北冰洋太平洋扇区沉积物放线菌多样性、次级代谢产物合成酶基因筛查及其抗菌活性研究

发布时间：2018-04-06 20:16

本文选题：北冰洋　切入点：海洋沉积物　出处：《中国海洋大学》2014年硕士论文

【摘要】：放线菌是天然产物的重要来源，随着放线菌素的发现，迄今为止从微生物中发现的33500种有生物活性的次级代谢产物中有13700种来自于放线菌，占所有生物活性物质的40%以上。近几年来，从海洋环境尤其是极端环境中分离放线菌及其活性物质成为必然的趋势。北冰洋气候寒冷，水温大部分时间在0℃以下，环境十分恶劣，面对如此极端环境，放线菌必须产生与之相适应的生理生化特征及代谢途径。因而北冰洋的放线菌可能会产生结构新颖、活性特殊的生物活性物质。本文对采集自北冰洋太平洋扇区的24份海洋沉积物样品进行放线菌的分离培养，通过16S rDNA序列进行系统发育分析；对分离到的菌株进行耐盐及需海水检测、次级代谢产物合成酶基因的筛查、抗菌活性研究；并对其中一株潜力菌株进行分子鉴定、系统发育分析、发酵条件优化及发酵液稳定性研究。通过分散差速离心法对样品进行处理,采用5种分离培养基从北冰洋太平洋扇区24份沉积物样品中分离出143株放线菌，经BOX-PCR排重后选取70株代表菌株进行16S rDNA测序分析，分离得到的放线菌归属于壤球菌属Agrococcus（1株）、微杆菌属Microbacterium（3株）、盐细菌属Salinibacterium（7株）、节细菌属Arthrobacter（5株）、柠檬球菌属Citricoccus（4株）、库克菌属Kocuria（6株）、红球菌属Rhodococcus（8株）、砷球菌属Arsenicicoccus（1株）、两面神菌属Janibacter（7株）、拟诺卡氏菌属Nocardiopsis（6株）、迪茨氏菌属Dietzia（5株）、短杆菌属Brevibacterium（3株）、糖多孢菌属Saccharopolyspora（1株）、链霉菌属Streptomyces（8株）、棒状杆菌属Corynebacterium（1株）、小月菌属Microlunatus（1株）、类诺卡氏菌属Nocardioides（1株）等17个属，大部分菌株属于稀有放线菌。其中菌株Y284与标准菌株Saccharopolyspora gregoriiNCIMB12823T(X76962)的相似度为97.771%，菌株Y25与标准菌株Nocardioideskribbensis KSL-2T(AY835924)的相似度为97.804%，二者可初步判断为放线菌的新种。放线菌在不同培养基上分离的情况差异较大，M7培养基的放线菌分离率（70.97%）以及所分离到属数都是最多的，因此分离效果最好。 70株放线菌的PKSⅠ、PKSⅡ、NRPS、phzE、dTGD、Halo、CYP基因筛查结果分别为：41株（58.6%）、27株（38.6%）、25株（35.7%）、16株（22.9%）、60株（85.7%）、27株（38.6%）、32株（45.7%）。抗菌活性检测的结果为：对白色念珠菌有抗性的放线菌有9株，分别属于Nocardiopsis和Streptomyces；可以抑制枯草芽孢杆菌的放线菌菌株有12株，分别属于Microlunatus、Nocardiopsis和Streptomyces；对大肠杆菌和金黄葡萄球菌有抗性的菌株分别只有一株，都属于Streptomyces。对其中一株潜力菌株Y18进行分子鉴定，发现与其亲缘关系最近的标准菌株为Nocardiopsis dassonvillei subsp. dassonvillei DSM43111T，16S rDNA序列比对结果显示相似性高达99.8%。适合Y18产抗枯草芽孢杆菌活性产物的发酵培养基配方为：蛋白胨2g，酪素水解物2g，酵母膏2g，葡萄糖1g，，海水1L；发酵条件为：发酵培养基初始pH值8.0，种子液接种量4%，发酵温度28℃，发酵时间6天，1000mL三角瓶装液量20%。对发酵液的稳定性研究表明，发酵液对温度具有较好的稳定性，对酸碱环境适应性较强，低温贮藏性较好。上述结果表明，北冰洋海洋沉积物中蕴含着丰富的放线菌资源，并具有产生多种生物活性次级代谢产物的潜力。本文的研究增加了我国海洋放线菌的资源储备，丰富了我们对海洋放线菌的认识，为极地海洋沉积物放线菌的研究、开发与利用提供依据和有价值的参考，具有巨大的科学价值。
[Abstract]:Actinomycetes are an important source of natural products, with the discovery of actinomycetes, 33500 kinds of bioactive secondary metabolites from microorganisms so far found in 13700 from actinomycetes, accounting for more than 40% of all bioactive substances. In recent years, especially from the marine environment actinomycetes and active substance separation has become an inevitable trend in extreme environments. The Arctic cold climate, most of the time the temperature is below 0 DEG C and the environment is very bad, in the face of such extreme environments, actinomycetes must produce physiological and biochemical characteristics and the metabolic pathway and the Arctic Ocean. Therefore actinomycetes may produce structure a novel, biologically active substances and special activity.
This paper collected from the Pacific sector of the Arctic Ocean 24 marine sediment samples were isolated from actinomycetes, phylogenetic analysis was carried out by 16S rDNA sequence; the isolated strains were resistant to salt and water detection, screening of secondary metabolites synthase gene, activity of anti bacteria; and one of these strains of potential strains molecular identification and phylogenetic analysis of stability, optimization of fermentation conditions and fermentation liquid.
