【作者】 刘冰；

【导师】 宗兆锋；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2006， 硕士

【摘要】 为了提高野生生防放线菌株的防病效果,克服以往生防菌应用方面存在的诸多弊端,探索简单易行的人工改良生防放线菌的途径,我们选用重寄生链霉菌F46和放线菌SC11作为亲本,进行了双亲原生质体融合研究,以期得到整合双亲多种优良性状的工程菌株。通过测量菌丝干重、显微观察计数和计算再生率的方法对双亲原生质体制备及再生条件进行了优化。研究结果表明,菌株F46和SC11分别在蔗糖浓度为20%和10%以及Gly浓度为1.5%和1.0%的培养液中培养96～100h,能够形成分散度好的菌丝体,酶解时会有大量再生率较高的原生质体形成;在35℃水浴条件下,菌株SC11选用浓度为5mg·mL-1的溶菌酶酶解90min,菌株F46选择10mg·mL-1溶菌酶与10mg·mL-1蜗牛酶混合,酶解时间以120min为佳。本研究以处理不同时间后原生质体的再生情况确定了双亲株原生质体的灭活条件。结果表明,在60℃条件下,菌株F46和SC11的原生质体热灭活时间分别以60min和15min适宜;如果在20W紫外灯下15cm处照射处理,菌株F46和SC11原生质体的灭活时间分别为15min和7min。在优化了双亲原生质体形成、再生以及灭活条件的基础上,经过敏感菌丝体培养,原生质体制备,融合及再生的重复试验,依据再生培养基上出现菌落的形态和颜色分离获得9株再生菌株。通过比较融合子与亲本的抑菌活性,筛选出抑菌效果优于亲本的融合菌株F-S5、F-S15、F-S16和F-S17。对于初筛获得的融合菌株,进一步通过抑菌机理研究、遗传稳定性检测、形态特征观察、培养性状多重比较、生理生化特性观察以及生长速率比较发现,融合菌株与双亲既有差别,又有相同之处,表明双亲原生质体融合后形成的融合菌株既保留了亲本的某些特性,又产生了一些新性状。利用细菌通用引物对融合菌株及其亲本16S rDNA进行扩增,均获得大小约1.5 kb的片段,用限制性内切酶对扩增产物进行酶切,酶切图谱中既具备与双亲相同的一些条带,也有完全不同于双亲的新条带,这可能与融合菌株产生的一些新性状相对应。总之,本研究得到了4株抑制病原真菌能力优于亲本的融合菌株,摸索出了一套简单易行的放线菌原生质体融合方法。更多还原

【Abstract】 The protoplast fusion between hyperparasitic Streptomyces strain F46 and Actinomyces strain SC11 was studied in order to improve the effect of wild actinomyces strains to control plant diseases, solve the problems in application of the biocontrol strains, explore the method of protoplast fusion and get the recombinants with some excellent characteristics of both parents.The conditions of protoplast formation and regeneration were optimized by measuring the dried mycelia, counting the protoplasts with microscope and calculating the regeneration rate. The results showed that strains F46 & SC11 can form high quantity and quality mycelia which were easy to prepare protoplast when they grew respectively in liquid medium with 20% & 10% sucrose and 1.5% & 1.0% Gly for 96-100h; High quality protoplasts of strains SC11 & F46 can be gained respectively in 5mg·mL-1 lysozyme and enzyme mixing 10mg·mL-1 lysozyme with 10mg·mL-1 snailase for 90min and 120min.Inactivating conditions of strains F46 & SC11 were determined by survival rate. In 60℃, strains F46 & SC11 protoplasts were treated for 60min and 15min respectively; In the distance of 15cm, 15min and 7min radiation treatments to strains F46 & SC11 protoplast with 20W UV-light were the best.Following all conditions optimized, protoplasts’preparation, fusion and regeneration were done and nine recombinant strains were selected according to colony characteristics of strains. Recombinant strains F-S5, F-S15, F-S16 and F-S17, which were better than strain SC11, were gained by comparing the suppressive ability with the parents to the target pathogens.According to the inhibiting mechanism to the target pathogens, the stability of fusants in inheritance, the morphological characteristics, the cultural properties on different media, biochemical and physiological characteristics and growth speed of mycelium, the fusants were identified if they were true. The result showed that there were the different as well as the same between the fusants and strains F46 & SC11. Conclusion was gotten that the recombinants inherited characteristics of the parents and produced some new properties. A single band about 1500bp were gained in all strains by amplifying 16S rDNAs with universal更多还原