【作者】 丁成翔；

【导师】 葛绍荣；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 稻瘟病对水稻生产危害极大，传统上化学防治已不能适应绿色农业发展要求，近年采用生物防治己成为主要研究方向。除了从土壤等环境中筛选稻瘟病菌的拮抗菌外，对已有拮抗菌株通过基因重组方法实现菌种改良也成为重要的研究方向。原生质体融合技术在细胞水平上进行微生物菌种改良。其理论基础是基因重组。近年来利用该技术，有效选育到许多抗生素生产菌。其优点是不受亲缘关系影响，遗传信息传递量大，不需要了解双亲菌株详细遗传背景，可以有目的的选择亲株以选育理想菌株。刺孢吸水链霉菌北京变种(streptomyces hygrospinosus var．beijingensis)，由中国农科院从棉花根部土壤分离。其所产抗生素农抗120，是一种广谱性抗真菌的核苷类抗生素，对花卉、经济农作物真菌病害有较好的防治效果。淡紫灰链霉菌(streptomyce lavendule)产N-糖苷类抗生素——中生菌素，对农作物真菌病害也有广谱性防治效果。采用原生质体融合技术融合这两个母本菌株优良特性，筛选稻瘟病强拮抗菌。通过对甘氨酸浓度、酶解温度、酶浓度、酶解时间等影响因素考察，及正交试验优化，确定了刺孢吸水链霉菌和淡紫灰链霉菌原生质体制备的最佳条件。实验结果表明，刺孢吸水链霉菌最佳原生质体制备条件是：甘氨酸浓度为0.5％，酶解温度28℃，溶菌酶浓度为3 mg／ml，酶作用时间60min；淡紫灰链霉菌：甘氨酸浓度为1.0％，酶解温度32℃，溶菌酶浓度4 mg／ml，酶作用时间60min。有效的原生质体保存条件是：-20℃，在5天内，原生质体再生率在15％以上。通过原生质体融合育种方法，刺孢吸水链霉菌和淡紫灰链霉菌原生质体融合，经染色体融合重组得到一批稳定的重组体，从中筛选强拮抗稻瘟菌菌株。融合体筛选标记，刺孢吸水链霉菌一方选择抗链霉素性，其链霉素最小抑制浓度(MIC)是33μg／mL；淡紫灰链霉菌原生质体选择原生质体灭活法，设置紫外灭活组和热灭活组，其中紫外辐射灭活最低时间选择60S，热灭活选择55℃水浴下的最小时间是105min，得到100％致死原生质体。两亲本菌株原生质体经40％PEG促融，紫外灭活组得到融合体381株，热灭活组得到78株。相比较，紫外灭活组比热灭活组得到更多抗链霉素融合体。随机选择50株融合体经自然分离得到557株较稳定重组子。平板对峙实验表明，重组子中拮抗稻瘟病菌的能力是亲本菌株刺孢吸水链霉菌拮抗能力的30％～60％的，占总分离子的0.16；60％～110％的占0.69；在110％以上的占0.15。其中，编号RH103菌，相对拮抗能力在167％以上。其拮抗稻瘟病菌菌丝扩散生长、稻瘟菌孢子萌发，稻瘟菌丝黑色素化的提早，均比亲本显著。对通过原生质体融合筛选的重组菌株和亲本菌株生理生化特性研究结果表明：重组菌株在PDA、高氏一号、和葡萄糖天门冬素培养基上的培养特征近似亲本刺孢吸水链霉菌，但其菌丝相比亲本不太茂盛，产孢子量少。在不同培养基上发酵液效价存在差别，但差别不大。相比发酵成本考虑，发酵培养基3性价比高。重组菌株相比亲本刺孢吸水链霉菌，效价提升30％以上。组分分析结果表明：重组菌株发酵液组分与亲本菌株存在差异，最明显的表现在薄层层析点A(0.62)，很值得深入研究。更多还原

【Abstract】 In production agriculture of rice, Fungal pathogen of Magnaporthegriseainfects plants and crops easily and often seriously, causing millions of dollars worthof economic damage each year. So, how to control this disease and maintain thenormal growth of rice is always the highlight. Application of chemical pesticides isthe effective measure to control the fungal and bacterial infection and plays animportant role in preventive defence. With the further knowledge to chemicalpesticides, however, many concerns, such as the pollution of soil, water andatmosphere, the increase of the leftover of germicides in produce which imperilshuman health and life directly, ecological environment resulting from thepesticides, phytopathogenic fungi and bacteria to germicides, the agriculturalunbalance of and resistance of have caused the withdrawal of many of them. So,new and safe alternative control measures are much sought after. Biologicalcontrol by natural antagonistic organisms is a potential nonchemical tool for cropprotection against phytopathogenic fungi and bacteria and offers a promising alternative to synthetic pesticides, in part because it is perceived as safe to theenvironment and human health and can avoid the appearance of drug-resistance.Protoplast fusion is in the level of cell, which can breed industrial bacterium.lots of heredity message are transferred by Protoplast fusion. It does not need toknow the heredity background of candidacy bacteriums. There is an aim to getpotential industrial bacterium.Agricultural antibiotic 120, being from streptomyces hygrospinosusvar. beijingensis is often used to control the fungal and bacterial infection in plantand flowers. Streptomyce lavendule is also an impotant bacterium which produceantibiotic 751. By the method of protoplast fusion, two kinds of streptomyces canbe fused to get a new streptomyce which is used to restrain Fungal pathogen ofMagnaporthegrisea efficiently.Conditions about protoplast optimum preparation of S. hygrospinousvar. Beijingensis and S. lavendule were studied by orthogonal test, including theconditions of concentration of Gly, lytic temperature, lysozyme concentration andlytic time. Experimental results indicated that the conditions of optimum preparationof S. hygrospinous var. Beijingensis are as following: gly 0.5%, lytic temperature28℃, lysozyme 3 mg/ml, lytic time 60 min. Those of S. lavendule are as following:gly 1.0%, lyric temperature 32℃, lysozyme 4mg/ml, lytic time 60 min. Theregenerated frequency of protoplasts could reach up to above 15% in the conditionsof -20℃, 5 days.The MIC of S. hygrospinosus was about 33μg/mL（streptomycin）. The minimumUV lethal time of S. lavendule’ protoplast was about 60S. Two kings of protoplastswere fused by 40% PEG. There were 381 colonies in the culture medium platescontaining streptomycin 33μg/mL. The regenerated rate was about 1.0*10^-2%. 50colonies were selected randomly and planted in the basic mediums and selectedones according to the methodology of natural isolation. There were 15heterocaryons, 30%. The stable haploid fusions was about 15% against Magnaporthegrisea remarkably. RH103, it was as 200% as S. hygrospinosus against Magnaporthe grisea remarkably.The physicochemical characteristics of the recombinant RH103 screened fromregenerated strains of fusiants between producing strains S. hygrospinosus andS. lavendule were studied. Through analysis the cultural characteristics of therecombinants in different medium in the process of culture, components andinhibition of its fermentation liquor to yeasts, the results showed that therecombinant RH103 is similarto the S. hygrospinosus. There are also differentfrom the original strains. Its mycelia are white color and produce little spores.更多还原