产苦马豆素疯草内生真菌Alternaria Section Undifilum oxytropis的诱变筛选

doi:10.3864/j.issn.0578-1752.2019.15.015

Abstract

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

HAO BaoCheng, SONG XiangDong, GAO Yan, WANG XueHong, LIU Yu, LI YuanXi, LIANG Yan, CHEN KeYuan, HU YuYao, XING XiaoYong, HU YongHao, LIANG JianPing. Mutagenesis and Screening of Endophytic Fungus Alternaria Section Undifilum oxytropis Producing Swainsonine from Locoweed[J].Scientia Agricultura Sinica, 2019, 52(15): 2716-2728.

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References 37

[1]	周启武, 赵宝玉, 路浩, 王姗姗, 张樑, 温伟利, 杨晓雯 . 中国西部天然草地疯草生态及动物疯草中毒研究与防控现状. 中国农业科学, 2013,46(6):1280-1296. doi: 10.3864/j.issn.0578-1752.2013.06.023
	ZHOU Q W, ZHAO B Y, LU H, WANG S S, ZHANG L, WEN W L, YANG X W . The research and control situation of ecology and animal poisoning of locoweed in western natural grassland of China. Scientia Agricultura Sinica, 2013,46(6):1280-1296. (in Chinese) doi: 10.3864/j.issn.0578-1752.2013.06.023
[2]	赵宝玉, 刘忠艳, 万学攀, 霍星华, 郭玺, 王建军, 刘忠艳, 孙莉莎, 史志诚 . 中国西部草地毒草危害及治理对策. 中国农业科学, 2008,41(10):3094-3103.
	ZHAO B Y, LIU Z Y, WAN X P, HUO X H, GUO X, WANG J J, LIU Z Y, SUN L S, SHI Z C . Damage and control of poisonous-weeds in Chinese western grassland. Scientia Agricultura Sinica, 2008,41(10):3094-3103. (in Chinese)
[3]	全国畜牧总站. 中国草业网, 草原毒害草: , 2011-02-08.
	The National Stockbreeding Station. China grass industry network, Grassland poison grass: , 2011-02-08.(in Chinese)
[4]	王雨梅, 韩国栋, 赵萌莉 . 疯草内生真菌共生体的研究与展望. 中国草地学报, 2007,29(4):99-103.
	WANG Y M, HAN G D, ZHAO M L . Prospects and research progress in locoweed fungal endophyte symbiont. Chinese Journal of Grassland, 2007,29(4):99-103. (in Chinese)
[5]	COOK D, MICHAEL H R, KEVIN D W, STEGELMEIER B L . Locoweed poisoning in livestock. Society for Range Management, 2009,2:16-20.
[6]	SINGH D, KAUR G . Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae. Journal of Basic Microbiology, 2011,10(5):1072-1078.
[7]	郝宝成, 武凡琳, 邢小勇, 项海涛, 温峰琴, 王学红, 权晓弟, 胡永浩, 梁剑平 . 苦马豆素抗牛病毒性腹泻病毒研究. 中国农业科学, 2014,47(1):170-180. doi: 10.3864/j.issn.0578-1752.2014.01.018
	HAO B C, WU F L, XING X Y, XIANG H T, WEN F Q, WANG X H, QUAN X D, HU Y H, LIANG J P . Study on inhibitory effect of the swainsonine from alkaloid of Astragalus strictus Grah. Ex bend on bovine viral diarrhea virus. Scientia Agricultura Sinica, 2014,47(1):170-180. (in Chinese) doi: 10.3864/j.issn.0578-1752.2014.01.018
[8]	GOSS P E, REID C L, BAILEY D, DENNIS J W . Phase IB clinical trial of the oligosaccharide processing inhibitor swainsonine in patients with advanced malignancies. Clinical Cancer Research, 1997,3(7):1077-1086.
[9]	萨如拉, 卢萍 . 内生真菌苦马豆素生物合成研究进展. 绿色科技, 2018,8:227-229.
	SARULA, LU P . Research progress of swainsonine biosynthesis in endophytic fung. Journal of Green Science and Technology, 2018,8:227-229. (in Chinese)
[10]	PHILIP E S, WALTER S, PAUL E, JENNIFER K, ERIC W, RONALD M B . Phase II study of the efficacy and safety of oral GD0039 in patients with locally advanced or metastatic renal cell carcinoma. Investigational New Drugs, 2005,23(6):577-581.
[11]	汪治刚, 曹师, 徐娜, 徐杉, 李彦忠 . 我国3种疯草的内生真菌培养特性研究. 草业学报, 2017,26(10):158-169.
