苹果硝态氮应答基因MdBT2调控苹果酸积累的分子机理

苹果是我国的第一大水果，其代谢中间产物苹果酸直接影响果实的风味和加工品质。过量施氮能够降低苹果果实中苹果酸的含量，然而具体机理尚不清楚。在前期研究中，申请人发现外源高浓度硝酸盐明显抑制苹果果实中苹果酸的积累；酵母双杂交筛库显示，硝酸盐诱导的编码BTB-TAZ类蛋白MdBT2可以与调控苹果酸积累的bHLH转录因子MdCIbHLH1蛋白相互作用；通过酵母双杂交、双分子荧光互补和Pull-down的技术进一步验证了两者蛋白间的互作。据报道，MdBT2可通过26S蛋白酶体途径参与下游靶蛋白的降解，因此我们推测MdBT2可能通过泛素化降解MdCIbHLH1蛋白调控苹果酸的积累。为证实该猜想，本研究利用Cell-free系统，拟检测MdBT2对MdCIbHLH1蛋白的泛素化调控，最终解析硝酸盐应答蛋白MdBT2通过泛素化降解MdCIbHLH1蛋白调控苹果酸积累的分子机理与调控途径。

Apple (Malus domestica Borth.) is one of the most economically important fruit crops worldwide, and its metabolite malate directly affects the flavor and processing quality of fruits. Excessive nitrogen application reduces malate contents of apple fruits, however, the specific mechanism remains largely unexplored. In our preliminary studies, it was found that high concentration of exogenous nitrate significantly inhibited malate accumulation in apple fruits; Yeast two-hybrid (Y2H) screening assays showed that nitrate-induced MdBT2 encoding BTB-TAZ protein interacted with MdCIbHLH1 protein, a bHLH transcription factor that regulates malate accumulation. The interactions between these two proteins were further verified by Y2H, bimolecular fluorescence complementation (BiFC) and pull-down assays. It is well known that BTB-TAZ skeleton protein MdBT2 is involved in the degradation of downstream target proteins through the 26S proteasome pathway, suggesting that MdBT2 may regulate malate accumulation through ubiquitinating MdCIbHLH1 protein. To verify this hypothesis, cell-free assay systems and a series of transgenic materials treated with the proteasome inhibitor MG132 will be used to confirm the ubiquitination regulation of MdBT2 on MdCIbHLH1 protein. Overall, Our findings will provide new insights into the mechanism of nitrate-responsive protein MdBT2 modulation of malate accumulation, which occur by directly ubiquitinating MdCIbHLH1 protein in response to a nitrate signal in apple.