腾冲嗜热厌氧杆菌EndoTT作用的分子机制研究

DNA endonuclease play important role in DNA replication, DNA recombination and DNA repair. EndoTT encoded by tte0829 of Thermoanaerobacter tencongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA, been a complicated functions in vitro. However, the biochemical and physiological functions of EndoTT have not been described in vivo yet. To elucidate the molecular mechanism of EndoTT function in vivo, the following works would be performed in detail in this proposal. Firstly, disruption, complementation and overexpression strains of endoTT is established integrallty in T. tencongensis. Then, we would be compared the phenotypic character between wild strain and disruption, complementation and overexpression strains of EndoTT in T. tencongensis. Secondly, to compare the transcriptome and proteome between wild strain and disruption strains of EndoTT in T. tencongensis by RNA sequencing and 2-D electrophoresis. Then, we would be analyzed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of differentially expressed genes (or proteins). Thirdly, we would be studied the function and interacting protein of EndoTT in T. tencongensis by co-immunoprecipitation and yeast two-hybrid. Finally, we would be confirmed the function of EndoTT at different temperatures. This proposal is aimed to elucidate the molecular mechanism of EndoTT function, which would disclose the molecular mechanism of EndoTT function in T. tencongensis in adaptation to the extreme environment.

DNA内切酶在细胞的DNA复制、重组和损伤修复中都起着非常重要的作用。在前期研究中我们发现，腾冲嗜热厌氧杆菌EndoTT有复杂的、受多环境因子调控的体外生化功能，它可以结合ssDNA、dsDNA和损失的dsDNA，也可以识别ssDNA上的特异位点或结构进行切割。但是EndoTT在体内发挥什么样的功能，我们仍然不清楚。为深入揭示EndoTT在体内作用的分子机制，本项目将开展以下研究：构建endoTT的阻断突变株、回补株和高表达株，观察与野生株的表型；通过RNA测序和2-D电泳技术比较野生型与endoTT的阻断突变株的转录组和蛋白质组，确定差异表达基因和蛋白，并对差异表达基因和蛋白进行GO分析和KEGG分析；利用免疫共沉淀技术和酵母双杂交系统确定EndoTT的互作蛋白。从而确定腾冲嗜热厌氧杆菌中EndoTT在不同温度下的功能，以及它的互作蛋白并揭示它们对EndoTT功能的影响。

腾冲嗜热厌氧杆菌EndoTT在体外同时具有单链DNA结合活性和单链DNA切割活性的双功能作用。然而腾冲嗜热厌氧杆菌EndoTT在体内有何生理功能及在热适应过程中发挥什么样的作用仍不清楚，为揭示EndoTT在不同温度下的功能及其互作蛋白对其影响。. 我们成功构建了endoTT突变株、EndoTT双功能回补株ΔendoTT::endoTT、单链DNA结合活性回补株ΔendoTT::endoTTB和单链DAN切割活性回补株ΔendoTT::endoTTS。利用 RNA 测序技术对腾冲嗜热厌氧杆菌野生株和ΔendoTT 的转录组进行了比较，结果显示：在50℃、60℃、75℃和80℃条件下分别有35、73、90和21个差异基因表达。这些基因参与了信号转导、磷酸转移酶、糖基化和 ABC 转运系统等代谢途径。利用 2-DE 电泳技术对腾冲嗜热厌氧杆菌野生株和 ΔendoTT 的蛋白质组进行了比较，结果显示 AphC 蛋白在 ΔendoTT 中不表达。Western blotting 结果也显示： SSB2 和 SSB3 在野生株中的表达水平低于 ΔendoTT。