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標題: 澎湖群島根瘤菌之分類及特性分析
Characterization and taxonomy of rhizobia strains isolated from Peng-Hu
作者: 陳柏沅
Chen, Po-Yuan
關鍵字: 根瘤菌
出版社: 土壤環境科學系
摘要: 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.
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