【作者】 赵志祥；

【导师】 刘二明； 谢丙炎；

【作者基本信息】 湖南农业大学 ， 微生物学， 2010， 博士

【摘要】 自然环境中的微生物资源极其丰富。然而,只有不到1%的微生物是可纯培养的,99%以上的微生物是不可纯培养的。长期以来,人们用传统可纯培养方法来研究环境样品微生物,温室土壤样品也不例外,这就阻碍了对微生物多样性的研究和有益微生物资源的挖掘利用。因此,为全面弄清温室土壤中微生物群落结构组成,了解微生物与温室主栽植物、植物病原根结线虫以及微生物内部之间的互作关系；同时,从温室作物病虫害的发生与防控关系最为密切的微生物中筛选出有用的基因资源,本研究采用非培养的手段对微生物的群落结构组成和微生物功能基因的挖掘,尤其是杀线虫蛋白酶基因的挖掘做了初步研究,具体研究结果有以下几点：第一,通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库来分析温室土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOUTER软件将古菌和真菌序列按照相似性97%的标准分成若干个可操作分类单元（OTUs）。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs；真菌克隆文库包括真菌界的大多数亚门真菌,共24个OTUs,未发现担子菌门真菌。古菌多样性比较丰富,且发现少量的广域古菌（甲烷菌）,出现这一情况的原因可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境中有关；土壤真菌的优势种群为子囊菌,占到土壤真菌的80%以上,这可能与大多数植物真菌性病害是通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。第二,采用16S rRNA基因克隆文库和宏基因组末端测序对温室黄瓜根围土壤细菌的多样性进行了初步分析。根据97%的序列相似性水平划分OUT,共有35个OUT分别属于γ-Proteobacteria、α-Proteobacteria、δ-Proteobacteria和Firmicutes,其中优势菌群是y-Proteobacteria,其次为Firmicutes, Bacillus为优势细菌。在优势种群上,末端测序结果与16S文库分析结果基本一致。但是,宏基因组末端测序包含了大多数的弱势种群,更能反映细菌多样性的真实水平。与露地土壤细菌16S文库相比较,土壤细菌多样性降低,这可能与温室多年连作,种植蔬菜种类单一直接相关。第三,本研究旨在通过非培养手段构建和筛选宏基因组文库,以求找到新型的杀线虫蛋白酶基因。采用密度梯度离心法提取和纯化温室土壤微生物总DNA,经平末端、连接、包装、转染后,构建宏基因组Fosmid文库。该文库库容31,008个克隆,平均插入片段36.5 kb,包含1.13 Gbp的微生物基因组信息,适合大规模的微生物功能基因筛选。同时,以脱脂奶为底物,以根结线虫为靶标,对文库进行功能初筛,共筛选到11个含蛋白酶基因的Fosmid克隆,其中,8个含杀线虫蛋白酶基因。最后,对P11做了进一步研究,构建和筛选出亚克隆（espl14a5）,通过对基因结构进行了初步分析发现：esprol14a5是一种分泌型胞外蛋白酶,与来自于Maricaulis maris MCS10 （accession no. YP₇56822 at NCBI）的丝氨酸蛋白酶S15仅有45%的同源性,有其保守的催化三元组：Asp469,His541和Ser348。杀线虫室内生物测定和盆栽实验表明：克隆发酵液在无需诱导剂,无需克隆载体上的启动子,就能起到杀线虫的作用。说明该蛋白酶基因有独立的启动子,同时,也说明该蛋白酶是一种分泌型胞外丝氨酸蛋白酶。更多还原

