玉米联合固氮菌Kosakonia radicincitans GXGL-4A的分离鉴定与固氮特性研究  

Isolation, identification and characterization of associative nitrogen-fixing endophytic bacterium Kosakonia radicincitans GXGL-4A in maize

作　　者：李琼洁 程杰杰[1] 孙帅欣 陈云鹏[1] 

机构地区：[1]上海交通大学农业与生物学院农业部都市农业(南方)重点实验室,上海200240

出　　处：《微生物学通报》2016年第11期2456-2463,共8页Microbiology China

基　　金：国家重点基础研究发展规划项目(973计划)(No.2015CB755702)~~

摘　　要：【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。[Objective] Nitrogen-fixing microorganisms are the main component of biological nitrogen fixation agent, and the breeding of nitrogen-fixing-bacterium provides a solid foundation for the investigation and application in biological nitrogen fixation field. A nitrogen-fixing bacterium was identified and furthermore its nitrogenase activities were determined. [Methods] Ashby medium is used for the isolation and purification of nitrogen-fixing-bacterium. Morphological, physiological and biochemical analyses were conducted combining with 16 S r RNA gene sequencing and genomic scanning to identify the bacteria. The ammonia-secreting capacity of bacterial cells was measured by indophenol blue spectrophotometric method and the nitrogenase activity was determined using the acetylene reduction assay（ARA）. [Results] Kosakonia radicincitans GXGL-4A, a Gram-negative nitrogen-fixing bacterium with the size of 1.5 μm×0.5 μm was finally isolated from maize root tissues. The strain were observed to show single cell distribution and joint of two tandem bacterial cells under a microscope. Phylogenic analysis based on 16 S r RNA gene sequence revealed that GXGL-4A shares 95% of DNA homology with the Kosakonia oryzae strain Fo8A1 d. Eventually, the isolate was identified as Kosakonia radicincitans and the stain was named as K. radicincitans GXGL-4A. The nitrogen-fixing gene, nif H was successfully amplified by PCR technique. The content of ammonia nitrogen secreted by GXGL-4A bacterial cells was assayed at 2.5 mg/L through indophenol blue spectrophotometric method. The results showed that this strain has a good capacity in ammonium secretion. The nitrogen-fixing bacterium GXGL-4A grown on nitrogen-free media can effectively restore acetylene, and its nitrogenase activity expressed in ethylene production capacity was evaluated at 232.94 nmol C2H4/（m L·h）. [Conclusion] K. radicincitans GXGL-4A will benefit for the research of microbial nitrogen fixation.

关 键 词：玉米 固氮菌 分离鉴定 Kosakonia radicincitans GXGL-4A 

分 类 号：S513[农业科学—作物学] S182