蔗糖响应的促分裂原活化蛋白激酶FvMPK1调控草莓果实着色的分子机理

Sugar is one of the most important factors which influence fruit yield and quality. Sucrose functions as a signal molecule involved in the regulation of strawberry ripening and fruit coloration, but the molecular mechanism is still unknown. A sucrose-induced gene of mitogen-activated protein kinase,FvMPK1 was isolated from strawberry fruit, which is also involved in the regulation of fruit coloration.It is interested that yeast two hybrid assay showed that FvMPK1 protein physically interacts with FvTTG1 which is a key activator for anthocyanin biosynthesis and fruit coloration in strawberry, suggesting that FvMPK1 may be involved in the regulation in anthocyanin biosynthesis and fruit coloration by modulating FvTTG1 in strawberry. To verify this hypothesis, we use diploid strawberry as a model for fruit developmental study. Firstly, bimolecular fluorescence complementation (BiFC) and Pull-Down assays are to be performed to check if FvMPK1 protein interacts with FvTTG1 in vivo in plant cells. Subsequently, Western bloting with specific antibodies will be conducted to determine if FvMPK1 influences the phosphorylation. Meanwhile, EMSA and ChIP-PCR assays will be carried out to make sure if FvMPK1 impacts on the specific binding of FvMPK1 protein to the promoters of downstream genes FvANS and Fv3-GT. Finally, test the regulation function of surcose to the interacted-protein to characterize the biological function of FvMPK1 in anthocyanin biosynthesis and fruit coloration in strawberry. This study aims to elucidate the mechanism and pathway by which FvMPK1 influences anthocyanin biosynthesis and fruit coloration in strawberry, maybe by the regulation of FvMPK1 in the function of sucrose-induced FvTTG1 protein.

糖是形成果实产量和品质的重要因子，蔗糖可作为信号分子参与草莓果实成熟着色调控，而其分子机理并不清楚。申请人从草莓中克隆到一个受蔗糖诱导并调控果实着色的促分裂原活化蛋白激酶FvMPK1，酵母双杂交表明FvMPK1能够与花青素苷合成调控蛋白FvTTG1相互作用，且FvTTG1蛋白有MAPK磷酸化位点，表明FvMPK1可能通过FvTTG1调控果实色泽形成。为验证该设想，本研究采用二倍体草莓，利用双分子荧光互补（BiFC）和Pull-Down方法，验证FvMPK1和FvTTG1在活体状态下的互作，并检测FvMPK1对FvTTG1的磷酸化影响，鉴定FvMPK1对FvTTG1蛋白结合下游基因启动子的影响，确定FvMPK1调控FvTTG1的方式。最后，鉴定蔗糖对互作蛋白的调控作用，确定FvMPK1在果实色泽形成中的生物学功能，解释FvMPK1影响蔗糖诱导的FvTTG1调控果实色泽形成的分子机理。

糖是形成果实产量和品质的重要因子，蔗糖可作为信号分子参与草莓果实成熟着色调控，而其分子机理并不清楚。本研究采用二倍体草莓，从果实中克隆到一个受蔗糖诱导并调控果实着色的促分裂原活化蛋白激酶FvMPK1，酵母双杂交表明FvMPK1能够与花青素苷合成调控蛋白FvTTG1相互作用，利用双分子荧光互补（BiFC）和Pull-Down实验，验证FvMPK1和FvTTG1在活体状态下的互作，并且发现FvMPK1同时与FvMYB10也存在着互作关系。进而研究发现FvTTG1蛋白有MAPK磷酸化位点，点突变实验表明FvMPK1可以通过磷酸化FvTTG1，调控花青素合成关键基因FvANS，进而调控果实色泽形成。瞬时过量表达FvMPK1及RNAi实验显示，过量表达FvMPK1可以促进果实着色，而抑制FvMPK1基因可以抑制果实着色，表明FvMPK1在草莓果实着色中确实起了重要作用。进而对的FvMPK1进行了稳定遗传转化研究，发现FvMPK1通过和FvTTG1互作，调控草莓FvANS基因，进而调控草莓果实着色的机理。.