产苦马豆素疯草内生真菌U.oxytropis的发酵培养及苦马豆素检测方法的建立
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摘要
通过对生长初期疯草内生真菌U.oxytropis菌丝复苏后连续培养32d,每隔4d取样1次,并设3个平行培养组,测定其不同培养时期菌丝干重变化,利用超声减压旋蒸法提取菌丝和发酵液中苦马豆素(SW),并分别利用薄层层析法、气相色谱法和α-甘露糖苷酶抑制法对菌丝、发酵液中SW含量进行测定。结果显示,经测定疯草内生真菌U.oxytropis菌丝干重质量,确定疯草内生真菌U.oxytropis生长周期为24d;薄层层析法、气相色谱法和α-甘露糖苷酶抑制法都可快速对菌丝和发酵液中提取的SW进行定性、定量检测,并比较3种检测方法,发现气相色谱法检测限低、灵敏度更高。本试验完成了疯草内生真菌U.oxytropis生长周期的测定,并建立了菌丝、发酵液中SW的有效、快速提取及检测方法,为下一步产苦马豆素疯草内生真菌U.oxytropis生物合成机理的研究奠定基础。
The sample was collected at 4 days intervals for 32 days after resuscitated the early growth of U.oxytropis mycelium.Three parallel culture groups were set up to determine the dry weight of mycelium in different culture periods.The swainsonine of mycelium and fermentation broth was extracted through ultrasonic vacuum rotary method.The content of swainsonine inmycelia and fermentation broth was determined through thin layer chromatography,gas chromatography and inhibition ofα-mannosidase.The results showed that the growth cycle of U.oxytropis was24 days.The swainsonine,extracted from mycelium and fermentation broth,can be quickly determined through the method of thin layer chromatography,gas chromatography andα-mannosidase inhibition,and gas chromatography method has lower detection limit and higher sensitivity than other methods.The experiment provides the theoretical basis for the next research of swainsonsine producing endophytic fungi biosynthesis mechanism of locoweed.
The sample was collected at 4 days intervals for 32 days after resuscitated the early growth of U.oxytropis mycelium.Three parallel culture groups were set up to determine the dry weight of mycelium in different culture periods.The swainsonine of mycelium and fermentation broth was extracted through ultrasonic vacuum rotary method.The content of swainsonine inmycelia and fermentation broth was determined through thin layer chromatography,gas chromatography and inhibition ofα-mannosidase.The results showed that the growth cycle of U.oxytropis was24 days.The swainsonine,extracted from mycelium and fermentation broth,can be quickly determined through the method of thin layer chromatography,gas chromatography andα-mannosidase inhibition,and gas chromatography method has lower detection limit and higher sensitivity than other methods.The experiment provides the theoretical basis for the next research of swainsonsine producing endophytic fungi biosynthesis mechanism of locoweed.
引文
[1]周启武,赵宝玉,路浩,等.苦马豆内生真菌分离鉴定与多样性分析[J].畜牧兽医学报,2013,44(3):465-474.
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[15]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].陕西杨凌:西北农林科技大学,2012.
[16]BRAUN K,ROMERO J,LIDDELL C,et al.Production of swainsonine by fungal endophytes of locoweed[J].Mycol Res,2003,107(8):980-988.
[17]王帅,廖秋萍,贾琦珍,等.小花棘豆中毒对和田羊内脏器官α-甘露糖苷酶的影响[J].湖北农业科学,2016,55(9):2308-2312.
[18]DENNIS J W,KOCH K,YOUSEFI S,et al.Growth inhibition of human melanoma tumor xenografts in athymic nude mice by swainsonine[J].Cancer Res,1990,50(6):1867.
[19]REN Z,SONG R,WANG S,et al.The biosynthesis pathway of swainsonine,a new anti-cancer drug from three endophytic fungi[J].J Microb Biol,2017(11):1897-1906.
[20]陈基萍,赵宝玉,路浩,等.产苦马豆素菌株Aspergillus ustus发酵工艺研究[J].中国兽医学报,2013,33(3):423-427.
[21]马尧,路浩,赵宝玉,等.产苦马豆素真菌的筛选与鉴定[J].畜牧兽医学报,2010,41(5):621-629.
[22]郭伟,李勤凡,孔祥雅,等.疯草内生真菌FEL3生产SW培养基的最佳碳氮比优化[J].草地学报,2011,19(4):674-677.
[23]LU H,QUAN H,ZHOU Q,et al.Endogenous fungi isolated from three locoweed species from rangeland in western china[J].African J Microbiol Res,2017,11(5):155-170.
[24]COOK D,BGG D,CREAMER R,et al.Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi[J].G3Genes Genetics,2017,7(6):1791-1797.
[25]MOLYNEUX R J,ROITMAN P N.Specific detection of pyrrlizidine alkaloids on thin layer chromatograms[J].J Chromatography,1980,195:412-415.
