【作者】 张戌升；

【导师】 李志勇；

【作者基本信息】 上海交通大学 ， 微生物学， 2007， 硕士

【摘要】 1950年以来,人们从海绵中发现了大量活性物质,使海绵成为海洋活性物质最丰富的来源。越来越多研究表明一些活性物质是由海绵共附生微生物产生的,但是还缺乏直接的证据。我们前期研究已经筛选到多株具有抗菌活性的海绵共附生微生物,开展相关功能基因研究,从基因水平揭示这些抗菌活性的分子基础具有重要价值。同时,由于绝大多数海绵共附生微生物属于难培养微生物,因此建立宏基因组文库将为利用难培养的海绵共附生微生物功能基因资源意义正大。尽管已有从海绵及其共附生微生物中分离得到具有抗肿瘤、抗菌等活性的聚酮化合物和非核糖体多肽的报道,但海绵及其共附生微生物中这两类化合物的合成基因(簇)研究非常少。本文目的在于利用宏基因组技术从海绵及其共附生微生物中筛选到聚酮合成酶(PKS)基因(簇)和非核糖体多肽合成酶(NRPS)基因(簇),为所发现的抗菌活性提供分子基础,为海绵活性物质微生物来源假说提供证据,同时建立包含大片段DNA的海绵宏基因组文库为基因步从海绵中直接筛选相关功能基因簇提供基础。实验室前期研究证明细薄星芒海绵(Stelletta tenui)共附生缓慢葡萄球菌A75具有广谱抗菌活性。本文提取缓慢葡萄球菌A75基因组DNA建立了Fosmid宏基因组文库,文库的插入片段在25-43kb之间。采用基于功能基因的PCR筛选技术和基于功能的抑菌实验对宏基因组文库进行了筛选,得到了一个针对枯草芽孢杆菌有抑制活性的阳性克隆。采用酶切测序、拼接的方法获得了一条长约26kb的核苷酸序列。对其进行比对分析发现该序列含有较为完整的约21.8kb的PKS基因簇,将其翻译成氨基酸序列后进行功能域分析,发现包含了DH、AT、ACP、KS、KR五个系列共22个功能域基因,推测该基因簇可以催化一个含有12C主链的聚酮化合物合成。实验室前期研究证明皱皮软海绵(Halichondria rugosa)共附生芽孢杆菌B114具有广谱抗菌活性。本文针对该菌构建了适合快速筛选的半流体Fosmid宏基因组文库。通过PCR在B114半流体宏基因组文库中扩增到了一段974bp基因片段,比对分析证明属于非核糖体多肽合成酶(NRPS)簇腺苷酰化结构域序列。利用PCR技术对采集自我国南海的细薄星芒海绵(Stelletta tenui),皱皮软海绵(Halichondria rugosa),贪婪倔海绵(Dysidea avara)和澳大利亚厚皮海绵(Craniella australiensis )4种海绵的宏基因组进行多种功能基因的筛选,从皱皮软海绵宏基因组中筛选得到了一段671bp的序列,BLAST对比结果表明该基因对应的氨基酸序列与红细菌属的Rhodobacterales bacterium的聚酮合酶基因的KS域有96%的同源性,证明了海绵的一些活性物质是来自于其共附生的微生物。通过系统发育分析推测该段序列所在的PKS基因属于trans-AT型。在此基础上为今后进一步筛选相关功能基因(簇),我们建立了皱皮软海绵的Fosmid宏基因组文库,文库插入片段均在26-39kb之间,可以满足大片段基因簇筛选的需要。在本研究中,(1)我们建立了细薄星芒海绵共附生缓慢葡萄球菌A75的宏基因组文库,并从中筛选得到一个完整的PKS基因簇,为以后该缓慢葡萄球菌聚酮类活性物质研究和组合生物合成提供了基础。(2)从皱皮软海绵共附生芽孢杆菌B114的宏基因组中发现了NRPS基因,为有目的开展利用该菌制备非核糖体多肽化合物提供了基础。(3)从皱皮软海绵宏基因组中发现了共附生微生物来源的PKS基因,为海绵活性物质微生物来源学说提供了有力的证据。(4)构建的包含大片段DNA(26-39kb)皱皮软海绵Fosmid宏基因组文库为功能基因簇的筛选提供了良好的资源库。海绵及其共附生微生物功能基因的研究在国际上才刚刚开始,本文研究具有创新性。更多还原

