草甘膦诱导的非编码RNA G42参与施氏假单胞菌固氮基因表达调控的分子机制

Glyphosate [N-(phosphonomethyl)-glycine] is a broad-spectrum, non-selective herbicide that is widely and the largest used in the world. A lots of studies showed that glyphosate negative effects on nitrogen fixation, but the molecular mechanism of the affection has not been made clear. pseudomonas stutzeri A1501 is a associative nitrogen fixation bacterium with high glyphosate tolerant, but the nitrogen fixing capacity decreased significantly under the glyphosate shock. Preliminary study shows that non-coding RNA G42(G42ncRNA) was upregulated under the glyphosate impact, but down-regulated under the nitrogen fixation conditions. Over-express of G42ncRNA in A1501 not only impairs the nitrogenase activity, but also decreases the expression of the nif genes, implies that G42ncRNA might be involved in the nitrogen fixation regulation process. In order to investigate regulatory mechanism of G42ncRNA involved in nitrogen fixation gene regulation process, we propose to carry out:(1) elucidating the biological functions and express characteristics of G42ncRNA; (2)exploring the regulatory mechanism of G42ncRNA involved in nitrogen fixation gene regulation. Ultimately, this work will lay the foundation for deep understand the regulation and expression of nif genes , and may help to construct a glyphosate-tolerant GM azotobacter.

草甘膦是目前世界上使用面积和使用量最大的除草剂，已有多项研究表明草甘膦会对固氮微生物固氮效率产生不利影响，但对这种影响的分子机制尚不清楚。联合固氮施氏假单胞菌A1501具有较强固氮能力和较高草甘膦耐受能力，但在草甘膦冲击下固氮酶活受到抑制。前期研究表明施氏假单胞菌中非编码RNA G42在草甘膦冲击下高表达，固氮条件下低表达。非编码RNA G42过表达降低了固氮酶活性和固氮酶结构基因、固氮调节基因表达水平，暗示非编码RNA G42可能受草甘膦诱导，以某种机制参与固氮基因表达调控。为揭示上述科学问题拟进行如下研究：（1）明确非编码RNA G42的生物学功能和表达特性；（2）探索非编码RNA G42参与固氮基因表达调控机制。研究结果将为深入了解固氮菌固氮基因表达调控机理奠定基础，同时为构建耐草甘膦高效固氮工程菌提供理论指导。

草甘膦是目前世界上使用面积和使用量最大的除草剂，已有多项研究表明草甘膦会对固氮微生物固氮效率产生不利影响。前期研究表明施氏假单胞菌中非编码RNA G42可能受草甘膦诱导，以某种机制参与固氮基因表达调控。本研究首先构建了G42ncRNA缺失突变株和过表达菌株，实验证明G42ncRNA对A1501菌固氮酶活起负调控作用。在线分析G42ncRNA二级结构，发现G42ncRNA具有典型的茎环结构，利用5’-RACE实验确定了该小RNA转录起始位点和方向；根据序列分析结果设计实验，体外结合（微量热泳动仪）和Western Blot 实验确定固氮酶结构基因nifK mRNA是G42ncRNA的靶标之一。推测G42ncRNA可能通过结合nifK mRNA阻止其翻译成蛋白，从而影响固氮酶的正确组装。研究结果为深入了解在特殊环境下固氮菌固氮基因表达调控机理奠定基础。

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