香根草内生固氮菌的分离、鉴定及其生长特性和回接效应的研究
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摘要
利用Ashby无氮培养基从香根草体内分离得到10株细菌,分别命名为MJ-1至MJ-10。采用乙炔还原法检测细菌的固氮酶活性,发现菌株MJ-2和MJ-4固氮酶活性较高。对这两株菌进行16S rRNA基因克隆,通过序列分析它们的系统发育地位,结果证实MJ-2为肠杆菌属阴沟肠杆菌菌种,MJ-4为克雷白氏杆菌属肺炎克氏杆菌菌种。同时我们还研究了不同环境因子对这两株固氮菌生长的影响,结果表明它们对环境的适应范围较广。采用三亲交配法将nifH-gfp基因导入固氮菌MJ-4中,限菌培养条件下用该菌接种香根草,激光共聚焦扫描电子2显微镜观察,结果表明该菌在香根草根组织中存在,并且对香根草的生长有一定的促进作用。
The 10 bacterial strains were isolated from Vetiveria zianioides on the nitrogen free Ashby media in this paper. Their nitrogenase activities were detected using the method of acetylene reduction activity and the results indicated that the nitrogenase activities in strains of MJ-2 and MJ-4 were higher than the others. Their 16S rRNA genes were cloned, their phylogenetic tree was made and the sequence analysis indicated that the strain MJ-2 belonged to Enterobacter cloacae, while the strain MJ-4 belonged to Klebsiella pneumoniae. The study was also carried out such as the effect of different environmental factors to the growth of the strains of MJ-2 and MJ-4, and the results showed that they adapted to the environment greatly. The plasmids harboring the reporter gene gfp fused to the promoter nifH were transformed into the bacterial strain MJ-4 by triparental crossing, the conjugated diazotrophic bacteria were inoculated into Vetiveria zianioides under aseptic condition. After inoculation, the observation was made under the confocal laser scanning microscope, the results indicated that the bacteria were existed in the root tissues of Vetiveria zianioides and the data analysis showed that the growth of Vetiveria zianioides was increased.
The 10 bacterial strains were isolated from Vetiveria zianioides on the nitrogen free Ashby media in this paper. Their nitrogenase activities were detected using the method of acetylene reduction activity and the results indicated that the nitrogenase activities in strains of MJ-2 and MJ-4 were higher than the others. Their 16S rRNA genes were cloned, their phylogenetic tree was made and the sequence analysis indicated that the strain MJ-2 belonged to Enterobacter cloacae, while the strain MJ-4 belonged to Klebsiella pneumoniae. The study was also carried out such as the effect of different environmental factors to the growth of the strains of MJ-2 and MJ-4, and the results showed that they adapted to the environment greatly. The plasmids harboring the reporter gene gfp fused to the promoter nifH were transformed into the bacterial strain MJ-4 by triparental crossing, the conjugated diazotrophic bacteria were inoculated into Vetiveria zianioides under aseptic condition. After inoculation, the observation was made under the confocal laser scanning microscope, the results indicated that the bacteria were existed in the root tissues of Vetiveria zianioides and the data analysis showed that the growth of Vetiveria zianioides was increased.
引文
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