampling location in this research, it issuggested that plant Sonneratia apetala tree changed the soil microbial communitystructure and decreased microbial diversity.更多还原