SnRK1蛋白激酶调控桃果实糖代谢的作用机理

Sugar content is an important agronomic trait of peach fruit, and SnRK1 protein kinase is a key switch protein kinase adjusting carbon and nitrogen metabolism. Recently, the applicant found that the overexpression of the gene, which encodes the α subunit of MhSnRK1 in M. hupehensis, could enhance the photosynthesis of tomato and increase starch and soluble sugar content in leaf and fruit. It suggested that SnRK1 protein kinase may be involved in the regulation of fruit glycometabolism. But it is still an open question concerning how SnRK1 protein kinase influences fruit glycometabolism. Therefore, on the basis of the preliminary study that sorbitol and trehalose could regulate the enzymatic activity of SnRK1 and effect fruit sugar content, by using peach fruit, the effects of sorbitol and trehalose treatments on encoding level of α subunit of PpSnRK1, the enzymatic activity of SnRK1 and key sugar metabolism enzymes, fruit sugar content are detected and analyzed in the process of fruit development. Yeast two hybrid, bimolecular fluorescence complementation (BiFC) and Pull-Down assays are to be performed to check if SnRK1 protein kinase could regulate glucose metabolism enzyme activity and effect fruit sugar accumulation by phosphorylating sorbitol dehydrogenase and sucrose phosphate synthase. This study aims to provide theoretical reference to target SnRK1 for regulating fruit sugar content.

含糖量是桃果实重要的农艺性状，SnRK1激酶是调节碳氮代谢的关键开关。申请人研究发现，平邑甜茶MhSnRK1α亚基编码基因超表达可促进番茄光合作用，提高叶片与果实淀粉和可溶性糖含量，表明SnRK1激酶可能参与调控果实糖代谢过程，但其作用机理并不清楚。为此，本项目在初步研究了山梨醇和海藻糖能够调控桃果实SnRK1激酶活性，并影响果实糖含量的基础上，进一步检测分析桃果实发育过程中山梨醇与海藻糖处理对SnRK1α亚基编码基因表达、SnRK1激酶与糖代谢关键酶活性及果实含糖量的影响，并采用酵母双杂交、BiFC和Pull-Down检测等技术研究SnRK1激酶是否通过磷酸化山梨醇脱氢酶，磷酸蔗糖合酶等果实糖代谢关键酶，调控糖代谢酶活性，进而影响果实糖分积累。为以SnRK1为靶点调控果实含糖量提供理论参考。

为明确SnRK1在果实发育和糖代谢过程中的调控作用，以 ‘瑞蟠17’（Prunus persica var ‘Ruipan 17’）为试材，研究了桃SnRK1蛋白激酶通过山梨醇脱氢酶及转录因子RIN蛋白的相互合作用关系，探究了其调控果实成熟发育的作用机理。结果如下：.1.桃SnRK1蛋白激酶在‘瑞蟠17’桃果实发育初期及两次快速生长期酶活性较高，在硬核期酶活性较低，这与其α催化亚基在转录水平上的基因表达较一致。.2. 外源100 mM海藻糖显著提高了桃果实中海藻糖-6-磷酸的含量，显著降低桃SnRK1酶活性，山梨醇脱氢酶（SDH）和蔗糖合酶合成方向酶活性也显著降低，而蔗糖磷酸合酶（SPS）的活性显著升高。外源山梨醇显著提高了桃SnRK1和SDH酶活以及桃果实中的果糖和蔗糖的含量。在两种不同的糖处理下，SDH酶活性始终与SnRK1酶活性变化规律一致。. 3. 通过酵母双杂交实验和原核诱导蛋白表达验证了桃SnRK1α与SDH和SPS蛋白存在互作关系，并且SnRK1可磷酸化SDH，说明SnRK1可能在蛋白水平上调节SDH酶活性参与山梨醇代谢。.4. 超表达PpSnRK1α番茄SnRK1酶活性在果实发育各时期均显著高于野生型；完熟期果实中的淀粉及可溶性糖含量显著提高，而可溶性蛋白及可滴定酸的含量明显降低；且果实比野生型提早10天成熟；通过酵母双杂交和分子荧光互补试验证明了PpSnRK1α和转录因子SIRIN蛋白间存在相互作用关系，且发生在细胞核内。.5. 超表达PpSnRK1α番茄果实中RIN的表达量从果实转色期开始均显著高于野生型。同时RIN下游成熟相关靶基因NOR、FUL1、ACS2、ACS4和E8的表达量也均不同程度的上调。在果实发育不同时期，超表达PpSnRK1α番茄株系果实中RIN下游成熟相关靶基因的表达量均显著高于野生型WT，且成熟果实中乙烯的释放量与野生型相比也显著提高。