	WANG Z G, CAO S, XU N, XU S, LI Y Z . Cultural characteristics of fungal endophyte from 3 locoweed species in China. Acta Prataculturae Sinica, 2017,26(10):158-169. (in Chinese)
[12]	BRAUN K, ROMERO J, LIDDELL C, CREAMER R . Production of swainsonine by fungal endophytes of locoweed. Mycological Research, 2003,107(8):980-988.
[13]	BRAUN K . Fungal endophyte infection and swainsonine toxicity in locoweed. New Mexico: New Mexico State University, 1999: 156-162.
[14]	LU H, QUAN H Y, ZHOU Q W, REN Z H, XUE R X, ZHAO B Y, Creamer R . Endogenous fungi isolated from three locoweed species from rangeland in western China. African Journal of Microbiology Research, 2017,11(5):155-170.
[15]	WU C C, HANT S, LU H, ZHAO B Y . The toxicology mechanism of endophytic fungus and swainsonine in locoweed. Environ Toxicology Pharmacology, 2016,47:38-46.
[16]	路浩, 李国中, 杨晓雯, 曹丹丹, 薛瑞旭, 权海云, 赵宝玉 . 甘肃棘豆内生真菌种群多样性. 中国兽医学报, 2014,34(7):1094-1099.
	LU H, LI G Z, YANG X W, CAO D D, XUE R X, QUAN H Y, ZHAO B Y . Diversity of endophytic fungi from Oxytropis kansuensis. Chinese Journal of Veterinary Sciences, 2014,34(7):1094-1099. (in Chinese)
[17]	薛瑞旭, 权海云, 任祯慧, 路浩, 赵宝玉 . 疯草内生真菌U. oxytropis次级代谢产物分析. 动物医学进展, 2016,37(7):64-70.
	XUE R X, QUAN H Y, REN Z H, LU H, ZHAO B Y . Analysis of secondary metabolites of Undifilum Oxytropis from locoweeds. Progress in Veterinary Medicine, 2016,37(7):64-70. (in Chinese)
[18]	周启武, 于龙凤, 路浩, 曹丹丹, 赵宝玉 . 小花棘豆和变异黄芪内生真菌显微分布及定量检测. 微生物学报, 2014,54(5):572-581.
	ZHOU Q W, YU L F, LU H, CAO D D, ZHAO B Y . Microscopic distribution and quantitative detection of endophytic fungus Undifilum oxytropis from Oxytropis glabra DC and Astragalus variabilis. Acta Microbiologica Sinica, 2014,54(5):572-581. (in Chinese)
[19]	黄鑫, 梁剑平, 高旭东, 郝宝成 . 苦马豆素的来源、药理作用及检测方法研究进展. 畜牧兽医学报, 2016,47(6):1075-1085. doi: 10.11843/j.issn.0366-6964.2016.06.001
	HUANG X, LIANG J P, GAO X D, HAO B C . Research advance on sources, pharmacological effects and detection methods of swainsonine. Acta Veterinaria et Zootechnica Sinica, 2016,47(6):1075-1085. (in Chinese) doi: 10.11843/j.issn.0366-6964.2016.06.001
[20]	HARRIS C M, CAMPBELL B C, MOLYNEUX R J, HARRIS T M . Biosynthesis of swainsonine in the Diablolocoweed. (Astragalns oxyphysus) . Tetrahedron letters, 1988,29:4815-4818.
[21]	MUKHERJEE S, DAWE A L, CREAMER R . Development of a transformation system in the swainsonine producing, slow growing endophytic fungus,Undifilum oxytropis. Journal of Microbiology Method, 2010,81(2):160-165.
[22]	杨国栋 . 疯草内生真菌合成苦马豆素的研究[D]. 杨凌: 西北农林科技大学, 2012.
	YANG G D . Study on biosynthesis of swainsonine by the locoweed's endophytic fungi[D]. Yangling: Northwest A&F University, 2012. (in Chinese)
[23]	LI H L, YU Y Y, GAO R, WANG J H, YANG G D, YANG Z J, BAUCOM D, CREAMER R . Analy sis of secreted proteins from Undifium cinereum by two dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry. Journal of Animal & Veterinary Advances, 2012,11(11):1881-1889.
[24]	李海利 . 产苦马豆素疯草内生真菌蛋白质双向电泳及蛋白质组学研究[D]. 杨凌: 西北农林科技大学, 2012.
	LI H L . Two dimensional electrophoresis and proteomics analysis of Producing swainsonine endophytic fungi proteins from locoweed[D]. Yangling: Northwest Agriculture and Forestry University, 2012. (in Chinese)
