玉米联合固氮菌Kosakonia radicincitans GXGL-4A转座突变体系的构建

【背景】长期以来,农业生产上过度施用化学氮肥造成了农田生态环境的破坏,引起土壤板结、次生盐渍化和重金属污染。此外,由于田间大量的氮素流失和淋溶导致水体富营养化和地下水污染,使得农产品硝酸盐含量超标,最终可能通过食物链危及人类的健康。因此,通过合理地开发和利用生物固氮菌,从而减少化学氮肥施用量,对于保护生态环境,促进农业的可持续生产具有十分重要的意义。【目的】对从玉米根部分离得到的联合固氮菌Kosakonia radicincitans GXGL-4A进行Tn5转座突变,从而创制大量的突变体,经筛选后应用于对固氮及其调控的分子机制和氮代谢调控网络解析等研究。【方法】以固氮菌K.radicincitans GXGL-4A为研究对象,通过PCR法克隆得到GXGL-4A菌株的亚硝酸还原酶基因nirBD。通过构建重组质粒pMOD-egfp-tet,并利用电击转化法将转座复合体导入GXGL-4A野生株,进行Tn5转座突变,从而获得大量突变菌株。【结果】筛选到4株亚硝酸盐还原酶活性显著降低的突变株,并克隆了突变株M36突变位点的侧翼序列。【结论】对玉米联合固氮菌K.radicincitans GXGL-4A进行Tn5转座突变是可行的,初步建立了该菌株稳定有效的插入突变技术体系。

Generation of Tn5 insertion mutations in nitrogen-fixing bacterium Kosakonia radicincitans GXGL-4A associated with maize

Background] The long-term excessive use of chemical nitrogen fertilizers in agriculture has already caused ecological destruction of farmland. The direct consequences are soil compaction, secondary salinization and heavy metal pollution. Moreover, a large amount of nitrogen leaching loss of field could lead to eutrophication and groundwater pollution. The nitrate content of agricultural products may exceed standard, and eventually human health would be threatened due to the food chain. Thus, developing the biological nitrogen-fixing (NF) bacterial agents to reduce the use of chemical nitrogen fertilizer is of great significance for environmental protection and sustainable agriculture. Objective] This study aims at creating abundant mutants of an associative NF bacterial strain, Kosakonia radicincitans GXGL-4A, isolated from maize root by Tn5 insertional mutagenesis and the results will benefit for further exploration of nitrogen fixation mechanism, nitrogen metabolism and related regulatory network, and lay a solid foundation for the application of GXGL-4A in agricultural production. Methods] The nitrite reductase gene nirBD was cloned from K. radicincitans GXGL-4A by PCR. Meanwhile, a Tn5 transposon plasmid pMOD-egfp-tet was constructed and then transferred into the wild type GXGL-4A bacterial cells by electroporation to generate Tn5 insertional mutants. Results] Large quantities of mutants were obtained, and four mutants with significant decrease in nitrite reductase activities comparing with the wild type strain GXGL-4A were identified. Subsequently, DNA sequence flanking integration site in the genome of mutant M36 were also isolated. Conclusion] It is feasible and applicable for Tn5 transposon mutagenesis in the associative NF K. radicincitans strain GXGL-4A, and an effective and stable insertional mutagenesis system of this microorganism has been successfully established in this study.