烟草内生真菌多样性及其细胞毒活性初步研究
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摘要
为研究烟草内生真菌的生物多样性,揭示其在烟草中的分布规律及种群结构,对烟草栽培种的内生真菌进行了系统的分离和鉴定,并对其发酵粗提物进行了初步的细胞毒活性筛选。结果发现,采用组织分离法从健康烟株的根、茎、叶中共分离纯化到62株内生真菌,形态学和分子生物学鉴定为4目7科9属。发现叶部内生真菌的分离率最高,其次为茎,根部最少。采用CCK-8法对内生真菌的体外细胞毒活性进行筛选,发现多数菌株对人肾癌Caki-1细胞和人肝癌Hep G2细胞均具有不同程度的抑制活性,菌株TE-6L的PDB发酵液及大米发酵物对Caki-1细胞48 h内的IC50分别为13.28和0.36μg/m L。结合形态学特征和分子生物学鉴定TE-6L菌株为Fusarium sambucinum。研究表明,烟草中蕴含丰富的具有细胞毒活性的内生真菌资源,为寻找细胞毒活性物质新来源、开发新型抗肿瘤药物提供了新的途径。
To investigate the biodiversity, distribution, and the microbial community structure of the endophytic fungi in Nicotiana tabacum, the endophytic fungi in different cultispecies were systematically isolated and identified. The preliminary screening of the cytotoxic activities was also performed. As a result, a total of 62 strains of the endophytic fungi were isolated and purified from the healthy tissues(roots, stems, and leaves) of tobacco using the tissue isolation method. The isolated strains were identified to belong to 4 orders, 7 families, and 9 genera based on their morphological and molecular characteristics. More species and strains were isolated from the leaves, followed by the stems, and those in the roots were the least. The cytotoxicity of the crude extracts was determined by CCK-8 assay. The results indicated that most of the strains were found to have some inhibitory activities on Caki-1 and Hep G2 cell lines. The IC50 s of the strain TE-6L to Caki-1 at 24 h and 48 h were 13.28 and 0.36 μg/m L, respectively. Combined with morphological characteristics and molecular analysis, this strain was identified as Fusarium sambucinum. The results from this study indicated that the tobacco possess abundant endophytic fungi, which provides a new path for searching for novel antitumor resources.
To investigate the biodiversity, distribution, and the microbial community structure of the endophytic fungi in Nicotiana tabacum, the endophytic fungi in different cultispecies were systematically isolated and identified. The preliminary screening of the cytotoxic activities was also performed. As a result, a total of 62 strains of the endophytic fungi were isolated and purified from the healthy tissues(roots, stems, and leaves) of tobacco using the tissue isolation method. The isolated strains were identified to belong to 4 orders, 7 families, and 9 genera based on their morphological and molecular characteristics. More species and strains were isolated from the leaves, followed by the stems, and those in the roots were the least. The cytotoxicity of the crude extracts was determined by CCK-8 assay. The results indicated that most of the strains were found to have some inhibitory activities on Caki-1 and Hep G2 cell lines. The IC50 s of the strain TE-6L to Caki-1 at 24 h and 48 h were 13.28 and 0.36 μg/m L, respectively. Combined with morphological characteristics and molecular analysis, this strain was identified as Fusarium sambucinum. The results from this study indicated that the tobacco possess abundant endophytic fungi, which provides a new path for searching for novel antitumor resources.
引文
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[19]王雅俊,孟志霞,于雪梅,等.促进金线莲生长发育的内生真菌筛选研究[J].中国药学杂志,2009,44(13):976-979.
[20]PATHANIA A S,GURU S K,UI ASHRAF N,et al.A novel stereo bioactive metabolite isolated from an endophytic fungus induces caspase dependent apoptosis and STAT-3 inhibition in human leukemia cells[J].European Journal of Pharmacology,2015,765(15):75-85.
[21]武文斌,刘雅莉,岳高超,等.蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究[J].微生物学通报,2013,40(12):2280-2287.
[22]STIERLE A,STROBEL G,STIERLE D.Taxol and taxane production by Taxomycetesandreanae,an endophytic fungus of Pacific Yew[J].Science,1993,260(5105):214-216.
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[25]Noh M J,Yang J G,Kim K S,et al.Isolation of a novel microorganism,Pestalotiaheterocornis,producing paclitaxel[J].Biotechnology and Bioengineering,64(5):620-623.
[26]刘宏玉.烟草内生真菌多样性及促生和抗重金属菌株的筛选[D].杭州:浙江大学,2014.
