土壤杆菌氧调控基因fnrN突变株发酵性能及基于RNA-Seq的基因表达

Fermentation characteristics of Agrobacterium sp. with oxygen regulation gene fnrN mutation and genes expression analysis based on RNA-Seq

【目的】研究Crp家族转录调节因子fnr N突变对土壤杆菌ATCC 31749发酵性能和基因表达的影响。【方法】利用三亲结合法将构建的自杀式质粒p JQ-fnr N-kan导入土壤杆菌ATCC31749中,从而获得fnr N基因突变株(Δfnr N);分析Δfnr N的发酵特性;基于RNA-Seq对产胶期土壤杆菌ATCC 31749野生菌和Δfnr N差异表达基因进行分析。【结果】fnr N的突变使土壤杆菌合成热凝胶能力下降了22.0%,转录组分析发现在Δfnr N中186个基因表达发生显著性变化(|log2(|fold change|)|≥1且q≤0.001),其中65%的基因表达上调。热凝胶合成的关键基因crd ASC的表达受到不同程度的抑制,fnr N的突变使编码σ因子的ecf R和编码生物膜合成调节因子的sin R显著下调;Δfnr N菌中与细胞色素有关基因cyd AB、cy2、fix NOPQ的转录水平下调2-13倍。【结论】fnr N通过调控ecf R和sin R的表达调控热凝胶合成,通过调控fix NOPQ等基因的表达参与氧信号调控,该研究有助于丰富对土壤杆菌氧调控系统的认识。

[Objective] To study the effect of Crp family transcriptional regulator gene fnrN mutation on the fermentation characteristics and genes expression in Agrobacterium sp. ATCC 31749. [Methods] A suicide plasmid pJQ-fnrN-kan was constructed and transformed into wild type strain by triparental conjugation method to obtain the fnrN mutation strain （ΔfnrN）. The cell growth and fermentation characteristics of ΔfnrN were compared with that of the wild strain, then comparative transcriptomes with RNA-Seq were used to analyze the expression changes of genes in curdlan-producing phase due to fnrN mutation. [Results] The level of curdlan production was reduced by 22.0% because of fnrN mutation. Transcriptomes analysis revealed that 186 genes expressed significantly different （｜log2（｜fold change｜）｜≥1 and q≤0.001）, and about 65% differently expressed genes were up-regulated. The key genes （crdASC） in curdlan synthesis were repressed in different degree, the fnrN mutation significantly down-regulated the expression of ecfR and sinR, coding ECF family RNA polymerase sigma factor and Fnr family regulator of biofilm formation respectively. The transcription levels of genes （cydAB, cy2 and fixNOPQ） related with cytochrome were down-regulated 2？13 times. [Conclusion] The fnrN regulates curdlan synthesis by controlling the expression of ecfR and sinR, and takes part in oxygen signal regulation through down-regulating the expression of fixNOPQ, which is helpful to enrich the understanding of oxygen regulation system in Agrobacterium sp.