苔藓植物类黄酮生物合成基因PpFLS1和PpANS1的光胁迫逆境应答机制研究

Sunlight provides the energy that drives photosynthesis，but exposure to excess light can cause light stress and damage the photosynthetic apparatus. Plants have evolved efficient mechanisms to cope with the oxidative damage caused by strong light. Flavonoids are one of the most important groups of secondary metabolites, involved in various biological processes such as UV-B stress response, through UV absorption and reactive oxygen species (ROS) scavenging. The light signals can regulate the biosynthesis of a variety of secondary metabolite including Flavonoids. In this research, the moss materials were treated with strong light and UV irradiation, then the flavonoids accumulation and the expression profiles of flavonoid biosynthetic genes were assayed. A flavonol synthase gene PpFLS1 and an anthocyanidin synthase gene PpANS1 were cloned and characterized in vitro and in vivo. The catalytic mechanisms of both enzymes were discussed based on the crystal structure and biochemical analyses. Meanwhile, the flavonoids metabolites and the gene expression profiles were measured in the overexpression and knockout plants，and the this research is helpful to understand the flavonoids biosynthesis in moss, and to uncover the mechanisms of these metabolites governing development and abiotic stress response in plants.

光是植物光合作用的能量来源，而过量的光照导致光胁迫，伤害植物光合器官。植物发展了多种保护机制，应对强光带来的氧化伤害。其中，植物黄酮类化合物是一类重要的次生代谢物，能够通过吸收UV、清除活性氧等参与UV-B胁迫应答过程，而且光照是类黄酮生物合成重要的调控因子。本课题拟以小立碗藓为材料，比较测定正常光照和强光、UV处理植株中类黄酮含量和合成相关基因表达谱。克隆类黄酮合成关键酶黄酮醇合成酶基因PpFLS1和花青素合成酶基因PpANS1，测定其编码蛋白的体外活性，研究酶催化底物的选择性及反应特点，并通过解析晶体结构、结合定点突变、分子对接手段等阐明酶催化反应的机制。同时研究其过表达和敲除对小立碗藓植株类黄酮合成、抗逆能力和发育的影响，以阐明苔藓植物中类黄酮生物合成途径应对光胁迫的响应机制、探讨类黄酮在发育和抗逆中的作用机制。

光是植物生长发育的能量来源，过量的光照导致光胁迫。本项目利用不同光照强度处理苔藓模式植物小立碗藓和地钱，测定不同处理时间光合生理指标。结果显示，高光强胁迫条件下，苔藓植物光合活性的参数仍维持较高的水平，并启动环式电子传递增加能量传递效率；并且随处理时间延长，其非光化学淬灭值逐渐降低，植物对高光强的适应性增强；植物积累较多的参与非光化学淬灭的玉米黄质；苔藓植物通过增加光能利用率、激发非光化学淬灭机制增加能量耗散抵抗高光强胁迫。同时，项目测定UVB胁迫处理的苔藓植物转录组，对差异表达基因进行KEGG富集分析发现，多个光保护机制相关通路显著性富集，包括“光合作用-天线蛋白”、“类胡萝卜素合成”、“类黄酮生物合成”等；其中，类黄酮合成途径多个关键基因表达上调，对差异表达基因进行qRT-PCR验证，结果与测序结果一致。以上结果表明，苔藓植物采用多种机制应对高光强和UVB辐射胁迫带来的伤害，其中类黄酮合成及其调控积极响应光胁迫逆境，是苔藓植物光保护机制的重要组成部分。另外，对前期筛选到的一个黄酮醇合成酶进行克隆及功能鉴定，其具有黄酮-3-羟化酶和黄酮醇合成酶功能。以上结果对阐明苔藓植物类黄酮合成途径响应光胁迫逆境机制具有重要意义。