番茄果实花青素合成调控基因ATROVIOLACIUM候选基因的功能验证与分析

Anthocyanins are naturally occurring pigments ubiquitously present in vegetable and fruit and, as such, part of the human diet. Owing to their biological activity, anthocyanins may offer protection against many diseases, including coronary heart diseases and cancer. In addition, anthocyanin can provide protection against biotic and abiotic stresses for plants. The applicant of this project has been studying the regulation of anthocyanin biosynthesis in tomato for many years. Recently, the applicant has already finished the fine mapping of ATROVIOLACIUM (ATV), a locus underlying anthocyanin biosynthesis in tomato fruit. Only one candidate gene for ATV was obtained, termed as SlMYBATV. SlMYBATV encodes an R3-MYB repressor. This project will (1) verify the biological function of SlMYBATV by gene editing system and ectopic expression; (2) figure out the molecular mechanism of ATV protein in the biological process of anthocyanin regulation. The results of this project will not only improve the regulation network of anthocyanin biosynthesis in solanaceous fruit vegetables, but also may ultimately be promising for further engineering of plant secondary metabolism.

花青素属于类黄酮化合物，广泛的存在蔬菜和水果中。长期食用花青素有利于人们改善视力，降低心血管疾病、癌症的发病率。花青素也可以提高植物对生物胁迫和非生物胁迫的抵抗能力。在前期工作中，申请人已经完成了番茄果实花青素合成调控基因ATROVIOLACIUM（ATV）的精细定位，获得了ATV唯一的候选基因SlMYBATV，该基因编码一个R3-MYB转录因子。以此为基础，本项目拟通过转基因的方法验证ATV候选基因的功能，结合转录组分析和蛋白互作研究揭示ATV蛋白质参与花青素合成调控的分子机理。研究结果将有助于阐明番茄果实中花青素合成调控的分子机理，为番茄以及茄果类其他蔬菜作物遗传改良提供重要的基因资源，同时也为基因编辑高品质番茄育种改良提供理论基础。

花青素广泛存在植物体内，不仅可以提高植物本身对非生物胁迫的抵抗能力，还有利于人类健康。因此，培育高花青素番茄不仅可以丰富现有番茄品种的颜色，还有提高番茄果实的营养价值，具有的意义。本项目以紫黑番茄Indigo Rose为材料，将花青素合成关键关键基因ATV精细定位到番茄7号染色体约5 kb的范围内，该区间仅含有一个基因，编码R3 MYB转录因子，故将该基因命名为SlMYBATV；利用CRIPSR/Cas9技术突变SlMYBATV基因，证实了SlMYBATV负调控番茄果实花青素的积累；酵母单杂试验、酵母双杂交试验和双荧光素酶报告系统试验表明，SlAN2-like可直接结合SlMYBATV基因的启动子激活SlMYBATV基因的表达，SlMYBATV蛋白则与SlAN2-like竞争性结合SlJAF13蛋白，进而抑制SlAN2-like基因的功能和番茄果实中花青素的积累；而当SlMYBATV基因突变后，SlMYBATV蛋白抑制功能丧失，SlAN2-like则与SlJAF13结合激活SlAN1和SlAN11等基因的表达，进而促进花青素合成结构基因的表达促进番茄果实中花青素的积累；以上研究结果于2020年在《New Phytologist》上发表，对于加速培育高花青素番茄新品种具有重要的指导意义。在该项目的资助下，我们还利用CRISPR/Cas9技术和基因共表达网络分析技术，发现了光诱导花青素合成途径中不依赖于HY5调控的候选转录因子，扩展了光参与花青素合成调控的分子机理；这一研究结果于2019年在《Plant and Cell Physiology》上发表。此外，我们还探究了SlAN2基因在紫番茄Indigo Rose中的功能，发现SlAN2仅参与番茄营养器官中花青素的合成调控，这一研究结果于2020年在《Plant Cell Reports》上发表。期间，授权国家发明专利2项。