【Abstract】 Since 1950s, many active materials have been found from marine sponges. It was suggested that some of active materials are from bacteria associated with sponge, but direct proof need to be found. Many nicrobes with antimicrobial function have been isolated from sponge, it is of great value to study functional gene (cluster) with the aim to reveal the molecular mechanism of the antimicrobial compounds. Meanwhile, because that the majority of sponge-associated microbes are still not culturable at present, therefore, the construction of metagenome library is valuable for the utilization of uncultured microbial functional gene.To date, researches on PKS and NRPS gene clusters are rarely found, although some PKS and NRPS compounds with anti-cancer, antimicrobe had been found from sponge. This paper aim to get PKS and NRPS gene (cluster) using metagenome-based technique in order to find molecular basis for antimicrobial function and proof to the sponge-associated microbial role in the synthesis of active compound. At the same time, the construction of sponge metagenome library containing large segment DNA will be helpful for the screening of functional gene cluster of uncultured microbes.Genomic DNA of Staphylococcus lentus A75, with broad anti-microbial function according to our previous investigation, was extracted for the construction of Fosmid library. The size of insert DNA is from 25kb to 43kb in this library. Through PCR and anti-microbial activity screening,a positive clone against Bacillus subtilis was isolated from metagenome library and a gene cluster about 26kb in length was sequenced. Finally, the entire PKS gene about 21.8kb in length that probably coded a polyketide synthase cotaining 22 DH, AT, ACP, KS, KR which catalyzed the synthesis of a polyketide with 12C in its main chain was obtained.Genomic DNA of Bacillus sp. B114, with broad anti-microbial function according to our previous study, was extracted for the construction of Fosmid library. Through PCR a NRPS gene fragment about 974bp in length, which belonged to Adenylation domain, was obtained. Metagenomic DNA of four sponges Stelletta tenui,Halichondria regosa,Dysidea avara and Craniella australiensis from the South China Sea were extracted for the screening functional genes by means of PCR. As a result, a KS gene about 671bp in length was obtained from Halichondria regosa. BLAST analysis showed that the homology of the amino acid sequence with that of the KS domain of PKS gene in Rhodobacterales bacterium was up to 96%. It provided powerful proof to the microbial origin hypothesis of sponge active compounds. Phylogenetic analysis indicated that the obtained PKS gene belongs to trans-AT KS domains. Consequently, metagenomic DNA of Halichondria regosa was extracted to construct Fosmid library. The size of insert DNA is from 26kb to 39kb in this library, that can meet the need of gene cluster screening.In this paper, (1) a Fosmid metagenome library of Stelletta tenui associated Staphylococcus lentus A75 was constructed and a 21.8kb PKS gene cluster cotaining AT,ACP,KS,KR was obtained, which lay a basis for PKS active compound study and its biological combined synthesis. (2) 974bp NRPS gene fragment belonging to Adenylation domain was found in the metagenome library of Bacillus sp. B114 associated with Halichondria regosa, which lay a basis for NRPS compound study. (3) The finding of KS domain of PKS gene in sponge Halichondria regosa provided powerful proof to the microbial origin hypothesis of sponge active compounds. (4) The construction of metagenomic Fosmid library of Halichondria regosa containing large segement DNA from 26kb to 39kb provide a resource for functional gene cluster screening.This investigation is with great innovation because that the study on the functional gene (cluster) of sponge and its associated microbes has just begun in the world.更多还原