【作者】 王晓青；

【导师】 曾洪梅；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2004， 硕士

【摘要】 农用抗生素2-16是从不吸水链霉菌黄山变种（Streptomyces ahygroscopicus var．huangshanensis）发酵产物中发现的一种具有广谱抗菌作用的抗生素。农抗2-16对多种作物病原真菌有较强抑制作用，对酵母菌及部分革兰氏阳性细菌和革兰氏阴性细菌也有一定的抑制作用。农抗2-16用于防治油菜菌核病，防效大于65％。在进行本研究之前农抗2-16的发酵单位仅有2000u／ml，相对较低。为提高农抗2-16产生菌的单位效价，本研究通过四种诱变方法选育出一株农抗2-16的高产菌株515^#，并利用SAS软件提供的Plackett-Burman设计和响应面分析法对该菌株的摇瓶发酵培养基进行了优化组合。研究结果如下： 1．由出发菌株0^#（效价2000 u／ml）依次进行原生质体再生处理、紫外线诱变、NTG诱变、低能碳离子注入处理，每步筛选出高产菌株用于下一步诱变处理。原生质体再生处理未获得高产菌株，紫外线诱变获得菌株301^#（效价2317u／ml），NTG诱变获得菌株442^#（效价4084u／ml），低能碳离子注入获得菌株515^#（效价6462u／ml）。最终获得的目标菌株515^#效价较出发菌株0^#提高223.10％。四种诱变方法中，低能碳离子注入的诱变贡献率最高，达到53.29％，NTG次之，达到39.60％，本研究中低能碳离子注入和NTG诱变的作用效率显著高于紫外线诱变和原生质体再生处理，是进行农抗2-16产生菌诱变育种的高效方法。 2．利用SAS软件中的Plackett-Burman设计和响应面分析法对515^#菌株的摇瓶发酵培养基进行优化组合，从九个考察因素中选出化合物K和化合物F为重要因素，通过响应面分析法建立模型Y=8914.80—0.2749 x₂—298.65 X₉—170.53x₂²—19.75 x₂ x₉—299.78 X₉²，推测出在化合物K浓度为0.018％、化合物F浓度为0.03％时预测的最大响应值为8989 u／ml，经摇瓶发酵实验证实此点的实测值为（8952±94）u／ml，证明模型是有效的，并且存在极大值点。采用优化培养基，515^#菌株效价较原始发酵培养基提高38.53％。 3．高效液相色谱分析发酵液组份表明农用抗生素2-16主要含有A、B两个组份，B组份含量高于A组份。菌株442^#、301^#的A组份含量高于出发菌株0^#，B组份含量低于出发菌株，515^#和515^#（优化发酵培养基）的B组份含量高于出发菌株0^#，A组份含量低于出发菌株。B组份的含量对效价的影响更大，B组份是决定农抗2-16效价高低的关键因素。更多还原

【Abstract】 Agro-antibiotic 2-16 is an agricultural antibiotic with extensive inhibition effect, which was discovered from the culture filtrate of Streptomyces ahygroscopicus var. huangshanensis. Agro-antibiotic 2-16 exhibits strong effects on many pathogenic fungi of crops, and also some extent of inhibition effects on yeast, some G+ bacteria and G bacteria. When agro-antibiotic 2-16 was used to control Sclerotinia sclerotiorum in the field test, the control effect was more than 65%. Before this study, the production of 2-16 is low, with the titre of only 2000u/ml. In order to improve its production, four mutagenic methods were used in this work. At last, a high-yield strain 515# was obtained. By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of 515# was optimized. The results were as follows:1 .The initial strain 0# (with titre of 2000u/ml) was treated by means of protoplast regeneration, ultraviolet radiation, NTG mutagenesis and low energy C+ ions implantation respectively. Every time, a strain with higher titre acqucired from one step was selected as the starting strain of the next step. No high-yield mutant was found after protoplast regeneration, strain 301# (with titre of 2317u/ml) was got by ultraviolet radiation, strain 442# (with titre of 4084u/ml) was got by NTG mutagenesis, strain 515# (with titre of 6462u/ml) was got by low energy C+ ions implantation. The production of 515# was increased by 223.10% in comparision with that of 0#. Low energy C+ ions implantation contributed most to the improvement of the production among the four methods, which was 53.29%, and NTG was the second one, which was 39.60 %. In this study, the effects of NTG mutagenesis and low energy C+ ions implantation were better than those of protoplast regeneration and ultraviolet radiation obviously, and were effective methods for the mutation and breeding of the agro-antibiotic 2-16 producing strain.2.By using Plackett-Burman design and Response Surface Analysis provided by SAS software, the cultivation condition of strain 515# was optimized. Compound K and compound F were chosen as the major components from nine variables, a regression model equation was obtained after regression analysis performed on the data from RSA experiment, which is: Y=8914.80-0.2749 x2-298.65 x9-170.53x22-19.75 x2 x9-299.78 x92. The optimum concentrations of compound K and compound F were predicted to be 0.018% and 0.03%, respectively, for the maximum antibiotic production, which is 8989 u/ml. The model was validated experimentally by the maximum production of (8952 94)u/ml. The agro-antibiotic 2-16 production was increased by 38.53% when the strain 515 #was cultivated in the optimum medium instead of the initial one.3.The analysis of the culture filtrates of different strains with High Performance LiquidChromatogram indicted that there were two main components--A and B in agro-antibiotic 2-16, andthe content of B was higher than that of A. The contents of A in the culture filtrate of strain 442# and strain 301# were both higher than that of the initial strain 0#, while the contents of B were both lower.However, when it came to strain 515# and 515# in the optimum medium, the case was the opposite one. The contents of B in the culture filtrates of strain 515# and 515# in the optimum medium were both higher than that of the initial strain 0#, while the contents of A were both lower. The content of B has greater impact on the titre of agro-antibiotic 2-16 than that of A, and was the key factor that dominated the titre of agro-antibiotic 2-16.更多还原