[25]	白晓南, 余永涛, 赵清梅, 何生虎 . 苦马豆素生物合成研究进展. 动物医学进展, 2016,37(5):92-98.
	BAI X N, YU Y T, ZHAO Q M, HE S H . Advances in biosynthesis of swainsonine. Progress in Veterinary Medicine, 2016,37(5):92-98. (in Chinese)
[26]	LU H, QUAN H Y, REN Z H, WANG S, XUE R X, ZHAO B Y . The genome of Undifilum oxytropis provides insights into swainsonine biosynthesis and locoism. Scientific Reports, 2016,6:30760.
[27]	REN Z, SONG R, WANG S, QUAN H, LU L Y, ZHAO B Y, LU H . The biosynthesis pathway of swainsonine, a new anticancer drug from three endophytic fung. Micrological Biotechnology, 2017,27(11):1897-1906.
[28]	武晓雨, 邓百万, 陈文强, 解修超 . 紫外诱变选育耐高温香菇新品种. 浙江农业学报, 2017,29(12):2015-2022.
	WU X Y, DENG B W, CHEN W Q, XIE X C . Breeding thermo-tolerant strains of lentinula edodes by UV mutation. Acta Agriculturae Zhejiangensis, 2017,29(12):2015-2022. (in Chinese)
[29]	乔桦, 陈波, 朱天骄, 张国刚, 李长伟, 崔承彬 . 产黄青霉S-3-25的硫酸二乙酯诱变突变株3d10-01的次级代谢产物研究. 国际药学研究杂志, 2017,9:871-877.
	QIAO H, CHEN B, ZHU T J, ZHANG G G, LI C W, CUI C B . The secondary metabolites from a mutant 3d10-01 derived from Penicillium chrysogenum S-3-25 by diethylsulfate mutagenesis. Journal of International Medical Research, 2017,9:871-877. (in Chinese)
[30]	左芳, 许超, 陈铭德 . 应用果皮提取液探究紫外线诱变原理的实验活动. 生物学通报, 2009,44(12):50-52.
	ZUO F, XU C, CHEN M D . Experimental activity of applicating pericarp extract to explore the principle of UV mutagenesis. Bulletin of Biology, 2009,44(12):50-52. (in Chinese)
[31]	程明, 崔承彬, 李长伟, 田从魁, 杜智敏 . 化学诱变技术在微生物育种研究中的应用. 国际药学研究杂志, 2009,36(6):412-417.
	CHENG M, CUI C B, LI C W, TIAN C K, DU Z M . Chemical mutation technique applied in microorganism breeding. Journal of International Pharmaceutical Research, 2009,36(6):412-417. (in Chinese)
[32]	李旭媛, 王刚, 费卓群, 陈光 . 紫外-亚硝基胍复合诱变筛选高产淀粉酶菌株. 中国生物制品学杂志, 2012,25(11):1543-1546.
	LI X Y, WANG G, FEI Z Q, CHEN G . Screening of high amylase-producing strains by mutagenesis with ultraviolet ray and nitrosoguanidine. Chinese Journal of Biologicals, 2012,25(11):1543-1546. (in Chinese)
[33]	李戈, 余凤玉, 曾会才, 王文华, 彭燕 . 链霉菌LA5高产菌株诱变育种研究初报. 微生物学杂志, 2007,27(5):10-13.
	LI G, YU F Y, ZENG H C, WANG W H, PENG Y . Preliminary research on mutation breeding of streptomyces sp. LA5 strain for high-yield antibiotics. Journal of Microbiology, 2007,27(5):10-13. (in Chinese)
[34]	ADELBERG E A, MANDEL M, CHEN G C . Optimal conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine in Escherichia coli K12. Biochemical & Biophysical Research Communications, 1965,18(5):788-795.
[35]	白晓南 . 变异黄芪内生真菌的诱变及SW合成突变菌株的筛选[D]. 银川: 宁夏大学, 2017.
	BAI X N . Mutagenesis of Astragalus variabills Bunge endophytic fungi and screening of SW mutant strains[D]. Yinchuan: Ningxia University, 2017. (in Chinese)
[36]	SANDESH K B, VIDHYAVATHI R, SARADA R, RAVISHANKAR G A . Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcuspluvialis mutants. Bioresource Technology, 2008,99(18):8667-8673.
[37]	易小畅, 蒋智, 李娜, 陆兵, 谌斌 . 红芝漆酶高产菌株的诱变选育. 食品研究与开发, 2014(24):139-142.
	YI X C, JIANG Z, LI N, LU B, CHEN B . Mutational screening of ganoderma lucidum strain with high yield of laccase. Food Research and Development, 2014(24):139-142. (in Chinese)

