發(fā)布時(shí)間：2018-04-06 20:16

  本文選題：北冰洋　切入點(diǎn)：海洋沉積物　出處：《中國(guó)海洋大學(xué)》2014年碩士論文

【摘要】：放線菌是天然產(chǎn)物的重要來(lái)源，隨著放線菌素的發(fā)現(xiàn)，迄今為止從微生物中發(fā)現(xiàn)的33500種有生物活性的次級(jí)代謝產(chǎn)物中有13700種來(lái)自于放線菌，占所有生物活性物質(zhì)的40%以上。近幾年來(lái)，從海洋環(huán)境尤其是極端環(huán)境中分離放線菌及其活性物質(zhì)成為必然的趨勢(shì)。北冰洋氣候寒冷，水溫大部分時(shí)間在0℃以下，環(huán)境十分惡劣，面對(duì)如此極端環(huán)境，放線菌必須產(chǎn)生與之相適應(yīng)的生理生化特征及代謝途徑。因而北冰洋的放線菌可能會(huì)產(chǎn)生結(jié)構(gòu)新穎、活性特殊的生物活性物質(zhì)。 本文對(duì)采集自北冰洋太平洋扇區(qū)的24份海洋沉積物樣品進(jìn)行放線菌的分離培養(yǎng)，通過(guò)16S rDNA序列進(jìn)行系統(tǒng)發(fā)育分析；對(duì)分離到的菌株進(jìn)行耐鹽及需海水檢測(cè)、次級(jí)代謝產(chǎn)物合成酶基因的篩查、抗菌活性研究；并對(duì)其中一株潛力菌株進(jìn)行分子鑒定、系統(tǒng)發(fā)育分析、發(fā)酵條件優(yōu)化及發(fā)酵液穩(wěn)定性研究。 通過(guò)分散差速離心法對(duì)樣品進(jìn)行處理,采用5種分離培養(yǎng)基從北冰洋太平洋扇區(qū)24份沉積物樣品中分離出143株放線菌，經(jīng)BOX-PCR排重后選取70株代表菌株進(jìn)行16S rDNA測(cè)序分析，分離得到的放線菌歸屬于壤球菌屬Agrococcus（1株）、微桿菌屬M(fèi)icrobacterium（3株）、鹽細(xì)菌屬Salinibacterium（7株）、節(jié)細(xì)菌屬Arthrobacter（5株）、檸檬球菌屬Citricoccus（4株）、庫(kù)克菌屬Kocuria（6株）、紅球菌屬Rhodococcus（8株）、砷球菌屬Arsenicicoccus（1株）、兩面神菌屬Janibacter（7株）、擬諾卡氏菌屬Nocardiopsis（6株）、迪茨氏菌屬Dietzia（5株）、短桿菌屬Brevibacterium（3株）、糖多孢菌屬Saccharopolyspora（1株）、鏈霉菌屬Streptomyces（8株）、棒狀桿菌屬Corynebacterium（1株）、小月菌屬M(fèi)icrolunatus（1株）、類諾卡氏菌屬Nocardioides（1株）等17個(gè)屬，大部分菌株屬于稀有放線菌。其中菌株Y284與標(biāo)準(zhǔn)菌株Saccharopolyspora gregoriiNCIMB12823T(X76962)的相似度為97.771%，菌株Y25與標(biāo)準(zhǔn)菌株Nocardioideskribbensis KSL-2T(AY835924)的相似度為97.804%，二者可初步判斷為放線菌的新種。放線菌在不同培養(yǎng)基上分離的情況差異較大，M7培養(yǎng)基的放線菌分離率（70.97%）以及所分離到屬數(shù)都是最多的，因此分離效果最好。 70株放線菌的PKSⅠ、PKSⅡ、NRPS、phzE、dTGD、Halo、CYP基因篩查結(jié)果分別為：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%）�？咕钚詸z測(cè)的結(jié)果為：對(duì)白色念珠菌有抗性的放線菌有9株，分別屬于Nocardiopsis和Streptomyces；可以抑制枯草芽孢桿菌的放線菌菌株有12株，分別屬于Microlunatus、Nocardiopsis和Streptomyces；對(duì)大腸桿菌和金黃葡萄球菌有抗性的菌株分別只有一株，都屬于Streptomyces。 對(duì)其中一株潛力菌株Y18進(jìn)行分子鑒定，發(fā)現(xiàn)與其親緣關(guān)系最近的標(biāo)準(zhǔn)菌株為Nocardiopsis dassonvillei subsp. dassonvillei DSM43111T，16S rDNA序列比對(duì)結(jié)果顯示相似性高達(dá)99.8%。適合Y18產(chǎn)抗枯草芽孢桿菌活性產(chǎn)物的發(fā)酵培養(yǎng)基配方為：蛋白胨2g，酪素水解物2g，酵母膏2g，葡萄糖1g，，海水1L；發(fā)酵條件為：發(fā)酵培養(yǎng)基初始pH值8.0，種子液接種量4%，發(fā)酵溫度28℃，發(fā)酵時(shí)間6天，1000mL三角瓶裝液量20%。對(duì)發(fā)酵液的穩(wěn)定性研究表明，發(fā)酵液對(duì)溫度具有較好的穩(wěn)定性，對(duì)酸堿環(huán)境適應(yīng)性較強(qiáng)，低溫貯藏性較好。 上述結(jié)果表明，北冰洋海洋沉積物中蘊(yùn)含著豐富的放線菌資源，并具有產(chǎn)生多種生物活性次級(jí)代謝產(chǎn)物的潛力。本文的研究增加了我國(guó)海洋放線菌的資源儲(chǔ)備，豐富了我們對(duì)海洋放線菌的認(rèn)識(shí)，為極地海洋沉積物放線菌的研究、開發(fā)與利用提供依據(jù)和有價(jià)值的參考，具有巨大的科學(xué)價(jià)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.

【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：Q178.53

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