蓖麻脂滴蛋白的结构与功能分析

	蓖麻脂滴蛋白的结构与功能分析
	巩宇豪
导师	伊廷双
关键词	脂滴蛋白，蛋白质相互作用，油脂，脂滴，蓖麻，种子 lipid droplet protein, protein-protein interactions, lipid, lipid droplets, castor bean, seed
摘要	植物脂滴(LDs)是一种细胞内保存中性脂质的细胞器，在植物生长发育和胁迫反应中发挥重要作用。脂滴复杂的生命活动都依赖于附着在其表面的脂滴蛋白。在植物种子中，主要由oleosin和caleosin两种脂滴蛋白组成脂滴的基本结构，在脂滴的稳定性、大小形成、油脂积累和逆境胁迫中发挥重要作用。然而，关于种子oleosin和caleosin这两类脂滴蛋白是如何发挥其功能的机理尚不明确。该研究以世界重要油料作物蓖麻为研究对象，利用生物信息学和分子生物学等手段，鉴别了蓖麻种子oleosin和caleosin两类脂滴蛋白，分析了他们的互作模式，并分别在结构和功能上初步揭示了两类蛋白在脂滴结构维持和油脂调控上的作用。首先基于基因组数据和实验验证的方法，对蓖麻种子中的oleosin和caleosin进行基因家族的鉴定分析。通过生物信息学、基因克隆和亚细胞定位分析，我们在蓖麻种子中一共鉴定到5个oleosin和2个caleosin基因，亚细胞定位分析发现这7个基因编码的蛋白均定位于脂滴。通过酵母双杂交和双分子荧光互补实验，我们发现蓖麻种子中的OLE4可以分别与OLE3和CLO1互作。为了进一步了解这些相互作用蛋白对脂滴结构的调控作用，我们建立了人工脂滴系统（Artificial oil bodies， AOBs），在体外条件下对这些蛋白及蛋白复合体在脂滴稳定性保持方面的作用展开验证。通过实验我们发现，脂滴蛋白OLE3和OLE4之间经相互作用形成的蛋白复合体可以在极端条件下（pH 10 和 70 ℃）显著地提升脂滴的稳定性。通过酵母异源表达实验发现，OLE3和OLE4的共表达可以显著提升酵母油脂含量，而OLE4和CLO1的共表达却使油脂含量降低。同时，蓖麻叶片的瞬时转化实验结果表明，OLE3与OLE4蛋白复合体可以使转化后的叶片中的脂滴数目增多，而OLE4与CLO1的组合会减少脂滴的数目。以上结果表明，oleosin和caleosin之间存在相互作用，且这些不同的互作模式对脂质代谢和脂滴结构的稳定性维持方面有不同的调节作用。我们的结果分别从结构和功能对oleosin和caleosin这两类蛋白进行了分析，这些数据为植物细胞中脂滴蛋白调控脂滴稳定性的机制提供了一个新的模型，为油料植物种子油脂的合成代谢和动员提供了新的视角。; Plant lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play an important role in growth, development, and stress response. These complicated functions rely on the multiple proteins coated on the surface of LDs and the interaction between proteins. However, how these lipid droplet proteins participate in lipid metabolism and lipid droplet stability regulation on lipid droplet surface is still poorly understood. Oleosin and caleosin are two widely researched gene families participate in LD stability and lipid regulation in seeds，the interaction and function of these proteins within and among gene families remain obscure. The important oil plant castor bean was selected as our research object, we firstly identified and characterized the oleosin and caleosin gene families in castor bean seeds, and analyzed the interactions between these proteins, then we investigated the roles of the two protein families in lipid droplet structure maintenance and lipid regulation in terms of structural and functional analyzation. Bioinformation analyzation showed that there were five oleosins and two caleosins in Ricinus communis L. genome, protein mass spectrometer identification of LD proteins and gene cloning from castor bean seeds experimentally validated the exists and relative abundance of these seven proteins. Subcellular location exhibited the proteins all localized to the surface of LDs. According to the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) results, here we show that OLE4 could interact with both OLE3 and CLO1. For further understanding the functions of theses interacted proteins in modulating lipid accumulation and lipid droplet stability maintenance, in vitro assay based on artificial oil bodies system was developed. The results showed that the interaction between OLE3 and OLE4 was found to be facilitate to LD stability maintaining. Heterologous expression in yeast cells demonstrated that OLE3 and OLE4 complex promoted the lipid accumulation, whereas, OLE4 and CLO1 co-expression reduced the lipid content. Moreover, more numbers of LDs were observed by transient transformation of both OLE3 and OLE4 into castor bean leaves, in contrast, co-expression of the OLE4 and CLO1 showed the reduction of LDs numbers. Our results indicated that the interaction of oleosins and caleosin differently modulate the lipid metabolism and the stability of LDs. These data provide a new model for lipid regulation by lipid droplet proteins in plant cells, which gives a new sight in lipid metabolism and mobilization by lipid droplet proteins in oilseed.
语种	中文
	2022-07
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75214
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	巩宇豪. 蓖麻脂滴蛋白的结构与功能分析[D]. 中国科学院大学,2022.

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