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【目的】固氮微生物是生物固氮的主体,其菌种的选育更是生物固氮研究的基础。本实验室分离鉴定出一株新的固氮菌,并对其固氮相关活性进行初步研究。【方法】固氮菌采用Ashby无氮培养基进行分离纯化。通过形态特征分析、生理生化特征分析、16S r RNA基因序列分析和基因组扫描对固氮菌进行鉴定。利用靛酚蓝-分光光度法检测固氮菌的泌铵能力。固氮酶活性的测定使用乙炔还原法。【结果】从玉米根部分离到一株固氮菌株GXGL-4A,菌体短杆状,大小约为1.5μm×0.5μm,单个或常见2个菌体细胞串联在一起,革兰氏染色结果为阴性。16S r RNA基因序列分析结果表明该菌株与Kosakonia oryzae Fo8A1d 16S r RNA基因序列有95%的相似性,结合形态特征、生理生化特征和基因组扫描,将其命名为K.radicincitans GXGL-4A。对其进行固氮基因nif H的检测,经PCR扩增得到预期的296 bp条带;采用靛酚蓝-分光光度法,测得发酵液中铵态氮含量为2.5 mg/L,表明该菌株具有较好的泌铵能力。乙炔还原法测其固氮酶活性,以乙烯生产量表示,结果显示GXGL-4A菌株在无氮培养基上能够有效地还原乙炔,达到232.94 nmol C2H4/(m L·h)。【结论】菌株GXGL-4A是一株可较好地分泌铵的新的固氮菌,具有很好的研究价值。
【Aim】 Nitrogen fixing microorganisms are the main body of biological nitrogen fixation. Breeding of their strains is the basis of biological nitrogen fixation. A new strain of nitrogen-fixing bacteria was isolated and identified in our laboratory, and its nitrogen-fixing activity was preliminary studied. 【Method】 Azotobacter was isolated and purified by using Ashby nitrogen-free medium. Azotobacter was identified by morphological analysis, physiological and biochemical characteristics analysis, 16S rRNA gene sequence analysis and genome scanning. Determination of ammonium secretion ability of Azotobacter by indophenol blue - spectrophotometry. Nitrogenase activity was measured using the acetylene reduction method. 【Result】 A strain of nitrogen-fixing GXGL-4A was isolated from the roots of maize. The cells were short rod with a size of 1.5μm × 0.5μm. Single or common cells were connected in series and Gram stain was negative . 16S rRNA gene sequence analysis showed that the strain was 95% similar to the Kosakonia oryzae Fo8A1d 16S rRNA gene sequence, and named K.radicincitans GXGL-4A in combination with morphological, physiological and biochemical characteristics and genome scanning. The nitrogen-fixing gene nif H was detected and the expected 296 bp band was amplified by PCR. The concentration of ammonium nitrogen in the fermentation broth was determined to be 2.5 mg / L by indophenol blue spectrophotometry, Better ability to produce ammonium. The acetylene reduction method was used to measure the nitrogenase activity and ethylene production. The results showed that the GXGL-4A strain could effectively reduce acetylene to 232.94 nmol C2H4 / (m L · h) in nitrogen-free medium. 【Conclusion】 Strain GXGL-4A is a new azotobacter capable of secreting ammonium and has good research value.