三酰甘油酯酶（TGL）在三角褐指藻中的功能及其对油脂积累和生长的调控研究

For the production of biodiesel a low-cost feedstock is vital. Marine diatoms have short life-cycle and high lipid content (40%-60%). Phaeodactylum tricornutum, a model species of diatom, is rich in C16 fatty acids (over 60%) and is acknowledged as a suitable renewable lipid source for biodiesel. However, microalgae tend to accumulate triacylglycerol (TAG) only under stress conditions that limit growth. TAG lipase (TGL) is involved in TAG hydrolysis. There are 14 putative TGL genes in the P. tricornutum genome, and their transcript abundance is not only high during cellular exponential growth (TAG does not accumulate) but also high under nutrient stress conditions (TAG accumulates) according to the 16 cDNA library data. It is indicated that growth and lipid accumulation were under the control of TGL. Traditionally, engineering efforts for lipid metabolism have focused on increasing lipid biosynthesis or blocking the competing pathways of carbohydrate formation. Although the strategies successfully increased lipids, all resulted in decreased growth in the engineered strains. As a relevant pathway for engineering, lipid catabolism has largely been ignored. This project is intended to identify TGL genes in P. tricornutum genome, detect their expression under different growth phases and nutrient conditions. We will knockdown or overexpress the specific PtTGL under different growth phases to increase lipid accumulation or enhance growth. Finally, two specific TGL genes are supposed to be transformed into P. tricornutum, in order to increase its oil accumulation and biomass thus obtaining a genetically engineered diatom which can serve as an economic bulk biodiesel feedstock.

发展生物柴油，原料是基础。海洋硅藻生产周期短，油含量高；作为硅藻研究模式的三角褐指藻富含16碳脂肪酸，被认为是最合适的生物柴油原料之一。然而，与其他产油微藻相同，油脂的大量积累通常仅在生长受限或完全停止的胁迫条件下发生。三酰基甘油酯酶（TGL）是参与甘油三酯（TAG）分解的脂肪酶。三角褐指藻基因组中预测有多达14个TGL基因，它们除了在无油脂积累的对数期有高的表达，在营养胁迫诱导的油脂积累期也有很高的表达。暗示藻的生长和油脂积累受TGL的严格调控。与传统通过针对增加TAG合成但多影响生长的遗传改造策略不同，本申请项目从分析多个PtTGL基因表达时序及对营养盐的响应差异出发，分别选定特定的PtTGL基因，通过在生长受限时减少PtTGL的表达，不影响生长且增加油脂积累；在生长旺盛时提高PtTGL的表达，促进生长。最后结合两个策略，用诱导型启动子，试图得到生长加速、油脂积累增加的转基因硅藻株。

三角褐指藻是硅藻研究的模式种，富含16碳脂肪酸，被认为是最合适的生物柴油原料之一。然而，与其他产油微藻相同，油脂的大量积累通常仅在生长受限或完全停止的胁迫条件下发生。三酰基甘油酯酶（TGL）是降解甘油三酯（TAG）的脂肪酶。根据Ensembl对三角褐指藻基因组最新的注释，该硅藻有多达18个TGL编码基因，它们除了在无油脂积累的对数期有高的表达，在营养胁迫诱导的油脂积累期也有很高的表达。暗示藻的生长和油脂积累受TGL的严格调控。本项目首先获得了三角褐指藻所有18个TGL基因（PtTGL）的全长编码序列，对它们进行了系统进化及功能分析，确定了它们的亚细胞定位，发现半数以上的PtTGL定位于叶绿体。指明了叶绿体作为TAG合成、分解发生的主要场所对于细胞TAG积累的重要贡献。随后，通过转录组揭示了所有酯酶在不同培养条件下的表达时序，并基于表达量和规律，选取了7个在藻细胞油脂积累中可能起作用的酯酶基因（ProtID_44028、37711、47614、47650、47445、45518和54974），构建了这7个酯酶基因的过量表达、敲降和/或敲除表达载体，获得了相应基因的过量表达和突变藻株，比较了野生型和突变株的生理表型。根据7个突变株的表型结果，确定了两个对藻细胞甘油三酯（TAG）积累有主要贡献、定位于叶绿体的酯酶PsTGL（ProtID_44028）和OmTGL（ProtID_37711），详细分析了PsTGL和OmTGL突变株的全脂质组学，描述了叶绿体TAG合成与分解代谢模型，提出了通过减少叶绿体TAG降解从而促进细胞TAG积累的调控策略。