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组别 Group	吸光值 Absorbance ($\bar{x}\pm s$)	SW含量 SW content (μg·mL^-1)	SW含量变化 Change of SW content (%)
Alternaria Section Undifilum oxytropis	0.6116±0.08621	2.6445±0.1416	—
U1	0.6110±0.00664	2.636±0.1075	-0.31%
U2	0.6431±0.00094	2.4600±0.1883	-6.98%*
U3	0.6265±0.03167	2.5520±0.0437	-3.50%
U4	0.5330±0.00175	3.0683±0.1604	+16.02%**

诱变处理时间 Mutagenesis time（min）	亚硝基胍剂量 Nitroguanidine dose (μL)						空白对照 Blank control
诱变处理时间 Mutagenesis time（min）	2	4	6	8	10	12	空白对照 Blank control
5	8.33±5.38	33.33±14.05	58.33±4.59	75.00±3.72	91.67±13.29	100.00±0.00	0
10	33.33±7.38	66.67±13.66	75.0±22.36	91.67±7.52	91.67±3.50	100.00±0.00	0
15	58.33±7.46	75.00±6.85	91.67±6.05	100.0±0.00	100.0±0.00	100.00±0.00	0
20	75.00±3.95	91.67±7.45	100.0±0.00	100.0±0.00	100.00±0.00	100.00±0.00	0
25	83.33±12.11	100.00±0.00	100.0±0.00	100.00±0.00	100.00±0.00	100.00±0.00	0
30	91.67±5.31	100.0±0.00	100.0±0.00	100.00±0.00	100.00±0.00	100.00±0.00	0

