荧光定量PCR分析土壤杆菌及其ntrC突变株对氮源的应答反应

Application of Real-Time PCR to Study the Responses of Agrobacterium sp.and the Mutant ntrC to Nitrogen Availablility

摘要:

热凝胶是土壤杆菌在环境氮源限制时产生的水不溶性多糖,其合成与氮源代谢调控相关,但其机理还不清楚。作者使用荧光定量PCR的方法比较了土壤杆菌野生菌株和其ntrC突变株在氮源充足和限制条件下氮源代谢相关基因和碳代谢相关基因的转录水平差异,探索了土壤杆菌对氮源限制环境的应答机制及氮源调控蛋白NtrC和热凝胶合成之间的关系。结果表明土壤杆菌在环境氮源限制时,ntrB,glnA,glnB,nifR等4种氮代谢相关基因和exoC,exoN,crd,cisY,ndk,glmU等6种碳代谢基因的相对转录水平都有不同程度的提高,表明当环境氮源缺乏时土壤杆菌调整细胞整体代谢(包括氮源吸收代谢、碳代谢、能量合成等),同时使碳代谢流进入热凝胶合成途径用于能量储存。NtrC蛋白参与调控热凝胶合成,而且热凝胶合成过程相关酶的调控,除了存在转录水平调控外,还可能还存在翻译水平的调控。ntrC突变株的热凝胶合成能力相对于野生菌显著降低,且热凝胶合成基因crd在氮源缺乏时的相对转录水平较野生菌株高,推测NtrC蛋白并非crd基因的转录因子。

Abstract:

Curdlan is water-insoluble polysaccharides from Agrobacterium sp.ATCC 31749 after cell growth has ceased due to nitrogen exhaustion.The involvement of nitrogen regulatory systems in curdlan production is not surprising,but the mechanism is not known.To explore homeostatic responses to N limitation and the relationships between nitrogen regulation protein NtrC and curdlan synthesis,differences of expression level between Agrobacterium sp.ATCC 31749 and the ntrC mutant under nitrogen sufficient and nitrogen limitation were correlated by real-time PCR.For Agrobacterium sp.ATCC 31749 wild type,four nitrogen metabolism-associated genes(ntrB,glnA,glnB,nifR)and six carbon metabolism-associated genes(exoC,exoN,crd,cisY,ndk,glmU)relative expression level were improved differently under nitrogen limitation.The results indicated that Agrobacterium sp.regulated the whole celluar metabolism(nitrogen assimilation、carbon metabolism and energy metabolism etc.) and channeled the carbon flux into curdlan synthesis for energy storage under adverse condition.NtrC protein was involved in regulating curdlan production,and curdlan synthesis-associated enzymes were regulated at not only transcriptional level but also translational level.Curdlan synthesis capacity of the ntrC mutant decreased severely while relative expression level of the crd gene increased as compared to the wild type under nitrogen-limited conditions.It is speculated that the NtrC protein may not be transcription factor of the crd gene.