尖孢链霉菌SN0280次生代谢产物分离鉴定及抑菌活性研究

发布时间：2018-01-10 15:11

本文关键词：尖孢链霉菌SN0280次生代谢产物分离鉴定及抑菌活性研究　出处：《沈阳农业大学》2017年博士论文　论文类型：学位论文

【摘要】：放线菌作为一类十分重要的微生物资源,其在微生物源农药中占有了不可替代的重要地位,带来了巨大的经济价值和广泛的实际用途。本文使用了大孔吸附树脂、硅胶柱层析、凝胶柱层析、高效液相制备等多种现代分离手段,以及紫外、红外、旋光核磁共振、高分辨质谱的多种波谱技术,对放线菌SN0280的次生代谢产物进行了分离提纯及结构鉴定;利用多种活性检测方法测定了这些代谢产物对多种植物病原菌的抑菌活性;系统研究了聚醚类化合物对植物病原细菌的抗菌活性情况。主要研究结果如下:结合抑菌活性和化学初筛,筛选得到既具有广谱和显著的抑菌活性、又可产生丰富代谢产物的最优菌株SN0280。对采自不同地区的29 土壤放线菌进行纯化、继代、保种,对其中生长的26株菌株进行抑菌活性初筛,有3株菌(SN0270、SN0277、SN0280)表现出了较为广谱且较强的抑菌活性,同时结合小发酵化学初筛结果,选取高抑菌活性、化合物产量丰富的菌株SN0280作为试验菌株。结合传统分类学和分子生物学方法,确定菌株SN0280为尖孢链霉菌。试验提取菌株SN0280的DNA,扩增后并进行序列测序,并与EzTaxon数据库中已有基因序列进行比对,结果显示SN0280菌株与尖抱链霉菌(StreptomycescuspidosporusNBRC 12378)最为相似,同源性为99.85%,建立的统发育树中二者亲缘关系最近,同时,SN0280菌株形态特征、培养特征以及生理生化特征,与Scuspidospor.基本一致,因此,最终鉴定SN0280菌株为尖抱链霉菌(S.cuspidosporus)结合多种分离纯化手段,采用活性追踪法,在放线菌SN0280代谢产物中分离提纯得到7个单体化合物。试验通过大孔吸附树脂对菌株SN0280发酵产物进行吸附,甲醇洗脱树脂,二氯甲烷萃取,得到红棕色粘稠状的粗提物4.36 g。对粗提物进行一级硅胶柱层析后分为6个组分:T1-T6,并通过对番茄细菌性斑点病菌和溃疡病菌的活性检测,对其中表现出较好抑菌活性的4个组分T2、T3、T4、T6进行进一步的分离纯化,结合薄层层析、硅胶柱层析、凝胶柱层析和高效液相制备等分离手段得到7个纯化合物:H1-H7。综合采用核磁共振、质谱、红外、旋光测定等多种结构鉴定手段对这7单体化合物进行结构鉴定,并进行了初步的活性检测。结合谱图检测数据和文献数据,确定这些化合物中,有5个新化合物,它们分别是:吲哚类衍生物H1(chloroindole),聚酮类化合物 H2、H3、H5(streptoone A,streptoone B,streptoone C),丁内酯衍生物 H4(streplactone);以及2个已知化合物:大环内酯类抗生素H6(X-14952B)和聚醚类化合物H7(1.264-B)。并测定了这些单体化合物对11株植物病原菌的抑菌活性,其中,H7对番茄细菌性斑点病菌表现出了最好的抑菌效果,同时还对番茄细菌性溃疡病菌和尖孢镰刀菌也有很好的抑制效果;H2对番茄溃疡病菌表现出了与链霉素相当的抑菌活性;H3对辣椒疫霉病菌,H4对瓜果腐霉病菌,H6对辣椒疫霉病菌和番茄灰霉病菌也表现出了较好的抑菌活性(MIC20μg/mL)。聚醚化合物H7对番茄细菌性斑点病具有非常好的活性,并对番茄植株表现出了很高的安全性。H7在外界环境温度低于60℃C时是很稳定的,其抗菌活性基本不受影响;当化合物浓度为1MIC(2 μg/mL)的对靶标菌表现出了很好的抑菌效果,菌落数与抑菌率基本处于稳定状态,浓度达到2MIC(4 μg/mL)时不仅表现出了很好的抑菌效果,还起到了杀菌作用;此外,通过测定H7对斑点病菌生长曲线的影响,对比3种不同浓度化合物对供试菌的生长曲线,表明其能使靶标细菌的分裂增殖受到抑制,且随着浓度的增加其抑制效果增强。
[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.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S476

【参考文献】