苹果氮响应蛋白MdBT4与MdJAZ2互作调控花氰苷合成和果实着色的研究

项目名称： 苹果氮响应蛋白MdBT4与MdJAZ2互作调控花氰苷合成和果实着色的研究

项目编号： No.31471854

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 由春香

作者单位： 山东农业大学

项目金额： 85万元

中文摘要： 过量施氮抑制苹果果实着色，但分子机理尚不清楚。前期研究发现，硝酸盐能抑制茉莉酸（JA）诱导的花氰苷积累，并上调氮响应基因MdBT4的表达水平；以MdBT4为诱饵蛋白进行酵母双杂交筛库，筛选到JA信号途径的阻遏蛋白MdJAZ2，表明氮素可能通过MdBT4调控JA信号，进而影响花氰苷合成和果实着色。为了验证该设想，本研究拟利用CoIP和Pull-Down等技术，进一步验证MdBT4和MdJAZ2的相互作用，然后检测MdBT4对MdJAZ2蛋白稳定性的影响，同时利用酵母三杂交等技术检测MdBT4对MdJAZ2与MdbHLH3互作的影响，确认MdBT4调控MdJAZ2的方式。最后，检测转基因苹果愈伤组织和植株以及瞬时表达MdBT4的果实中花氰苷的积累量，确定MdBT4在苹果果实着色中的功能，最终解析氮信号通过MdBT4影响MdJAZ2介导的JA信号、进而调控果实着色的调控途径和分子机理。

中文关键词： 苹果；氮信号；茉莉酸信号；花氰苷合成；果实着色

英文摘要： 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 values and reducing disease resistance in apple trees. It is still an open question concerning how nitrogen influences fruit quality at molecular level. Our preliminary studies demonstrated that nitrate treatment inhibited the JA-induced anthocyanins accumulation, while upregulated the expression level of a nitrate-responsive gene MdBT4. When MdBT4 was used as a bait to screen an apple fruit peel cDNA library with a yeast two-hybrid method, it was found that MdBT4 preyed MdJAZ2, which is a inhibitor protein in JA signaling pathway. These results suggest that nitrate regulates anthocyanins biosynthesis and fruit coloration probably by modulating MdJAZs-mediated JA signaling. To verify this hypothesis, firstly, CoIP and Pull-Down assays are to be performed to check if MdBT4 protein interacts with MdJAZ2 in vivo and in vitro. Subsequently, Western blotting with specific antibodies will be conducted to determine if MdBT4 influences the stability of MdJAZ2 protein. Meanwhile, yeast three-hybrid assay will be carried out to make sure if MdBT4 impacts on the specific interaction between MdJAZ2 and MdbHLH3. These assays are used to make sure by which way MdBT4 regulates the function of MdJAZ2. Finally, transgenic apple calluses and plantlets, as well as apple fruits transiently expressing MdBT4 and MdJAZ2 together or each alone with a VIGS vector-mediated approach, are to be used to characterize the biological function of MdBT4 in anthocyanins biosynthesis in apple. This study aims to elucidate the molecular mechanism and regulatory pathway by which nitrogen influences anthocyanin biosynthesis and fruit coloration in apple, maybe by the regulation of MdBT4 in the stability and function of MdJAZ2 protein.

英文关键词： Apple;Nitrogen signal;JA signal;Anthocyanin biosynthesis;Fruit coloration