乙醇发酵途径关键基因Thadh4调控中山杉406耐水淹胁迫的分子机理

水库消落带等湿地环境迫切需要通过植被恢复来重构生态系统，而水淹是该类型地区植物遭受的主要逆境。目前，耐淹能力突出的植物尤其是木本植物匮乏，中山杉具有极强的耐淹能力。乙醇发酵途径在中山杉406耐淹过程中发挥着重要作用，但其关键基因Thadh4的表达调控机制尚不明确。基于中山杉406乙醇发酵生理机制以及Thadh4功能与启动子分析的研究基础，本项目拟（1）采用Thadh4启动子片段缺失识别水淹诱导核心元件，通过酵母单杂交体系筛选该元件结合的上游转录因子；完善中山杉遗传转化体系，通过中山杉转基因植株鉴定关键上游转录因子功能，解析Thadh4上游转录调控机制；（2）通过酵母双杂交体系筛选Thadh4互作蛋白，利用中山杉遗传转化体系鉴定关键候选基因功能，明确Thadh4调控基因与蛋白网络。本项目的实施，可以揭示Thadh4调控中山杉耐淹的分子机理，对于培育耐淹林木，改善涝渍地区生态环境具有重要意义。

Wetland ecosystem, e.g. hydro-fluctuation belt of Three Gorges Reservoir Areas needed desperately to be recovered by vegetation restoration. Waterlogging is the main stress for plant survival in wetland. So far, plants resisting to waterlogging stress, especially woody plants are scarce. Taxodium hybrid ‘zhongshanshan’ [T. mucronatum Tenore. ×T. distichum（Linn.）Rich.] bred by the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences has been proved to have remarkable waterlogging tolerance. As we known, through adaptive evolutionary process, various strategies would be used by plants to adapt long- or short-term waterlogging stress. However, molecular mechanisms underlying T. hybrid ‘zhongshanshan’ waterlogging tolerance remain largely obscure. For this purpose, the RNA-Seq was used previously to analyze genome-wide transcriptome changes of T. hybrid ‘zhongshanshan406’ (T. hybrid 406) treated with three months of control, 1/2 flooding (30 cm), and complete flooding (submerge 30 cm) stress. We noted in a preliminary analysis that many unigenes annotated as encoding enzymes involved in glycolysis and fermentation pathway differentially expressed between treated and control plants. Consequently, we demonstrated the mechanism of the ethanol fermentation in the flooded T. hybrid 406, and successfully cloned four genes, e.g. Thadh1, Thadh4, Thpdc2 and Thldh1 related to waterlogging tolerance in ethanol fermentation pathway. Then, the genes expression pattern and subcellular localization of the proteins were disclosed. Moreover, the positive function of Thadh4 was identified by an experiment with over-expression transgenic plants. Based on these results, firstly, we will obtain 5-8 key candidate genes related to the expression and regulation of Thadh4 through the screening of yeast single hybrid and yeast two hybrid systems, as well as the verification of chromatin immunoprecipitation assay (ChIP), electrophoretic mobility shift assay (EMSA), co-immunoprecipitation (CoIP) and glutathione-s-transferase-pull down (GST-pull down). Secondly, the key candidate genes will be cloned, and their expression pattern and subcellular localization of the proteins will be determined by the qRT-PCR and protoplast transient expression system, respectively. Finally, we will optimize the waterlogging experimental platform, and construct a genetic transformation system via Agrobacterium of T. hybrid ‘zhongshanshan’. In terms of these two platforms, an experiment with over-expression transgenic plants would be conducted to determine the function of key candidate genes. In short, this information is of great help towards the understanding of molecular regulatory mechanisms of Thadh4 in the waterlogging tolerance of T. hybrid ‘zhongshanshan’ and lays foundation for future molecular breeding.