本文關(guān)鍵詞：尖孢鏈霉菌SN0280次生代謝產(chǎn)物分離鑒定及抑菌活性研究　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 土壤放線菌 尖孢鏈霉菌 結(jié)構(gòu)鑒定 最小抑菌濃度 聚醚化合物 番茄細(xì)菌性斑點(diǎn)病菌

【摘要】：放線菌作為一類十分重要的微生物資源,其在微生物源農(nóng)藥中占有了不可替代的重要地位,帶來(lái)了巨大的經(jīng)濟(jì)價(jià)值和廣泛的實(shí)際用途。本文使用了大孔吸附樹(shù)脂、硅膠柱層析、凝膠柱層析、高效液相制備等多種現(xiàn)代分離手段,以及紫外、紅外、旋光核磁共振、高分辨質(zhì)譜的多種波譜技術(shù),對(duì)放線菌SN0280的次生代謝產(chǎn)物進(jìn)行了分離提純及結(jié)構(gòu)鑒定;利用多種活性檢測(cè)方法測(cè)定了這些代謝產(chǎn)物對(duì)多種植物病原菌的抑菌活性;系統(tǒng)研究了聚醚類化合物對(duì)植物病原細(xì)菌的抗菌活性情況。主要研究結(jié)果如下:結(jié)合抑菌活性和化學(xué)初篩,篩選得到既具有廣譜和顯著的抑菌活性、又可產(chǎn)生豐富代謝產(chǎn)物的最優(yōu)菌株SN0280。對(duì)采自不同地區(qū)的29 土壤放線菌進(jìn)行純化、繼代、保種,對(duì)其中生長(zhǎng)的26株菌株進(jìn)行抑菌活性初篩,有3株菌(SN0270、SN0277、SN0280)表現(xiàn)出了較為廣譜且較強(qiáng)的抑菌活性,同時(shí)結(jié)合小發(fā)酵化學(xué)初篩結(jié)果,選取高抑菌活性、化合物產(chǎn)量豐富的菌株SN0280作為試驗(yàn)菌株。結(jié)合傳統(tǒng)分類學(xué)和分子生物學(xué)方法,確定菌株SN0280為尖孢鏈霉菌。試驗(yàn)提取菌株SN0280的DNA,擴(kuò)增后并進(jìn)行序列測(cè)序,并與EzTaxon數(shù)據(jù)庫(kù)中已有基因序列進(jìn)行比對(duì),結(jié)果顯示SN0280菌株與尖抱鏈霉菌(StreptomycescuspidosporusNBRC 12378)最為相似,同源性為99.85%,建立的統(tǒng)發(fā)育樹(shù)中二者親緣關(guān)系最近,同時(shí),SN0280菌株形態(tài)特征、培養(yǎng)特征以及生理生化特征,與Scuspidospor.基本一致,因此,最終鑒定SN0280菌株為尖抱鏈霉菌(S.cuspidosporus)結(jié)合多種分離純化手段,采用活性追蹤法,在放線菌SN0280代謝產(chǎn)物中分離提純得到7個(gè)單體化合物。試驗(yàn)通過(guò)大孔吸附樹(shù)脂對(duì)菌株SN0280發(fā)酵產(chǎn)物進(jìn)行吸附,甲醇洗脫樹(shù)脂,二氯甲烷萃取,得到紅棕色粘稠狀的粗提物4.36 g。對(duì)粗提物進(jìn)行一級(jí)硅膠柱層析后分為6個(gè)組分:T1-T6,并通過(guò)對(duì)番茄細(xì)菌性斑點(diǎn)病菌和潰瘍病菌的活性檢測(cè),對(duì)其中表現(xiàn)出較好抑菌活性的4個(gè)組分T2、T3、T4、T6進(jìn)行進(jìn)一步的分離純化,結(jié)合薄層層析、硅膠柱層析、凝膠柱層析和高效液相制備等分離手段得到7個(gè)純化合物:H1-H7。綜合采用核磁共振、質(zhì)譜、紅外、旋光測(cè)定等多種結(jié)構(gòu)鑒定手段對(duì)這7單體化合物進(jìn)行結(jié)構(gòu)鑒定,并進(jìn)行了初步的活性檢測(cè)。結(jié)合譜圖檢測(cè)數(shù)據(jù)和文獻(xiàn)數(shù)據(jù),確定這些化合物中,有5個(gè)新化合物,它們分別是:吲哚類衍生物H1(chloroindole),聚酮類化合物 H2、H3、H5(streptoone A,streptoone B,streptoone C),丁內(nèi)酯衍生物 H4(streplactone);以及2個(gè)已知化合物:大環(huán)內(nèi)酯類抗生素H6(X-14952B)和聚醚類化合物H7(1.264-B)。并測(cè)定了這些單體化合物對(duì)11株植物病原菌的抑菌活性,其中,H7對(duì)番茄細(xì)菌性斑點(diǎn)病菌表現(xiàn)出了最好的抑菌效果,同時(shí)還對(duì)番茄細(xì)菌性潰瘍病菌和尖孢鐮刀菌也有很好的抑制效果;H2對(duì)番茄潰瘍病菌表現(xiàn)出了與鏈霉素相當(dāng)?shù)囊志钚?H3對(duì)辣椒疫霉病菌,H4對(duì)瓜果腐霉病菌,H6對(duì)辣椒疫霉病菌和番茄灰霉病菌也表現(xiàn)出了較好的抑菌活性(MIC20μg/mL)。聚醚化合物H7對(duì)番茄細(xì)菌性斑點(diǎn)病具有非常好的活性,并對(duì)番茄植株表現(xiàn)出了很高的安全性。H7在外界環(huán)境溫度低于60℃C時(shí)是很穩(wěn)定的,其抗菌活性基本不受影響;當(dāng)化合物濃度為1MIC(2 μg/mL)的對(duì)靶標(biāo)菌表現(xiàn)出了很好的抑菌效果,菌落數(shù)與抑菌率基本處于穩(wěn)定狀態(tài),濃度達(dá)到2MIC(4 μg/mL)時(shí)不僅表現(xiàn)出了很好的抑菌效果,還起到了殺菌作用;此外,通過(guò)測(cè)定H7對(duì)斑點(diǎn)病菌生長(zhǎng)曲線的影響,對(duì)比3種不同濃度化合物對(duì)供試菌的生長(zhǎng)曲線,表明其能使靶標(biāo)細(xì)菌的分裂增殖受到抑制,且隨著濃度的增加其抑制效果增強(qiáng)。
[Abstract]:Actinomycetes is a very important kind of microbial resources, plays an irreplaceable important role in microbial pesticides, has brought huge economic value and a wide range of practical applications. In this paper, using macroporous resin, silica gel column chromatography, gel column chromatography, HPLC preparation and other modern separation means, as well as the ultraviolet, infrared, optical magnetic resonance spectroscopy, high resolution multiple mass spectrometry, secondary metabolites of actinomycetes SN0280 of purification and isolation; antifungal activity of these metabolites on various plant pathogens were measured using a variety of active detection method; system of polyether compounds of plants the antibacterial activity of pathogenic bacteria. The main results are as follows: the combination of antimicrobial activity and chemical screening, screening has broad-spectrum antibacterial activity and significantly, and can produce abundant metabolites The optimal SN0280. strains collected from different regions of 29 actinomycetes were purified, subcultured, Paul, of which the growth of 26 strains of antibacterial activity screening, 