橡胶树乳管蔗糖降解关键转化酶HbNIN2的互作蛋白筛选与功能分析

In rubber tree (Hevea brasiliensis), natural rubber biosynthesis occurs in a specialized cell type called laticifers, with sucrose as the precursor molecule. In the laticifers, the efficiency of sucrose catabolism is a rate-limiting step affecting latex (cytologically, the cytoplasm of laticifers) metabolism and rubber productivity. Previously, an alkaline/neutral invertase (A/N-Inv), HbNIN2, was identified as the key enzyme responsible for sucrose catabolism in the laticifers. In the roots of Arabidopsis, phosphatidylinositol monophosphate 5-kinase (PIP5K) and 14-3-3 protein directly interact with A/N-Invs and regulate their enzymatic activity. It remains to learn whether there are new proteins interacting with A/N-Invs or novel regulation modes in different plant species or different tissue/cell types. This proposal aims to screen and identify the HbNIN2-interacting proteins with combining the techniques of Y2H, CoIP and BiFC, investigate their regulations on the enzymatic properties of HbNIN2, and learn the correlation of such regulations in the regulating latex invertase and latex metabolism. The results expected will help elucidate the molecular mechanisms underlying sucrose catabolism in the laticifers of rubber tree, expand the researches in the molecular regulation of A/N-Invs in plants, and give hints to developing novel techniques to stimulate latex production.

橡胶生物合成以蔗糖为原料、在特化的乳管细胞中进行。乳管细胞中蔗糖的降解效率是决定胶乳代谢活性、影响橡胶产量的关键因素。在前期研究中，我们分离和鉴定了负责乳管蔗糖降解利用的中/碱性转化酶（A/N-Inv）HbNIN2。拟南芥根中，磷脂酰肌醇磷酸激酶（PIP5K）和14-3-3蛋白能直接同A/N-Inv互作并调控其酶活性，但不清楚在不同植物或不同组织（或细胞）中是否有新的A/N-Inv互作蛋白或存在不同的调控模式。本项目以HbNIN2为靶标，将酵母双杂交、免疫共沉淀和双分子荧光互补技术相结合筛选和鉴定胶乳中HbNIN2的互作蛋白，同时利用体外酶学测定和胶乳分析法，研究互作蛋白对HbNIN2的酶学调控模式，以及这种调控与胶乳转化酶和胶乳代谢调控的关系。研究结果将有助于阐明橡胶树乳管蔗糖降解调控的分子机制，拓展植物A/N-Inv分子调控研究的领域，为胶乳代谢人工调控新技术的研发提供理论指导。

我们的前期工作证明HbNIN2是决定橡胶树乳管蔗糖降解利用和橡胶产量形成的关键酶，并且已有研究显示该酶的表达调控主要在翻译后水平，但具体调控机制不清楚。本项目以酵母双杂交筛选HbNIN2互作蛋白，进而研究互作蛋白调控HbNIN2酶活的机制，取得的主要进展为：筛选到14-3-3和DnaJ等多个HbNIN2互作蛋白，体外重组蛋白的共孵育显示DnaJ和14-3-3蛋白均可显著提高HbNIN2酶活，DnaJ还可以提高HbNIN2 酶活的热稳定性，减轻重金属离子胁迫对HbNIN2 酶活的抑制作用；进一步研究了HbNIN2与14-3-3蛋白的互作机制，确定第550-555位的氨基酸（K550RSSSW555）为HbNIN2与14-3-3互作的核心基序，Ser554磷酸化是HbNIN2和14-3-3互作的必要条件，在胶乳中14-3-3显著提高HbNIN2酶活。项目研究为阐明胶乳转化酶活性调控的分子机制奠定了基础，为通过分子改良手段调控橡胶树产胶碳库分配提供了新思路。项目研究已发表论文6篇（SCI收录4篇），授权国家发明专利1项，1人获国家杰出青年科学基金项目资助，培养研究生7名（博士4名），应邀在国内外学术会议上做学术报告7个。