【作者】 陈丽萍；

【导师】 宗兆锋；

【作者基本信息】 西北农林科技大学 ， 有害生物治理生态工程， 2008， 硕士

【摘要】 植物内生放线菌作为新的微生物资源具有广泛的应用前景,无论在控制植物病害的生物防治领域,还是从中寻找新型杀菌剂或药物先导化合物方面都极具开发价值。为了提高植物内生放线菌的抗菌活性,将本研究室分离保存的2株植物内生放线菌SF4、SG2和1株具有较好抗菌活性的生防放线菌SC1作为亲本,开展双亲和三亲本的原生质体融合,通过研究,获得了综合亲本优良性状的新型高效菌株。通过蔗糖质量浓度、Gly质量浓度、亲本菌株对数生长期、酶的种类与浓度、酶解时间与温度以及亲本菌株灭活条件的确定,以原生质体形成率和再生率为指标,确定及优化了亲本菌株的融合条件。实验得出,亲本菌株SF4和SG2的融合条件为:蔗糖质量浓度:SF4为30﹪,SG2为20﹪;Gly质量浓度:SF4和SG2均为0.5﹪;对数生长期:SF4为48～81h,SG2为33～72h;酶的种类:溶菌酶;酶的浓度:SF4为15 mg·mL-1,SG2为10 mg·mL-1;酶解温度:均为35℃;酶解时间:SF4为90min,SG2为60min;热灭活温度:均为55℃;热灭活时间:SF4为15min ,SG2为10min;紫外线灭活时间:SF4为4min,SG2为2min。根据以上优化条件,以2个亲本或3个亲本菌株进行原生质体融合,获得了25株融合子。通过遗传稳定性、菌落形态特征以及皿内抑菌活性测定进行融合子筛选,共获得6株整体性能稳定且有较好抑菌活性的融合菌株。通过抗菌机理、形态与培养特征、产孢量和菌丝生长量、生理生化特征、内生性以及多胺测定对融合菌株进行进一步的检测,发现融合菌株与亲本菌株既有差别,又有相同之处,说明亲本原生质体融合后形成的融合菌株既保留了亲本的某些性状,而其自身又产生了一些新的性状。最后通过分子生物学方法对亲本菌株及其融合菌株进行检测,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1.5kb的片段,用限制性内切酶对扩增产物进行酶切,以亲本菌株为对照,酶切图谱中既具备与亲本相同的一些条带,也有完全不同于亲本的新条带,这可能与融合菌株产生的一些新性状相对应。总之,本研究利用原生质体融合技术,实现了植物内生放线菌之间以及植物内生放线菌与土壤放线菌之间的融合,为开发和利用植物内生放线菌资源提供了新思路,同时也为拓宽原生质体融合技术的应用范围提供了新的证明。更多还原

【Abstract】 As a new microbial resource, the plant endophytic actinomycetes have wide application prospects. The plant endophytic actinomycetes have a high potential not only as bio-control agents to control plant diseases, but also to find novel pesticides or new medicines produced as secondary metabolites. Improving the biocontrol character of endophytic actinomycetes, the protoplast fusion between endophytic actinomycetes SF4, SG2 and actinomycetes SC1 was studied for getting the recombinants recombined excellent characteristics of both parents.The conditions of protoplast formation and regeneration were optimized by the concentration of Sucrose, concentration of Gly, the growth logarithm period of the parents、the kind and the concentration of the enzyme, the time and the temperature of lysing, the time of thermal and UV light inactivation. Concentration of Sucrose: SF4-30﹪, SG2-20﹪; Concentration of Gly: 0.5﹪; The growth logarithm period: SF4-48-81h, SG2-33-72h; The kind of enzyme: lysozyme; Concentration of lysozyme: SF4-15 mg·mL-1, SG2-10 mg·mL-1; Temperature of lysing: 35℃; Time of lysing: SF4-90min, SG2-60min; Temperature of thermal inactivation: 55℃; Time of thermal inactivation: SF4 -15min, SG2-10min; Time of UV light inactivation: SF4 -4min, SG2-2min;Two parents and three parents were used to fusion, following all conditions optimized, and 25 recombinants were selected according to colony characteristics, and 6 recombinants were selected basied on succession culture of seven generations、antibiotic activity in vitro and growth speed of mycelia.According to the inhibiting mechanism to the target pathogens, the morphological characteristics, the culture properties on different media, biochemical and physiological characteristics and growth speed of mycelium, the recombinants were identified if they were true. The result showed that there were the different as well as the same between the recombinants and strains SF4, SG2 and SC1. 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 rDNA with universal primers of bacteria, and were digested with endoenzyme respectively, and the different patterns were obtained for each enzyme. In the patterns, some new stripes which were different from the parents’were found.In this study, achieving protoplast fusion between endophytic antinomycetes and biocontrol antinomyectes by the technology of protoplast fusion, which providing a new idea for exploring and utilizing the resources of endophytic actinomycetes, and broadening application area of the technology of protoplast fusion at the same time.更多还原