【作者】 曹艳茹；

【导师】 和文祥； 姜怡；

【作者基本信息】 西北农林科技大学 ， 资源环境生物学， 2012， 博士

【摘要】 无论从过去的抗生素筛选还是从近年来微生物基因组学的研究成果，放线菌在天然产物的产生与筛选中都占有重要地位。近年，耐药性的不断增加、顽固疾病和新疾病的不断上升，急需有效新药的问世，这使得科学家们将目光转向一些新型的放线菌资源。动物肠道是一类典型而又独特的环境，过去将粪便放线菌作为资源研究的极少。本研究以多种动物粪便为对象，通过免培养和纯培养的研究手段分析粪便放线菌群落组成，从中发掘有开发潜力的菌种资源和新物种资源；研究了菌株的活性及代谢产物,以期开辟一条放线菌资源开发利用的新途径。本研究首先选择了7种动物（三种灵长目动物、两种熊科动物及一种专性肉食和一种杂食性动物）的粪便，通过高通量454焦磷酸测序技术，进行了细菌的多样性调查。共得到42540条细菌16S rRNA基因V3区片段有效序列，在100%和97%的序列相似度水平下对序列进行归并，分别获得2979条不同的序列和876个OTUs（可操作分类单元），其类群多达13个门，分别是厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）、梭杆菌门（Fusobacteria）、变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、螺旋体门（Spirochaetes）、蓝藻门（Cyanobacteria）、纤维杆菌门（Fibrobacteres）、柔膜菌门（Tenericutes）、酸杆菌门（Acidobacteria）、脱铁杆菌门（Deferribacteres）、疣微菌门（Verrucomicrobia）和暂定的TM7门。其中，厚壁菌门和拟杆菌门广泛存在于各样品中，并且占绝对优势。在门水平未能鉴定的序列数为1034，占合并后序列总数的34.7%，可见在动物粪便中蕴藏着数量巨大的分类地位较高的未知细菌类群。此外，经分析共有116个细菌属。多样性指数分析发现，杂食性动物川金丝猴、白眉长臂猿、黑猩猩和蓝孔雀的细菌类群多样性较高，而熊科的亚洲黑熊、大熊猫和专性肉食动物东北虎的类群多样性相对较低。另外，同属于灵长目的川金丝猴、白眉长臂猿和黑猩猩的多样性较接近；熊科的亚洲黑熊和大熊猫的多样性较相近。各多样性指数曲线均表明杂食性动物蓝孔雀和专性肉食动物东北虎的类群多样性差异最大。这表明，分类地位近且食性相近的动物，其粪便菌类群也较相近，反之则差异较大。随后，单独对放线菌类群进行分析发现，放线菌门的序列占总测序量的3.2%，分属于103个OTUs。在7份样品中，东北虎的放线菌门序列数最多达15%，其中的未知放线菌类群也最多。获得纯培养是研究微生物生物学功能及开发利用它们的最基本前提。本工作重点研究了粪便放线菌的分离方法。结果表明，将粪便样品干热处理并配合适宜的稀释度，使用半合成培养基，添加“高浓度”的抑制剂，可以获得较好的分离效果。在此基础上，从28种动物粪便样品中获得923株放线菌，从中选取344株进行分类鉴定，它们分属于35个属，其中一个新属。粪便放线菌中，产丝的放线菌以链霉菌属分布最普遍，数量达107cfu/g；红球菌是另一个分布较广的放线菌类群，有2/3的粪便样品分离到这个属；其它非产丝放线菌以微球菌科成员占优势。本研究只分离好氧放线菌，不涉及厌氧菌。通过形态学、生理生化、细胞化学和分子分类等分析的多相分类方法，确定了YIM100590的分类地位。该菌株是微球菌科的一个新属，被命名为Enteractinococcus gen.nov.，典型种为Enteractinococcus coprophilus sp. nov.。对344株粪便放线菌进行的活性筛选实验发现，有76株菌抑制慢性粒细胞白血病癌细胞株（K562），71株菌抑制急性白血病细胞株（HL60）；有26株菌具较强的广谱抗菌活性；有90%以上的供试菌株有碱性磷酸盐酶、酸性磷酸酶、白氨酸芳胺酶和萘酚-AS-BI-磷酸水解酶酶活，有103株菌具纤维素酶活，174株有几丁质酶活；通过PCR检测，获得PKS I（聚酮合酶I）基因阳性菌株34株，PKS II（聚酮合酶II）阳性菌株30株，NRPS（非核糖体多肽合成酶）阳性菌株58株，CYP（细胞色素P450同工酶）阳性菌株22株。将抗肿瘤活性强的菌株YIM100525发酵后，分离了其中的次生代谢产物，共获得9个化合物。采用波谱学技术确定化合物1、2和3为新化合物。从以上结果总结出以下结论：第一，粪便中蕴藏着数量巨大且种类繁多的细菌类群，并且有很多分类地位较高的未知类群；第二，粪便放线菌的种类非常丰富,其中以链霉菌和红球菌占优势，微球菌亚目的成员也广泛分布，这是粪便放线菌组成的显著特征，并且存在大量的未知放线菌；第三，粪便放线菌的抗肿瘤活性、抗菌活性、酶活性比例较高，尤其是具有抗肿瘤活性的菌株较多，活性也很高，推测它们在宿主代谢和免疫活动中可能起着重要作用。深入研究这些作用的机制，对于宿主的健康研究具有重要意义；第四，由粪便放线菌分离到了结构多样、活性广泛的化合物，说明粪便放线菌也是一类产生活性先导化合物的重要资源。因此，粪便放线菌与来自土壤、海洋及植物的放线菌一样，是开发药物和其他产品的重要来源之一，应当加强粪便放线菌的研究和保护利用。本研究为粪便放线菌资源的开发利用奠定了基础。更多还原