The dispersion and differential centrifugation of the samples, using 5 kinds of isolation medium were isolated 143 strains of actinomycetes from the Pacific sector of the Arctic Ocean in 24 sediment samples by BOX-PCR after re scheduling from the 70 representative isolates were analyzed by 16S rDNA sequencing, the isolated actinomycetes belonging to the genus Agrococcus (1 strains in soil Microbacterium strain), Microbacterium (3 strains), salt bacteria of the genus Salinibacterium (7 strains), Arthrobacter Arthrobacter (5 strains), lemon Citricoccus Staphylococcus (4 strains), Kocuria Cook spp. (6 strains), Rhodococcus Rhodococcus spp. (8 strains), Staphylococcus aureus (1 strains of arsenic Arsenicicoccus Janibacter), Janus spp. (7 strains), Nocardiopsis Nocardia (6 strains), Dietzia Dietz's genus (5 strains), Brevibacterium Brevibacterium (3 strains), Saccharopolyspora genus Saccharopolyspora (1 strains), Streptomyces Streptomyces (8 strains), Corynebacterium Corynebacterium (1 strains), Ozuki bacteria The genus Microlunatus (1 strains), Nocardioides Nocardia (1 strains), 17 genera, and most of the strains belong to rare actinomycetes. The Y284 strain and standard strain Saccharopolyspora gregoriiNCIMB12823T (X76962) and the similarity is 97.771%, and the standard strain Y25 strain Nocardioideskribbensis KSL-2T (AY835924) and the similarity is 97.804%, two preliminary for the judgment of new species of actinomycetes. Difference of Actinomyces in different culture medium large, medium M7 actinomycetes isolation rate (70.97%) and the isolated genera are the most, so the best separation effect.
PKS I, 70 strains of actinomycetes PKS II, NRPS, phzE, dTGD, Halo, CYP gene screening results were 41 strains (58.6%), 27 strains (38.6%), 25 strains (35.7%), 16 strains (22.9%), 60 strains (85.7%), 27 strains (38.6%), 32 strains (45.7%). The antibacterial activity test results for Candida albicans resistant actinomycetes and 9 strains belonged to Nocardiopsis and Streptomyces; can inhibit Bacillus subtilis strains of actinomycetes and 12 strains, respectively belonging to Microlunatus, Nocardiopsis and Streptomyces; for Escherichia coli and Staphylococcus aureus resistant strains were only one strain, belong to Streptomyces.
One potential strain strain Y18 by molecular identification and phylogenetic found recently for the Nocardiopsis dassonvillei standard strain subsp. dassonvillei DSM43111T and 16S rDNA gene sequence comparison showed high similarity of 99.8%. suitable for the fermentation of Y18 producing Bacillus subtilis anti metabolite medium was peptone 2G, yeast extract, casein hydrolysate 2G 2G, glucose 1g, water 1L; fermentation conditions for fermentation medium initial pH value 8, seed inoculation amount 4%, fermentation temperature 28, fermentation time 6 days, on the stability of 1000mL flask fluid volume 20%. of the fermentation broth showed that fermentation liquid has good stability to temperature, adaptability to acid-base environment low temperature storage, better.
The results show that the Arctic marine sediment contains actinomycete resource rich, and has produced a variety of bioactive secondary metabolites. The potential of this study increased our country marine actinomycetes resource reserves, enriches our knowledge of marine actinomycetes, for the study of polar marine sediments actinomycetes the development and utilization, and provide valuable reference, has great scientific value.

【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q178.53

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