苹果硝态氮应答基因MdBT2调控叶片衰老的分子机理研究

As well known, nitrogen is one of the most important nutrient factors which influence leaf senescence in plants, although it is not clear how nitrogen involves in the modulation of this process. Our preliminary studies demonstrated that nitrate treatment inhibited leaf senescence and upregulated the expression level of a nitrate-responsive gene MdBT2. When MdBT2 was used as a bait to screen through an apple cDNA library with a yeast two-hybrid method, it was found that MdBT2 preyed MdEIN3, which is a crucial component in ethylene signaling pathway. These results suggest that nitrate regulates leaf senescence probably by recruiting MdEIN3-mediated ethylene signaling. To verify this hypothesis, BiFC, CoIP and Pull-Down assays are to be conducted to verify the in vivo and in vitro interaction between MdBT2 and MdEIN3 proteins. Subsequently, Western blotting will be performed with specific antibodies to examine if MdBT2 involves in the ubiquitination modification and the resultant stability of MdEIN3 proteins. Finally, transgenic apple calluses and plantlets, as well as apple materials transiently expressing MdBT2 and MdEIN3 together or each alone with a VIGS vector-mediated approach, are to be used to characterize the biological function of MdBT2 with leaf senescence in apple. This study aims to elucidate the molecular mechanism and regulatory pathway by which nitrogen-responsive protein MdBT2 recruits MdEIN3-mediated ethylene signaling to control leaf senescence in apple.

众所周知，氮素的供应水平影响植物叶片衰老，但分子机理至今不清楚。前期研究发现，硝酸盐能够抑制叶片衰老，并上调氮应答基因MdBT2的表达水平；转基因研究表明，MdBT2参与了硝态氮对叶片衰老的调控；以MdBT2为诱饵蛋白进行酵母双杂交筛库，筛选到乙烯信号途径的重要蛋白MdEIN3，表明硝态氮可能通过MdBT2调控乙烯信号，进而参与叶片衰老的调控。为了验证该假设，本研究拟利用BiFC、CoIP和Pull-Down等技术，进一步验证MdBT2和MdEIN3的相互作用，然后检测MdBT2对MdEIN3蛋白的泛素化修饰及其稳定性的影响。最后，检测转基因苹果叶片的衰老症状，确定MdBT2在苹果叶片衰老中的功能，最终解析氮信号通过MdBT2征集MdEIN3介导的乙烯信号，进而调控叶片衰老的调控途径和分子机理。

氮素的供应水平影响植物叶片衰老，但相关信号调控通路并不清晰。前期研究表明，硝酸盐响应基因MdBT2参与了氮素对叶片衰老的调控。本研究以MdBT2为诱饵蛋白进行酵母双杂交筛库，筛选到乙烯信号途径的重要转录因子MdEIL1，并利用一系列生化实验手段，验证了MdBT2和MdEIL1的相互作用。体外降解实验证实MdBT2负调控MdEIL1蛋白积累水平，表达分析显示MdBT2负调控叶片衰老相关基因表达。进一步通过酵母实验证实，MdBT2能够与茉莉酸信号关键组分MdMYC2互作，并促进MdMYC2蛋白的泛素化和降解，进而负调控MdMYC2介导的叶片衰老过程，从而建立了MdBT2-MdMYC2互作调控茉莉酸介导的叶片衰老信号通路。最后，还筛选到一个与MdBT2互作的MdZAT10蛋白，MdZAT10与MdABI5互作，并增强其转录活性，而MdBT2与MdZAT10互作，抑制了其蛋白稳定性，进而抑制了MdZAT10-MdABI5介导的叶片衰老过程。综上，本研究发现MdBT2能够与乙烯、茉莉酸、脱落酸信号通路互作，通过调控不同靶基因的蛋白稳定性，进而影响这些激素介导的叶片衰老过程。

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