百合双色花转录因子LhMYB305控制花青素苷合成的上游调控机理研究

Lily (Lilium L.) is a worldwide important ornamental plant. Lily hybrids with bicolor flowers have high ornamental values due to their unique color patterning. We have elucidated LhMYB305 gene regulates the development of lily bicolor tepals by regulating the anthocyanin biosynthesis pathway. But the mechanism involved in regulating the spatial-temporal expression of LhMYB305 is still unknown. In this present proposal, we will carry out the following study：firstly, bisulfite sequencing PCR will be used to uncover whether the methylation levels of LhMYB305 promoter regulates the spatial-temporal expression of LhMYB305 in tepals at four development stages. Secondly, upstream genes, determining the spatial-temporal expression of LhMYB305, will be screened via yeast one-hybrid analysis. And then, electrophoretic mobility shift assay and dual-luciferase reporter assay system will be used to confirm the interaction of candidate genes with LhMYB305 promoter and the transcriptional activity of candidate genes, respectively. The function of the genes will be determined by subcellular localization, expression analysis, and overexpression in tobacco plants. The present proposal will elucidate the mechanism involved in regulating the spatial-temporal expression of LhMYB305, which will provide some important candidate genes for breeding to change flower colors in lily cultivars. Therefore, the present proposal is of both practical and theoretical significance.

百合是世界重要观赏植物，其双色花类型极具观赏价值，探明双色花形成和调控的分子机理对百合花色改良具有重要意义。项目组前期研究发现：LhMYB305基因调控着百合双色花中花青素苷的生物合成，但调控LhMYB305基因特异性表达的上游调控机制尚未揭示。因此，本申请拟在已有研究基础上，测定花蕾不同发育时期花被片白色和紫红色区LhMYB305基因启动子甲基化动态变化，明确甲基化与LhMYB305基因表达的关系；通过酵母单杂交筛选直接调控LhMYB305基因时空表达的上游转录因子，然后运用凝胶迁移（EMSA）技术对候选上游转录因子与LhMYB305基因启动子进行体外结合试验，并运用双荧光素酶报告基因检测技术检测候选转录因子转录活性，再通过转化烟草对候选转录因子进行功能鉴定。以此，阐明调控LhMYB305基因表达的上游直接调控机制，明确百合双色花形成的分子机理，为百合花色分子改良提供基因储备和理论依据。

百合双色花极具观赏价值，探明其形成和调控的分子机理对百合花色改良具有重要意义。项目组前期研究发现：LhMYB305（LhMYB12-lat）基因调控着百合双色花中花青素苷的生物合成，但调控该基因特异性表达的上游调控机制尚未揭示。本项目研究通过染色体步移克隆获得该基因上游3222 bp的启动子序列。在此基础上，测定了不同时期双色花上下部该基因启动子甲基化程度，结果表明其启动子甲基化程度不参与调控该基因特异性表达；通过酵母单杂交筛选到13个直接调控该基因时空表达的上游转录因子，亚细胞定位结果表明候选转录因子定位于细胞核；运用双荧光素酶报告基因检测技术检测结果表明候选转录因子LhBED、LhARR、LhMYB、LhAUX具有强激活该基因表达活性、候选转录因子LhARF具有抑制其表达的活性；酵母单杂交试验表明LhBED、LhARR、LhMYB、LhAUX候选转录因子可以直接结合该基因启动子上。在此基础上，进一步构建过了表达载体转化烟草和拟南芥进行了功能验证。通过本项目的实施进一步阐明了百合双色花形成的分子机理，为百合花色分子改良提供基因储备和理论依据。发表SCI论文6篇，中文核心1篇。培养博士研究生三名，硕士研究生两名。