产苦马豆素疯草内生真菌Alternaria SectionUndifilum oxytropis的诱变筛选
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摘要
【背景】疯草是黄芪属和棘豆属有毒植物的统称,疯草内生真菌Alternaria Section Undifilum oxytropis是从疯草中分离获得的一种具有产生苦马豆素(swainsonine,SW)能力的真菌。因其产物SW一方面体现出良好的抑制肿瘤细胞生长、浸润和转移作用及潜在的抗艾滋病病毒等多种药用活性,另一方面牛羊因大量误食疯草能导致中毒,对草原畜牧业健康发展造成了严重的危害,受到研究人员的广泛关注。但由于疯草内生真菌Alternaria Section Undifilum oxytropis产苦马豆素生物合成机制尚不清楚,严重制约了后续通过生物发酵大量获得SW用以抗肿瘤机制研究与临床应用,以及通过基因工程手段对疯草进行脱毒育种,使疯草成为牛羊可食用、无毒的天然牧草。【目的】利用有效的研究手段,阐明Alternaria Section Undifilum oxytropis产苦马豆素生物合成机制。【方法】以Alternaria Section Undifilum oxytropis为出发菌株,分别采用紫外辐照诱变、亚硝基胍化学诱变、紫外辐照与亚硝基胍复合诱变的3种诱变方式,在不同诱变时间、诱变剂量等条件下对其进行了诱变筛选研究。通过测定不同诱变方式和作用条件下对菌株的致死率,经发酵培养、连续5代传代培养、并采用α-甘露糖苷酶活性分析法检测诱变菌株产苦马豆素含量变化,优化确定了不同诱变方式下最佳诱变条件,并将优势突变菌株接种马铃薯葡萄糖琼脂培养基(potato dextrose agar,PDA)和改良察式液体培养基,连续培养32 d,测定并绘制了突变菌株D4和UD1的生长周期曲线。【结果】经上述3种诱变方式处理后,分别获得1株产苦马豆素含量变化较大、能稳定连续传代培养的突变菌株U4、D4、UD1。其优化的诱变条件,紫外辐照为辐照处理160 s,亚硝基胍化学诱变为亚硝基胍诱变剂量6μL、诱变处理时间5 min,紫外辐照与亚硝基胍复合诱变为紫外辐照20 s,亚硝基胍诱变剂量2μL,诱变处理时间5 min。突变菌株U4、D4、UD1较原始菌株,菌落形态偏小、中间凸起,菌落颜色呈微粉色或白色,且其产苦马豆素含量均有显著变化,其中U4突变菌株产苦马豆素量平均增加16.02%(P<0.01)、D4突变菌株产苦马豆素量平均降低23.58%(P<0.01),UD1突变菌株SW产量平均增加21.87%(P<0.01),但D4、UD1突变菌株生长周期与出发菌株一致,均为24 d。【结论】通过紫外辐照诱变、亚硝基胍化学诱变、紫外辐照与亚硝基胍复合诱变方法,成功筛选出产苦马豆素含量变化较原始菌株差异较大的3株突变菌株U4、D4、UD1,这为后续利用高通量测序等分子生物学手段,在分子水平上阐释疯草内生真菌Alternaria Section Undifilum oxytropis关键基因、关键酶在其生物合成苦马豆素机制关系方面奠定研究基础。
【Background】 Locoweed is referred to as the Astragalus and Oxytropis of poisonous plants, Alternaria Section Undifilum oxytropis is a kind of fungus with the ability to produce swainsonine(SW) isolated from locoweed. On one hand,Swainsonine exhibits good inhibition of tumor cell growth, invasion and metastasis, and potential anti-HIV and other medicinal activities. On the other hand, cattle and sheep can be poisoned by eating a large number of locoweed grass by mistake, which has caused serious harm to the healthy development of grassland animal husbandry and attracted extensive attention of researchers.However, the biosynthesis mechanism of producing SW in the endophytic fungus Alternaria Section Undifilum oxytropis is not clear, which seriously restricts the subsequent research and clinical application of swainsonine for anti-tumor mechanism by biological fermentation. And by means of genetic engineering of locoweed detoxification breeding, make locoweed edible and non-toxic natural forage for cattle and sheep.【Objective】it is urgent to clarify the biosynthesis mechanism of swainsonineproducing of Alternaria Section Undifilum oxytropis by using effective research methods.【Method】 Using Alternaria Section Undifilum oxytropis as the starting strain, ultraviolet irradiation mutagenesis, chemical mutagenesis of nitrosoguanidine and ultraviolet irradiation-NTG mutagenesis were used respectively. The mutagenic screening was carried out under the conditions of different mutagenesis time and mutagenesis dosage. By measuring the lethal rate of strains under different mutagenesis conditions,fermentation culture, continuous subculture of 5 generations and using alpha-mannosidase activity analysis method to detected the changes of swainsonine content, the optimal mutation conditions of different mutagenesis methods were optimized. The dominant mutant strains were inoculated into Potato Dextrose Agar(PDA) and Modified Czapek-Dox Medium, and the growth cycle curves of mutant strains D4 and UD1 were determined and plotted.【Result】After treatment with the above three mutagenesis methods,three mutant strains of U4, D4 and UD1 were obtained, which can be cultured steadily and continuously, and the content of swainsonine varies greatly. The optimum mutagenesis conditions were as follows: ultraviolet irradiation for 160 seconds, chemical mutagenesis of nitrosoguanidine for 6 μL and 5 min, ultraviolet irradiation-NTG mutagenesis for 20 seconds and 2 μL for 5 min.U4, D4 and UD1 mutant strains were smaller in colony morphology and protruded in the middle than original strains, and the color of colony was pink or white. And the content of swainsonine in the production of swainsonine had significant changes. Among them, the production of swainsonine of U4 mutant strain increased by 16.02%(P<0.01), D4 mutant strain decreased by 23.58%(P<0.01), and UD1 mutant strain increased by 21.87%(P<0.01). However, the growth cycle of D4 and UD1 mutant strains were the same as that of the original strain, which were 24 days.