微拟球藻中C2H2型锌指蛋白对三脂酰甘油合成途径的调控机制研究

Algal breeding is the key bottleneck in algal biofuel industry. However, due to the lack of experimental validation, the transcriptional regulatory mechanism in lipid production in microalgae remains largely unknown. Our previously established spatial-temporal model of Transcriptome-Lipidome dynamics in lipid production in Nannochloropsis oceanica IMET1 suggested two C2H2-type Zinc-finger proteins (ADR1 and MIG1) were involved in the regulation of triacylglycerol (TAG) biosynthesis. To probe "how ADR1 and MIG1 regulate the TAG biosynthesis pathway, I will employ the wild oleaginous microalgae Nannochloropsis oceanica IMET1 as the research model, to investigate the interaction of ADR1 and MIG1 with their predicted binding cites in upstream of several key genes related to TAG biosynthesis that include LPAT, PAP and certain DGAT genes. In addition, the effects in transcripts and metabolites related to lipid metabolism will be tracked in transgenic microalgae where the two transcriptional factors are over-expressed or knocked down/out. Furthermore, we will identify and validate potential targeted genes of ADR1 and MIG1 in the whole genome using mRNA-Seq or Chip-Seq, so as to construct their genome-wide regulatory network and explore their roles in regulation in carbon flux allocation in oil production. This work should provide new insights to the diversity and conservation in regulatory mechanism of lipid production between microalgae and higher plants, and also enable rational engineering of oleaginous traits.

藻种选育是微藻能源产业化的主要瓶颈之一。但因缺少关键实验证据，野生高产油藻产油性状的转录调控机制研究仍知之甚少。申请人基于前期首个微藻产油过程中转录组-脂组时空代谢模型的构建，提出C2H2型锌指蛋白ADR1和MIG1参与调控三脂酰甘油（TAG）的合成。本项目以微拟球藻为研究模型，针对ADR1和MIG1“如何调控TAG合成途径”及其“全基因组调控网络”这两个关键问题，研究他们与TAG合成关键基因LPAT、PAP及多个DGAT基因上游结合位点的相互作用，验证ADR1或MIG1过表达或敲除后对相关基因的转录和油脂代谢的影响；同时在野生/转基因藻中利用mRNA-Seq和Chip-Seq在全基因组范围鉴定和验证靶基因，构建其全基因组调控网络，从而以这两个关键调控因子为切入点，探索微藻产油过程中代谢流分配的调控机制。这些努力有助于阐明微藻和高等植物产油调控机制的特性和共性，以及高产油性状的理性构建。

高产油微拟球藻是发展藻基柴油的最有潜力的野生藻种之一。解析微藻油脂合成的转录调控机制是优化改良关键产油性状的重要前提之一。本项目针对C2H2型等多种转录因子，通过遗传改造方法鉴定参与调控油脂代谢途径的关键调控元件。已分别构建两个转录因子的过表达转基因藻株，二者均生长缓慢，油脂含量降低，其中长链脂肪酸组分减少，而C13:0含量增多，暗示这两个转录因子可能参与调节油脂合成途径中对长链脂肪酸的偏好性。对于下游靶基因的鉴定及分子调控机制的解析工作在开展中。由于项目负责人的依托单位变动带来研究领域和方向上的改动，本项目在继续推进原申请研究内容的基础上，同时开展磷酸烯醇式丙酮酸羧化酶（PEPC）在微拟球藻缺氮下的碳硫分配与油脂合成中的生物学功能研究，以及经济海藻红毛菜高产二十碳五烯酸（EPA）的遗传基础和低温适应机理的研究。已克隆PEPC基因，并采用RNAi方法敲低PEPC表达。在两株PEPC敲低藻株中，PEPC的转录水平均显著降低，藻株生长速率有所降低，但油脂含量提高约20%。此部分工作准备投稿。此外，重要的经济海藻红毛菜富含EPA，且在低温下累积EPA。针对遗传信息缺乏的瓶颈，我们完成了红毛菜的转录组测序，重构EPA合成代谢途径，并根据低温下的基因表达谱测序和脂肪酸代谢分析，解析红毛菜高产EPA的遗传基础，并阐明红毛菜中在低温下累积EPA的分子机理，此部分工作已发表。项目负责人同时承担大型经济海藻紫菜基因组的系统生物学研究及逆境下蛋白质组及修饰蛋白质组学研究，并获得良好进展，2018年会有更多可视化成果展现。