小叶榕内生菌分离纯化方法的建立
|
摘要
【目的】探索并建立小叶榕茎段和叶片内生菌的分离纯化方法,尤其是表面消毒方法。【方法】茎段和叶片均用75%酒精-0.1%升汞法表面消毒,茎段用组织块法分离内生菌,叶片用组织匀浆法分离内生菌。设计两种消毒剂作用时间组合,找到合适的表面消毒方法。【结果】对茎段的表面消毒,两种消毒剂浸泡维持时间有两种方案:75%酒精浸泡(1-1.5)min,0.1%升汞浸泡1min;或者75%酒精浸泡1min,0.1%升汞浸泡(1-1.5)min。对叶片的表面消毒,两种消毒剂漂洗维持时间分别为75%酒精(1.5-2.0)min,0.1%升汞1.5min。采用上述方法,从小叶榕植物样本中分离得到3株丝状真菌,2株细菌。【结论】建立了小叶榕茎段和叶片的分离纯化方法,为小叶榕抗菌活性内生菌的筛选、鉴定、诱变育种、培养条件优化、活性代谢物提取等后续工作奠定了良好的基础。
[Objective] To explore and establish a method for the isolation and purification of endophytes from the stems and leaves of Ficus microphylla, especially the method of surface disinfection.[Methods] Both stems and leaves were disinfected by75% alcohol and 0.1% corrosive sublimate. Endophytes were isolated from stems by tissue block method, and isolated from leaves by tissue homogenization method. The combination of the two kinds of disinfectant were designed to find a suitable surface disinfection method. [Results] The surface disinfection methods of the stems were soaking for(1.0-1.5) min with 75% alcohol,1.0 min with 0.1% corrosive sublimate; or soaking for 1.0 min with 75% alcohol,(1.0-1.5) min with 0.1% corrosive sublimate.The surface disinfection method of the leaves was poaching for(1.5-2.0)min with 75% alcohol, 1.5 min with 0.1% corrosive sublimate.Five strains of endophytes were isolated from Ficus microphylla,which including three strains of filamentous fungi and two strains of bacteria.[Conclusion]A method for the isolation and purification of endophytes from Ficus microphylla was established,which laid a good foundation for the forwarding research,such as screening, identification, mutation breeding, culture conditions optimization and active metabolites extraction of the endophytes.
[Objective] To explore and establish a method for the isolation and purification of endophytes from the stems and leaves of Ficus microphylla, especially the method of surface disinfection.[Methods] Both stems and leaves were disinfected by75% alcohol and 0.1% corrosive sublimate. Endophytes were isolated from stems by tissue block method, and isolated from leaves by tissue homogenization method. The combination of the two kinds of disinfectant were designed to find a suitable surface disinfection method. [Results] The surface disinfection methods of the stems were soaking for(1.0-1.5) min with 75% alcohol,1.0 min with 0.1% corrosive sublimate; or soaking for 1.0 min with 75% alcohol,(1.0-1.5) min with 0.1% corrosive sublimate.The surface disinfection method of the leaves was poaching for(1.5-2.0)min with 75% alcohol, 1.5 min with 0.1% corrosive sublimate.Five strains of endophytes were isolated from Ficus microphylla,which including three strains of filamentous fungi and two strains of bacteria.[Conclusion]A method for the isolation and purification of endophytes from Ficus microphylla was established,which laid a good foundation for the forwarding research,such as screening, identification, mutation breeding, culture conditions optimization and active metabolites extraction of the endophytes.
引文
[1]黎万奎,胡之璧.内生菌与天然药物[J].中国天然药物,2005,3(4)193-199.
[2]邓雪萍,杨博.药用植物内生菌及其产生的活性成分研究现状[J].安徽农学通报.2010,16(1):78-80.
[3]韦锦斌,黄仁彬,林军,等.小叶榕水提取物和醇提取物止咳平喘作用的比较研究[J].广西中医药,2006,29(4):58.
[4]中华人民共和国卫生部药政局.中华人民共和国卫生部药品标准,中药成方制剂,第14册[S].1997:108-110.
[5]邓振山,魏婷婷,苏瑞,等.一株具有抑菌活性的酸枣内生菌的分离[J].浙江农业学报,2017,29(12):2068-2076.
[6]邓雪萍.抗菌活性植物内生菌的分离、筛选及发酵条件优化[D].华南理工大学.2010:8-13.
[7]李晓红,傅本重,麻春花,等.不同表面消毒方法对核桃叶内生菌分离效果的比较[J].中国农学通报.2012,2(28):163-168.
[8]竺锡武,陈集双,陈海敏.用于分离植物内生菌的消毒方法:中国,200610049933.5[P].2008-01-23.
[9]陶诗,刘一令,黄小香,等.春兰菌根真菌的分离及培养特征研究[J].北方园艺,2010,1(16):108-111.
[10]胡青,诸葛增玉,任超.中药黄芪内生菌的分离鉴定及方法分析[J].中国兽医杂志,2016,2(2):59-60.
[11]陈至里,沈娟,王影姣,等.美洲大蠊肠道内生细菌的分离及其初步抑菌活性研究[J].生物学通报,2016,51(11):54-57.
[12]周晓坤,陈钧,韩邦兴,等.水菖蒲内生菌分离与抗菌活性菌株筛选[J].食品科学,2010,31(9):211-215.
[2]邓雪萍,杨博.药用植物内生菌及其产生的活性成分研究现状[J].安徽农学通报.2010,16(1):78-80.
[3]韦锦斌,黄仁彬,林军,等.小叶榕水提取物和醇提取物止咳平喘作用的比较研究[J].广西中医药,2006,29(4):58.
[4]中华人民共和国卫生部药政局.中华人民共和国卫生部药品标准,中药成方制剂,第14册[S].1997:108-110.
[5]邓振山,魏婷婷,苏瑞,等.一株具有抑菌活性的酸枣内生菌的分离[J].浙江农业学报,2017,29(12):2068-2076.
[6]邓雪萍.抗菌活性植物内生菌的分离、筛选及发酵条件优化[D].华南理工大学.2010:8-13.
[7]李晓红,傅本重,麻春花,等.不同表面消毒方法对核桃叶内生菌分离效果的比较[J].中国农学通报.2012,2(28):163-168.
[8]竺锡武,陈集双,陈海敏.用于分离植物内生菌的消毒方法:中国,200610049933.5[P].2008-01-23.
[9]陶诗,刘一令,黄小香,等.春兰菌根真菌的分离及培养特征研究[J].北方园艺,2010,1(16):108-111.
[10]胡青,诸葛增玉,任超.中药黄芪内生菌的分离鉴定及方法分析[J].中国兽医杂志,2016,2(2):59-60.
[11]陈至里,沈娟,王影姣,等.美洲大蠊肠道内生细菌的分离及其初步抑菌活性研究[J].生物学通报,2016,51(11):54-57.
[12]周晓坤,陈钧,韩邦兴,等.水菖蒲内生菌分离与抗菌活性菌株筛选[J].食品科学,2010,31(9):211-215.