甘薯IbOr蛋白参与调控类胡萝卜素裂解途径的机制研究

Carotenoids and apocarotenoids play essential roles in the formation of pigments, aromas and nutritional quality in fruit or tuber, and also in improving human immunity and anti-aging. We previously reported that IbOr functions as a molecular chaperone in regulating carotenoid accumulation in sweetpotato. Preliminary result suggest that IbOr might be involved in carotenoid cleavage via interacting with carotenoid cleavage dioxygenases 4 (IbCCD4) identified by co-IP-MS. Here, we will confirm the binding region of IbCCD4 in IbOr through Y2H and BiFC; and to reveal the role of IbOr on IbCCD4, we will introduce plasmids expressing recombinant IbOr and/or IbCCD4 proteins into strains of E. coli BL21 with plasmids for the production of β-carotene; and to examine whether the IbOr-IbCCD4 protein complex affect carotenoids cleavage function of IbCCD4 in sweetpotato plants, we will develop the transgenic sweetpotato plants with over-expressed IbOr and IbCCD4, and plants with over-expressed IbOr but down-regulated IbCCD4, the carotenoids and apocarotenoids in these plants will be measured upon exposure to carotenoid inhibitor NFZ treatment. We expect to provide theory basis for further understanding the regulatory mechanism of carotenoid metabolism in sweetpotato and developing high-quality cultivars of sweetpotato plants.

类胡萝卜素及其裂解物在果实或薯块色泽、风味和营养品质的形成，以及提高人体免疫力和延缓衰老等方面起着重要作用。已知甘薯IbOr蛋白能通过其分子伴侣活性调控类胡萝卜素的生物合成。项目申请人前期通过免疫共沉淀联合质谱技术发现IbOr可能通过与类胡萝卜素裂解双加氧酶4（IbCCD4）相互作用参与调控类胡萝卜素裂解途径。本项目拟通过酵母双杂交和双分子荧光互补技术，确定IbOr对IbCCD4的结合区域；通过在用于类胡萝卜素合成的工程菌株中表达IbOr和IbCCD4，揭示IbOr对IbCCD4活性的影响；分别培育在过量表达IbOr的转基因甘薯中过量或RNAi干扰IbCCD4表达的甘薯株系，通过分析它们在哒草伏（类胡萝卜素抑制剂）处理下类胡萝卜素及其裂解物的变化，明确IbOr通过IbCCD4调控类胡萝卜素裂解途径的机制。本项目的研究结果为丰富甘薯类胡萝卜素代谢调控网络和培育高品质甘薯品种提供理论基础。

类胡萝卜素及其裂解物在果实或薯块色泽、风味和营养品质的形成，以及提高人体免疫力和延缓衰老等方面起着重要作用。项目申请人前期通过免疫共沉淀联合质谱技术发现IbOr可能通过与类胡萝卜素裂解双加氧酶4（IbCCD4）相互作用参与调控类胡萝卜素裂解途径。本项目通过酵母双杂交和双分子荧光互补试验，发现IbOr主要通过其氨基端与IbCCD4进行互作，并且作用位置主要位于叶绿体内；通过在用于类胡萝卜素合成的工程菌株中表达IbOr和IbCCD4，以及培育在过量表达IbOr的转基因甘薯中过量或基因组编辑IbCCD4表达的甘薯株系，分析它们的类胡萝卜素及其裂解物含量变化，明确IbOr 通过与IbCCD4 互作主要对玉米黄素裂解途径进行负调控，而非作用于IbCCD4的主要底物β-胡萝卜素的降解。本研究丰富了甘薯类胡萝卜素代谢调控网络，并为培育高品质甘薯品种奠定理论基础。