艾丁湖可培养嗜盐菌多样性及功能酶、抗菌活性筛选
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摘要
【目的】探究艾丁湖可培养嗜盐菌的多样性、功能酶活性以及抗菌活性,进一步了解其次级代谢产物情况,为新型生物活性物质的发掘提供依据。【方法】选用以20种糖及糖的衍生物作为唯一碳源的寡营养培养基从艾丁湖5个样点中分离得到298株嗜盐菌,根据形态特征去重复后,选取62株菌运用16S r RNA基因系统发育分析的方法研究样品中嗜盐菌的多样性;从不同类群选取22株代表菌株,采用点接法进行3种功能酶的筛选,运用平板对峙法检测代表菌株对12种病原菌的抗菌活性。【结果】从5%、10%和15%3个盐浓度中分别分得221、54和23株嗜盐菌,获得的嗜盐菌分布在9个科18个属;其中放线菌分布于4个属,细菌分布于14个属;Nocardiopsis和Pontibacillus属为艾丁湖可培养嗜盐菌的优势类群,分别占17.7%和16.1%;有15株嗜盐菌相似性低于98.5%,可能为潜在新种。所选取的22株代表菌株中,分别有68.2%、22.7%和72.7%的实验菌株具有蛋白酶、淀粉酶和酯酶活性;45.5%的代表菌株对12种病原菌表现出了抗至少1种病原菌的活性,其中一株Nocardiopsis属放线菌能抗9种病原菌,表现出了广谱的抗菌活性。【结论】新疆艾丁湖土样中嗜盐菌的多样性较丰富,而且具有较好的生物活性,为后续进一步研究其次级代谢产物提供了依据。
[Objective] To explore secondary metabolites and new bioactive substances, the diversity, functional enzyme activity and antimicrobial activity of halophilic bacteria from Aiding Lake were studied. [Methods] A total of 20 sugars and sugar derivatives were selected as the sole carbon source of medium, and 298 halophilic bacteria were isolated from 5 samples of Aiding Lake. Based on the morphological characteristics, 62 representative strains were selected for 16 S r RNA gene sequencing and phylogenetic analysis. Twenty-two halophilic bacteria were chosen from different species to screen functional enzyme activity and antimicrobial activity. Activity for 3 functional enzyme were tested by noculating single colony method. Antimicrobial activity for 12 pathogenic bacteria and fungus were tested by plate confront method. [Results] There were 221, 54, 23 halophilic bacteria isolated from 5%, 10%, 15% salt concentration respectively. They were distributed in 9 families, 18 genera. Actinobacteria were distributed in 4 genera, and bacteria were distributed in 14 genera. Nocardiopsis and Pontibacillus were dominant groups of culturable halophilic bacteria from Aiding Lake, comprising 17.7% and 16.1% respectively. The 16 S r RNA gene sequence similarity of 15 halophilic bacteria were lower than 98.5%, which may present potential novel species. Among the 22 representative strains, 68.2%, 22.7%, 72.7% of them showed positive result for protease activity, amylase activity and asterase activity respectively. And 45.5% of the tested strains showed at least one kind of microbial activity against 12 pathogenic bacteria and fungus. Especially, one Nocardiopsis strain exhibited extensive antagonism against 9 pathogenic bacteria and fungus. [Conclusion] The halophilic bacteria of Aiding Lake in Xinjiang has abundant diversity and good biological activity, which provides the basis for further study on secondary metabolites.
[Objective] To explore secondary metabolites and new bioactive substances, the diversity, functional enzyme activity and antimicrobial activity of halophilic bacteria from Aiding Lake were studied. [Methods] A total of 20 sugars and sugar derivatives were selected as the sole carbon source of medium, and 298 halophilic bacteria were isolated from 5 samples of Aiding Lake. Based on the morphological characteristics, 62 representative strains were selected for 16 S r RNA gene sequencing and phylogenetic analysis. Twenty-two halophilic bacteria were chosen from different species to screen functional enzyme activity and antimicrobial activity. Activity for 3 functional enzyme were tested by noculating single colony method. Antimicrobial activity for 12 pathogenic bacteria and fungus were tested by plate confront method. [Results] There were 221, 54, 23 halophilic bacteria isolated from 5%, 10%, 15% salt concentration respectively. They were distributed in 9 families, 18 genera. Actinobacteria were distributed in 4 genera, and bacteria were distributed in 14 genera. Nocardiopsis and Pontibacillus were dominant groups of culturable halophilic bacteria from Aiding Lake, comprising 17.7% and 16.1% respectively. The 16 S r RNA gene sequence similarity of 15 halophilic bacteria were lower than 98.5%, which may present potential novel species. Among the 22 representative strains, 68.2%, 22.7%, 72.7% of them showed positive result for protease activity, amylase activity and asterase activity respectively. And 45.5% of the tested strains showed at least one kind of microbial activity against 12 pathogenic bacteria and fungus. Especially, one Nocardiopsis strain exhibited extensive antagonism against 9 pathogenic bacteria and fungus. [Conclusion] The halophilic bacteria of Aiding Lake in Xinjiang has abundant diversity and good biological activity, which provides the basis for further study on secondary metabolites.
