跳到主要內容

臺灣博碩士論文加值系統

(44.192.20.240) 您好!臺灣時間:2024/02/24 02:19
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳柏沅
研究生(外文):Po-Yuan Chen
論文名稱:澎湖群島根瘤菌之分類及特性分析
論文名稱(外文):Characterization and taxonomy of rhizobia strains isolated from Peng-Hu
指導教授:蘇孝華楊秋忠楊秋忠引用關係
指導教授(外文):Shiawhwa-Paul SuChiu-Chung Young
學位類別:碩士
校院名稱:國立中興大學
系所名稱:土壤環境科學系
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:122
中文關鍵詞:根瘤菌
相關次數:
  • 被引用被引用:8
  • 點閱點閱:405
  • 評分評分:
  • 下載下載:73
  • 收藏至我的研究室書目清單書目收藏:6
摘 要
豆科植物約有750屬,包含16,000 ~ 19,000種分布世界各地,為所有的植物種類中最大的族群之一,並為人類重要之植物性蛋白質的來源。而土壤中根瘤菌能在豆科植物的根系或莖上著生根瘤,藉由共生固氮(symbiotic nitrogen— fixation)轉換大氣中的氮氣(N2)供給豆科植物利用。氮素為作物生長所需的主要元素之一,也是土壤中的重要養分,本研究從澎湖群島採集豆科植物之根瘤,分離純化出澎湖本土根瘤菌株,以下列方法應用於根瘤菌的分類及特性研究 : (1)生理生化特性(耐鹽性、溫度、產酸產鹼型) (2) 16S rDNA sequencing (3) 16S rDNA限制酵素DNA片段長度多樣性分析(Amplified 16S rDNA restriction analysis) (4) 蛋白質電泳圖譜分析(SDS-PAGE)。以所得資料探討澎湖群島根瘤菌的分類、多樣性及親緣關係。根據16S rDNA sequencing研究之結果,八株菌為Rhizobium,二株菌為Sinorhizobium,一株菌為Mesorhizobium,共計11株根瘤菌,可分成三個群集(GroupⅠ、Ⅱ、Ⅲ)。16S rDNA限制酵素DNA片段長度多樣性分析(amplified 16S rDNA restriction analysis)結果澎湖群島分離之根瘤菌Rhizobium屬菌株B2B2N1(2)、RB1N1(2)、Q3S2N1(2)及QB2N1(2)和其他同群菌株間差異大,相似度在0.47~0.69,Sinorhizobium屬菌株G1S1N1(2)及G1S1N1(3)間只有些微差異,相似度為0.90。蛋白質電泳圖譜(SDS-PAGE)分析顯示Rhizobium屬RB1N1(2)、RB1N1(2)”、S3L1N1(1)及B2B2N1(2)與其同群的根瘤菌株差異較大,Sinorhizobium屬菌株G1S1N1(2)及G1S1N1(3)間只有些微差異,相似度為0.87。生理生化特性測試結果顯示除了G1S1N1(3)及B2B2N1(1)之外其他根瘤菌株皆可於高鹽( 4% NaCl)濃度下生存,所有菌株可在50℃之高溫下生存,且除了W1B1N1(4)與RB1N1(2)”無法在pH4狀態下生存外,其餘9株根瘤菌耐pH之範圍極廣(pH4~pH12),以生理生化特性為基礎之親緣關係圖顯示各菌株間差異不大,相似度最低為0.90,推測是其所存在之海島地理環境而造成,且菌株之抗逆境能力極強,值得進一步應用。
Abstract
Legumes which contain 750 genera about 16,000 ~ 19,000 species in the world is one of the largest communities of all plant and supply proteins for humen. The rhizobias in soil can form nodules with legumes in root or stem and convert dinitrogen(N2) into ammonia(NH3) for legumes to use. Nitrogen is major element for plant growth and in soil. In this study rhizobias were isolated from native legume plants in Peng-Hu islands. The nodules of these lesumes were removed and sterilized. Then rhizobias in the nodules were separated, screened to obtain purified isolates. And methods which use in rhizobia taxonomy and characterization were : (1)Physiological and biochemical properties. (2)16S rDNA sequencing. (3) Amplified 16S rDNA restriction analyses(ARDRA). (4)Proteins SDS-PAGE. According to 16S rDNA sequencing, 11 rhizobias can separate into 3 groups(Group I, II and III), 8 strains belong to Rhizobium, 2 strains belong to Sinorhizobium and 1 strain is Mesorhizobium. The results of ARDRA showed that Rhizobium strains B2B2N1(2), RB1N1(2), Q3S2N1(1) and QB2N1(2) are extremely diverse, with similarity range between 0.47~0.69. Sinorhizobium strains G1S1N1(2) and G1S1N1(3) have similarity 0.90. Protein SDS-PAGE showed that Rhizobium strains RB1N1(2), RB1N1(2)’’, S3L1N1(1) and B2B2N1(2) are pretty diverse. Sinorhizobium strains G1S1N1(2) and G1S1N1(3) have similarity 0.87. The results of physiological and biochemical properties showed that all strains can tolerate 4 % NaCl (G1S1N1(3) and B2B2N1(2) are excepted) and grow well in temperature 50℃. The test of pH found that except W1B1N1(4) and RB1N1(2)’’ all strains can grow in pH between 4.0 ~ 12. The similarity base on physiological and biochemical properties are very similar, with similarity range between 0.90 ~ 1.00, suggest that strains live in the same environment and result in ability of resist osmostic stress.
