摘要
为了从磷矿区土壤分离高效溶磷菌,为开发施用于矿区复垦的高效微生物解磷菌肥提供试验基础,利用磷酸钙培养平板筛选磷矿区土壤中的解磷菌,采用PCR法扩增16S rDNA序列进行鉴定,探讨不同碳源、氮源、起始pH值和温度下菌株的生长情况,另外还进一步克隆表达该菌株的碱性磷酸酶。结果显示,经筛选得到1株高活性的解磷菌,16S rDNA鉴定其为成团泛生菌(Pantoea agglomerans);该菌株最佳碳源为甘油、氮源为NH4Cl类无机氮,起始pH值为9,培养温度为33℃;在最适培养条件下,有效溶磷量达到了287.48 mg/L;成功克隆了该菌株中的碱性磷酸酶基因,并在大肠杆菌中实现了异源表达,重组菌裂解液活性为原始菌株裂解液的32.77倍。分离得到的菌株及重组表达的碱性磷酸酶在矿区复垦、农业种植和工业上均具有广泛的应用前景。
To isolate high-efficient phosphorus-solubilizing bacteria from the soil of phosphorus mining area,and to provide a basis for improving the serious phosphorus deficiency in the soil of phosphorus mining area,phosphorus-solubilizing bacteria were screened by calcium phosphate culture plate,and the 16S rDNA sequences were amplified by PCR to identify the bacteria.The growths of strains under different carbon and nitrogen sources,initial pH and temperature were detected,and alkaline phosphatase of the strain was cloned and expressed.The results showed that a highly active strain of phosphorus-solubilizing bacteria was isolated and was identified as Pantoea agglomerans by 16S rDNA.Glycerol was the preferred carbon source,and inorganic nitrogen,such as NH4Cl,was the preferred nitrogen source.The initial pH value was 9,and the cultune temperature was 33℃.Under the optimum culture conditions,the effective phosphorus solubility reached 287.48 mg/L.The alkaline phosphatase gene of the strain was successful cloned,and was heterologous expressed in Escherichia coli.The activity of recombinant bacterial lysate was 32 times higher than that of the original strain.The strains isolated from this experiment and the recombinant expressed alkaline phosphatase will have wide application prospects in mining area reclamation,agricultural cultivation and industry.
作者
宁清
刘迪
靳翔
弓亚杰
薛智权
吕建华
NING Qing;LIU Di;JIN Xiang;GONG Yajie;XUE Zhiquan;LYU Jianhua(College of Life Sciences,Shanxi Agricultural University,Taigu 030801,China)
出处
《山西农业科学》
2019年第11期1974-1979,共6页
Journal of Shanxi Agricultural Sciences
基金
山西省高等学校大学生创新创业训练计划项目(2018114)
山西农业大学引进人才博士科研启动经费项目(2013YJ35,2013YJ38)
关键词
解磷菌
NBRIP平板筛选
钼锑抗法
碱性磷酸酶
16S
RDNA
phosphate-solubilizing bacteria
NBRIP plate screening
Molybdenum antimony resistance method
alkaline phosphatase
16S rDNA
