ZAT5介导ROS诱导红梨花青苷合成的分子机制研究

ROS可以诱导植物花青苷积累，但其机制仍有待解明。申请者前期从红梨中鉴定得到受ROS信号诱导的ZAT5基因，转入‘茄梨’愈伤组织中过表达可诱导花青苷的合成；同时用ROS处理过表达ZAT5的‘茄梨’愈伤组织进一步促进花青苷积累。本项目将进一步通过同源转基因，瞬时表达等技术进一步研究梨ZAT5在调控花青苷合成中的功能；通过ChIP-Seq结合RNA-Seq筛选ZAT5的下游靶基因，并对靶基因调控花青苷合成的功能进行研究。此外，筛选ZAT5上游的调控基因并进行功能验证；从已经筛选到的互作激酶MAPK6入手，探究磷酸化对ZAT5蛋白的影响;通过筛库技术筛选ZAT5其他伴侣蛋白且进行功能验证和作用方式验证。通过上述分析揭示ROS通过调控ZAT5基因进而诱导花青苷合成的分子机制。研究结果有助于完善红梨着色的分子调控网络，为红梨的遗传改良提供重要的基因资源，也为开发促进红梨着色的技术提供理论依据。

ROS signal has proved to induce anthocyanin accumulation in plants. However, the mechanism underlying ROS-induced anthocyanin biosynthesis is still unknown. In our previous studies, we identified a ZAT gene in red pear, namely ZAT5, which was found to be induced by ROS signal. Overexpression of ZAT5 in ‘Clapp’s Favorite’ pear calli increased anthocyanin biosynthesis. Meanwhile, the anthocyanin accumulation in ZAT5 transgenic pear calli was further improved by ROS treatments. In this project, the function of ZAT5 will be clarified via homologous transgenic technology and transiently expression technology in pear fruits. Besides, we will screen the ZAT5-targeted downstream genes by using ChIP-Seq and RNA-Seq analysis. The function of the identified target genes in regulating anthocyanin biosynthesis will be further verified. Furthermore，we will elucidate how ROS affects the expression of ZAT5. The promoter region of ZAT5 will be cloned and analyzed, and then the promoter will be used as a bait to screen upstream transcription factors. The function of these transcription factors will be clarified via transgenic technology. Moreover, the partner kinase MAPK6 has been previously found to interact physically with ZAT5. In this project, the function and action mode of MAPK6 will be further studied. In addition, ZAT5 will be used as a bait to screen other partner proteins, of which the function and action modes will be verified. Taken together, the molecular mechanism of ROS-induced anthocyanin synthesis by regulating ZAT5 gene will be revealed. This project will not only provide a novel gene for genetic improvement of red pear, but also contribute to the development of technologies for improving coloration of red pears.