目錄
中文摘要……………………………………………………………………………I
英文摘要……………………………………………………………………………II
目錄…………………………………………………………………………………Ⅲ
表次…………………………………………………………………………………Ⅳ
圖次…………………………………………………………………………………Ⅴ
壹、前言……………………………………………………………………………1
貳、前人研究..................................................................................................3
一、細菌分類學簡介....................................................................................3
二、細菌的分類特徵……………………………………………………………4
三、根瘤菌之分類及背景………………………………………………......11
四、根瘤菌耐受性……………………………………………………………14
參、材料與方法………………………………………………………………………17
一、澎湖豆科植物種類及根瘤菌的分離與純化………………………………17
二、土壤基本性質測定…………………………………………………………21
三、根瘤菌的分類特性…………………………………………………………22
(一)生理生化特性分析…………….....………………………..…….……18
(二)16S rDNA序列分析………………….………….………...…………..25
(三) Amplify 16S rDNA restriction analysis……...………….……………31
(四)蛋白質特性分析(SDS-PAGE)…….……..………….…...……………34
肆、結果與討論……………………………………………………………………36
一、澎湖豆科植物與根瘤菌……………………………………………………36
二、土壤基本性質測定…………………………………………………………37
三、根瘤菌的分類特性…………………………………………………………41
(一)生理生化特性分析…………….....……………………..……….……41
(二)16S rDNA序列分析………………….………….……………...……..54
(三)Amplify 16S rDNA restriction analysis……...…………………………59
(四)蛋白質特性分析(SDS-PAGE)…….……..……………………………71
伍、結論……………………………………………………………………………76
陸、參考文獻………………………………………………………………………78
染、附錄……………………………………………………………………………83
王孟群。1983。實用微生物學實驗。九州出版社。
林良平。1987。土壤微生物學。南山堂出版社。
葉茂生、鄭隋和。1991。臺灣豆類植物資源彩色圖鑑。行政院農業委員會。
土壤分析手冊。1993。中華土壤肥料學會、臺灣省政府農林廳。
劉瑞美。1995。臺灣根瘤菌溶解無機磷之特性及接種研究。國立中興大學土壤學
研究所博士論文。
楊秋忠。1997。土壤與肥料。農世股份有限公司。
楊麗聲。1997。細菌分類學。中國農業大學出版社。
趙震慶。1998。土壤微生物實習手冊。國立中興大學。P.60-74。
曾文聖。1997。台灣地區本土性快生型大豆根瘤菌之數值分類。國立台灣大學農
業化學研究所碩士論文。
Stryer。2000。大學生物化學(上、下)。藝軒圖書公司。
朱玉賢、李毅。2001。現代分子生物學。藝軒圖書公司。
洪美華。2002。台灣本土豆科植物根瘤菌分離及特性研究。國立中興大學土壤環境科學研究所碩士論文。
李炎。2002。基礎生物資訊學實務。藝軒圖書公司。
陳仁炫、林正金芳、郭惠千。2003。土壤肥力因子之分級標準彙集。國立中興大學土壤環境科學系。
劉瑞美、楊秋忠。2001。逢機複製多型性DNA技術分析溶磷與不溶磷根瘤的基因親緣性。土壤與環境。4:193-204。
Acosta-Duran C., and E. Martinez-Romero. 2002. Diversity of rhizobia from nodules of the leguminous tree Gliricidia sepium, a natural host of Rhizobium tropici. Arch. Microbiol. 178:161-164.
