产银杏内酯内生真菌的分离与鉴定
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摘要
利用组织块培养法从健康的银杏根、茎中分离得到116株内生真菌。通过高效液相色谱(HPLC)法对内生真菌的代谢产物进行分析,最终筛选出1株能够产银杏内酯的内生真菌,利用标准曲线法测定其发酵液和菌丝体中总内酯产量约为11.4mg/L。形态学特征和真菌rDNA间隔序列(internal transcribed spacer,ITS)的系统进化分析结果表明,该菌株属于毛霉属,为产银杏内酯内生真菌的首次报道。
In the present study,the method of tissue culturing was used to isolate the endophytic fungi from healthy ginkgo roots and stems.After repeated separation and purification,116 Ginkgo endophytes were isolated.Metabolites of these fungi were preliminarily analyzed by high-performance liquid chromatography(HPLC),and a ginkgolide producing endophyte strain was finally screened out.The production of ginkgolides by this strain was more than 11.4 mg/L.The endophytic fungi strain was identified as Mucor spp.by morphology characteristics and phylogenetic analysis of internal transcribed spacer(ITS),and It was the first report in the ginkgolides producing endophytic fungus from Ginkgo biloba.
In the present study,the method of tissue culturing was used to isolate the endophytic fungi from healthy ginkgo roots and stems.After repeated separation and purification,116 Ginkgo endophytes were isolated.Metabolites of these fungi were preliminarily analyzed by high-performance liquid chromatography(HPLC),and a ginkgolide producing endophyte strain was finally screened out.The production of ginkgolides by this strain was more than 11.4 mg/L.The endophytic fungi strain was identified as Mucor spp.by morphology characteristics and phylogenetic analysis of internal transcribed spacer(ITS),and It was the first report in the ginkgolides producing endophytic fungus from Ginkgo biloba.
引文
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[8]张灿,宫彩娥,范龚健,等.酶解银杏蛋白制备α-葡萄糖苷酶抑制肽的研究[J].食品与机械,2016,32(11):137-141.
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[11]杜慧竟,苏静,余利岩,等.药用植物内生放线菌的分离和生物学特性[J].微生物学报,2013,53(1):15-23.
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[15]包飞,樊明涛,贺江.产银杏内酯B内生真菌的分离与筛选[J].西北农业学报,2008,17(3):328-331.
[16]CUI Yu-na,YI Da-wei,BAI Xiu-feng,et al.Ginkgolide B produced endophytic fungus(Fusarium oxysporum)isolated from Ginkgo biloba[J].Fitoterapia,2012,85(3):913-920.
[17]席晓圆,赵盛英,张鹏,等.产紫杉醇内生真菌N-15的分离及鉴定[J].天然产物研究与开发,2016,28(8):1 219-1 222,1 283.
[18]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1982:60-75.
[19]MAROPOLA M K,RAMOND J B,TRINDADEA M.Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor(L.Moench)[J].Journal of Microbiological Methods,2015,112:104-117.
[20]GONZALEZ-TEUBER M,VILO C,BASCUNAN-GODOY L.Molecular characterization of endophytic fungi associated with the roots of Chenopodium quinoainhabiting the Atacama Desert,Chile[J].Genomics Data,2017,11:109-112.
[21]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[22]杜若源,谢晶,王婷,等.超声波辅助提取银杏叶中总黄酮的工艺优化[J].食品与机械,2015,31(1):167-170.
[23]喻林华,李晓烈,蓝华,等.银杏叶提取物对柑橘青霉病菌的抑菌效果[J].食品与机械,2010,26(6):60-62.
[2]袁志辉,蒋琼凤,何福林.植物内生微生物及其“暗物质”开发技术的研究进展[J].天然产物研究与开发,2016,28(7):1 156-1 163.
[3]YAN J F,BROUGHTON S J,YANG S L,et al.Do endophytic fungi grow through their hosts systemically[J].Fungal Ecology,2015,13:53-59.
[4]刘颖,魏希颖.内生菌对植物次生代谢产物的转化[J].天然产物研究与开发,2014,26(2):300-303.
[5]王兴红.内生真菌与天然药物研究进展[J].中草药,2007,38(1):140-143.
[6]STROBEL G,DAISY B.Bioprospecting for microbial endophytes and their natural products[J].Microbiology and Molecular Biology Review,2003,67(4):491-502.
[7]韩宏伟,谢博文,许旦,等.漂烫对真空油炸银杏果品质的影响[J].食品与机械,2016,32(1):179-182,212.
[8]张灿,宫彩娥,范龚健,等.酶解银杏蛋白制备α-葡萄糖苷酶抑制肽的研究[J].食品与机械,2016,32(11):137-141.
[9]雷爱秋,华洋林,孙为正.含银杏叶提取物胶囊中黄酮类化合物检测方法改进[J].食品与机械,2016,32(4):88-91.
[10]QIAN Yi-xin,KANG Ji-chuang,LUO Yi-kai,et al.A bilobalide-producing endophytic fungus,pestalotiopsis uvicola from medicinal plant Ginkgo biloba[J].Current Microbiology,2016,73(2):280-286.
[11]杜慧竟,苏静,余利岩,等.药用植物内生放线菌的分离和生物学特性[J].微生物学报,2013,53(1):15-23.
[12]严铸云,庞蕾,罗静.银杏内生真菌中产生银杏内酯类菌株的筛选[J].华西药学杂志,2007,22(5):491-493.
[13]严铸云,罗静,郭晓恒,等.产银杏内酯内生真菌的筛选及培养条件研究[J].天然产物研究与开发,2007,19(4):554-558.
[14]罗静,严铸云,郭晓恒,等.产银杏内酯内生真菌的选育及发酵条件研究[J].四川大学学报:自然科学版,2009,46(4):1 161-1 165.
[15]包飞,樊明涛,贺江.产银杏内酯B内生真菌的分离与筛选[J].西北农业学报,2008,17(3):328-331.
[16]CUI Yu-na,YI Da-wei,BAI Xiu-feng,et al.Ginkgolide B produced endophytic fungus(Fusarium oxysporum)isolated from Ginkgo biloba[J].Fitoterapia,2012,85(3):913-920.
[17]席晓圆,赵盛英,张鹏,等.产紫杉醇内生真菌N-15的分离及鉴定[J].天然产物研究与开发,2016,28(8):1 219-1 222,1 283.
[18]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1982:60-75.
[19]MAROPOLA M K,RAMOND J B,TRINDADEA M.Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor(L.Moench)[J].Journal of Microbiological Methods,2015,112:104-117.
[20]GONZALEZ-TEUBER M,VILO C,BASCUNAN-GODOY L.Molecular characterization of endophytic fungi associated with the roots of Chenopodium quinoainhabiting the Atacama Desert,Chile[J].Genomics Data,2017,11:109-112.
[21]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[22]杜若源,谢晶,王婷,等.超声波辅助提取银杏叶中总黄酮的工艺优化[J].食品与机械,2015,31(1):167-170.
[23]喻林华,李晓烈,蓝华,等.银杏叶提取物对柑橘青霉病菌的抑菌效果[J].食品与机械,2010,26(6):60-62.