【Abstract】 In the natural environment, microbial resources is extremely rich. However, only less than 1% of microorganisms can be cultured, over 99% of microorganisms cannot be cultured by the traditional culturable methods. For a long time, the traditional cultureable methods could be used to study the environmental samples, greenhouse soil samples were not exception, so it hindered the study of microbial diversity, it also restricted the excavation and utilization of benefical microbial resources. In order to understand the greenhouse soil microbial community composition fully, find out the interaction between microorganisms and greenhouse plant cultivars, plant pathogenic root-knot nematodes and among of themselves; at the same time, and to screen useful genetic resources from the microorganisms which related directly to the occurrence and the prevention of greenhouse crop diseases and pests. In these studies, uncultured methods could be used to survey the microbial community composition and to explore the microbial functional genes, especially the development and utilization of the nem-aticidal protease genes. The results were showed as follow:Firstly, in order to develope and utilize rich microbial resources in the particular greenhouse ecological environment, and to understand the interaction between micro-bes and plants, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constru-cted to analyze the composition of archaeal and fungal community structure in green-house soil samples. Total greenhouse soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS heating treatment and treatment of CTAB. Archaeal and fungal universal primers were used to amplify their specific ge-nes. After retrieving, ligating, transforming, screening of white clones, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constructed. The sequences of archa-ea and fungi were defined into operational taxonomic units(OTUs) according to 97% similarity threshold for OTU assignment using the software program DOTUR. Phylo-genetic analysis showed that Crenarchaeota and unidentified-Archaea were the prima-ry two sub-groups and only a few of Euryarchaeota existed in the archaeal clone libra-ry, total 45 OTUs. All the Crenarchaeota belonged to Thermoprotei; except Basidiom- ycota, the other four sub-group fungi were discovered in the fungal library, total 24 OTUs. The diversity of archaea was very abundant and a few Euryarchaeota (methan-ebacteria) existed in the archaeal clone library. It is may be directly related to the long-term high temperature, high humidity, high content of organic matter environ-memt. The limitation of oxygen was the other reason to cause this phenomenon; Asc-omycotina(over 80%) were the dominant subgroups in fungal library. It was because most of the plant fungus diseases belonged to soil-borne diseases which gone through the winter by the ways of scierotium or perithecium and became the source of primary infection.Secondly, in order to study the bacterial diversity in cucumber rhizosphere soil samples,16S rRNA gene clone library and metagenomic end rondom sequencing were combined to analyze the bacterial diversity preliminarily.35 OTUs were obtained fro-m the library according to sequences similarity of 97%, which affiliated toγ-Proteob-acteria,α-Proteobacteria,δ-Proteobacteria,Firmicutes and so on.γ-Proteobacteria we-re the dominant bacterial groups followed by Firmicutes, and Bacillus were the domi-nant bacterial species.In terms of dominant bacteria, the results revealed by metagen-omic end rondom sequencing were similar to that of 16S rRNA gene library. However, method of metagenomic end rondom sequencing contained most of the vulnerable populations, so it could reflect the true level of bacterial diversity. Comparing to the exposed soil bacterial 16S rRNA gene library, the bacterial diversity was very low. This phenomenon may be directly related to Continuous cultivation for many years and plant a single vegetable species.Thirdly, in order to find out the novel nematicidal protease genes, a metagenomic fosmid library was constructed and screened by uncultured method. Method of dens-ity gradient centrifugation was used to extract and purify total greenhouse soil micro-bial DNA. After end-repair, ligation, packing and transformation, metagenomic fos-mid library was constructed. This library contained 31,008 clones with the average in-sert fragment of 36.5 kb, including 1.13Gbp microbial genetic information, so it was suitable for large-scale microbial functional gene screening. At the same time,in order to screen the library, function-driven screening was used as a potential strategy, skim milk was served as the substrate and root-knot nematodes were served as targets,11 Fosmid clones which contained putative protease genes could be screened, among them,8 Fosmid clones contained the putative nematicidal protease genes, included p11 and p7.Finally, further research was done on P11. The subclone library was constructed and the subclone espl14a5 which could degraded the protein was screened. After ana-lysis of gene structure, espl14a5 is a secreted extracellular protease and a database search for homologies revealed it possessed 45% identities with peptidase S15 from Maricaulis maris MCS10(accession no. YP 756822 at NCBI).It is a novel serine protease. Besides these, it has the serine protease-conserved catalytic triad residues, Asp469, His541 and the catalytic nucleophile Ser348.Indor biological nematicidal and potted tests showed that cloning broth could kill the nematodes without inducer and the promoter which existed in the clone plasmids. So this protease gene had its own promoter. At the same time,this protease is a secreted extracellular serine protease.更多还原