关键词: Alternaria Section Undifilum oxytropis, 疯草内生真菌, 苦马豆素, 诱变筛选, 生物合成机制

Abstract:

【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 swainsonine- producing 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.

Key words: Alternaria Section Undifilum oxytropis, locoweed endophytic, Swainsonine (SW), mutation screening, biosynthesis mechanism