观赏海棠花色素苷代谢中RNA介导的DNA甲基化修饰McCOP1途径研究

Exploiting the anthocyanin biosynthesis in crabapple during the leaf development is important to reveal the molecular mechanism of color formation of Malus. Several researches showed that anthocyanin regulation transcription factor MYB could be bound by the E3 ubiquitin ligase COP1, and COP1 may be modified by RNA-directed methylation pathway. In this study, we plan to reveal that McCOP1 regulate anthocyanin biosynthesis which is mediated by the mechanism of RNA-directed DNA methylation in Malus crabapple. Our study includes two aspects: Firstly, to analyze the relationship between anthocyanin accmulation and the methylation of McCOP1, we will use ever-color-leaf cultivars as materials to analyze the methyaltion and accmulation level of McCOP1; Secondly, BiFC, genetic transformation and yeast one hybrid were used to determine the regulation mechanism of RNA-directed DNA methylation on anthocyanin biosynthesis by the methylation of McCOP1. Ultimately, we expect to provide theoretic and technical basis in the color formation of Malus crabapple.

探索观赏海棠叶片发育过程中花色素苷代谢分子机理，对揭示苹果属植物彩叶成色机理和分子育种意义重大。近年来的研究表明，E3泛素化连接酶COP1能够特异结合调控花色素苷生物合成的重要MYB转录因子，同时COP1可能被RNA介导的DNA甲基化途径修饰。因此，本课题旨在揭示泛素化基因McCOP1的上游修饰调控机制即RNA介导的DNA甲基化途径能否通过修饰McCOP1基因来调控花色素苷代谢的机理。研究包括两方面内容，第一方面主要通过分析MYB10特异结合的E3泛素连接酶McCOP1的积累和甲基化水平，确定McCOP1甲基化是否影响观赏海棠花色素苷代谢；第二方面主要通过对RNA介导的DNA甲基化途径关键基因进行蛋白互作、遗传转化、蛋白与靶基因启动子结合研究，确定观赏海棠中RNA介导的DNA甲基化途径通过修饰McCOP1调控花色素苷代谢。以期待为观赏海棠叶色发育、彩色品种的遗传改良提供理论基础和技术支撑。

研究发现COP1蛋白可以作为花色素苷的调控者，并推测COP1基因启动子可能被RNA介导的DNA甲基化途径进行甲基化修饰。本研究以‘王族’、‘绚丽’、‘火焰’3个观赏海棠品种10个不同发育时期的叶片为材料，研究观赏海棠中RdDM途径如何通过修饰McCOP1基因启动子进而调控花色素苷生物合成代谢。结果表明，McCOP1s 基因启动子发生甲基化修饰，以CHH型为主；在一定程度上，McAGO4-A、McAGO4-Like、McDRM2、McRDM1与McCOP1s的基因表达水平呈负相关。BiFC结果表明McAGO4s、McDRM2二者与McRDM1形成蛋白复合体；同时Y1H、EMSA试验结果显示，McAGO4s能够结合McCOP1基因启动子。沉默McRDM1能够使‘王族’叶片和苹果果皮花色素苷含量降低，以上结果证明：在RNA介导的DNA甲基化途径中，McAGO4、McDRM2 、McRDM1形成RdDM蛋白复合体后甲基化修饰观赏海棠McCOP1s基因启动子，抑制基因表达；继而通过调控下游靶蛋白McMYB10积累水平，影响花色素苷生物合成。