[27]金慧清,程昌合,徐清泉,等.烟草内生真菌对烟草生长和烟叶重金属含量的影响[J/OL].菌物学报,http://www.cnki.net/kcms/detail/11.5180.Q.20160317.1051.005.html
[28]裴洲洋.烟草内生真菌种群多样性及烟草赤星病生防内生菌的筛选[D].郑州:河南农业大学,2009.
[29]杨明俊,李娟,薛鸿燕,等.水仙内生真菌的分离及抑菌和抗肿瘤活性研究[J].中草药,2014,45(6):819-824.
[30]HUANG J,GE X,SUN M.Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA[J].Biotechniques,2000,28(3):432-434.
[31]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR protocols:a guide to methods and applications,1990,18(1):315-322.
[32]布芳芳,余仲东,侯姣姣,等.古侧柏鳞叶内生真菌分离及其抑菌活性研究[J].中国森林病虫,2016,35(3):12-16.
[33]万文婷,李宁,刘静,等.CCK-8法与MTT法检测人前列腺癌PC3细胞活性的比较研究[J].时珍国医国药,2010,21(12):3046-3048.
[34]彭细桥,刘红艳,罗宽,等.烟草内生青枯病拮抗细菌的筛选和初步鉴定[J].中国烟草科学,2007,28(2):38-40.
[35]雷丽萍,夏振远,郭荣君,等.非硝酸盐还原细菌K18降低TSNA机理的初步研究[J].中国烟草科学,2009,30(5):54-57.
[36]李文君.云南大理不同烟草产区烟叶内生菌群及其动态差异研究[D].昆明:云南大学,2011.
[37]陆荣,刘小娟,樊美珍,等.喜树内生真菌的分离及其抗肿瘤活性菌株的筛选[J].安徽农业大学学报,2008,35(1):76-79.
[38]宋萍,洪伟,吴承祯,等.雷公藤内生真菌的分离及抗肿瘤活性研究[J].北华大学学报(自然科学版),2009,10(4):310-313.
[2]张传博,陈荣林,殷幼平,等.金荞麦和苦荞麦抗菌活性内生真菌的筛选及鉴定[J].微生物学通报,2011,38(1):70-77.
[3]李秀璋,姚祥,李春杰,等.禾草内生真菌作为生防因子的潜力分析[J].植物生态学报,2015,39(6):621-634.
[4]金文进,李春杰,王正凤.禾草内生真菌的多样性及意义[J].草业学报,2015,24(1):168-175.
[5]WARDECKI T,BR?TZ E,DE FORD C,et al.Endophytic Streptomyces in the traditional medicinal plant Arnica Montana L.:secondary metabolites and biological activity[J].Antonie van Leeuwenhoek,2015,108(2):391-402.
[6]李瑾,张慧茹,刘诺阳,等.金银花内生真菌的分离鉴定及抑菌活性研究[J].中国抗生素杂志,2010,35(3):236-237.
[7]毕江涛,李萍,马飞,等.濒危药用植物沙冬青内生真菌分离及其抑菌活性初步研究[J].中国农学通报,2012,28(30):40-48.
[8]周雅琴,谭小明,陈娟,等.南方红豆杉内生真菌的分离及抗菌活性筛选研究[J].中国药学杂志,2015,50(1):19-22.
[9]DEVI P,RODRIGUES C,NAIK C G,et al.Isolation and characterization of antibacterial compound from a mangrove-endophytic fungus,PenicilliumchrysogenumMTCC 5108[J].India Journal of Microbiology,2012,52(4):617-623.
[10]陈亮亮,王佩,王昊,等.角果木内生真菌Coriolopsissp.J5代谢产物及活性研究[J].中国海洋药物,2016,35(2):7-12.
[11]MORITA S,AZUMA M,AOBA T,et al.Induced systemic resistance of Chinese cabbage to bacterial leaf spot and Alternaria leaf spot by the root endophytic fungus,Heteroconiumchaetospira[J].Journal of General Plant Pathology,2003,69(1):71-75.
[12]BAN Y,TANG M,CHEN H,et al.The response of dark septate endophytes(DSE)to heavy metals in pure culture[J].PLo S One,2012,7(10):e47968.
[13]?ELMULLER R,SHERAMETI I,TRIPATHI S,et al.Piriformosporaindica,a cultivable root endophyte with multiple biotechnological applications[J].Symbiosis,2009,49(1):1-17.
