丁香假单胞菌GntR/HutC类转录调控因子InpR调控网络解析与调控冰核基因分子机制研究

Pseudomonas syringae, a leading plant-pathogenic bacterium, has been drawn a great attention worldwide for its distinctive infestation efficacy and interaction with the plant hosts. However, little studies have been conducted to investigate the whole-cell regulatory network and molecular mechanism of ice-nucleation activity caused by the ice nucleation protein so far. In this proposal, we focus on probing and elucidating the whole-cell regulating target gene network, the regulatory activity, binding sites, influence factors as well as the action mechanism towards the specific ice-nucleation gene (inaQ) by a HutC-like transcriptional regulating factor (InpR) from a highly nucleating active strain P. syringae MB03. A whole-cell chromatin immunoprecipitation (ChIP) sequencing and transcriptome sequencing will be initially performed to obtain the whole cell-wide InpR-regulating target gene network. The preliminarily screened target genes will be further confirmed by in vitro ChIP experiments, gene-interrupt/expression compensation and real time-quantitative PCR experiments. A followed in vitro multiple experiments, including electrophoretic mobility shift assay (EMSA), DNase I footprinting, site-directed mutagenesis, isothermal titration calorimetry (ITC)/surface plasmon resonance (SPR), homology modeling and molecular docking, etc., will be performed to identify the binding activity and binding sites of the inaQ promoter, as well as the molecular effecters that modulating the regulating activity of InpR, such as temperature, pH, metal ions and those from the bacterial host extract juice. Subsequently, the study will be further undertaken to construct the mutant P. syringae strains that expressing various mutant InpR proteins with different truncated N-termnal domains or C-terminal domains, and to heterologously express these InpR mutant proteins through E. coli expression systems, to investigate the binding activity of InpR and the inaQ promoter, which aims at elucidate the possible action mechanisms of InpR in regulating the inaQ gene in the host strain P. syringae MB03.

以一株高冰核活性丁香假单胞菌MB03中HutC亚家族调控因子InpR为对象，拟研究InpR调控作用的全细胞靶基因网络和调控冰核基因inaQ启动子作用的分子机制。首先通过全细胞ChIP测序和转录组测序获得InpR调控靶基因的全细胞网络后，经扩增靶基因、体外ChIP试验和基因中断与表达互补鉴定后，对初筛靶基因与数据库比对确定InpR全细胞靶基因调控网络；再利用EMSA、DNase I足迹、位点特异性突变、ITC/SPR等试验，测定InpR与冰核基因inaQ启动子的精细结合部位，并研究温度、pH值、金属离子、宿主细胞提取物中的分子效应物对InpR与靶基因结合的影响；最后构建不同截断InpR片段突变株和InpR突变蛋白，分析其在体内外与靶基因inaQ的结合性能，解析InpR调控inaQ基因的可能作用模式和分子机制。

丁香假单胞菌（Pseudomonas syringae）所产生的冰核蛋白（Ice nucleation protein）可引发宿主植物发生冻害，但迄今国内外对冰核蛋白的表达合成调控仍缺乏深入研究。本项目以一株高冰核活性丁香假单胞菌MB03菌株中GntR/HutC家族转录调控因子InpR为对象，研究了InpR调控作用的全细胞靶基因网络和调控冰核基因inaQ启动子的分子机制。使用转录组测序、全细胞ChIP测序、靶基因motif基序在全基因组中搜寻和VirulentPred在线预测等复合筛选，并结合体外ChIP 实验和EMSA试验，从MB03中共确定了103个受InpR调控的细胞内靶基因（包括冰核基因inaQ），建立了MB03细胞内的InpR调控网络。使用体外EMSA、DNase I足迹、位点特异性突变进一步对InpR结合inaQ启动子的活性进行了鉴定。对InpR进行截断和位点特异性突变鉴定到Arg45和Arg49是InpR与DNA结合的关键氨基酸位点。体外EMSA和ITC试验进一步发现柠檬酸、4-羟基-3-硝基苯乙酸、3,5-二硝基水杨酸、赖氨酸、精氨酸、Cu2+和Zn2+对InpR与冰核蛋白InaQ启动子的结合活性有抑制作用。发现了一种新型受InpR直接调控的转录调控蛋白CbrB。通过DNAase I足迹、回文序列突变、ITC实验以及Western blot等实验，发现cbrB启动子与InpR的结合位点为一段回文序列ATGGAATAGACCAG，且这种结合是特异性的，InpR是cbrB基因的阻遏调控因子，可进一步调控MB03的细胞毒性。利用VirulentPred毒性蛋白数据库、转录组学分析、文献报道的杀线虫蛋白序列比对和转座文库筛选等方式筛选了MB03菌株的杀线虫蛋白，鉴定了其中重要的32种蛋白，并对其中高活性的PtxA和一种甲基趋化蛋白MCP03通过酵母双杂交、GST-Pull down和Western blot等，鉴定其在秀丽隐杆线虫体内的受体分别为核糖体40S小亚基上的结构蛋白Rps9以及线粒体内信号转导复合体COP9的一个亚基csn5。研究了PtxA和MCP03与线虫受体发生相互作用后的虫体病理变化等。同时，分别利用RNAi技术和代谢组测定对PtxA/MCP03作用于线虫受体的分子机制进行了探讨，为深入了解丁香假单胞菌致病机理提供新的重要的研究基础。