固氮模块在耐辐射异常球菌底盘中的表达特性分析

Analyses of Deinococcus radiodurans Chassis Expressing a Nitrogen-fixing Module

利用生物技术方法将固氮模块转入真核细胞实现真核生物自主固氮具有重大科学意义和应用价值,迄今为止始终未能获得具有自主固氮能力的真核生物。为了探究固氮模块在真核底盘中的表达适配性,以进化地位介于原核生物和真核生物之间的极端微生物耐辐射异常球菌(Deinococcus radiodurans)R1为底盘,将类芽孢杆菌(Paenibacillus polymyxa)WLY78的固氮模块导入耐辐射异常球菌中获得重组耐辐射异常球菌R78,并利用qPCR(quantitative real-time PCR)和Western Blot等方法,同时结合转录组分析,对重组耐辐射异常球菌的表达特性进行探究。结果表明,重组耐辐射异常球菌固氮模块中的9个固氮基因均能够正常转录,但是固氮酶铁蛋白的翻译受到影响;转录组测序结果显示,参与能量传递、氮代谢以及铁硫转运的相关基因的表达变化可能是影响重组菌株中固氮酶表达的限制因子。以上研究结果为进一步的固氮模块设计和微生物底盘优化,并最终构建人工高效固氮装置奠定了理论基础。

Transferring the nitrogen fixation module into a eukaryotic cell to accomplish autonomous nitrogen fixation in eukaryotes via biotechnology is of enormous scientific significance and application benefit.So far,no eukaryotes have been discovered that can fix nitrogen on their own.In order to explore the expression adaptability of nitrogen fixation modules in eukaryotic chassis,the nitrogen-fixing module of Paenibacillus polymyxa WLY78 was introduced into the Deinococcus radiodurans R1,which was a gram-positive model bacterium with an evolutionary position between prokaryotes and eukaryotes,to obtain the Deinococcus radiodurans R78.The expression characteristics of the recombinant Deinococcus radiodurans were investigated by qPCR(quantitative real-time PCR)and Western Blot analysis.The results showed that the genes related to the nine genes in nitrogen fixation module of the recombinant Deinococcus radiodurans could be transcribed normally,but the translation of nitrogenase ferritin genes was affected.Transcriptome results showed that the expression changes of related genes involved in energy transfer,nitrogen metabolism,iron and sulfur transport might be the limiting factors affecting the expression of nitrogenase in the recombinant strain.Above results laid a theoretical foundation for further design of nitrogen fixation module and chassis optimization,and finally built artificial high-efficiency nitrogen fixation device.