组别 Group	吸光值 Absorbance ($\bar{x}$±s)	SW含量 SW content (μg·mL^-1)	SW含量变化 Change of SW content（%）
Alternaria Section Undifilum oxytropis	0.5916±0.0353	2.7890±0.1870	-
D1	0.5714±0.0029	2.8597±0.0082	+2.53%
D2	0.5213±0.0088	3.1364±0.0385	+12.46%**
D3	0.5876±0.0413	2.7662±0.2250	-1.18%
D4	0.7031±0.0033	2.1314±0.0180	-23.58%**
D5	0.5634±0.0029	2.9038±0.0180	+4.11%
D6	0.5811±0.0085	2.8062±0.0473	+0.61%

紫外辐照时间 Ultraviolet irradiation time (s)	亚硝基胍剂量 Nitroguanidine dose (μL)	诱变时间 Mutagenesis time (min)	诱变后菌株生长数 Number of strains after mutagenesis						对照组 Control group	致死率 Lethal rate (%)
20	2	5	6	5	6	6	4	5	13	58.98±6.28
40	2	5	3	2	2	4	3	3	12	76.39±6.27
80	2	5	1	0	1	0	0	2	12	94.45±6.80
160	2	5	0	0	2	0	0	1	13	96.15±6.43
320	2	5	0	0	0	0	0	0	11	100.00±0.00
640	2	5	0	0	0	0	0	0	13	100.00±0.00

组别 Group	吸光值 Absorbance($\bar{x}$±s)	SW含量 SW content (μg·mL^-1)	SW含量变化 Change of SW content (%)
Alternaria Section Undifilum oxytropis	0.6313±0.0116	2.5806±0.06237	—
UD1	0.2321±0.0203	3.0774±0.02356	+21.87%**
UD2	0.6848±0.0354	2.2293±0.02997	-11.72%**
UD3	0.6311±0.0639	2.5256±0.07710	-0.01%

Mutagenesis and Screening of Endophytic Fungus Alternaria Section Undifilum oxytropis Producing Swainsonine from Locoweed

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辐照时间 Ultraviolet irradiation time (s)	辐照后菌株生长数 Number of strains after ultraviolet irradiation						对照组生长数 Number of strains in control group	平均致死率 Average mortality rate（%）
20	12	11	13	10	12	11	13	11.54±8.07
40	6	8	6	7	8	8	12	40.28±8.20
80	2	3	1	1	3	2	12	83.33±7.46
160	1	0	0	1	1	1	13	94.87±3.97
320	0	0	0	0	0	0	11	100.00±0.00
640	0	0	0	0	0	0	13	100.00±0.00

时间 Time (d)	菌丝干重 The dry mycelial weight (g)			SW含量 The content of SW (μg·mL^-1)
时间 Time (d)	D4	UD1	Alternaria Section Undifilum oxytropis	D4	UD1	Alternaria Section Undifilum oxytropis
4	0.231±0.003	0.361±0.003	0.341±0.003	0.2660±0.034	0.2671±0.041	0.2743±0.003
8	0.503±0.023	0.613±0.002	0.491±0.061	1.2776±0.019	1.2775±0.016	1.7201±0.052
12	0.800±0.011	0.770±0.012	0.792±0.026	2.1141±0.011	1.8384±0.009	2.4970±0.038
16	1.015±0.05	1.103±0.047	1.033±0.049	3.0571±0.010	3.2219±0.019	3.0724±0.052
20	1.793±0.084	1.691±0.082	1.693±0.081	3.7713±0.019	5.4179±0.044	4.7585±0.011
24	1.986±0.018	2.105±0.025	2.100±0.033	4.1852±0.053	5.8580±0.005	5.1561±0.011
28	1.836±0.049	1.976±0.041	1.963±0.083	3.9043±0.009	5.8357±0.003	5.0102±0.010
32	1.021±0.031	1.433±0.027	1.230±0.026	2.2371±0.016	3.1252±0.045	3.2580±0.004