Aguilar O.M., D.H. Grasso, P.M. Riccillo, M.V. Lopez, and E. Szafer. 1997. Rapid identification of bean rhizobium isolates by a nifH gene-PCR assay. Soil Biol. Biochem. 30:1655-1661.
Allen O.N. and E.K. Allen. 1981. The leguminosae: A source book of characteristics, uses, and nodulation. University of Wisconsin press, Madison, WI.
Bala A., P. Murphy, and K.E. geller. 2002. Occurrence and genetic diversity of rhizobia nodulating sesbania sesban in African soils. Soil Biol. Biochem. 34:1759-1768.
Bolanos L., E. Esteban, C de Lorenzo, M. Fernandez-Pascual, M.R. de Felipe, A. Garate, and I. Bonilla. 1994. Ewwcntiality of boron for symbiotic dinitrogen fixation in pea (Pisum sativum)Rhizobium nodules. Plant Physiol. 104:85-90.
Bostsford J.L. Osmoregulation in Rhizobium meliloti: inhibition of growth by salts. Arch. Microbiol. 137:124-127.
Botsford J.L., and T.A. Lewis. 1990. Osmoregulation in Rhizobium meliloti: production of glutamic acid in rdsponse to osmotic stress. Appl. Environ. Microbiol. 56:488-494.
Bremner, J. M., and C. S. Mulvaney. 1982. Nitrogen-total. In Page et al.(ed.) Methods of soil analysis, part 2. 2nd ed. Agronomy Monograph no. 9:595-624.
Caudry-Reznick S., D. Prevost, and H.M. Schulman. 1986. Some properties of arctic rhizobia. Arch. Microbiol. 146:12-18.
Chen H., A.E. Richardson, and E. Gartner. 1991. Construction of an acid-tolerant Rhizobium leguminosarum biovar trifolii strain with enhanced capacity for nitrogen fixation. Appl. Environ. Microbiol. 57:2005-2011.
Chen W.M., S. Laevens, T.M. Lee, T. Coenye, P. De Vos, M. Mergeay, and P. Vandamme. 2001. Ralstonia taiwanensis sp. Nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int. J. Syst. Bacterol. 51:1729-1735.
Chen W.M., and T.M. Lee. 2001. Genetic and phenotypic diversity of rhizobial isolates from sugarcane-Sesbania cannabina-rotation fields. Viol. Fertil. Soils. 34:14-20.
Chen W.M., T.M. Lee, C.C. Lan, and C.P. Cheng. 2000. Characterization of halotolerant rhizobia isolated from root nodules of Canavalia rosea from seaside areas. FEMS Microbiol. Ecology. 34:9-16.
Chen W.X., Z.Y. Tan, J.L. Gao, Y. Li, and E.T.Wang. 1997. Rhizobium hainanense sp. Nov., isolated from tropical legumes. Int. J. Syst. Bacteriol. 47:870-873.
Davies B.E. 1974. Loss-on-ignition as an estimate of soil organic matter. Soil Sci. Soc. Am. J. 38: 150-151.
Doignon-bourcier F., A. Willems, R. Coopman, G. Laguerre, M. gillis, and P. Lajudie. 2000. Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses. Appl. Environ. Microbiol. 66:3987-3997.
De Lajudie P., E. Laurent-Fulele, A. Willems, U. Torck, R. Coopman, M.D. Collins, K. Kersters, B. Dreyfus, and M. Gillis. 1998. Allorhizobium undicola gen. nov., sp. Nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal. Int. J. Syst. Bacteriol. 48:884-890.
Eardly B.D., J.P.W. Young, and R.K. Selander. 1992. Phylobenetic position of rhizobium sp. Strain Or191, a symbiont of both medicago sativa and phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes. Appl. Environ. Microbiol. 58:1809-1815.
Elkan G.H., and C.R. Bunn. 1991. The rhizobia. InBalows, A.H., G. Truper, M. Dworkin, W. Harder, and K.H. Schleifer(ed.) The Prokaryotes. 2nd ed. A Handbook on the Biology of Bacteria:Ecophysiology, Isolation, Identification, Applications. p.2197-2213.