引文
[1]Guan TW.Diversity analysis and polyphasic taxonomy of actinobacteria of sediment samples from Lop Nur Salt Lake in Xinjiang,China[D].Chengdu:Doctoral Dissertation of Sichuan Agricultural University,2012(in Chinese)关统伟.新疆罗布泊盐湖放线菌多样性及多相分类[D].成都:四川农业大学博士学位论文,2012
[2]Han J.Study on biodiversity and enzyme-producing characteristic of halophilic bacteria from Lop Nur Region in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2008(in Chinese)韩剑.新疆罗布泊嗜盐细菌的多样性及产酶特性研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2008
[3]Liu BB.Diversity and functional enzyme screening of cultured halophilic archaea in Lop Nur Region[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)刘冰冰.新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选[D].阿拉尔:塔里木大学硕士学位论文,2011
[4]Shi R.Diversity of culturable actinobacteria and PKS I gene from Qijiaojing salt lake in Xinjiang[D].Kunming:Master’s Thesis of Yunnan University,2011(in Chinese)石蓉.新疆七角井盐湖可培养放线菌及其I型PKS基因多样性研究[D].昆明:云南大学硕士学位论文,2011
[5]Chen C.Diversity of culturable bacteria from Qijiaojing Lake in Xinjiang and their ability to produce enzymes[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)陈超.新疆七角井盐湖可培养细菌多样性及其功能酶筛选[D].阿拉尔:塔里木大学硕士学位论文,2011
[6]Zhang ZY.Study on secondary metabolites of a halophilic bacterium[D].Kunming:Master’s Thesis of Yunnan Normal University,2015(in Chinese)张正芸.一株嗜盐菌次生代谢产物研究[D].昆明:云南师范大学硕士学位论文,2015
[7]Cao LL.Study on diversity and polyphasic taxonomy of actinomycetes isolated from Hami Salt Lakes in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2009(in Chinese)曹兰兰.新疆哈密地区盐湖放线菌多样性及多相分类研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2009
[8]Izzat O.SD modeling of Turpan oasis socia~ecological system drawing on Luhhmann’s social theory[D].Urumqi:Master’s Thesis of Xinjiang University,2014(in Chinese)依再提·乌斯满.基于卢曼社会理论的吐鲁番绿洲社会~生态系统动态仿真研究[D].乌鲁木齐:新疆大学硕士学位论文,2014
[9]Tang SK.Diversity of culturable actinomycetes in hypersaline environment of Xinjiang[D].Kunming:Doctoral Dissertation of Yunnan University,2007(in Chinese)唐蜀昆.新疆高盐环境可培养放线菌多样性研究[D].昆明:云南大学博士学位论文,2007
[10]Zhang YJ.A new study for isolation of actinomycetes in salt environment based on Biolog technology[D].Kunming:Master’s Thesis of Yunnan University,2012(in Chinese)张芸娇.基于Biolog技术对盐环境放线菌分离方法的新探究[D].昆明:云南大学硕士学位论文,2012
[11]Jia M.Mining of actinomycetales recources in hypersaline environment from Aidinglake[D].Kunming:Master’s Thesis of Yunnan University,2014(in Chinese)贾鳗.艾丁湖地区高盐环境放线菌物种资源的挖掘[D].昆明:云南大学硕士学位论文,2014
[12]Liu J.Isolation,diversity and antibiotic activity of actinomycetes from Apis cerana cerana Fabricius[D].Chengdu:Master’s Thesis of Sichuan Agricultural University,2015(in Chinese)刘娟.中华蜂内共生放线菌的分离、多样性及抗菌活性研究[D].成都:四川农业大学硕士学位论文,2015
[13]Liao P,Zhao C,Zhang HN,et al.Diversity and antimicrobial activity of endophytic actinobacteria isolated from Melia toosendan Sieb.et Zucc[J].Microbiology China,2016,43(11):2374-2385(in Chinese)廖萍,赵翀,张瀚能,等.川楝内生放线菌多样性及抗菌活性筛选[J].微生物学通报,2016,43(11):2374-2385
[14]Zhang YQ,Li WJ,Chen GZ,et al.Comparison of five PCR template preparation methods for fast identification of actinobacteria[J].Biotechnology,2004,14(5):37-39(in Chinese)张玉琴,李文均,陈国忠,等.PCR法快速识别Actinobacteria的五种模板制备方法的比较[J].生物技术,2004,14(5):37-39
