中山杉耐水淹胁迫的分子机理研究

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’ [Taxodium. mucronatum Tenore. ×Taxodium 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, mechanisms underlying Taxodium hybrid ‘zhongshanshan’ waterlogging tolerance remain largely obscure. For this purpose, the RNA-Seq was used previously to analyze genome-wide transcriptome changes of Taxodium hybrid ‘zhongshanshan406’ roots and shoots 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. Based on this, we will determine the contents of starch and soluble sugar, and measure the activities of lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), and also investigate the amounts of alcohol, lactic acid and alanine in the roots of Taxodium hybrid ‘zhongshanshan406’ under control, 1/2 flooding (30 cm), and complete flooding (submerge 30 cm) stress. And then, the physiological regulation pattern of alcoholic fermentation pathway in the waterlogging tolerance in Taxodium hybrid ‘zhongshanshan’ would be disclosed. This information is of great help towards the understanding of mechanisms of the waterlogging tolerance in Taxodium hybrid ‘zhongshanshan’ and lay 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，中山杉具有很强的耐淹能力，在三峡库区消落带等湿地中已得到广泛应用，但其耐淹机制尚不明确。基于水淹处理下中山杉406转录组测序结果等研究基础，本项目拟围绕乙醇代谢途径，研究中山杉406根系的淀粉等碳水化合物、乙醇脱氢酶等能量代谢酶以及乙醇等代谢产物对不同水淹胁迫处理的响应，揭示乙醇代谢途径在中山杉耐淹过程中的生理调控机制。本项目的实施，可以深入揭示中山杉耐水淹胁迫的乙醇代谢生理机制，对于进一步揭示其相关分子机理以及利用转基因技术培育耐淹林木，改善涝渍地区生态环境具有重要意义。

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，而中山杉406具有很强的耐淹能力，在三峡库区消落带等湿地构建中已得到广泛应用，但其耐淹机制尚不明确。本研究设置一半淹水和没顶淹水处理，表明通气组织、肥大皮孔和不定根的形成，细胞间隙的增加以及较强的光合适应性，有利于半淹水处理下中山杉406的生长。而没顶淹水下，中山杉406通过减缓生长的策略来保存能量，从而增加成活率。此外，与对照相比，淹水胁迫下，中山杉406根系的淀粉和可溶性糖等碳水化合物含量显著降低，同时，乙醇发酵和乳酸发酵途径的关键蛋白编码基因adh4，pdc2和ldh1的表达量上调，乙醇脱氢酶，乳酸脱氢酶和丙酮酸脱羧酶等能量代谢酶活性显著升高，而植物体内的乙醇和乳酸等有毒物质并未积累，可见乙醇发酵途径和乳酸发酵途径在植物耐淹水过程中发挥着重要的作用。. 本研究对中山杉在非生物胁迫下内参基因表达稳定性作综合分析，通过2个不同软件的数据分析，PP2A和APRT可作为相对稳定的内参基因来使用，适量增加内参基因可以将qRT-PCR结果标准化，为后续研究落羽杉属植物基因表达提供了依据。. 基于前期淹水条件下，中山杉406转录组生物信息学分析以及关于乙醇发酵途径和乳酸发酵途径能量代谢酶活性和关键基因表达的研究，本研究筛选出中山杉糖酵解代谢途径中关键蛋白编码基因thadh1、thadh4和thpdc2，克隆出其全长，并表明thadh1、thadh4和其他物种的adh基因高度同源均属于adh分支，thpdc2也和其他物种的pdc基因高度同源均属于pdc分支。此外，研究表明中山杉406的thadh1、thadh4蛋白为细胞核定位，而thpdc2蛋白在细胞质，细胞核以及细胞膜上都会表达。通过对转thadh4基因杨树21d没顶淹水后的观察，发现对照植株叶片萎蔫腐烂，而转基因植物产生适应性变异，株高增加，叶片完好且茎上长出不定根，这表明thadh4可以显著提高植株的耐水淹能力。. 本研究揭示了中山杉406耐水淹胁迫的生理生态以及乙醇发酵途径的关键作用机制，为后续研究耐淹相关功能基因研究及耐淹中山杉品种的选育奠定了坚实的基础。