马铃薯转化酶复合体中SnRK1α的磷酸化机理及块茎低温糖化抗性形成机制解析

Accumulation of reducing sugars in potato cold stored tubers, termed as cold-induced sweetening, is a serious post-harvest problem for potato tubers, which results in poor-quality fried potato products. Sucrose cleavage by vacoular invertase StvacNV1 plays vital roles in the cold-induced sweetening. We previously identified a protein complex (IRPC), which was composed of StvacINV1, its specific inhibitor StInvInh2B, and the α and β subunits of the kinase SbSnRK1. We further confirmed that the phosphorylation status of SbSnRK1α determined the activity of StvacINV1. However, the phosphorylation mechanism of SbSnRK1α and the interactions among IRPC components remains elusive. This proposed project plans to identify the genes controlling SbSnRK1α phosphorylation by protein interaction screening, in vitro and transgenic functional verification. Meanwhile, other members of SnRK1β subunits, as well as SnRK1γ, will be cloned and their function will be dissected through expression analysis, subcellular localization, heterologous expression, in vitro enzyme assays and genetic transformation. By employing homology modeling and molecular docking, the binding sites of IRPC will be predicted. By using point mutation and in vitro functional test, the predicted results will be confirmed. Finally, the structural basis of invertase regulation by IPRC will be illustrated. Collectively, these results will ultimately uncover the regulator mechanism of invertase activity, deepen our understanding for the formation of potato cold-induced sweetening, and also provide potential applicable genetic resources and theoretical basis for improving the quality of fried potato products.

马铃薯块茎低温贮藏后还原糖的累积是影响油炸加工品质的重要因素，液泡转化酶控制的蔗糖分解对低温糖化起着重要作用。我们前期研究证明，蛋白复合体IRPC精确调控了转化酶活性，其中SbSnRK1α磷酸化与否决定了转化酶活性高低，但其磷酸化机制及IRPC组分间互作的机理尚不清楚。本项目拟通过互作蛋白分离SbSnRK1α磷酸化的调控基因，利用体外活性测定和转基因等技术，明确其功能，解析SbSnRK1α磷酸化的调控机制。通过基因表达分析、亚细胞定位、体外添加活性测定以及遗传转化等手段，明确SbSnRK1的β和γ的在转化酶活性调控中的功能。利用同源建模及分子对接等生物信息学手段，对IRPC的互作模式进行模拟，进一步通过体外蛋白活性及互作验证，解析IRPC对转化酶活性调控的分子结构基础。研究结果将深入揭示马铃薯转化酶活性调控的分子模式，解析块茎低温糖化抗性的形成机理，为其加工品质改良提供理论指导和基因资源。

马铃薯块茎低温贮藏后还原糖的累积是影响油炸加工品质的重要因素，液泡转化酶控制的 蔗糖分解对低温糖化起着重要作用。我们前期研究证明，蛋白复合体IRPC精确调控了转化酶活性，其中SbSnRK1α磷酸化与否决定了转化酶活性高低。在此基础上，通过同源比对鉴定到了激酶StGRIK1为其潜在的上游激酶，通过StGRIK1异源表达和对StSnRK1α体外激酶活性，证实了StGRIK1能直接磷酸化SbSnRK1α，亚细胞定位发现StGRIK1定位于液泡膜。抑制StGRIK1的表达会导致光照后马铃薯叶片淀粉和还原糖的含量降低和SnRK1α内源的磷酸化水平降低，表明StGRIK1可能是马铃薯中SnRK1上游唯一的激酶。通过序列比对鉴定到了马铃薯SnRK1γ亚基，qRT-PCR表达分析表明在不同抗性材料中都受低温诱导。进一步通过酵母点对点验证了StSnRK1γ与SbSnRK1α间确实存在互作。亚细胞定位结果显示StSnRK1γ定位于细胞质和细胞核。干涉StSnRK1γ能显著降低低温贮藏后块茎的还原糖含量，酸性转化酶活性测定结果显示干涉StSnRK1γ亦能显著抑制低温贮藏后块茎的酸性转化酶活性，明确了SnRK1γ对IRPC中转化酶活性的调控功能。利用基于神经网络开发并可提供原子级精度的蛋白质结构预测工具AlphaFold2分别对IRPC复合体各组分进行了蛋白质结构解析。并进一步在StSnRK1α-StSnRK1β、StvacINV1-StInvInh2B和StSnRK1β-StInvInh2B三对二聚体鉴定到了8对氨基酸位点互作对，互作对可能是介导IRPC形成的重要位点并可能在调控转化酶活性过程中起关键作用。项目研究成果揭示马铃薯转化酶活性调控的分子模式，为马铃薯加工品质改良提供理论指导和基因资源。