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 LIU Xiaoyun,ZHANG Bin,WU Wei & LI Qiaoxian.Verification of Nodulation Ability of Strains Isolated from Mimosa spp. Nodules Using Whole Cell Protein SDS-PAGE Molecular Marker Method[J].Chinese Journal of Applied & Environmental Biology,2009,15(05):719-723.[doi:10.3724/SP.J.1145.2009.00719]





Verification of Nodulation Ability of Strains Isolated from Mimosa spp. Nodules Using Whole Cell Protein SDS-PAGE Molecular Marker Method
(1河北大学生命科学学院,河北省微生物多样性研究与应用实验室 保定 071002)
(2西南林学院资源学院,西南地区生物多样性保育国家林业局重点实验室 昆明 650224)
(3西南林学院保护生物学院 昆明 650224)
(4云南省草地动物科学研究院 昆明 650212)
LIU Xiaoyun ZHANG Bin WU Wei & LI Qiaoxian
(1College of Life Sciences, Hebei University, Key Laboratory of Microbial Diversity Research and Application of
Hebei Province, Baoding 071002, Hebei, China)
(2College of Resources, Southwest Forestry College, Key Laboratory of Biodiversity Conservation in Southwest China of
State Forestry Administration, Kunming 650224, China)
(3College of Conservation Biology, Southwest Forestry College, Kunming 650224, China)
(4Academy of Grassland and Animal Science of Yunnan, Kunming 650212, China)
Mimosa rhizobia molecular marker whole cell protein pattern SDS-PAGE
对云南省热带及亚热带地区的含羞草根瘤菌进行了分离,选择其中40株菌为接种菌株,通过结瘤试验并采用全细胞蛋白SDS-PAGE分子标记方法研究了其结瘤能力. 经过结瘤试验,发现除菌株SWF66075和SWF66093没有结瘤外,其它38株菌株均与含羞草植物结瘤,结瘤率为95%. 从结瘤试验所获根瘤中,分离得到结瘤菌株,采用全细胞蛋白SDS-PAGE分子标记对结瘤菌株与接种菌株进行了比较研究. 蛋白图谱及聚类分析显示,26株接种菌株与其结瘤菌株的全细胞蛋白分子图谱完全相同,在100%的相似水平上与其结瘤菌株聚在一起,说明宿主植物所形成的根瘤确系接种菌株侵入所致,因而可将这些菌株确认为根瘤菌菌株;而SWF66012、SWF66029、SWF66044和SWF66058等12株菌株的结瘤菌株与其各自接种菌株的全细胞蛋白图谱存在较大差异,推测这12株接种菌株与其结瘤菌株可能不是同一菌株,尚不能确定它们与含羞草植物的结瘤能力,这些菌株是否为根瘤菌菌株仍需进一步验证. 研究结果表明,全细胞蛋白SDS-PAGE分子标记技术是一种快速、准确地验证根瘤菌结瘤能力的方法. 该方法进一步完善了结瘤试验,并初步揭示了根瘤菌的竞争结瘤能力,适用于对大量根瘤内分离菌株进行根瘤菌的证实研究. 图2 参28
The nodules of Mimosa spp. were collected from some areas in Yuanjiang and Xishuangbanna, Yunnan, China. Bacteria were isolated from these nodules with modified medium, and 40 strains were selected to carry out the nodulation test. The result showed that 38 strains could nodulate M. pudica. The nodulation rate was approximately 95%. 38 re-isolated strains and their whole cell protein profiles were obtained. The clustering analysis demonstrated that 26 re-isolated strains had a protein pattern identical to that of their original strains at the similarity of 100%, indicating that these 26 strains could establish symbiosis with M. pudica. The whole cell protein profiles of 12 strains were different from those of the re-isolated strains, indicating that the nodules were not formed by them and further research is needed. This study shows that the whole cell protein SDS-PAGE can be used in inoculation test, and it is a time-saving and accurate molecular marker to identify rhizobia, especially for characterizing plenty of isolates from nodules of various plants. Also, this study preliminarily reveals that the competitive nodulation ability differs among rhizobial strains. Fig 2, Ref 28


1 Wang LL, Wang ET, Liu J, Li Y, Chen WX. Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens. Microbial Ecol, 2006, 52: 436~443
2 Wang ET, Rogel MA, Garcia-de los Santos A, Martinez-Romero J, Cevallos MA. Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis. Int J Syst Evol Microbiol, 1999, 49: 1479~1491
3 Chen WM, Laevens S, Lee TM, Coenye T, De Vos P, Mergeay M, Vandamme P. Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol, 2001, 51: 1729~1735
4 Chen WM, Moulin L, Bontemps C, Vandamme P, Bena G, Boivin-Masson C. Legume symbiotic nitrogen fixation by β-proteobacteria is widespread in nature. J Bacteriol, 2003b, 185 (24): 7266~7272
