氮素是影响苹果果实产量和品质的重要营养因素，在果树生产中，过量施氮负调控果实品质形成，导致品质下降，但分子机理至今不清楚。在前期研究中，申请人从苹果中克隆到一个氮响应基因MdBT1，酵母双杂交表明MdBT1能够与花氰苷合成调控蛋白MdMYB1相互作用，暗示氮信号可能通过MdBT1和MdMYB1调控果实色泽形成。为了验证该设想，本研究拟利用BiFC和Pull-Down检测，验证MdBT1和MdMYB1在活体状态下的互作，然后检测MdBT1对MdMYB1蛋白泛素化的影响，同时检测MdBT1对MdMYB1蛋白结合下游基因启动子的影响，确认MdBT1调控MdMYB1的方式。最后，检测转基因苹果愈伤组织和组培苗以及瞬时表达MdBT1的果实中的花氰苷合成，确定MdBT1在花氰苷合成和果实色泽形成中的生物学功能，最终解释氮信号通过MdBT1影响MdMYB1调控果实色泽形成的分子机理和调控途径。

Nitrogen is one of the most important nutrient factors which influence fruit yield and quality in apple industry. Generally, nitrogen application improves fruit yield, however, oversupply of nitrogen negatively contributes to fruit quality formation, resulting in fruits with low commodity and market values in apple trees. It is still an open question concerning how nitrogen influences fruit quality in molecular level. Recently, the applicant isolated a nitrogen-responsive gene MdBT1 from apple fruit peel. It encodes a predicted polypeptide containing conserved BTB-TAZ and CaMBD domains. Interestingly, yeast two hybrid assay showed that MdBT1 protein physically interacts with MdMYB1 which is a key activator for anthocyanin biosynthesis and fruit coloration in apple, suggesting that MdBT1 may be involved in the regulation of nitrogen in anthocyanin biosynthesis and fruit coloration by modulating MdMYB1 in apple. To verify this hypothesis, firstly, bimolecular fluorescence complementation (BiFC) and Pull-Down assays are to be performed to check if MdBT1 protein interacts with MdMYB1 in vivo in plant cells. Subsequently, Western blotting with specific antibodies will be conducted to determine if MdBT1 influences the ubiquitination and degradation of MdMYB1 protein. Meanwhile, EMSA and ChIP-PCR assays will be carried out to make sure if MdBT1 impacts on the specific binding of MdMYB1 protein to the promoters of downstream genes MdDFR and MdUFGT. These assays are used to make sure by which way MdBT1 regulates the function of MdMYB1. Finally, transgenic apple calluses and plantlets, as well as apple fruits transiently expressing MdBT1 and MdMYB1 together or each alone with a VIGS vector-mediated approach, are used to characterize the biological function of MdBT1 in anthocyanin biosynthesis and fruit coloration in apple. This study aims to elucidate the mechanism and pathway by which nitrogen influences anthocyanin biosynthesis and fruit coloration in apple, maybe by the regulation of MdBT1 in the function of MdMYB1 protein.