Elkan G.H. 1992. Taxonomy of the rhizobia. Can. J. Microbiol. 38:446-450.
Goss T.J., G.W. O’Hara, M.J. Dilworth, and A.R. Glenn. 1990. Cloning, characterization and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419. J. Bacteriol. 172:5173-5179.
Graham P.H., M.J. Sadowsky, H. H. Keyser, Y.M. Barnet, R.S. Bradley, J.E. Cooper, D.J. deLey, B.D.W. Jarvis, E.B. Roslycky, B.W. Strijdom, and J.P. W. Young. 1991. Proposed minimal standard for the description of new genera and species of root-nodulating and stem-nodulating bacteria. Int. J. Syst. Bacteriol. 41:582-587.
Hall T.A. 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98.
Hameed S., S. Yasmin, K.A. Malik, Y. Zafar, and F.Y. Hafeez. 2004. Rhizobium, bradyrhizobium and agrobacterium strains isolated from cultivated legumes. Biol. Fertil. Soils. 39:179-185.
Hardy R.W.F. 1993. Ecology and agricultural applications of nitrogen-fixing system. In agriculture and environmental challenges. p. 109-117. World Bank, USA.
Hennecke H., K. Kaluza, B. Thony, M. Fuhrmann, W. Ludwig, and E. Stackebrandt. 1985. Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen-fixing bacteria. Arch. Microbiol. 142:342-348.
Heyndrickx M., L. Vauterin, P. Vandarmme, K. Kersters, and P. De Vos. 1996. Applicability of combined amplified ribosomal DNA restriction analysis(ARDRA) patterns in bacterial phylogeny and taxonomy. J. Micorbiol. Meth. 26:247-259.
Hungria M., D.S.Andrade, L.M.O. Chueire, A. Probanza, F.J. Guttierrez-Manero, and M. Megias. 2000. Isolation and characterization of new efficient and competitive bean (Phaseolus vulgaris L.) rhizobia from Brazil. Soil Biology and Biochemistry. 32:1515-1528.
Jenkins M.B. Rhizobial and bradyrhizobial symbionts of mesquite from the Sonoran desert:salt tolerance, facultative halophily and nitrate respiration. 2003. Soil Biology and Biochemistry. 35:1675-1682.
Jordan D.C. 1984. Family III. Rhizobiaceae. In Bergey’s manual of systematic bacteriology. Vol. 1. 32-242.
Kafkafi U. 1991. Plant roots/the hidden half. Edited by Waisel, Y., A. Eshel and U. Kafkafi. pp.671-702. Marel Dekker, Inc. New York.
Kajalainen S. and K. Linstron. 1989. Restriction fragment length polymorphism analysis of Rhizobium galedae strains. J. Bacteriol. 171:5561-5566.
Lafay B., and J.J. Burdon. 1998. Molecular Diversity of rhizobia occurring on native shrubby legumes in southeastern Australia. Appl. Environ. Microbiol. 61:3989-3997.
Legocki A., H. Bothe, and A. Puhler. 1997. Biological fixation of nitrogen for ecology and sustainable agriculture.
Le Rudulier D., A.R. Strom, A.M. Dandekar, L.T. Smith, and R.C. Valentine. 1984. Molecular biology of osmoregulation. Science. 224:1024-1028.
Lloret J., L. Bolanos, M.M. Lucas, J.M. Peart, N.J. Brewin, I. Bonilla, and R. Rivilla. 1995. Ionic stress and osmotic pressure induce different alterations in the lipopolysaccharide of a Rhizobium meliloti strain. Appl. Environ. Microbiol. 61:3701-3704.
McLean, E. O. 1982. Soil pH and lime requirement. In Page et al.(ed.) Methods of soil analysis, Part 2. 2nd ed. Agronomy Monograph no. 9:199-224.
Mehlich, A. 1978. New extractant for soil test evaluation of phos- phorus, potassium, magnesium, calcium, sodium, manganese and zinc. Commun. In Soil Sci. Plant Anal. 9:477-492.
Mhamdi R., M. Jebara, M.E. Aouani, R. Ghrir, and M. Mars. 1999. Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of phaseolus vulgaris L. grown in Tunisian soils. Biol. Fertil. Soils. 28:313-320.
Munevar F., and A.G. Wollum. 1981. Growth of Rhizobium japonicum strains at temperatures above 27 ℃. Appl. Environ. Microbiol. 42:272-276.