[15]Zhou SQ,Huang XL,Huang DY,et al.A rapid method for extracting DNA from actinomycetes by Chelex-100[J].Biotechnology Bulletin,2010(2):123-125(in Chinese)周双清,黄小龙,黄东益,等.Chelex-100快速提取放线菌DNA作为PCR扩增模板[J].生物技术通报,2010(2):123-125
[16]Hall TA.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic Acids Symposium Series,1999,41(41):95-98
[17]Lu KY,Zhang YF,Jia M,et al.Bacteria distribution diversity in tea rhizospheric soil from different habitats at nannuo mountain based on 16S r RNA sequence analysis[J].Journal of Kunming University of Science and Technology(Natural Science Edition),2016,41(2):89-95(in Chinese)卢开阳,张云峰,贾鳗,等.基于16S r RNA序列分析南糯山不同生境茶树根际土壤细菌群落分布多样性[J].昆明理工大学学报:自然科学版,2016,41(2):89-95
[18]Bascom-Slack CA,Ma C,Moore E,et al.Multiple,novel biologically active endophytic actinomycetes isolated from upper Amazonian rainforests[J].Microbial Ecology,2009,58(2):374-383
[19]Tamura K,Dudley J,Nei M,et al.MEGA4:Molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599
[20]Saitou N,Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425
[21]Zhang WQ,Zhang YG,Fang BZ,et al.Studies on diversity of culturable bacteria in karst cave soil of Xingyi,Guizhou and their protease and amylase activities[J].Microbiology China,2016,43(5):955-964(in Chinese)张万芹,张永光,房保柱,等.贵州兴义喀斯特洞穴土可培养细菌多样性及其产蛋白酶、淀粉酶活性筛选[J].微生物学通报,2016,43(5):955-964
[22]Yang YL,Zhao C,Liao P,et al.Diversity and antimicrobial activity of endophytic actinobacteria from Glycyrrhizain flata Bat[J].Microbiology China,2016,43(10):2138-2147(in Chinese)杨雅琳,赵翀,廖萍,等.塔里木盆地胀果甘草内生放线菌多样性及抗菌活性分析[J].微生物学通报,2016,43(10):2138-2147
[23]Zhao LF,Xu YJ,Peng DH,et al.Screening and inhibitory effect of antagonistic endophytic bacteria associated with soybean root nodules against Magnaporthe Grisea[J].Microbiology China,2016,43(5):998-1008(in Chinese)赵龙飞,徐亚军,彭顶华,等.稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果[J].微生物学通报,2016,43(5):998-1008
[24]Tan L,Yuan B,Qin S,et al.Isolation,screening and preliminary identification of endophytic actinobacteria from Ginkgo biloba[J].Microbiology China,2015,42(6):1043-1051(in Chinese)谭力,袁博,秦盛,等.邳州银杏内生放线菌分离、筛选及活性菌株鉴定[J].微生物学通报,2015,42(6):1043-1051
[25]Dilbar T,Xu XJ.The numerical distribution of halophilic bacteria and halotolerant bacteria in Aydin Lake and the surrounding area[J].Acta Ecologica Sinica,2001,21(8):1388-1391(in Chinese)迪丽拜尔·托乎提,徐晓晶.新疆艾丁湖及邻近地区嗜盐菌和耐盐菌的数量分布[J].生态学报,2001,21(8):1388-1391
[26]Dilbar T,Xu XJ,Jiamila M.A study of halobacteria in the extreme environment arount the Ayding Lake[J].Arid Zone Research,1999,16(1):25-28(in Chinese)迪丽拜尔·托乎提,徐晓晶,加米拉·买买提.艾丁湖极端环境中嗜盐菌的研究[J].干旱区研究,1999,16(1):25-28
[27]Dong XH.Diversity of halophilic bacteria in Xinjiang two salt lakes using an integrated approach of BIOLOG carbon source screening and culture-dependent methods[D].Urumqi:Master’s Thesis of Xinjiang University,2013(in Chinese)董秀黄.结合BIOLOG碳源筛选研究新疆两盐湖可培养嗜盐菌多样性[D].乌鲁木齐:新疆大学硕士学位论文,2013
