【作者】 黄云鹏；

【导师】 黄建忠； 朱宝利；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 海洋微生物是潜在的新物种和天然产物的重要来源。为了研究台西南海盆北部陆坡区的微生物多样性,构建了16S rDNA基因文库并进行了PCR-RFLP的分析,然后对160个细菌克隆和50个古菌克隆进行了测序,对测序的结果进行了相应的分析,所分析的细菌群体总共有83个OTUs,在群体结构上,Proteobacteria和Planctomycetes是细菌群体中的优势类群,其他的细菌类群分别是Acidobacteria,Actinobacteria,Chloroflexi,Chlamydiae和Firmicutes。利用测序的细菌序列构建进化树上,发现其中有三个潜在的新的细菌类群。在所研究的古菌群体中,所有的克隆都来源于Crenarchaeota门,其中属于Marine GroupⅠCrenarchaeota的序列最多。为研究和挖掘群体中的基因信息和基因资源,利用总沉积物DNA构建了元基因组fosmid文库,从文库中选取了一些克隆进行了RFLP预分析,以观察文库克隆的多样性,随机选取了大约200个fosmid克隆进行了末端测序。所构建的fosmid文库大约含有1,000,000个克隆,文库克隆的平均插入片段为38kb,末端测序的结果显示文库中包含了丰富且多样的基因信息,如碳水化合物代谢、转运因子等相关的基因信息,这些基因信息为研究微生物群体和环境之间的关系提高了重要的线索。利用功能为基础的筛选方法,从元基因组文库中筛选获得6个酯裂解基因,其中5个基因是新的酯酶基因,并有一个新的酯酶家族,对酯酶Lip_Y1和Lip_Y18进行了克隆和原核表达,两者都表现出了酯裂解活性,编码的蛋白经SDS-PAGE检测约为32kDa。本实验研究了台西南盆地微生物群体的多样性,揭示了该微生物群体的基因和遗传信息,也揭示了微生物群体基因信息与环境之间可能的联系,从该群体中挖掘得到了有用的活性基因资源,具有重要的生态学和环境微生物学意义。更多还原

【Abstract】 Marine microbes are potential sources of biological active compounds and novelorganisms. In this study, 16S rDNA gene clone library and PCR-RFLP technologies wereused to investigate the diversity of bacterial and archaeal communities in marine sedimentwhich collected from the South China Sea. A total of 150 bacterial Clones and 50 archaealclones were sequenced, the results of sequencing were checked and analyzed. There were83 OTUs in bacterial communities, and the dominant phylotypes were corresponded tothe Proteobacteria and Planctomycetes, followed by Acidobacteria, Actinobacteria,Chloroflexi, Chlamydiae and Firmicutes. Three new bacterial divisions were observed onthe phylogenetic level. The detected archaeal sequences were grouped into the phylaCrenarchaeota, the majority of them were closely related with the uncultured MarineGroup I Crenarchaeota. A fosmid library was constructed; the randomly selected clonesfrom the library were end-sequenced and characterized by RFLP analysis. The fosmidlibrary, with average insert size about 38 kb, was consisted of approximately 1 millionclones. The results of end-sequencing exposed some useful genes and unknown geneticinformation, such as carbohydrate metabolism related genes and transpoter genes, all ofwhich giving the view of microbial community functions in marine sediment. 6 lipolyticrelated genes were screened from metagenomic library using the function based approach,5 of them were seem to be novel, also a novel esterase family was observed. Cloned andexpressed the genes of Lip_Yl and Lip_Y18, they were all active proteins with thesimilar molecular weights about 32kDa according to the results of SDS-PAGE. Ourfindings provided the insight into prokaryotic biodiversities in Taixinan Basin, revealedthe importance of deep-sea microbes as the source of novel genetic information, and theymay contribute to the balance of deep sea sediment ecosystems.更多还原