【作者】 甘邱锋；

【导师】 关雄； 黄必旺； 张晓琳；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 多杀菌素是由刺糖多孢菌(Saccharopolyspora spinosa)经有氧发酵后产生的次级代谢产物,兼有生物农药的安全性和化学合成农药的速效性,作用机理独特,对哺乳动物、昆虫天敌和环境安全。开发该产品具有很好的经济效益和社会效益。本文研究了刺糖多孢菌原生质体制备与再生的条件,通过原生质体诱变、刺糖多孢菌与阿维链霉菌科间原生质体融合、刺糖多孢菌种内原生质体融合选育多杀菌素高产菌株。主要研究结果如下:1.通过考察不同条件对刺糖多孢菌原生质体制备和再生的影响,确定了菌体在添加0.3%甘氨酸的EHC培养基中培养72h,用2mg/mL溶菌酶32℃酶解40min后,于R6再生培养基上再生,原生质体制备率超过99%,再生数达107cfu/mL。原生质体可置于4℃短期保存72h,长期保存需要放置于-80℃条件下。2.通过原生质体紫外诱变获得遗传稳定的多杀菌素高产菌株1株,多杀菌素产量较出发菌株提高了近40 %。3.通过双亲灭活原生质体法筛选融合子。刺糖多孢菌NU-92采用紫外灭活,灭活时间为120s,刺糖多孢菌C3-10-4采用热灭活,灭活温度60℃,灭活时间为50min。阿维链霉菌76-02-e原生质体也采用相同温度进行热灭活,灭活时间30min。4.通过电融合技术将阿维链霉菌与刺糖多孢菌进行原生质体融合,考察了排列电场以及脉冲电场条件对原生质体融合率的影响,最终确定了排列电压7V、持续时间50s、脉冲电压200V、脉冲时间40μs、脉冲次数2次时融合率最佳,但是融合子产素水平不高,未筛选到遗传稳定的高产菌株。刺糖多孢菌种内原生质体融合率要显著高于科间的原生质体融合,但是所获融合子产素水平未有提高。5.利用PEG介导的化学融合方法进行刺糖多孢菌科间以及种内的原生质体融合。选用50%的PEG1000作为促融剂,融合时间为2-5min时融合率最高。但是阿维链霉菌与刺糖多孢菌科间原生质体融合负突变率高达94.7%,未筛选到高产菌株。刺糖多孢菌种内的原生质体融合正突变率达75%,获得多杀菌素高产菌株1株,多杀菌素产量较对照菌株提高了近200 %,且遗传稳定性良好。更多还原

【Abstract】 Spinosyns and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. Its many excellent features, containing favorable to mammalian and environmental, insect selectivity, unique mechanism of action and outstanding efficacy, lead to high benefit of economy and society. In this paper, we have tested the optimal conditions for the formation, regeneration and preservation of protoplasts, selected high spinosad-producing strain using mutation and interfamial/intraspecific protoplast fusion. The main results were as follows:1. All factors were tested on protoplast formation and regeneration including the preparation medium, regeneration medium, spawn age, glycine concentration and the treatment conditions of lysozyme. The maximum rate of protoplast formation and regeneration were under these conditions: the collected mycelia grown in EHC medium with 0.3% glycine for 72 h were treated by 2mg/mL lysozyme at 32℃for 40min, then plated on the R6 medium. The rate of protoplasts formation was more than 99% and the number of regeneration protoplast was up to 107cfu/mL. Method of short-term storage for protoplasts was 4℃for 72h, long-term storage needed -80℃.2. With UV mutation, one high spinosad-producing strain was achieved, which was 40% higher than the initial strain.3. Screen the recombinants by inactivatting parental strain protoplasts. S.spinosa NU-92 was inactivated by UV for 120s. S.spinosa C3-10-4 was heat-inactivated for 50min at 60℃. S.avermitilis 76-02-e was heat-inactivated for 30min at 60℃.4. The best conditions of pre-alignment electric field and pulse electric field in the electrofusion were tested between S.spinosa and S.avermitilis.The maximum rate of protoplast fusion were under these conditions: pre-alignment voltage 7V, time 50s, pulse voltage 200V, pulse time 40μs, pulse number 2. But the high spinosad-producing strain was non-achieved. Although the fusion rate of intraspecific was much higher, the spinosad-producing wasn’t improved.5. The maximum rate of protoplast chemical fusion between interfamial/intraspecific protoplasts were achieved by using 50% PEG1000 and the fusion time was 2-5min. The rate of negative mutation was 94.7% in the protoplast fusion between S.spinosa and S.avermitilis.The positive mutation was 75% in the intraspecific protoplasts fusion. One high spinosad-producing strain was achieved, which was near to 200% higher than the initial strain.更多还原