矮牵牛PhAAE13b及其泛素化修饰参与花色素苷合成的功能及其分子机制研究

Flower color is one of important qualities of ornamental plants. Anthocyanins are important pigments of many ornamental plants. In the previous study, we found that PhAAE13a is the key enzyme of the formation of malonyl-CoA, the precursor of anthocyanin biosynthesis. PhAAE13b, another member of PhAAE13 subfamily, can be modified by ubiquitin. The expression of PhAAE13b is closely related to anthocyanin biosynthesis. In this project, we will analyze the molecular mechanism of involvement of PhAAE13b and its ubiquitination in anthocyanin biosynthesis. (1) The function of PhAAE13b will be identified by VIGS silencing, CRISP/Cas9 knockout and over expression technology. (2) The E3 ubiquitin ligase interacting with PhAAE13b will be obtained by yeast two hybrid, BiFC and Co-IP method. (3) The spatio-temporal expression and subcellular localization of the E3 ubiquitin ligase will be analyzed. (4) The function of E3 ubiquitin ligases will be identified by VIGS silencing, CRISP/Cas9 knockout and overexpression techniques. (5) Western blotting technique is used to further determine the enzyme - substrate relationship of PhAAE13b and E3 ubiquitin ligases. In this project, anthocyanin biosynthesis is studied from the perspective of ubiquitination modification of protein. It is of great significance to enrich and develop the molecular mechanism of anthocyanin biosynthesis, and might provide a new path for flower color improvement.

花色素苷是许多观赏植物呈色的重要色素。本课题组前期发现形成矮牵牛花色素苷的丙二酰CoA由PhAAE13a催化苹果酸形成，而PhAAE13亚家族另一成员PhAAE13b能够被泛素化修饰，PhAAE13b表达与花色素苷合成的关系密切。本项目将分析PhAAE13b及其泛素化修饰参与花色素苷合成的分子机制：(1) CRISP/Cas9敲除等技术鉴定PhAAE13b的功能。(2) 酵母双杂交等方法获取PhAAE13b的互作E3泛素连接酶。(3) 所获E3的时空表达及亚细胞定位分析。(4) VIGS沉默、CRISP/Cas9敲除和超表达技术鉴定E3的功能。(5) Western Blotting技术等进一步确定E3与PhAAE13b的酶-底物关系。本项目从泛素化修饰的角度研究花色素苷合成，对丰富和发展花色素苷合成的分子机理具有重要意义，同时可能为花色调节提供新的路径。

花色是园林植物的主要观赏性状之一。花色素苷是植物花瓣呈色的主要色素。本项目对矮牵牛PhAAE13b、PhACLs、PhCSs和PhRSs在花色素苷生物合成中的功能进行研究：（1）PhAAE13b沉默后花色没有发生变化。（2）PhACLB1-B2沉默后植株矮化，植株的枝条茎间变短，叶片变小，叶色加深，花变小，花色变浅，花冠中的乙酰CoA含量和花青苷含量均明显降低。（3）PhmCS沉默植株的幼叶黄化，花色变浅，略微偏蓝，花冠和叶片中的柠檬酸含量降低，pH值升高，氨基酸含量发生变化。（4）PhRHM1-RHM2沉默导致叶片皱缩变黄、花色由紫红色变为红色，叶绿素和总花色素苷含量显著下降，并有6种含有鼠李糖基团的花色素苷被明显下调。本项目研究发现PhACLs、PhmCS和PhRHMs均参与了矮牵牛花瓣花青苷的生物合成，对矮牵牛花色素苷的生物合成路径进行了补充，并为观赏植物的花色改良提供新思路。