黄丝藻棕榈油酸生物合成途径及关键环境因子调控机制研究

Plamitoleic acid (C16:1Δ9) is one of omega-7(ω-7) fatty acids, and palmitoleic acid is highly valued in pharmaceutical, aquaculture and industrial applications. Palmitoleic acid is mainly from biosynthesis of living things, however, it is quite rare in plants. Microalgae Tribonema sp. is often used in regulating water quality in the early stage of shirmp and crab farming. Studies reported that Tribonema cells could accumulate large amounts of lipid, and the ratio of palmitoleic acidin the cells is quite high, up to ca. 50% in total fatty acids, much higher than most common oleaginous microalgae. Therefore, oleaginous Tribonema could be used as the ideal feedstock for palmitoleic acid production, however, the synthesis pathway and regulation mechanism of palmitoleic acid in Tribonema is still unkown..In the study, on the basis of present work, the function of two different Δ9D genes will be validated via heterologous expression. Combined with the palmitoleic acid lipid molecular form, the biosynthesis pathway of palmitoleic acid will be clarified. Different environmental factors will be used to culture Tribonema cells, and the biomass, lipid content and fatty acid profiles will be analyzed, and the environment factors which have effect on the palmitoleic acid content will be grasped. As follows, the transcriptomic data of Tribonema which contains different content of palmitoleic acid will be compared, and the differentially expressed genes which are related the palmitoleic acid biosynthesis will be coloned. According to their function, the molecular regulation mechanism will be clarified. The study will lay the groundwork for the process optimization of palmitoleic acid from Tribonema and provide valued gene and technique pathway for oil crops.

棕榈油酸（C16:1Δ9）是ω-7单不饱和脂肪酸的一种，在医药、水产及工业领域具有重要应用价值。棕榈油酸主要来源于生物合成，但其在普通油料作物种子中含量甚微。黄丝藻是在虾蟹养殖初期用于调节水质的微藻之一，近年来发现其细胞内能够积累大量油脂，且棕榈油酸比例占总脂肪酸的50%以上。黄丝藻具有作为棕榈油酸生产原料的巨大潜力，然而其细胞内棕榈油酸的合成调控机制目前尚不清楚。.本项目拟在已有基础之上，对不同载体类型的Δ9去饱和酶的底物特异性展开研究，结合棕榈油酸脂质分子存在形式，阐明黄丝藻细胞棕榈油酸的生物合成途径；改变培养条件，通过对细胞生物量、总脂及脂肪酸的分析，掌握影响棕榈油酸含量的关键环境因素，并借助比较转录组学手段，对关键基因与转录调控因子进行筛选、克隆、表达及功能分析，搞清棕榈油酸合成调控机制。为黄丝藻棕榈油酸生产的调控策略提供依据，也为普通油料作物棕榈油酸遗传改造奠定基础。

棕榈油酸（C16:1Δ9）是ω-7单不饱和脂肪酸的一种，在医药、水产及能源领域都具有重要的应用价值。黄丝藻是目前发现的细胞内棕榈油酸含量最丰富的物种之一，具有作为棕榈油酸生产原料的巨大潜力。阐明黄丝藻细胞内棕榈油酸的合成调控机制，不仅能够为黄丝藻棕榈油酸生产调控策略，也为普通油料作物棕榈油酸遗传改造提供依据。本项目首先利用脂质组学对棕榈油酸脂质分子的存在形式进行分析，根据目前已有基础，阐明黄丝藻细胞内棕榈油酸的生物合成存在两条途径，分别位于质体与胞质中。在此基础上，对影响黄丝藻细胞内总脂含量以及脂肪酸比例的营养与环境因子进行分析，结果显示CO2浓度、光照与温度是影响黄丝藻油脂含量的关键因素，其中光照强度与温度的改变也作用于油脂中脂肪酸的比例。在此基础上，利用低温低光以及高温高光对黄丝藻进行培养，获取棕榈油酸比例显著差异的黄丝藻样品并进行转录组测序，从结果中挖掘到15个与油脂合成以及棕榈油酸合成相关的表达差异基因。以pYES2.0质粒为模板，构建包括不同载体类型的Δ9去饱和酶基因(Des)等黄丝藻关键候选基因的异源表达载体，进行酿酒酵母BY4741外源转化，并对酵母细胞油脂含量以及脂肪酸组分进行分析，结果摆明MDH2基因，ACACA基因及Des的过表达能够提高油脂的产量，而且Des基因还能够提高棕榈油酸的产量。通过以上研究，搞清了影响黄丝藻棕榈油酸合成代谢的关键环境因子与分子调控机制，为棕榈油酸生产应用奠定重要基础。