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

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.

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。

中山杉406（Taxodium hybrid ‘zhongshanshan 406’）为落羽杉属树木种间杂交优良无性系，具有极强的耐淹能力，已广泛应用于包括三峡库区消落带在内的众多湿地生态系统的恢复与构建。前期研究发现，ThADH4是中山杉406乙醇发酵途径中的重要低氧供能基因，其过表达可显著提高杨树的耐淹性，但其调控机制未知。本项目（1）采用ThADH4启动子片段缺失识别的方法，识别出中山杉406水淹诱导核心元件在GCCC-box及W box区域，且这两个区域包括厌氧诱导所需的顺式调节元件；（2）通过酵母单杂交体系筛选出核心元件结合的上游转录因子6个，并通过凝胶阻滞技术（EMSA）再次验证，获得上游与其互作的关键转录因子ThOEEP1；（3）利用酵母双杂交系统筛选出9个ThADH4互作候选蛋白，进一步通过GST 融合蛋白沉降技术（GST Pull-down）、双分子荧光互补技术（BiFC）共同验证ThADH4与候选蛋白的相互作用的准确性，最终筛选出2个与ThADH4互作的重要蛋白，ThRAP2.1及ThgapCpSh。（4）对筛选基因进行功能验证后发现，过表达ThRAP2.1可以显著提高植物的耐淹性，ThRAP2.1是耐淹正调控因子；综合转基因植株水淹胁迫后的生理指标及基因表达特性，表明ThADH4与 ThRAP2.1，ThADH4 与ThOEEP1在中山杉406耐淹调控通路中存在显著正相关。本项目的实施深化了对ThADH4调控中山杉耐淹分子机制的理解，为林木抗逆基因工程研究提供基因储备和理论指导，将来对于培育耐淹林木，改善涝渍地区生态环境具有重要意义。