3 strains (SN0270, SN0277, SN0280) showed a broad-spectrum and strong antibacterial activity, combined with small chemical fermentation the results of screening, selection of high yield rich compound antibacterial activity, strain SN0280 as test strains. Combined with the traditional taxonomy and molecular biology method, strain SN0280 was identified as Fusarium. Streptomyces strain SN0280 DNA extraction, amplification and sequence sequencing, and compared with the existing EzTaxon database of gene sequences, the results show that SN0280 hold the tip strain and Streptomyces (StreptomycescuspidosporusNBRC 12378) is the most similar to that of 99.85% homology, phylogenetic relationship of two phylogenetic trees established recently, at the same time, SN0280 morphological, cultural characteristics Physiological and biochemical characteristics, consistent with Scuspidospor., therefore, the final strain SN0280 was identified as Streptomyces with tip (S.cuspidosporus) purification combined with a variety of means, using active tracking method, separation and purification of 7 monomer compounds in actinomycetes metabolites of SN0280. The adsorption test of strain SN0280 fermentation product by macroporous adsorption resin, methanol elution resin, dichloromethane, red brown viscous crude extract of 4.36 g. grade silica gel column chromatography of the crude extract was divided into 6 components: T1-T6, and the activity detection of tomato bacterial speck and canker, of which 4 groups showed good antibacterial the activity of T2, T3, T4, T6 to further purify, combined with thin layer chromatography, silica gel column chromatography, gel column chromatography and HPLC preparation and separation method obtained 7 purified compounds: H1-H7. comprehensive use NMR, mass spectrometry, infrared polarimetry, and other identification approaches for the structure identification of the 7 compounds, and the activity was detected preliminarily. Combined with the spectrum detection data and literature data, identify these compounds, 5 new compounds, they are: indole derivatives (chloroindole, H1) polyketide compounds H2, H3, H5 (streptoone A, streptoone B, streptoone C), y-butyrolactone derivatives H4 (streplactone); and 2 known compounds: macrolide antibiotic H6 (X-14952B) and polyether compound H7 (1.264-B). The antibacterial activity of these compounds, 11 strains of plant pathogens and the determination of the H7 of the tomato bacterial speck showed the best antibacterial effect, but also to tomato bacterial canker and Fusarium oxysporum also had good inhibitory effect on tomato bacterial canker; H2 showed with chain The antibacterial activity of fosfomycin equivalent; H3 of Phytophthora capsici, H4 against Pythium aphanidermatum, H6 of Phytophthora capsici and Botrytis cinerea also showed better antibacterial activity (MIC20 g/mL). H7 polyether compound has good activity against tomato bacterial speck, and tomato the.H7 plants showed very high security in the external environment temperature lower than 60 DEG C C is very stable, its antibacterial activity is basically not affected; when the compound concentration is 1MIC (2 g/mL) of the target bacteria showed good antibacterial effect, and the inhibitory rate of colony number is basically in a stable state and the concentration of 2MIC (4 g/mL) not only showed good antibacterial effect, but also play a bactericidal effect; in addition, the effect of H7 on the growth curve determination spot, growth curve comparison of 3 different concentrations of compounds were tested for, that it can make the target bacteria The proliferation was inhibited, and the increase of the concentration of the inhibitory effect is enhanced.