Munevar F., and A.G. Wollum. 1981. Effect of high root temperature and Rhizobium strain on nodulation, nitrogen fixation, and growth of soybeans. Soil Sci. Soc. Am. J. 45:1113-1120.
Olsen, S. R., and L. E. Sommers. 1982. Phosphorus. In Page et al. (ed.) Methods of soil analysis, Part 2. 2nd ed. Agronomy Monograph no. 9:403-430.
Page R.D.M. 1996. Treeview: an application to display phylogenetic trees onf personal computers. Comput. Appl. Biosce. 12:357-358.
Peter X. and W.J. Broughton. 1998. Rapid identification of Rhizobium strains by targeted PCR fingerprinting. Plant and Soil. 204:21-34.
Rhoades, J. D. 1982. Soluble salts. In Page. et al.(ed.) Methods of soil analysis, Part 2. 2nd ed. Agronmy Monogrph no. 9:167-179.
Saleena L.M., P. Loganathan, and S.R.S. Nair. 2001. Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea. Biol. Fertil. Soils. 34:276-281.
Sexena D., M. Amin, and S. Khanna. 1996. Modulating of protein profiles in Rhizobium sp. under salt stress. Can. J. Microbiol. 42:617-620.
Shenbagarathai R. 1993. Isolation and characterization of mutants of Rhizobium SBS-R100 symbiotic with Sesbania procumbens. Soil Biol. Biochem. 25:1339-1342.
Singleton P.W., S.A. EL Swaify, and B.B. Bohlool. 1982. Effect of salinity on Rhizobium growth and survival. Appl. Environ. Microbiol. 44:884-890.
Smith L.T., and G.M. Smith. 1989. An osmoregulated dipeptide in stressed Rhzobium meliloti. J. Bacteriol. 171:4714-4717.
Steinborne J., and R.J. Roughley. 1975. Toxicity of sodium chloride ions to Rhizobium spp. in broth and peat culture. J. Appl. Bacteriol. 39:133-138.
Surange S., A.G. Wollim II, N. Kumar, and C.S. Nautiyal. 1997. Characterization of Rhizobium from root nodules of leguminous trees growing in alkaline soils. Can. J. Microbiol. 43:891-894.
Tan Z.Y., E.T. Wang, G.X. Peng, M.E. Zhu, E.M. Romero, and W.X. Chen. 1999. Characterization of bacteria isolated from wild legumes in the north-western regions of China. Int. J. Syst. Bacteriol. 49:1457-1469.
Thompson J.D., D.G. Higgins, and T.J. Gibson. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
Ueda T., U. Suga, N. Yahiro, and T. Matsuguchi. 1995. Remarkable N2-fixing bacterial diversity detected in rice roots by Molecular evolutionary analysis of nifH gene sequences.J. Bacteriol. 177:1414-1417.
Unni S., and K.K. Rao. 2001. Protein and lipopolysaccharide profiles of a salt-sensitive Rhizobium sp. and its exopolysaccharide-ceficient mutant. Soil Biol. Biochem. 33:111-115.
Vincent J.M. 1970. Amanual for the pratical study of root —nodule bacteria. IBP handbook no.15. Black well sci. Oxford. Great Britian.
Weisburg W.G., S.M. Barns, D.A. Pelletier, and D.J. Lane. 1990. 16S ribosomal DNA Amplification for phylogenetic study. J. of Bacteriol. 173:697-703.
Yap S.F., and S.T. Lim. 1983. Response of Rhizobium sp. UMKL 20 to sodium chloride stress. 135:224-228.
Young C.C., J.Y. Chang, and C.C. Chao. 1988. Physiological and symbiotic characteristics of Rhizobium fredii isolated from subtropical-tropical soils. Biol. Fertil. Soils. 5:350-356.
Zahran H.H., and J.I. Sprent. 1986. Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum. Planta. 167:303-309.
Zarucchi J.L. 1989. Advances in legume biology. Edited by Stirton C.H., and J.L. Zarucchi. p. 545-557. Missouri Botanical Gardon. New York.
Zhang X.P., R. Harper, M. Karsisto, and K. Lindstrom. 1991. Diversity of Rhizobium bacteria islated from the root nodule of leguminous trees. Int. J. Syst. Bacteriol. 41:104-113.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top