四川白三叶根瘤菌遗传多样性及系统发育研究
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摘要
为阐明四川部分地区野生白三叶根瘤菌的遗传多样性及系统发育地位,对分离自四川雅安、康定、泸定、西昌、成都和乐山6个地区白三叶根瘤的69株菌进行系统研究。采用16SrDNA限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP)和16SrDNA基因、持家基因(recA、atpD、glnII)、结瘤基因(nodC)、固氮基因(nifH)系统发育分析的方法进行了研究。结果表明,16SrDNA PCR-RFLP中所有供试菌株产生了4种酶切图谱类型,表现出较为丰富的遗传多样性。持家基因与16SrDNA基因系统发育分析结果基本一致,9株代表菌株主要分布在α-变形菌纲(Alpha-Proteobacteria)的根瘤菌属(Rhizobium),并与豌豆根瘤菌三叶草生物型(R.leguminosarumbv.trifolii)ATCC 14480T的亲缘关系较近。PCR可扩增出nodC和nifH基因片段,但从属于土壤杆菌属(Agrobacterium)的菌株LS1105中则扩增不出这两个基因。所有供试菌株被鉴定到了种的水平,证实了68株为白三叶根瘤菌,并通过不同采样地点菌株之间的比较,发现白三叶与根瘤菌的共生关系因地理分布不同而具有多样性,对于丰富白三叶根瘤菌资源及其开发利用具有重要意义。
The objective of this study was to determine the genetic diversity and phylogeny of 69 isolates of symbiotic bacteria associated with wild white clover obtained from six sites in Sichuan Province,China:Ya'an,Kangding,Luding,Xichang,Chengdu and Leshan.Polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)and phylogenetic analyses of the 16 SrDNA gene,housekeeping genes(recA,glnII and atpD),nodulation gene(nodC)and nitrogen-fixing gene(nifH)were conducted in the research.Four PCR-RFLP fingerprint patterns were identified among the 69 isolates from the 16 SrDNA analysis,indicating relatively high genetic diversity.The results of phylogenetic analysis of 16 SrDNA and housekeeping genes were similar;nine representative strains mainly belonged to the genus Rhizobium in Alpha-Proteobacteria were identified.In addition,nodCand nifH genes from strains from the Rhizobium genus were successfully amplified by PCR,but strains belonging to the Agrobacteriumgenus were not able to be amplified.All of the isolates could be identified by species with sixty-eight strains confirmed as white clover rhizobia.The diversity of white clover rhizobia differed at different sites,indicating the potential to increase the availability of beneficial rhizobia strains.
The objective of this study was to determine the genetic diversity and phylogeny of 69 isolates of symbiotic bacteria associated with wild white clover obtained from six sites in Sichuan Province,China:Ya'an,Kangding,Luding,Xichang,Chengdu and Leshan.Polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP)and phylogenetic analyses of the 16 SrDNA gene,housekeeping genes(recA,glnII and atpD),nodulation gene(nodC)and nitrogen-fixing gene(nifH)were conducted in the research.Four PCR-RFLP fingerprint patterns were identified among the 69 isolates from the 16 SrDNA analysis,indicating relatively high genetic diversity.The results of phylogenetic analysis of 16 SrDNA and housekeeping genes were similar;nine representative strains mainly belonged to the genus Rhizobium in Alpha-Proteobacteria were identified.In addition,nodCand nifH genes from strains from the Rhizobium genus were successfully amplified by PCR,but strains belonging to the Agrobacteriumgenus were not able to be amplified.All of the isolates could be identified by species with sixty-eight strains confirmed as white clover rhizobia.The diversity of white clover rhizobia differed at different sites,indicating the potential to increase the availability of beneficial rhizobia strains.
引文
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[3]马霞,王丽丽,李卫军,等.不同施氮水平下接种根瘤菌对苜蓿固氮效能及种子生产的影响[J].草业学报,2013,22(1):95-102.
[4]李智燕,邢学峰,唐华,等.铝和酸胁迫对苜蓿根瘤菌生长和抗氧化酶系的影响[J].草业学报,2013,22(3):146-153.
[5]王卫栋,杨培志,张攀,等.共生根瘤菌对NaCl胁迫下紫花苜蓿抗氧化和渗透调节能力的影响[J].草业学报,2013,22(5):120-127.
[6]贾慎修,祝廷成,黄文惠,等.中国饲用植物志[M].北京:农业出版社,1987:321-337.