[28]Xia ZF.Studies on the actinobacterial diversity in aiding lake sediment[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)夏占峰.艾丁湖沉积物放线菌多样性研究[D].阿拉尔:塔里木大学硕士学位论文,2011
[2]Han J.Study on biodiversity and enzyme-producing characteristic of halophilic bacteria from Lop Nur Region in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2008(in Chinese)韩剑.新疆罗布泊嗜盐细菌的多样性及产酶特性研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2008
[3]Liu BB.Diversity and functional enzyme screening of cultured halophilic archaea in Lop Nur Region[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)刘冰冰.新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选[D].阿拉尔:塔里木大学硕士学位论文,2011
[4]Shi R.Diversity of culturable actinobacteria and PKS I gene from Qijiaojing salt lake in Xinjiang[D].Kunming:Master’s Thesis of Yunnan University,2011(in Chinese)石蓉.新疆七角井盐湖可培养放线菌及其I型PKS基因多样性研究[D].昆明:云南大学硕士学位论文,2011
[5]Chen C.Diversity of culturable bacteria from Qijiaojing Lake in Xinjiang and their ability to produce enzymes[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)陈超.新疆七角井盐湖可培养细菌多样性及其功能酶筛选[D].阿拉尔:塔里木大学硕士学位论文,2011
[6]Zhang ZY.Study on secondary metabolites of a halophilic bacterium[D].Kunming:Master’s Thesis of Yunnan Normal University,2015(in Chinese)张正芸.一株嗜盐菌次生代谢产物研究[D].昆明:云南师范大学硕士学位论文,2015
[7]Cao LL.Study on diversity and polyphasic taxonomy of actinomycetes isolated from Hami Salt Lakes in Xinjiang[D].Urumqi:Master’s Thesis of Xinjiang Agricultural University,2009(in Chinese)曹兰兰.新疆哈密地区盐湖放线菌多样性及多相分类研究[D].乌鲁木齐:新疆农业大学硕士学位论文,2009
[8]Izzat O.SD modeling of Turpan oasis socia~ecological system drawing on Luhhmann’s social theory[D].Urumqi:Master’s Thesis of Xinjiang University,2014(in Chinese)依再提·乌斯满.基于卢曼社会理论的吐鲁番绿洲社会~生态系统动态仿真研究[D].乌鲁木齐:新疆大学硕士学位论文,2014
[9]Tang SK.Diversity of culturable actinomycetes in hypersaline environment of Xinjiang[D].Kunming:Doctoral Dissertation of Yunnan University,2007(in Chinese)唐蜀昆.新疆高盐环境可培养放线菌多样性研究[D].昆明:云南大学博士学位论文,2007
[10]Zhang YJ.A new study for isolation of actinomycetes in salt environment based on Biolog technology[D].Kunming:Master’s Thesis of Yunnan University,2012(in Chinese)张芸娇.基于Biolog技术对盐环境放线菌分离方法的新探究[D].昆明:云南大学硕士学位论文,2012
[11]Jia M.Mining of actinomycetales recources in hypersaline environment from Aidinglake[D].Kunming:Master’s Thesis of Yunnan University,2014(in Chinese)贾鳗.艾丁湖地区高盐环境放线菌物种资源的挖掘[D].昆明:云南大学硕士学位论文,2014
[12]Liu J.Isolation,diversity and antibiotic activity of actinomycetes from Apis cerana cerana Fabricius[D].Chengdu:Master’s Thesis of Sichuan Agricultural University,2015(in Chinese)刘娟.中华蜂内共生放线菌的分离、多样性及抗菌活性研究[D].成都:四川农业大学硕士学位论文,2015
[13]Liao P,Zhao C,Zhang HN,et al.Diversity and antimicrobial activity of endophytic actinobacteria isolated from Melia toosendan Sieb.et Zucc[J].Microbiology China,2016,43(11):2374-2385(in Chinese)廖萍,赵翀,张瀚能,等.川楝内生放线菌多样性及抗菌活性筛选[J].微生物学通报,2016,43(11):2374-2385
[14]Zhang YQ,Li WJ,Chen GZ,et al.Comparison of five PCR template preparation methods for fast identification of actinobacteria[J].Biotechnology,2004,14(5):37-39(in Chinese)张玉琴,李文均,陈国忠,等.PCR法快速识别Actinobacteria的五种模板制备方法的比较[J].生物技术,2004,14(5):37-39
