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【目的】分离纯化青藏高原东麓(四川甘孜藏族自治州)高山豆根瘤菌,揭示其遗传多样性。【方法】采用纯培养法从该地区高山豆植物根瘤中分离纯化根瘤菌;通过BOXAIR、16S rDNA-RFLP及PCA(PrincipalComponent Analysis)来分析高山豆根瘤菌的遗传多样性;通过16S rDNA序列同源性确定菌株的系统发育地位;通过测定菌株的耐盐性、初始pH生长范围及生长温度范围来分析高山豆根瘤菌的抗逆性。【结果】从8个县12个采样点共分离纯化出22个菌株。22个菌株在16S rDNA PCR-RFLP分析中聚成4个遗传群,在BOX-PCR分析中则聚成9个遗传群。高山豆根瘤菌16S rDNASimpson遗传多样性指数D=0.872。22个菌株分别属于Rhizobium(11/22株)、Mesorhizobium(4/22株)、Rhizobium-Agrobacterium(7/22株)3个属。生理性状测定试验表明,所有菌株均能在1%NaCl的YMA培养基上生长,大多数(15/22株)菌株能在4%NaCl的YMA培养基上生长,其中,SCAU679、SCAU694、SCAU706等3个菌株能在7%NaCl的培养基上生长,SCAU689能在8%NaCl的培养基上生长;15/22的菌株能在pH4-11的培养基上生长;16/22的菌株能在4-45℃条件下生长,所有菌株能在60℃(处理10 min后置28℃)条件下生长。【结论】青藏高原东麓(四川甘孜州)高山豆根瘤菌具有丰富的遗传多样性。大多数菌株对高盐、高温、低温及过酸过碱环境均具有很强的耐受能力。
【Objective】 Isolation and purification of Rhizobium japonicum from the eastern foot of the Tibetan Plateau (Ganzi Tibetan Autonomous Prefecture, Sichuan Province) revealed the genetic diversity. 【Method】 Rhizobium was isolated and purified from root nodules of alpine soybean plants in this area by pure culture method. The genetic diversity of Rhizobium japonicum was analyzed by BOXAIR, 16S rDNA-RFLP and Principal Component Analysis (PCA). By 16S rDNA sequence homology To ascertain the phylogenetic position of the strain, and to analyze the salt tolerance of the strain, the initial pH range of growth and the range of growth temperature to analyze the resistance of the strain Takakodonin. 【Result】 Twenty-two strains were isolated and purified from 12 sampling sites in 8 counties. Twenty-two strains clustered into four genetic groups in 16S rDNA PCR-RFLP analysis and nine genetic groups in BOX-PCR analysis. Rhizobium japonicus 16S rDNASimpson genetic diversity index D = 0.872.22 strains belong to Rhizobium (11/22), Mesorhizobium (4/22), Rhizobium-Agrobacterium (7/22) three genera. Physiological test showed that all the strains could grow on YMA medium of 1% NaCl. Most strains (15/22 strains) could grow on YMA medium of 4% NaCl. Among them, SCAU679, SCAU694, SCAU706, etc. Three strains were able to grow on 7% NaCl medium, SCAU689 on 8% NaCl medium; 15/22 strains were able to grow on medium pH4-11; 16/22 strains were able to grow at 4 -45 ℃ growth, all strains can grow at 60 ℃ (10 min after treatment set 28 ℃) conditions. 【Conclusion】 Rhizobium japonicum is rich in genetic diversity at the eastern foot of the Tibetan Plateau (Ganzi Prefecture, Sichuan Province). Most strains are highly tolerant to high salt, high temperature, low temperature and peracid environments.