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[11]吕飞,蒋欣,徐佳洁,等.新疆和陕西三叶草属根瘤菌16SrDNA多态性及系统发育研究[J].草地学报,2009,17(3):304-309.
[12]吕飞.新疆和陕西部分地区三叶草属根瘤菌多样性及系统发育研究[D].西安:西北农林科技大学,2009.
[13]刘晓云,戴燕燕,郭振国,等.三叶草根瘤菌SDS-PAGE分析及结瘤试验分子验证[J].草业科学,2010,27(1):79-84.
[14]Woese C R.Bacterial evolution[J].Microbiological Reviews,1987,51(2):221.
[15]Vandamme P,Pot B,Gillis M,et al.Polyphasic taxonomy,a consensus approach to bacterial systematics[J].Microbiological Reviews,1996,60(2):407-438.
[16]王风芹,张勇法,刘杰,等.合欢,金合欢和银合欢根瘤菌系统发育研究方法比较[J].微生物学报,2008,48(1):1-7.
[17]Gevers D,Cohan F M,Lawrence J G,et al.Re-evaluating prokaryotic species[J].Nature Reviews Microbiology,2005,3(9):733-739.
[18]窦雅静,陆俊锟,康丽华,等.黑木相思根瘤菌遗传多样性[J].微生物学报,2012,52(12):1439-1448.
[19]Martens M,Dawyndt P,Coopman R,et al.Advantages of multilocus sequence analysis for taxonomic studies:a case study using 10housekeeping genes in the genus Ensifer(including former Sinorhizobium)[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(1):200-214.
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[21]韦革宏,刘虎岐,孙科技,等.河南省豆科植物根瘤菌表型多样性研究[J].西北农林科技大学学报(自然科学版),2002,30(3):71-75.
[22]Bergersen F J.The growth of Rhizobiumin synthetic media[J].Australian Journal of Biological Sciences,1961,14(3):349-360.
[23]陈强,张小平,李登煜,等.从豆科植物的根瘤中直接提取根瘤菌DNA的方法[J].微生物学通报,2002,29(6):63-67.
[24]谭志远,牛天贵,陈文新.天山根瘤菌(R.tianshanense)模式菌株16SrDNA全序列测定及其系统发育[J].高技术通讯,1997,1:5-8.
[25]Selenska-Pobell S,Evguenieva-Hackenberg E,Radeva G,et al.Characterization of Rhizobium‘hedysari’by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting[J].Journal of Applied Microbiology,1996,80(5):517-528.
[26]Thompson J D,Gibson T J,Plewniak F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(24):4876-4882.
[27]Van de Peer Y,De Wachter R.TREECON for Windows:a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment[J].Computer Applications in the Biosciences:CABIOS,1994,10(5):569-570.
[28]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.
[29]Islam M S,Kawasaki H,Muramatsu Y,et al.Bradyrhizobium iriomotense sp.nov.,isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan[J].Bioscience,Biotechnology,and Biochemistry,2008,72(6):1416-1429.
[30]Li M,Li Y,Chen W F,et al.Genetic diversity,community structure and distribution of rhizobia in the root nodules of Caragana spp.from arid and semi-arid alkaline deserts,in the north of China[J].Systematic and Applied Microbiology,2012,35(4):239-245.
[31]谭志远,陈文新.根瘤菌新类群代表菌株的16SrDNA全序列测定及其系统发育地位[J].微生物学报,1997,37(6):411-416.
[32]Chen W X,Wang E T,Chen W F.The relationship between the symbiotic promiscuity of rhizobia and legumes and their geographical environments[J].Scientia Agricultura Sinica,2004,37(1):81-86.
[33]Tian C F,Zhou Y J,Zhang Y M,et al.Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations[J].Proceedings of the National Academy of Sciences,2012,109(22):8629-8634.
[34]杨亚珍,韦革宏,万晓红,等.西北地区甘草根瘤菌的表型多样性研究[J].微生物学通报,2004,31(2):20-25.
[35]何恒斌,贾昆峰,贾桂霞,等.沙冬青根瘤菌的抗逆性[J].植物生态学报,2006,30(1):140-146.
[36]阎爱民,陈文新.苜蓿,草木樨,锦鸡儿根瘤菌的表型多样性分析[J].生物多样性,1999,7(2):1-8.
[37]冀玉良,李堆淑,赵龙飞,等.陕西商洛部分地区刺槐根瘤菌的遗传多样性和系统发育[J].农业生物技术学报,2013,21(11):1373-1383.
