光调控苹果褪黑素合成的转录机制

Melatonin (N-acetyl-5-methoxytryptamine) is the effective scavenger of free radicals, and also a beneficial chemical for human health. The synthetic pathway of melatonin has been well elucidated, but its transcriptional regulation remains unknown in plants. We have isolated melatonin synthetase genes in apple. Different from animals, melatonin synthesis in plants was induced by high intensity of light. Apple, as perennial plant, grows under the annual cyclic light, and has more complicated mechanism regulated by light. We have isolated many candidate transcription factors, which co-expressed with melatonin synthetase genes under light, which is confirmed by EMSA in vitro. In addition, we will further explore the direct regulation by candidate transcription factors in the transient system of callus in vivo. In transgenic apple plants/callus overexpressing/RNAi transcription factors, the regulation in situ will be examined by ChIP-qPCR, the detection of melatonin and the expression of synthetase genes. Taken together, we will uncover how the transcription factors regulate the melatonin synthesis, through controlling directly the expression of key synthetase genes under light. Our research on the transcriptional regulation of melatonin synthesis in apple under light, will fill in gaps in the transcriptional regulation of melatonin synthesis, and contribute to engineering apples with high melatonin content.

褪黑素（俗称“脑白金”）是高效的活性氧清除剂，也是苹果中有益于人类健康的活性物质。褪黑素的合成路径已知，但植物褪黑素合成的转录调控机制仍是空白。项目组已从苹果中分离褪黑素关键合成酶基因，并发现，不同于动物，褪黑素的合成受强光诱导。苹果植株在周年周期性变化的光照下，可能具有更为精细复杂的光下调控机制。申请人已经分离了多个光下与褪黑素合成酶基因共表达的候选转录因子，并进行了体外EMSA检测。进一步，将在体内愈伤瞬时系统中，验证候选转录因子对合成酶基因的直接调控。并在超表达/RNA干扰转录因子的转基因苹果植株/愈伤中，进行ChIP-qPCR、褪黑素测定与合成酶表达的检测，原位印证调控关系。最终揭示，光下转录因子通过直接控制关键合成酶基因表达，调控褪黑素合成的机制。本项目对光下苹果褪黑素合成转录调控机制的揭示，将填补植物褪黑素合成调控领域的空白，为通过分子育种培育“褪黑素强化”的苹果奠定基础。

褪黑素（N-乙酰-5-甲氧基色胺）是有效的活性氧清除剂，在植物逆境响应中发挥重要作用。同时，它也是重要的活性物质，调控植物的生长发育。在植物中，褪黑素的合成路径已被解析，且褪黑素的合成受强光和黑暗诱导，但褪黑素合成调控的分子机制仍不清楚。因此，本研究首先分离并克隆了明显响应光/暗的关键褪黑素合成酶基因MzASMT3、AtSNAT6和AtASMT8，在体内外证明了它们具有合成褪黑素的生物活性。在苹果及模式植物拟南芥中发现，光/暗通路中的重要成员HY5可能是调控褪黑素合成的重要转录因子。通过体外EMSA和体内ChIP-qPCR分析，证明了HY5直接结合这3个关键合成酶基因的启动子，抑制其转录。进一步，对HY5转录因子调控褪黑素合成转录的机制及其功能进行研究，发现黑暗下HY5转录因子的蛋白水平下降，对关键褪黑素合成酶基因启动子的结合减少，对关键合成酶基因表达的抑制也减少，关键合成酶基因的表达上调，褪黑素合成增加。褪黑素作为一种光周期信号，抑制子叶开张，而HY5能减少褪黑素对子叶开张的抑制。本研究为进一步揭示褪黑素合成的调控机制，以及通过分子育种培育“褪黑素强化”的苹果奠定了基础。