【Abstract】 According to the antibiotics screening or research achievements of microbe genomerecently, actinomycetes remain a rich source for natural products screening. Nowadays, newdrugs, especially antibiotics, are urgently needed to counter and reverse the spread ofantibiotic resistant pathogens and to combat life-threatening diseases as well as new diseases.All of these compel the scientists to search some actinomycetes from new sources. Animals’gut is a typical and unique environment, few researches study the fecal actinobacteria assource. In this study, we researched the diversity of fecal actinobacteria usingculture-independent and culture-dependent approaches, and got new species by polyphasictaxonomic research, and further screened and evaluated their bioactivities againstpathogenetic microorganisms and tumor cell and enzymatic activities. One strain with highantitumor activity was further studied on its secondary metabolites. The current study willprovide a new way to exploit and utilize the actinomycetes sources.We studied the diversity of bacteria16S rRNA gene in seven fecal samples collectedfrom Yunnan Safari Park by454sequencing. A total of42540bacteria V3sequences of16SrRNA genes were obtained and clustered into2979(a threshold of100%identity) uniquesequences and876(a threshold of97%identity) OTUs (operational taxonomic units), andthey distributed at Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, Actinobacteria,Spirochaetes, Cyanobacteria, Fibrobacteres, Tenericutes, Acidobacteria, Verrucomicrobia,Deferribacteres and TM7, total13phylums. Amoung them, Firmicutes and Bacteroideteswere dominant in all fecal samples. However,34.7%of the bacterial sequences can not beidentified into any of the known phylum, indicating that these sequences represented somenew species. The total OTUs were ditributed in116genera. Diversity Index curves showedthat the diversity in omnivorous Rhinopithecus roxellanae, Hlobates hoolock, Pan troglodytesand Pavo cristatus samples was high, but was relative low in Ursus thibetanus, Ailuiopodidaemelanoleuca and Panthera tigris altaica samples. The diversity from Primates’ fecal sampleswere similar, and Ursidae’s fecal samples were similar, too. The difference of the diversitybetween omnivorous Pavo cristatus and carnivorous Panthera tigris altaica was very large.These indicated the bacteria diversity was associated with phylogenetic relationship and feeding habit. Thereinto,3.2%sequences belong to Actinobacteria, including103OTUs. For7samples, the proportion of Actinobacteria in Panthera tigris altaica was the largest,accounting for15%, the novel Actinobacteria was the most, too.Attaining culturable strains is essential for studying and utilizing the microorganisms,thus the isolation method of fecal Actinobacteria was researched. We found that drying andheating up the fecal samples can greatly increase the rate of the actinobacteria. Moreover,many kinds of inhibitors and chemical defined media were suitable for isolation of fecalactinobacteria. Base on this,923purified strains of actinobacteria were isolated from28animal feces samples, and344strains of them were identified as35genera, one of them isnew genus candidate. Streptomyces was the primary filamentous actinobacteria group and upto107colony forming unit (CFU) per gram of dry samples. Rhodococcus was second mostabundant, and this group appeared in2/3samples; The members of suborder Micrococcineaewere main non-filamentous actinobacteria. This study aimed at aerobic actinobacteria, notrefer to anaerobic actinobacteria.The new genus candidate was subjected to polyphasic taxonomy study including analysisof morphological characteristics, physiological and biochemical tests, chemotaxonomicproperties, phylogenetic analysis based on16S rRNA gene sequences. Strain YIM100590was identified as a member of the novel genus of the family Micrococcaceae, the nameEnteractinococcus gen. nov. was proposed. Enteractinococcus coprophilus sp. nov. is the typespecies of new genus.Through screening of bioactivities on the fecal Actinobacteria,76strains showed activityagainst K562cell and71strains showed activity against HL60cell by using MTT assay;26strains showed antimicrobial activity using the agar plate diffusion method; More than90%tested strains were positive for alkaline phosphatase, acid phosphatase, leucine arylamidaseand naphthol-AS-BI-phosphohydrolase activity,103could hydrolyze cellulose and174showed keratin activity; The functional gene screening results showed that34strains hadpolyketide synthase I (PKS I) gene,30strains had PKS II gene,58strains had non-ribosomalpeptide synthetase (NRPS) gene and22strains had cytochrome P450hydroxylase (CYP)gene.Nine compounds were isolated and purified from80liter fermentation broth of strainYIM100525, which had high antitumor activity. The structure of compounds1-9wereconfirmed by spectroscopic analysis, and compounds1-3had novel structures.The results of this work firstly showed that fecal samples harbored huge quantity ofbacteria, and many groups at phylum level were novel. Secondly, fecal actinobacteria wereabundant. The strains of genera Streptomyces and Rhodococcus were dominating, the members of suborder Micrococcineae were also abundant. This is a distinct feature for groupcomposition of fecal actinobacteria. Many novel actinobacteria existed in feces, too. Thirdly,the proportion of bioactive strains was large. We infered fecal actinobacteria was veryimportant from a nutritional, physiological and immune point of view. Further studyingmechanisms of these function of actinobacteria is significant for the health of host. Fourthly,abundant compounds with different activities were obtained from the fecal actinobacteria,which showed fecal actinobacteria was an important resource for seeking active leadcompound. In conclusion, like soil, marine and endophytic actinobacteria, fecal actinobacteriais a significant source for developing drug and other manufacture. Thus, the research,protection and utilization of fecal actinobacteria should be strengthened. This study has layedthe foundations for development and utilization of fecal actinomycete resources.更多还原