[38]Gaunt M W,Turner S L,Rigottier-Gois L,et al.Phylogenies of atpDand recAsupport the small subunit rRNA-based classification of rhizobia[J].International Journal of Systematic and Evolutionary Microbiology,2001,51(6):2037-2048.
[39]Fox G E,Wisotzkey J D,Jurtshuk P.How close is close:16SrRNA sequence identity may not be sufficient to guarantee species identity[J].International Journal of Systematic Bacteriology,1992,42(1):166-170.
[40]Liu X Y,Wang E T,Li Y,et al.Diverse bacteria isolated from root nodules of Trifolium,Crotalaria and Mimosa grown in the subtropical regions of China[J].Archives of Microbiology,2007,188(1):1-14.
[41]Mhamdi R,Mrabet M,Laguerre G,et al.Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains[J].Canadian Journal of Microbiology,2005,51(2):105-111.
[42]Mhamdi R,Laguerre G,Aouani M E,et al.Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils[J].FEMS Microbiology Ecology,2002,41(1):77-84.
[43]Wang L L,Wang E T,Liu J,et al.Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens[J].Microbial Ecology,2006,52(3):436-443.
[44]常佳丽.三叶草根瘤中根瘤菌与土壤杆菌关系的初步研究[D].西安:西北农林科技大学,2010.
[2]陈文新,汪恩涛.中国根瘤菌[M].北京:科学出版社,2011.
[3]马霞,王丽丽,李卫军,等.不同施氮水平下接种根瘤菌对苜蓿固氮效能及种子生产的影响[J].草业学报,2013,22(1):95-102.
[4]李智燕,邢学峰,唐华,等.铝和酸胁迫对苜蓿根瘤菌生长和抗氧化酶系的影响[J].草业学报,2013,22(3):146-153.
[5]王卫栋,杨培志,张攀,等.共生根瘤菌对NaCl胁迫下紫花苜蓿抗氧化和渗透调节能力的影响[J].草业学报,2013,22(5):120-127.
[6]贾慎修,祝廷成,黄文惠,等.中国饲用植物志[M].北京:农业出版社,1987:321-337.
[7]李州,彭燕,苏星源.不同叶型白三叶抗氧化保护及渗透调节生理对干旱胁迫的响应[J].草业学报,2013,22(2):257-263.
[8]Buchanan R E,Gibbonsbian N E.伯杰氏鉴定细菌学手册(第8版)[M].北京:科学出版社,1974:658.
[9]Jordan D C.Family III.Rhizobiaceae[A].In:Krieg N R,Holt J G.Bergey’s Manual of Systematic Bacteriology[M].The Williams and Wilkins Co.,Baltimore,Md,1984:234-242.
[10]刘晓云,陈文新.三叶草、猪屎豆和含羞草植物根瘤菌16SrDNA PCR-RFLP分析和数值分类研究[J].中国农业大学学报,2003,8(3):1-6.
[11]吕飞,蒋欣,徐佳洁,等.新疆和陕西三叶草属根瘤菌16SrDNA多态性及系统发育研究[J].草地学报,2009,17(3):304-309.
[12]吕飞.新疆和陕西部分地区三叶草属根瘤菌多样性及系统发育研究[D].西安:西北农林科技大学,2009.
[13]刘晓云,戴燕燕,郭振国,等.三叶草根瘤菌SDS-PAGE分析及结瘤试验分子验证[J].草业科学,2010,27(1):79-84.
[14]Woese C R.Bacterial evolution[J].Microbiological Reviews,1987,51(2):221.
[15]Vandamme P,Pot B,Gillis M,et al.Polyphasic taxonomy,a consensus approach to bacterial systematics[J].Microbiological Reviews,1996,60(2):407-438.
[16]王风芹,张勇法,刘杰,等.合欢,金合欢和银合欢根瘤菌系统发育研究方法比较[J].微生物学报,2008,48(1):1-7.
[17]Gevers D,Cohan F M,Lawrence J G,et al.Re-evaluating prokaryotic species[J].Nature Reviews Microbiology,2005,3(9):733-739.
[18]窦雅静,陆俊锟,康丽华,等.黑木相思根瘤菌遗传多样性[J].微生物学报,2012,52(12):1439-1448.
[19]Martens M,Dawyndt P,Coopman R,et al.Advantages of multilocus sequence analysis for taxonomic studies:a case study using 10housekeeping genes in the genus Ensifer(including former Sinorhizobium)[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(1):200-214.