【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李增政;林滕;常永芳;周琴;劉俊林;;一株植物內(nèi)生放線菌次級(jí)代謝產(chǎn)物的提取及抑菌活性研究[J];科技視界;2016年11期

2 陳國(guó)棟;肖高鏗;姚新生;高昊;;電子圓二色譜計(jì)算方法在天然產(chǎn)物絕對(duì)構(gòu)型確定中的應(yīng)用[J];國(guó)際藥學(xué)研究雜志;2015年06期

3 蔣忠科;張洋;郭連宏;姜蓉;孫承航;;鏈霉菌I06A-03304產(chǎn)生的二酮哌嗪類化合物的純化及結(jié)構(gòu)鑒定[J];中國(guó)醫(yī)藥導(dǎo)報(bào);2014年18期

4 羅紅麗;關(guān)菲菲;沈蘭;黃長(zhǎng)武;李丹;胡昌華;廖國(guó)建;;南方紅豆杉內(nèi)生放線菌的分離鑒定和次級(jí)代謝潛力評(píng)估[J];中國(guó)抗生素雜志;2014年02期

5 白寶平;;晶體表征在無(wú)機(jī)化學(xué)研究中的應(yīng)用統(tǒng)計(jì)報(bào)告[J];太原城市職業(yè)技術(shù)學(xué)院學(xué)報(bào);2013年08期

6 張正玉;吳綿斌;;抗生素分離純化技術(shù)研究進(jìn)展[J];中國(guó)生物工程雜志;2012年06期

7 魏賽金;張智平;涂曉嶸;何健;涂國(guó)全;;鏈霉菌702抗尖孢鐮刀菌次級(jí)代謝產(chǎn)物的分離鑒定及活性研究[J];江西農(nóng)業(yè)大學(xué)學(xué)報(bào);2011年05期

8 石妞妞;王浩鑫;魯春華;劉最;沈月毛;;海洋放線菌Streptomyces sp. LZ35中的安莎霉素類化合物[J];中國(guó)藥學(xué)雜志;2011年17期

9 黃素芳;車建美;劉波;史懷;蘇明星;陳崢;;短短芽胞桿菌FJAT-0809-GXL代謝產(chǎn)物活性物質(zhì)提取方法的優(yōu)化[J];福建農(nóng)業(yè)學(xué)報(bào);2011年04期

10 牛四文;李蘇梅;田新朋;胡濤;鞠建華;楊曉紅;張Pr;張長(zhǎng)生;;海洋放線菌Streptomyces sp.SCSIO 1934中次生代謝產(chǎn)物的分離和鑒定[J];中國(guó)中藥雜志;2011年13期

，

本文編號(hào)：1405741