苹果氮响应基因MdBT1调控花氰苷合成和果实着色的分子机理研究

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.

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。

果实色泽是苹果等果树重要的农艺性状，苹果果皮的颜色是由花青苷决定的，其中MdMYB1在果实色泽形成过程中起着关键作用。在果树生产中，过量施氮导致果实着色降低。为了研究氮素调控果实色泽形成的分子机制，本研究首先通过体外实验，利用苹果组培苗和苹果果实，证明了氮素能够抑制苹果花青苷积累与果实着色。基因表达分析结果表明，硝酸盐在转录水平调控MdMYB1及其下游结构基因的表达，同时，体外蛋白降解与体内泛素化试验证明，硝酸盐在翻译后水平调控MdMYB1蛋白的稳定性。因此，硝酸盐能够在转录和翻译后水平调控MdMYB1蛋白，进而影响花青苷积累和果实色泽。.为进一步研究硝酸盐促进MdMYB1降解的分子机理，本研究通过酵母双杂筛库，从苹果cDNA文库中筛选到一个氮响应蛋白MdBT1。然后，Co-IP和Pull-Down检测进一步表明，BTB-TAZ家族的MdBT1和MdBT2蛋白均能够与MdMYB1相互作用，且MdBT2与MdMYB1互作的强度高于MdBT1。随后，检测了MdBT2对MdMYB1蛋白泛素化的影响，结果表明，MdBT2能够促进MdMYB1蛋白的泛素化降解。随后，本实验通过蛋白降解实验和竞争性结合实验，证明了MdBT2对MdMYB1蛋白的稳定性调控不依赖于MdCUL3。.进一步研究发现，MdBT2不仅在翻译后水平上对MdMYB1蛋白稳定起调控作用，两个基因在响应外界各种环境因子（硝酸盐、糖、光环境等）也存在相反的表达响应模式，表明MdBT2与MdMYB1同时在转录和翻译水平上响应外界硝酸盐等环境，最终影响苹果果实着色和花青苷积累。.最后，为了验证MdBT1和MdBT2在活体条件下是否影响了花青苷的积累，我们将MdBT1正义与反义表达载体分别转化并获得了苹果愈伤组织，对转基因愈伤进行表型观察和分析，结果表明，MdBT1和MdBT2负调控愈伤组织中的花青苷积累。同时，我们将MdBT2的反义表达载体遗传转化并获得了转基因苹果组培苗，表型分析表明， MdBT2沉默表达明显促进了花青苷合成结构基因的表达，最终增加了转基因草料中的花青苷含量。.综合上述结果，我们证明了MdBT1和MdBT2能够响应外界硝酸盐信号，它们通过与MdMYB1蛋白相互作用，将MdMYB1蛋白泛素化修饰，进而促进其降解，最终影响花青苷合成和果实着色。