[26]LU H,WANG S S,ZHAO B Y.Isolation and identification of swainsonine from oxytropis glabra and its pathological lesions to SD rats[J].Asian J Anim Vet Adv,2012,7(9):822-831.
[2]马尧,路浩,赵宝玉,等.产苦马豆素真菌的筛选与鉴定[J].畜牧兽医学报,2010,41(5):621-629.
[3]曹丹丹,路浩,赵宝玉,等.变异黄芪内生真菌种群分布特征[J].畜牧兽医学报,2014,42(12):2057-2066.
[4]COLEGATE S M,DORLING P R,HUXTABLE CR,et al.A spectroscopic investigation of swainsonine:anα-mannosidase inhibitor isolated from swainsona canescens[J].Aust J Chem,1979,32(10):2257-2264.
[5]高新磊.小花棘豆内生真菌Undifilum oxytropis与苦马豆素关系研究[D].内蒙古呼和浩特:内蒙古农业大学,2011.
[6]TULSIANI D R,TOUSTER O.Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism[J].JBiol Chem,1987,262(14):6506-6514.
[7]DORLING P R,HUXTABLE C R,COLEGATE SM.Inhibition of lysosomal alpha-mannosidase by swainsonine,an indolizidine alkaloid isolated from swainsona canescens[J].Biochemical J,1980,191(2):649-651.
[8]MCLAIN-ROMERO J,CREAMER R,ZEPEDA H,et al.The toxicosis of embellisia fungi from locoweed(Oxytropis lambertii)is similar to locoweed toxicosis in rats[J].J Anim Sci,2004,82(7):2169-2174.
[9]RALPHS M H,COOK D,GARDNER D R,et al.Transmission of the locoweed endophyte to the nextgeneration of plants[J].Fungal Ecol,2011,4(4):251-255.
[10]COOK D,GARDNER D R,RALPHS M H,et al.Swainsoninine concentrations and endophyte amounts of Undifilum oxytropis in different plant parts of Oxytropis sericea[J].J Chem Ecol,2009,35(10):1272-1278.
[11]郝宝成,武凡林,邢小勇,等.苦马豆素抗牛病毒性腹泻病毒的研究[J].中国农业科学,2014,47(1):170-181.
[12]HARRIS C M,CAMPBELL B C,MOLYNEUX R J,et al.Biosynthesis of swainsonine in the diablo locoweed(Astragalus oxyphyrus)[J].Tetrahedron Lett,1988,29(38):4815-4818.
[13]MUKHERJEE S,DAWE A L,CREAMER R.Potential role for saccharopine reductase in swainsonine metabolism in endophytic fungus,Undifilum oxytropis[J].Fungal Biol K,2012,116(8):902-909.
[14]杨国栋.疯草内生真菌合成苦马豆素的研究[D].陕西杨凌:西北农林科技大学,2012.
[15]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].陕西杨凌:西北农林科技大学,2012.
[16]BRAUN K,ROMERO J,LIDDELL C,et al.Production of swainsonine by fungal endophytes of locoweed[J].Mycol Res,2003,107(8):980-988.
[17]王帅,廖秋萍,贾琦珍,等.小花棘豆中毒对和田羊内脏器官α-甘露糖苷酶的影响[J].湖北农业科学,2016,55(9):2308-2312.
[18]DENNIS J W,KOCH K,YOUSEFI S,et al.Growth inhibition of human melanoma tumor xenografts in athymic nude mice by swainsonine[J].Cancer Res,1990,50(6):1867.
[19]REN Z,SONG R,WANG S,et al.The biosynthesis pathway of swainsonine,a new anti-cancer drug from three endophytic fungi[J].J Microb Biol,2017(11):1897-1906.
[20]陈基萍,赵宝玉,路浩,等.产苦马豆素菌株Aspergillus ustus发酵工艺研究[J].中国兽医学报,2013,33(3):423-427.
[21]马尧,路浩,赵宝玉,等.产苦马豆素真菌的筛选与鉴定[J].畜牧兽医学报,2010,41(5):621-629.
[22]郭伟,李勤凡,孔祥雅,等.疯草内生真菌FEL3生产SW培养基的最佳碳氮比优化[J].草地学报,2011,19(4):674-677.
[23]LU H,QUAN H,ZHOU Q,et al.Endogenous fungi isolated from three locoweed species from rangeland in western china[J].African J Microbiol Res,2017,11(5):155-170.
[24]COOK D,BGG D,CREAMER R,et al.Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi[J].G3Genes Genetics,2017,7(6):1791-1797.
[25]MOLYNEUX R J,ROITMAN P N.Specific detection of pyrrlizidine alkaloids on thin layer chromatograms[J].J Chromatography,1980,195:412-415.
[26]LU H,WANG S S,ZHAO B Y.Isolation and identification of swainsonine from oxytropis glabra and its pathological lesions to SD rats[J].Asian J Anim Vet Adv,2012,7(9):822-831.