5 Elliott GN, Chen WM, Chou JH, Wang HC, Sheu SY, Perin L, Reis VM, Moulin L, Simon MF, Bontemps C, Sutherland JM, Bessi R, De Faria SM, Trinick MJ, Prescott AR, Sprent JI, James SEK. Burkholderia phymatum is a highly effective nitrogenfixing symbiont of Mimosa spp. and fixes nitrogen ex planta. New Phytol, 2007, 173: 168~180
6 Chen WM, James EK, Coenye T, Chou JH, Barrios E, De Faria SM, Elliott GN, Shih YS, Sprent JI, Vandamme P. Burkholderia mimosarum sp. nov., isolated from root nodules of Mimosa spp. from Taiwan and South America. Int J Syst Evol Microbiol, 2006, 58: 1847~1851
7 Chen WM, De Faria SM, James EK, Elliott GN, Lin KY, Chou JH, Sheu SY, Cnockaert M, Sprent JI, Vandamme P. Burkholderia nodosa sp. nov., isolated from root nodules of the woody Brazilian legumes Mimosa bimucronata and Mimosa scabrella. Int J Syst Evol Microbiol, 2007, 57: 1055~1059
8 Chen WM, De Faria SM, Chou JH, James EK, Elliott GN, Sprent JI, Bontemps C, Young JP, Vandamme P. Burkholderia sabiae sp. nov., isolated from root nodules of Mimosa caesalpiniifolia. Int J Syst Evol Microbiol, 2008, 58: 2174~2179
9 Parker MA, Wurtz AK, Payter Q. Nodule symbiosis of invasive Mimosa pigra in Australia and in ancestral habitats: A comparative analysis. Biol Inv, 2007, 9: 127~138
10 Chen WM, James EK, Prescott AR, Kierans M, Sprent JI. Nodulation of Mimosa spp. by the β-proteobacterium Ralstonia taiwanensis. Mol Pl Micr Interact, 2003a, 16: 1051~1061
11 Chen WM, De Faria SM, Straliotto R, Pitard RM, Simoes-Araujo JL, Chou JH, Chou YJ, Barrios E, Prescott AR, Elliott GN, Sprent JI, Young JPW, James EK. Proof that Burkholderia strains form effective symbioses with legumes: A study of novel mimosa-nodulating strains from South America. Appl Environ Microbiol, 2005a, 71 (11): 7461~7471
12 Chen WM, James EK, Chou JH, Shih YS, Yang SZ, Sprent JI. β-rhizobia from Mimosa pigra, a newly discovered invasive plant in Taiwan. New Phytol, 2005b, 168: 661~675
13 Tan ZY, Hurek T, Vinuesa P, Muller P, Ladha JK, Reinhold-Hurek B. Specific detection of Bradyrhizobium and rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR. Appl Environ Microbiol, 2001, 67 (8): 3655~3664
14 Pan F (潘峰), Wang P (王平), Hu ZJ (胡正嘉), He SJ (何绍江), Feng XM (冯新梅). Phylogenic study of rhizobia strains isolated from rice bean (Vignaum bellata L.) in Hubei by 16S-23S rDNA RFLP and 16S rRNA gene sequencing. Chin J Appl Environ Biol (应用与环境生物学报), 2007, 13 (1): 78~82
15 Zeng ZH (曾昭海), Chen WX (陈文新), Hu YG (胡跃高), Sui XH (隋新华), Chen DM (陈丹明). Study on competitive nodulation ability of Rhizobium melilotiin field test by using RAPD Molecular Marker Method. Acta Ecol Sin (生态学报), 2004, 24 (7): 1341~1345
16 Gao JL (高俊莲), Chen WX (陈文新), Kristina L, Terefework Z. Study on genotypic diversity of the rhizobia isolated from Astragalus adsurgens by AFLP fingerprinting. Chin J Appl Environ Biol (应用与环境生物学报), 1999, 5(4): 387~395
17 Tan ZY (谭志远), Chen WX (陈文新). SDS-PAGE of whole cell protein of new rhizobial groups and 16S rDNA sequencing of their representatives. Chin J Appl Environ Biol (应用与环境生物学报), 1998, 4 (1): 65~69
18 Vincent JM. A Manual for the Practical Study of Root Nodule Bacteria. Edinburgh: Blackwell Scientific Pubilications, 1970
19 Gu JG (顾金刚). Rulers for the Collection Characteration Preservation Management of Microorganism Resources No. 1. Beijing, China (北京): Chinese Agricultural Press, 2005
20 Somasegaran P, Hoben HJ. Handbook for Rhizobia. Methods in Legume-Rhizobium Technology. New York: Springer – Verlag, 1994
21 Guo YJ (郭尧君). Experimental Techniques of Protein Electrophoresis. Beijing, China (北京): Science Press, 1999
22 Liu XY, Wang ET, Li Y, Chen WX. Diverse bacteria isolated from root nodules of Trifolium, Crotalaria and Mimosa grown in the subtropical regions of China. Arch Microbiol, 2007, 188: 1~14
