【作者】 汪志芸；

【导师】 吴雪昌；

【作者基本信息】 浙江大学 ， 遗传学， 2003， 硕士

【摘要】 本文以一株抗生素产生菌AP19-1为出发菌株，对其进行了系统分类学以及UV诱变育种的研究。 形态学特征、生理生化性状和16S rDNA核苷酸序列分析表明，菌株AP19-1属于链霉菌属(Streptomyces sp.)。 用紫外线对AP19-1的孢子及原生质体分别进行诱变育种，获得了产抗生素的高产菌株。经纸片法和最小抑菌浓度(MIC)测定，其中一株由原生质体诱变得到的菌株，其发酵液中抗生素的相对效价是原始菌株的4倍，且有较高的遗传稳定性。实验结果表明，原生质体的紫外诱变比孢子的紫外诱变更易得到抗生素相对效价提高较多的突变株。 我们同时还研究了不同的培养温度对孢子的紫外诱变正向突变率的影响。将UV诱变后的AP19-1孢子，分别置于适宜的生长温度27℃与接近抑制生长的胁迫温度33℃下培养，结果表明：在33℃下培养获得的子代菌株中，产抗生素水平超过其出发菌株的正向突变体所占的比例，明显高于在27℃下培养的结果。27℃下培养，正向突变体占总子代菌株数的百分率为25.8％，而在33℃下培养则为58.1％。用17种10bp的随机引物对出发菌株与UV诱变后的子代菌株进行总DNA的RAPD测验证明，在接近抑制生长的胁迫温度33℃下培养获得的子代，发生在其DNA水平上的变异程度要比在27℃培养下的子代高得多，与对照菌株RAPD条带的相似性从27℃的66.4％下降到了33℃的57.4％。这一方法较大幅度地提高了链霉菌AP19-1紫外诱变育种的工作效率，同时也为链霉菌紫外诱变后突变形成机制的进一步研究提供了新途径。更多还原

【Abstract】 Studies on UV mutagenic breeding and identification of an antibiotic-producing strain AP19-1 were carried out.Based on the results of phenotypic characteristics and 16S rDNA sequence analysis, the isolate was assigned to the genus Streptomyces.In order to improve the antibiotic-producing trait of strain AP19-1, we exposed its protoplasts and spores to UV and isolated a mutant strain Y207. The antibiotic’s relative liter of the mutant’s fermented broth was 4 times as high as the parent strain through MIC experiment. And its high capacity for producing antibiotic remained stable after 4 times of subcultures. It was also found that the effect of protoplasts’ UV mutagenic breeding was better than spores’.UV irradiated spores of strain AP19-1 were incubated at 27℃ and 33℃ close to inhibiting growth temperature. Results showed that among the offspring from UV irradiated spores grown at 33℃ , the amount of forward mutants whose antibiotics-producing ability were over the original strain’s were many more than that at 27℃. The percentage of the forward mutants was 25.8% at 27℃ and 58.1% at 33 ℃ respectively. Total DNA of the progeny strain and the original strain was tested by RAPD with 17 primers. It was demonstrated that the variations occurred in the chromosomal DNA of the progeny strains grown at 33℃ were more than that at 27 ℃. This method improved the efficiency of UV mutagenic breeding and put forward a new way in researching into the mechanisms of mutation formation in Streptomyces sp. cells irradiated by UV.更多还原