低温介导MdSIZ1调控苹果果实色泽品质形成的分子机理研究

Fruit coloration is an important agronomic trait for fruit tree. Anthocyanin and its derivatives are the main pigments that determine the fruit coloration. Temperature is one of the most important enviromental factors that induce anthocyanin biosynthesis, however, the regulating molecular mechanism remains unclear. Recently, the appliant isolated a low-temperature-responsive gene MdSIZ1 from apple fruit peel. MdSIZ1 encodes an E3 SUMO ligase gene, which mediates many downstream transcription factors sumoylation to regulate their protein stability. Intrestingly, yeast-two-hybrid assay showed that MdSIZ1 protein physically interacts with MdMYB1, which is a key activator for anthocynin biosynthesis and fruit coloration in apple, suggesting that MdSIZ1 may be involved in the regulation of apple fruit coloration by modulating MdMYB1. To verify this hypothesis, firstly, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, and pull-down assays are to be performed to check if MdSIZ1 protein interacts with MdMYB1 in vivo in plant cells. Secondly, transgenic and sumoylation techniques will be used to test the sumoylation of MdMYB1 by MdSIZ1. Finally, transgenic apple callus and plantlets, as well as apple fruits transiently expressing MdSIZ1 with a VIGS vector-mediated approach, are used to charaterize the biological function of MdSIZ1 in anthocyanin biosynthesis and fruit coloration in apple. This study aims to clarify the molecular mechanism of low-temperature-induced MdSIZ1 mediated apple fruit coloration.

果实色泽是果树的重要农艺性状，苹果果皮的颜色主要由花青素及其衍生物决定，温度是影响花青素合成的重要环境因素，但调控的分子机理至今不清楚。在前期研究中，申请人从苹果中克隆了响应低温信号的重要基因MdSIZ1，它是一个E3 SUMO连接酶，可将下游很多转录因子SUMO化，影响其蛋白稳定性；酵母双杂交证实MdSIZ1和MdMYB1能够互作，推测MdSIZ1可能通过调控MdMYB1蛋白稳定性影响花青素积累。为了验证该假想，本研究拟利用双分子荧光互补、免疫共沉淀和Pull-down技术验证MdSIZ1与MdMYB1的相互作用，并通过转基因和SUMO化分析等技术检测MdSIZ1对MdMYB1的SUMO化作用。最后，检测转基因苹果愈伤、组培苗，以及瞬时表达MdSIZ1的苹果果实中花青素的积累，揭示低温介导的MdSIZ1调控苹果果实色泽形成的分子机理。

苹果果实的色泽是重要的外观品质性状，直接决定其市场价值。花青苷是决定苹果果实颜色的主要次生代谢产物，其合成受温度、光照、营养物质以及植物激素的调控。转录因子MdMYB1在调控花青苷合成方面发挥重要作用，但是关于翻译后层面的调控机理罕见报道。在本研究中，我们用MdMYB1蛋白进行酵母双杂交筛库，找到了可能与MdMYB1互作的蛋白MdSIZ1。.苹果MdSIZ1的基因全长3141 bp，编码一条含1047个氨基酸残基的蛋白，分子量约115 KD，MdSIZ1的表达受到低温、高温、强光和低磷等多种胁迫环境的诱导。将苹果MdSIZ1基因转化拟南芥siz1-2突变体，发现其可以部分恢复拟南芥突变体的缺陷表型，以及SUMO结合水平，证明MdSIZ1是拟南芥SIZ1的同源基因，与AtSIZ1具有类似的功能。.酵母双杂交、Pull-Down和Co-IP试验证明MdMYB1与MdSIZ1直接相互作用。活体和离体的SUMO化试验发现，MdSIZ1作为SUMO E3连接酶能够调控MdMYB1蛋白的SUMO化，被SUMO化修饰的MdMYB1蛋白稳定性提高。而MdSIZ1调控MdMYB1的稳定性，是通过抑制其发生26S蛋白酶体途径的降解实现的。.最后，通过MdSIZ1转化苹果愈伤组织和VIGS载体注射苹果果实的方法，发现在苹果愈伤和果实中过量表达MdSIZ1能够促进花青苷的生物合成和色泽形成，且调控作用是依赖于MdMYB1的。.综上所述，MdSIZ1在苹果花青苷的积累和色泽形成中发挥重要作用，因此明确MdSIZ1调控着色的分子机理对于改善苹果的品质具有一定的指导意义，并且为通过基因工程改善和促进果实的着色提供理论依据。