越橘VcMYB调控因子对低温诱导花青苷合成的作用机制

Blueberry is the world's leading nutritional fruit because of its high anthocyanin content. Low temperature affects anthocyanin synthesis. Generally, Anthocyanin synthesis were induced by MYB/bHLH/WD protein complexes, which regulated the expression of structural genes involved in anthocyanin synthesis in low temperature. However, the molecular mechanism of R2R3-MYB negatively regulates anthocyanin synthesis in low temperature was unclear. Recently, the applicant isolated a VcMYB protein in blueberry leaves, which is negative regulator of anthocyanin synthesis. The expression of VcMYB was inhibited and anthocyanin synthesis was promoted by low temperature. To clarify VcMYB gene by which several structural genes regulates the blueberry anthocyanin synthesis, heterologous expression of VcMYB in Arabidopsis thaliana are to be performed to identify the target genes of VcMYB gene negative regulation. Yeast one hybrid and electrophoretic mobility shift assay (EMSA) are to be performed to check if VcMYB gene regulates expression of target genes. To clarify VcMYB protein interacts with which several proteins to regulate expression of structural genes involved in anthocyanin synthesis in blueberry, yeast two hybrid library will be established to screen interacting proteins of VcMYB. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation are to be performed to check if VcMYB protein interacts with screened proteins. At the same time, interaction proteins are transferred to Arabidopsis thaliana to verify their function. The results will prove theoretically basis to known blueberry anthocyanin synthesis mechanism in low temperature from transcriptional negative regulation level.

越橘果实以其较高的花青苷含量，成为世界上主要的营养保健果品。低温下，MYB同bHLH和WD40蛋白形成蛋白复合体共同诱导花青苷合成相关基因的表达，但低温下MYB负调控因子对花青苷合成的作用机制并不清楚。前期研究中，申请人获得了调控越橘花青苷合成的负调控蛋白VcMYB, 低温抑制其表达，促进花青苷的合成。为了明确在低温下VcMYB基因是通过调控哪些结构基因的表达来调控花青苷的合成, 将VcMYB基因在拟南芥中异源表达，初步确定VcMYB基因负调控的靶基因，采用酵母单杂交和凝胶迁移试验对其进行验证。为了确定低温下哪些蛋白同VcMYB蛋白互作，共同调控花青苷合成，建立了酵母双杂交文库，筛选VcMYB互作蛋白，采用双分子荧光互补和免疫共沉淀对互作蛋白进行验证。将互作蛋白转入拟南芥，对其功能进行验证。研究将为从转录负调控水平认识低温下越橘花青苷合成的调控机制提供理论。

花青苷作为一种类黄酮化合物对提高果实品种至关重要。本项目主要研究了越橘R2R3-MYB转录因子VcMYB如何在低温条件下负调控花青苷的合成。项目主要研究成果如下：①对VcMYB转录因子氨基酸序列分析显示，该基因属于负调控因子，其表达量与花青苷合成负相关。将该基因转入拟南芥中发现，低温抑制了转基因拟南芥花青苷的合成。因此得出结论，VcMYB蛋白在低温下受到抑制，因此促进了花青苷的合成。②低温处理下，对蓝莓花青苷合成的相关基因的表达分析，以及转基因拟南芥花青苷合成途径的花青苷合成基因的表达量分析显示，VcMYB蛋白主要通过结合花青苷合成基因的启动子序列，来调控花青苷合成，并采用酵母单杂交和凝胶迁移试验进一步证明VcMYB蛋白主要通过调控ANS启动子序列来抑制花青苷的合成。③建立了蓝莓酵母双杂文库，对VcMYB的互作蛋白进行筛选，结果显示VcMYB不是通过与bHLH和WD40蛋白互作来调控花青苷的合成，而是通过直接绑定目的基因的启动子序列，来抑制花青苷的合成，属于AtMYB4-like型的负调控因子