三角褐指藻LACS调控油脂合成的分子功能与调控机制研究

真核微藻质体输出的游离脂肪酸通常需要被长链酰基CoA合成酶(LACS)激活为脂肪酰基CoA的硫酯形式，然后参与脂肪酸相关的代谢途径例如甘油三酯(TAG)的合成，因此LACS可能在细胞油脂合成中起重要调控作用。海洋硅藻三角褐指藻细胞在氮源缺乏条件下积累大量TAG，然而目前关于TAG的合成途径及其调控机制仍未阐明。在前期研究中，我们鉴定出三角褐指藻LACS蛋白家族的5个成员（ptACSL1-5）。本项目采用体外实验确定每个ptACSL蛋白的底物特异性，采用eGFP报告基因融合方法、GFP自组装方法以及胶体金标记免疫电镜技术研究其亚细胞定位，采用TALEN基因编辑技术针对质体、细胞质或油滴定位的LACS蛋白构建其编码基因的敲除突变株，并研究油脂积累条件下突变株脂质组的变化以及酰基CoA库中脂肪酸组成和含量的变化规律，综合这些研究结果以鉴定出参与调控细胞油脂合成的LACS蛋白并阐明其调控机制。

Free fatty acids exported from the plastid of eukaryotic microalge generally need to be re-activated to form fatty acyl-CoA thioesters by long-chain acyl CoA synthetases (LACS), and then participate in fatty acid-related metabolic pathways such as triacylglycerol (TAG) synthesis. Thus, LACS possibly plays an important role in lipid synthesis in the cell. The marine diatom Phaeodactylum tricornutum cells accumulate large amounts of TAGs upon nitrogen deprivation. However, TAG synthesis pathway and the mechanism by which TAG synthesis is regulated remain largely unknown at present. In the previous study, we have identified five members (ptACSL1-5) of the LACS protein family in P. tricornutum. In this proposal, we will use in vitro assay to determine substrate specificity of each ptACSL. We will use eGFP reporter gene fused with ptACSL, GFP self-assembly approach and immune-electron microscopy to determine subcellular localization of each ptACSL. For ptACSL proteins localized in the plastid, cytosol or lipid droplet, we will construct gene knockout mutants using TALEN-based gene editing technology. We will perform lipidomic analysis and profiling of acyl composition and content of the acyl-CoA pools in the generated mutants, which are grown under the condition of lipid accumulation. Based on the results from these experiments, we attempt to identify ptACSL(s) participating in regulation of lipid synthesis and reveal the related regulatory mechanism.