一种大豆LEA4蛋白PM1与AE7/MIP18相互作用对植物细胞质铁硫簇组装系统CIA功能的影响

Plant late embryogenesis abundant (LEA) proteins will highly express and accumulate at the late stage during seeds developing, and protect the biomolecule while the dehydration process. Iron-sulfur protein, a protein family which each contains a Fe-S cluster center, plays an important role in many metabolism pathways. Lost function of members in the Fe-S cluster assembly system will cause plants embryonic death. We have found that soybean LEA protein PM1 interacted with cytoplasmic iron-sulfur cluster assembly (CIA) system protein AE7/MIP18 using yeast two-hybrid (Y2H) experiment. Base on this result, we will analyze the relationship between PM1 and protein members of CIA complex by Y2H, pull down, BiFC and Co-IP experiments. Use tolerance to DNA damage as a phenotype for structural and functional integrity of CIA complex. Try to reveal the protective function of LEA protein in dehydration stage of seed developing from the angle of protein-protein interaction. Enrich the knowledge about the functions of intrinsically disordered proteins during stress resistance.

植物种子发育晚期将经历一个脱水阶段，LEA家族成员在此阶段表达富集，参与保护细胞中生物大分子。对生物体功能有重要作用的铁硫蛋白，其组装复合体成员的缺失往往导致植株胚胎致死，因此对其分子活性的保护是至关重要的。在前期研究中，我们利用酵母双杂交系统筛选并证明了大豆PM1蛋白与大豆细胞质铁硫簇组装系统复合体成员AE7/MIP18间存在相互作用。本项目拟在此基础之上，根据PM1蛋白自身的序列特征，进一步采用酵母双杂交、Pull Down、双分子荧光互补及免疫共沉淀实验等技术，研究PM1与AE7/MIP18间的相互作用关系，并通过检测转基因植株对于DNA损伤的耐受性来验证CIA复合体的功能，证明PM1蛋白对于CIA复合体功能的影响。从蛋白质间相互作用的角度阐释LEA蛋白在种子发育脱水阶段对生物分子的保护功能及其分子机理，进一步揭示固有无序蛋白在植物逆境应答中的功能，为作物抗逆分子育种提供理论依据。

植物无序蛋白LEA家族成员在种子发育晚期的脱水阶段表达富集参与保护细胞中生物大分子。铁硫蛋白参与生物体如DNA损伤修复等重要生理过程，其在细胞内的装配过程至关重要。本项目通过酵母双杂交系统筛选到了找到了大豆LEA蛋白PM1的相互作用蛋白之一，CIA复合体成员AE7/MIP18，通过酵母双杂交、Pulldown实验及烟草瞬时表达体系BiFC实验证明PM1与其存在相互作用，且这种相互作用会竞争AE7/MIP18与同复合体成员CIA1之间的互作，进而对于与DNA合成及损伤修复过程密切相关的铁硫蛋白的装配产生影响。为进一步揭示固有无序蛋白在植物逆境应答中的功能、为作物抗逆分子育种提供理论依据。

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