[14]李玲玲,罗合春,张先淑.柴胡内生真菌鉴定与抑菌活性筛选[J].生物技术通报,2015,31(10):165-170.
[15]PATIL M P,PATIL R H,MAHESHWARI V L.Biological activities and identification of bioactive metabolite from endophytic Aspergillus flavus L7isolated from Aegle marmelos[J].Current Microbiology,2015,71(1):39-48.
[16]史佳琴,周松林,王梅霞,等.槐树内生真菌抗氧化活性的初步研究[J].食品科学,2007,28(8):250-253.
[17]钱一鑫,康冀川,耿坤,等.青蒿内生真菌的抗肿瘤抗氧化活性[J].菌物研究,2014,12(1):44-50.
[18]侯晓强,郭顺星.铁皮石斛促生长内生真菌的筛选与鉴定[J].中国中药杂志,2014,39(17):3232-3237.
[19]王雅俊,孟志霞,于雪梅,等.促进金线莲生长发育的内生真菌筛选研究[J].中国药学杂志,2009,44(13):976-979.
[20]PATHANIA A S,GURU S K,UI ASHRAF N,et al.A novel stereo bioactive metabolite isolated from an endophytic fungus induces caspase dependent apoptosis and STAT-3 inhibition in human leukemia cells[J].European Journal of Pharmacology,2015,765(15):75-85.
[21]武文斌,刘雅莉,岳高超,等.蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究[J].微生物学通报,2013,40(12):2280-2287.
[22]STIERLE A,STROBEL G,STIERLE D.Taxol and taxane production by Taxomycetesandreanae,an endophytic fungus of Pacific Yew[J].Science,1993,260(5105):214-216.
[23]LI J Y,SIDHU R S,BOLLON A,et al.Stimulation of taxol production in liquid cultures of Pestalotiopsismicrospora[J].Mycol Res,102(4):461-464.
[24]STROBEL G,YANG X S,SEARS J,et al.Taxol from Pestalotiopsismicrospora,an endophytic fungus of Taxuswallachiana[J].Microbiology,142:435-440.
[25]Noh M J,Yang J G,Kim K S,et al.Isolation of a novel microorganism,Pestalotiaheterocornis,producing paclitaxel[J].Biotechnology and Bioengineering,64(5):620-623.
[26]刘宏玉.烟草内生真菌多样性及促生和抗重金属菌株的筛选[D].杭州:浙江大学,2014.
[27]金慧清,程昌合,徐清泉,等.烟草内生真菌对烟草生长和烟叶重金属含量的影响[J/OL].菌物学报,http://www.cnki.net/kcms/detail/11.5180.Q.20160317.1051.005.html
[28]裴洲洋.烟草内生真菌种群多样性及烟草赤星病生防内生菌的筛选[D].郑州:河南农业大学,2009.
[29]杨明俊,李娟,薛鸿燕,等.水仙内生真菌的分离及抑菌和抗肿瘤活性研究[J].中草药,2014,45(6):819-824.
[30]HUANG J,GE X,SUN M.Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA[J].Biotechniques,2000,28(3):432-434.
[31]WHITE T J,BRUNS T,LEE S,et al.Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics[J].PCR protocols:a guide to methods and applications,1990,18(1):315-322.
[32]布芳芳,余仲东,侯姣姣,等.古侧柏鳞叶内生真菌分离及其抑菌活性研究[J].中国森林病虫,2016,35(3):12-16.
[33]万文婷,李宁,刘静,等.CCK-8法与MTT法检测人前列腺癌PC3细胞活性的比较研究[J].时珍国医国药,2010,21(12):3046-3048.
[34]彭细桥,刘红艳,罗宽,等.烟草内生青枯病拮抗细菌的筛选和初步鉴定[J].中国烟草科学,2007,28(2):38-40.
[35]雷丽萍,夏振远,郭荣君,等.非硝酸盐还原细菌K18降低TSNA机理的初步研究[J].中国烟草科学,2009,30(5):54-57.
[36]李文君.云南大理不同烟草产区烟叶内生菌群及其动态差异研究[D].昆明:云南大学,2011.
[37]陆荣,刘小娟,樊美珍,等.喜树内生真菌的分离及其抗肿瘤活性菌株的筛选[J].安徽农业大学学报,2008,35(1):76-79.
[38]宋萍,洪伟,吴承祯,等.雷公藤内生真菌的分离及抗肿瘤活性研究[J].北华大学学报(自然科学版),2009,10(4):310-313.