【Conclusion】By using ultraviolet irradiation, chemical mutagenesis of nitroguanidine and ultraviolet irradiation-NTG mutagenesis, U4, D4 and UD1 of mutant strains were successfully screened out,whose content of swainsonine was different from that of the original strain. This provides a basis for the subsequent use of molecular biological means to explain the key genes of Endophytic Alternaria Section Undifilum oxytropis and key enzymes in its biosynthesis mechanism of swainsonine.
【Background】 Locoweed is referred to as the Astragalus and Oxytropis of poisonous plants, Alternaria Section Undifilum oxytropis is a kind of fungus with the ability to produce swainsonine(SW) isolated from locoweed. On one hand,Swainsonine exhibits good inhibition of tumor cell growth, invasion and metastasis, and potential anti-HIV and other medicinal activities. On the other hand, cattle and sheep can be poisoned by eating a large number of locoweed grass by mistake, which has caused serious harm to the healthy development of grassland animal husbandry and attracted extensive attention of researchers.However, the biosynthesis mechanism of producing SW in the endophytic fungus Alternaria Section Undifilum oxytropis is not clear, which seriously restricts the subsequent research and clinical application of swainsonine for anti-tumor mechanism by biological fermentation. And by means of genetic engineering of locoweed detoxification breeding, make locoweed edible and non-toxic natural forage for cattle and sheep.【Objective】it is urgent to clarify the biosynthesis mechanism of swainsonineproducing of Alternaria Section Undifilum oxytropis by using effective research methods.【Method】 Using Alternaria Section Undifilum oxytropis as the starting strain, ultraviolet irradiation mutagenesis, chemical mutagenesis of nitrosoguanidine and ultraviolet irradiation-NTG mutagenesis were used respectively. The mutagenic screening was carried out under the conditions of different mutagenesis time and mutagenesis dosage. By measuring the lethal rate of strains under different mutagenesis conditions,fermentation culture, continuous subculture of 5 generations and using alpha-mannosidase activity analysis method to detected the changes of swainsonine content, the optimal mutation conditions of different mutagenesis methods were optimized. The dominant mutant strains were inoculated into Potato Dextrose Agar(PDA) and Modified Czapek-Dox Medium, and the growth cycle curves of mutant strains D4 and UD1 were determined and plotted.【Result】After treatment with the above three mutagenesis methods,three mutant strains of U4, D4 and UD1 were obtained, which can be cultured steadily and continuously, and the content of swainsonine varies greatly. The optimum mutagenesis conditions were as follows: ultraviolet irradiation for 160 seconds, chemical mutagenesis of nitrosoguanidine for 6 μL and 5 min, ultraviolet irradiation-NTG mutagenesis for 20 seconds and 2 μL for 5 min.U4, D4 and UD1 mutant strains were smaller in colony morphology and protruded in the middle than original strains, and the color of colony was pink or white. And the content of swainsonine in the production of swainsonine had significant changes. Among them, the production of swainsonine of U4 mutant strain increased by 16.02%(P<0.01), D4 mutant strain decreased by 23.58%(P<0.01), and UD1 mutant strain increased by 21.87%(P<0.01). However, the growth cycle of D4 and UD1 mutant strains were the same as that of the original strain, which were 24 days.【Conclusion】By using ultraviolet irradiation, chemical mutagenesis of nitroguanidine and ultraviolet irradiation-NTG mutagenesis, U4, D4 and UD1 of mutant strains were successfully screened out,whose content of swainsonine was different from that of the original strain. This provides a basis for the subsequent use of molecular biological means to explain the key genes of Endophytic Alternaria Section Undifilum oxytropis and key enzymes in its biosynthesis mechanism of swainsonine.
引文
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