23 Chen DM (陈丹明). Screening and molecular labeling of highly effective symbiotic rhizobia on alfalfa: [Master’s Degree Dissertation]. Beijing, China (北京): China Agricultural University, 2002
24 Qi J (祁娟). Screening endogenous rhizobia from alfalfa seeds and their promoting alfalfa seedlings growth property: [Master’s Degree Dissertation]. Lanzhou, China (兰州): Gansu Agricultural University, 2006
25 Benhizia Y, Benhizia H, Benguedouar A, Muresu R, Giacomini A, Squartini A. Gamma proteobacteria can nodulate legumes of the genus Hedysarum. Syst Appl Microbiol, 2004, 27: 462~468
26 Li JH, Wang ET, Chen WF, Chen WX. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem, 2008, 40: 238~246
27 Muresu R, Polone E, Sulas L, Baldan B, Tondello A, Delogu G, Cappuccinelli P, Alberghini S, Benhizia Y, Benhizia H, Benguedouar A, Mori B, Calamassi R, Dazzo FB, Squartini A. Coexistence of predominantly nonculturable rhizobiawith diverse, endophytic bacterial taxa within nodules of wild legumes. Microbial Ecol, 2008, 63: 383~400
28 Liu XY (刘晓云), Chen WX (陈文新), Zhang B (张斌). β-proteorhizobia and nonrhizobial species—A review. Acta Microbiol Sin (微生物学报), 2008, 48 (10): 1408~1412


 Wang Suying,Chen Wenxin.PARTIAL 16S rRNA GENE SEQUENCING OF RHIZOBIAL STRAINS CA8561 AND JL84 ISOLATED FROM ASTRAGALUS[J].Chinese Journal of Applied & Environmental Biology,1996,2(05):393.
[2]张小平,李登煜,K. Lindstrom,等.花生根瘤菌的生长特性和脂肪酸组成研究[J].应用与环境生物学报,1997,3(01):44.
 Zhang Xiaoping,Li Dengyu,Giselle Nick,et al.GROWTH CHARACTERISTICS AND FATTY ACID ANALYSES OF BRADYRHIZOBIUM STRAINS ISOLATED FROM THE ROOT NODULES OF PEANUT(ARACHIS)[J].Chinese Journal of Applied & Environmental Biology,1997,3(05):44.
 CHEN Qiang,et al..Genetic Diversity of Rhizobia Isolated from Some Legumes in Sichuan, China[J].Chinese Journal of Applied & Environmental Biology,2008,14(05):83.
 CHEN Wenfeng,et al..Cloning, Sequencing and Phylogenetic Analysis of sinR Gene in Three Rhizobia Associated with Alfalfa[J].Chinese Journal of Applied & Environmental Biology,2008,14(05):240.
[5]王树和 王昶 王晓娟 金樑.根瘤菌、丛枝菌根(AM)真菌与宿主植物共生的分子机理*[J].应用与环境生物学报,2008,14(05):721.
 LU Junkun,XU Daping,YANG Zengjiang,et al.Isolation and Identification of Slow-growing Rhizobium DG and Its Symbiosis with Dalbergia odorifera[J].Chinese Journal of Applied & Environmental Biology,2011,17(05):379.[doi:10.3724/SP.J.1145.2011.00379]
[7]杨姗姗,张红蕊,伏圣秘,等.一株产聚羟基脂肪酸酯根瘤菌Rhizobium sp. H2-5的筛选与鉴定[J].应用与环境生物学报,2013,19(02):375.[doi:10.3724/SP.J.1145.2013.00375]
 YANG Shanshan,ZHANG Hongrui,FU Shengmi,et al.Screening and Identification of a Polyhydroxyalkanoate Producing Strain Rhizobium sp. H2-5[J].Chinese Journal of Applied & Environmental Biology,2013,19(05):375.[doi:10.3724/SP.J.1145.2013.00375]
 LI Yanmei,ZHONG Yuzhou,TAN Yuan,et al.Diversity of rhizobia nodulating Astragalus sinicus, Medicago sativa and Trifolium repens in nodulated soybean rhizosphere soil in Sichuan[J].Chinese Journal of Applied & Environmental Biology,2015,21(05):234.[doi:10.3724/SP.J.1145.2014.08021]
[9]康 夏 郑雯文 瞿 奎 崔永亮 李艳梅 陈 强 余秀梅.钒钛磁铁废矿土壤中根瘤菌的捕获及其共生固氮效应*[J].应用与环境生物学报,2016,22(02):230.[doi:10.3724/SP.J.1145.2015.09025]
 KANG Xia,ZHENG Wenwen,QU Kui,et al.The trapping of rhizobia in waste V-Ti magnetite mine soils and theirsymbiotic nitrogen-fixing effects*[J].Chinese Journal of Applied & Environmental Biology,2016,22(05):230.[doi:10.3724/SP.J.1145.2015.09025]


国家自然科学基金项目(No. 30570001)和国家林业局重点实验室开放基金(No. KL200703)资助 Supported by the National Natural Science Foundation of China (No. 30570001) and the Fund of Key Laboratory of the State Forestry Administration-Laboratory for Biodiversity Preservation in Southwest China (No. KL200703)
更新日期/Last Update: 2009-10-26