亚硝酸盐还原酶转录调控蛋白NsrR去阻遏机制研究

Nitrite sensitive repressor, NsrR, which is widely existed in bacteria during transcription of the nitrite reductase expression. It is a member of the Rrf2 family of transcription factors. NsrR can widely regulate nitrite reductase and nitric oxide reductase transcription in E. coli. NsrR binds to the upstream regions of the promoter, when nitric oxide or nitrite exists in the environment; NsrR relaeased from the promoter reigion and its repression has been removed. Many researches about the NsrR regulation focus on the types and sequences of the binding promoters, but the research on the derepression recognition domain of NsrR is still blank .This study proposed by constructing NsrR homologous or heterologous expression system, using in silico screening and in vivo verification of the NsrR mutants on GFP expression with its repression promoter. The research scheme include several steps: Establishing a NsrR deletion host for heterologous expression; In sillico screening of NsrR mutants, and in vivo gene complementation for mutants verification with GFP expression ; Illustrating the NsrR derepression mechanism at amino acids levels; Obtaining NsrR specific mutants towards the nitrobenzene pollutants for its monitoring and biodegradation. On one side, we find the E. coli NsrR key amino acids in the transactivation domain; on the other side, we explore the derepression mechanism of NsrR at the amino acid level. Finally we expect to obtain specific NsrR mutants towards the nitrobenzene pollutants for its accurate monitoring and biodegradation.

亚硝酸盐还原酶转录阻遏蛋白NsrR是一类广泛存在于细菌中的Rrf2家族转录阻遏蛋白，其能够广泛调控亚硝酸盐还原酶或一氧化氮还原酶的转录翻译，NsrR对基因表达的去阻遏不仅可以通过感应亚硝酸盐或一氧化氮而实现，硝基化合物亦能实现NsrR的去阻遏调控。因此NsrR去阻遏位点的分析及其调控模式在硝基化合物降解酶的表达中具有应用前景。本研究拟通过建立NsrR缺失表达体系，利用“目的基因敲除-目的基因体外突变体回补”的方式，结合计算机虚筛与体外实验，验证NsrR对其阻遏启动子下游GFP表达的影响，进而对NsrR效应结构域位点予以鉴定和分析。具体方法拟采用：建立目的基因缺失突变体表达宿主；利用计算机虚拟筛选方式获得NsrR效应结构域关键氨基酸突变体，并回补表达验证；对NsrR的去阻遏位点予以验证并获得针对硝基苯污染物的NsrR突变体，实现利用NsrR精确控制降解硝基苯污染物的目的。

硝基苯化合物在工业中得到了广泛应用，是一种典型污染物，长期受化工污染的环境中会存在大量的硝基污染物。其致癌性和致突变性会对人体健康及生态环境造成很大的危害。现有的处理方法主要为辐照、氧化或者电化学处理，不仅成本较高，而且易造成二次污染。所以使用微生物降解具有更高的可变性与适应性。NsrR阻遏蛋白存在于E.coli MG1655中，能够广泛调控亚硝酸盐还原酶或一氧化氮还原酶的转录翻译，它感应NO的胁迫而表达去阻遏基因。对其位点进行鉴定可以为之后利用此类模式生物奠定理论依据。本实验利用大肠杆菌的Red重组系统，通过质粒pKD46及辅助质粒pCP20表达相关酶敲除NsrR基因，构建NsrR基因缺失的E.coli MG1655ΔNsrR工程菌株，建立NsrR缺失表达体系，利用“目的基因敲除-目的基因体外突变体回补”的方式，计算机虚筛结合体外实验，验证NsrR对其阻遏启动子下游GFP表达的影响，进而对NsrR效应结构域位点予以鉴定和分析。结合NsrR的蛋白序列，通过i-Tasser的在线模拟分析及预测，NsrR具有典型的HTH型DNA结合蛋白结构特征，α6螺旋结构的D8和半胱氨酸loop中的C91，C96，C102同Fe-S簇的形成和稳定有关，且D8，R12同感应NO关系较为密切。因此，我们推测NO胁迫相应蛋白通过NsrR结构中Fe-S簇的改变来感应NO或亚硝酸基团，调控DNA序列的转录，而其中改变D8和半胱氨酸loop中非半胱氨酸类的氨基酸将有助于NsrR效应物的改变。对NsrR相应的结构域进行改造可以实现单一调控因子进行生物强化去除环境中的含硝基污染物。利用所得到的NsrR缺陷突变株受不同含硝基污染物影响时报告基因的表达情况，可以获得感应特征污染物的专一突变体，从而精准检测和降解硝基污染物。