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

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.

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。

氮素是一种重要的营养元素，在调控苹果花青苷积累、果实色泽形成与植物衰老过程中发挥着关键作用。在果树生产中，过量施氮导致果实着色降低，但是其中的调控机制并不十分清楚。之前的研究表明，茉莉酸（JA）是一种重要的植物激素，能够诱导苹果花青苷积累、果实着色与叶片衰老等。氮素是否通过JA信号调控果实着色与叶片衰老，其分子机制目前并不清楚。.我们前期筛选到氮素响应蛋白MdBT2和MdBT4等，过表达MdBT2能调控苹果花青苷积累与果实着色，表达分析显示MdBT2除了受到硝酸盐诱导外，还能够响应外源JA和ABA信号，并调控苹果花青素积累。为了鉴定MdBT2在花青苷合成中的生物学功能及其分子机理，本研究首先确定MdBT2对茉莉酸的表达响应；同时利用苹果组培苗和转基因拟南芥，检测MdBT2对花青苷和叶片衰老的调控；进一步通过酵母双杂技术，结合Co-IP和Pull-Down等体内体外实验，证实MdBT2与茉莉酸信号转导途径中的阻遏蛋白JAZs互作；随后检测MdBT2对MdJAZs蛋白泛素化的影响，体外蛋白降解实验证实，MdBT2能够提高MdJAZ2的蛋白稳定性；进一步研究发现，MdBT2也可以与花青苷合成调控因子MdbHLH3/茉莉酸信号下游关键转录因子MdMYC2、ABA应答因子MdbZIP44分别互作，调控硝酸盐、茉莉酸和ABA介导的花青苷积累与叶片衰老；最后，我们研究还发现MdJAZ2能够与MdHIR4互作，调控苹果花青苷积累与果实着色。本研究最终解析氮信号通过MdBT2-MdJAZs及相关互作蛋白调控果实着色和叶片衰老的途径和分子机理。

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