[20]Vincent J M.The cultivation,isolation and maintenance of rhizobia[A].In:Vincent J M.A Manual for the Practical Study of the Root-nodule Bacteria[M].Oxford and Edinburgh:Blackwell Scientific Publications,1970:1-13.
[21]韦革宏,刘虎岐,孙科技,等.河南省豆科植物根瘤菌表型多样性研究[J].西北农林科技大学学报(自然科学版),2002,30(3):71-75.
[22]Bergersen F J.The growth of Rhizobiumin synthetic media[J].Australian Journal of Biological Sciences,1961,14(3):349-360.
[23]陈强,张小平,李登煜,等.从豆科植物的根瘤中直接提取根瘤菌DNA的方法[J].微生物学通报,2002,29(6):63-67.
[24]谭志远,牛天贵,陈文新.天山根瘤菌(R.tianshanense)模式菌株16SrDNA全序列测定及其系统发育[J].高技术通讯,1997,1:5-8.
[25]Selenska-Pobell S,Evguenieva-Hackenberg E,Radeva G,et al.Characterization of Rhizobium‘hedysari’by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting[J].Journal of Applied Microbiology,1996,80(5):517-528.
[26]Thompson J D,Gibson T J,Plewniak F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(24):4876-4882.
[27]Van de Peer Y,De Wachter R.TREECON for Windows:a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment[J].Computer Applications in the Biosciences:CABIOS,1994,10(5):569-570.
[28]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.
[29]Islam M S,Kawasaki H,Muramatsu Y,et al.Bradyrhizobium iriomotense sp.nov.,isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan[J].Bioscience,Biotechnology,and Biochemistry,2008,72(6):1416-1429.
[30]Li M,Li Y,Chen W F,et al.Genetic diversity,community structure and distribution of rhizobia in the root nodules of Caragana spp.from arid and semi-arid alkaline deserts,in the north of China[J].Systematic and Applied Microbiology,2012,35(4):239-245.
[31]谭志远,陈文新.根瘤菌新类群代表菌株的16SrDNA全序列测定及其系统发育地位[J].微生物学报,1997,37(6):411-416.
[32]Chen W X,Wang E T,Chen W F.The relationship between the symbiotic promiscuity of rhizobia and legumes and their geographical environments[J].Scientia Agricultura Sinica,2004,37(1):81-86.
[33]Tian C F,Zhou Y J,Zhang Y M,et al.Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations[J].Proceedings of the National Academy of Sciences,2012,109(22):8629-8634.
[34]杨亚珍,韦革宏,万晓红,等.西北地区甘草根瘤菌的表型多样性研究[J].微生物学通报,2004,31(2):20-25.
[35]何恒斌,贾昆峰,贾桂霞,等.沙冬青根瘤菌的抗逆性[J].植物生态学报,2006,30(1):140-146.
[36]阎爱民,陈文新.苜蓿,草木樨,锦鸡儿根瘤菌的表型多样性分析[J].生物多样性,1999,7(2):1-8.
[37]冀玉良,李堆淑,赵龙飞,等.陕西商洛部分地区刺槐根瘤菌的遗传多样性和系统发育[J].农业生物技术学报,2013,21(11):1373-1383.
[38]Gaunt M W,Turner S L,Rigottier-Gois L,et al.Phylogenies of atpDand recAsupport the small subunit rRNA-based classification of rhizobia[J].International Journal of Systematic and Evolutionary Microbiology,2001,51(6):2037-2048.
[39]Fox G E,Wisotzkey J D,Jurtshuk P.How close is close:16SrRNA sequence identity may not be sufficient to guarantee species identity[J].International Journal of Systematic Bacteriology,1992,42(1):166-170.
[40]Liu X Y,Wang E T,Li Y,et al.Diverse bacteria isolated from root nodules of Trifolium,Crotalaria and Mimosa grown in the subtropical regions of China[J].Archives of Microbiology,2007,188(1):1-14.
[41]Mhamdi R,Mrabet M,Laguerre G,et al.Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains[J].Canadian Journal of Microbiology,2005,51(2):105-111.
[42]Mhamdi R,Laguerre G,Aouani M E,et al.Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils[J].FEMS Microbiology Ecology,2002,41(1):77-84.
[43]Wang L L,Wang E T,Liu J,et al.Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens[J].Microbial Ecology,2006,52(3):436-443.
[44]常佳丽.三叶草根瘤中根瘤菌与土壤杆菌关系的初步研究[D].西安:西北农林科技大学,2010.