[15]Zhou SQ,Huang XL,Huang DY,et al.A rapid method for extracting DNA from actinomycetes by Chelex-100[J].Biotechnology Bulletin,2010(2):123-125(in Chinese)周双清,黄小龙,黄东益,等.Chelex-100快速提取放线菌DNA作为PCR扩增模板[J].生物技术通报,2010(2):123-125
[16]Hall TA.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic Acids Symposium Series,1999,41(41):95-98
[17]Lu KY,Zhang YF,Jia M,et al.Bacteria distribution diversity in tea rhizospheric soil from different habitats at nannuo mountain based on 16S r RNA sequence analysis[J].Journal of Kunming University of Science and Technology(Natural Science Edition),2016,41(2):89-95(in Chinese)卢开阳,张云峰,贾鳗,等.基于16S r RNA序列分析南糯山不同生境茶树根际土壤细菌群落分布多样性[J].昆明理工大学学报:自然科学版,2016,41(2):89-95
[18]Bascom-Slack CA,Ma C,Moore E,et al.Multiple,novel biologically active endophytic actinomycetes isolated from upper Amazonian rainforests[J].Microbial Ecology,2009,58(2):374-383
[19]Tamura K,Dudley J,Nei M,et al.MEGA4:Molecular evolutionary genetics analysis(MEGA)software version 4.0[J].Molecular Biology and Evolution,2007,24(8):1596-1599
[20]Saitou N,Nei M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425
[21]Zhang WQ,Zhang YG,Fang BZ,et al.Studies on diversity of culturable bacteria in karst cave soil of Xingyi,Guizhou and their protease and amylase activities[J].Microbiology China,2016,43(5):955-964(in Chinese)张万芹,张永光,房保柱,等.贵州兴义喀斯特洞穴土可培养细菌多样性及其产蛋白酶、淀粉酶活性筛选[J].微生物学通报,2016,43(5):955-964
[22]Yang YL,Zhao C,Liao P,et al.Diversity and antimicrobial activity of endophytic actinobacteria from Glycyrrhizain flata Bat[J].Microbiology China,2016,43(10):2138-2147(in Chinese)杨雅琳,赵翀,廖萍,等.塔里木盆地胀果甘草内生放线菌多样性及抗菌活性分析[J].微生物学通报,2016,43(10):2138-2147
[23]Zhao LF,Xu YJ,Peng DH,et al.Screening and inhibitory effect of antagonistic endophytic bacteria associated with soybean root nodules against Magnaporthe Grisea[J].Microbiology China,2016,43(5):998-1008(in Chinese)赵龙飞,徐亚军,彭顶华,等.稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果[J].微生物学通报,2016,43(5):998-1008
[24]Tan L,Yuan B,Qin S,et al.Isolation,screening and preliminary identification of endophytic actinobacteria from Ginkgo biloba[J].Microbiology China,2015,42(6):1043-1051(in Chinese)谭力,袁博,秦盛,等.邳州银杏内生放线菌分离、筛选及活性菌株鉴定[J].微生物学通报,2015,42(6):1043-1051
[25]Dilbar T,Xu XJ.The numerical distribution of halophilic bacteria and halotolerant bacteria in Aydin Lake and the surrounding area[J].Acta Ecologica Sinica,2001,21(8):1388-1391(in Chinese)迪丽拜尔·托乎提,徐晓晶.新疆艾丁湖及邻近地区嗜盐菌和耐盐菌的数量分布[J].生态学报,2001,21(8):1388-1391
[26]Dilbar T,Xu XJ,Jiamila M.A study of halobacteria in the extreme environment arount the Ayding Lake[J].Arid Zone Research,1999,16(1):25-28(in Chinese)迪丽拜尔·托乎提,徐晓晶,加米拉·买买提.艾丁湖极端环境中嗜盐菌的研究[J].干旱区研究,1999,16(1):25-28
[27]Dong XH.Diversity of halophilic bacteria in Xinjiang two salt lakes using an integrated approach of BIOLOG carbon source screening and culture-dependent methods[D].Urumqi:Master’s Thesis of Xinjiang University,2013(in Chinese)董秀黄.结合BIOLOG碳源筛选研究新疆两盐湖可培养嗜盐菌多样性[D].乌鲁木齐:新疆大学硕士学位论文,2013
[28]Xia ZF.Studies on the actinobacterial diversity in aiding lake sediment[D].Alar:Master’s Thesis of Tarim University,2011(in Chinese)夏占峰.艾丁湖沉积物放线菌多样性研究[D].阿拉尔:塔里木大学硕士学位论文,2011