微藻甘油三酯代谢的表观遗传调控机制研究

Microalgae, generally referring to a group of specific eukaryotic microbes capable of photosynthesis, have been considered as promising feedstocks for biofuels. Many challenges remain to be addressed in the field of microalgal lipids, though a great deal of achievements have been obtained. How to further increase microalgal lipid content via genetic engineering, is one of the major challenges in microalgal biotechnology for biofuels uses. This would benefit from the in-depth understanding of regulatory mechanisms for lipid metabolism in microalgae. Previous publications indicated that epigenetic regulation may be involved in microalgal lipid metabolism under abiotic stress conditions. Our unpublished work in the green alga Chlamydomonas reinhardtii suggested that an insertional mutation in the histone demethylase, CrHDM1 (Cre17.g709550), led to a significant decrease in triacylglycerol content, further evidencing the involvement of epigenetic regulation in lipid metabolism. This proposal intends to focus on studying the regulatory mechanisms of CrHDM1 for lipid metabolism in C. reinhardtii. Firstly, the insertional mutant, the complemented line of the insertional mutant, and the overexpression lines transformed with various tag (GFP, TAP, HA, etc) fusions will be generated and characterized. By employing these algal lines as research materials, 1) high-throughput RNA-Seq will be employed to analyze the gene regulatory network of CrHDM1 in triacylglycerol metabolism, 2) high-throughput ChIP-Seq and qPCR will be used to study the target genes of CrHDM1, and 3) tandem affinity purification (TAP) analysis and Co-IP will be applied to evaluate the interacting proteins of CrHDM1, thereby elucidating the regulatory mechanisms of CrHDM1 in microalgal lipid metabolism. This will not only helps understand the epigenetic regulation of microalgal lipid metabolism but also provides valuable implications into future engineering of microalgae for improved lipid production.

微藻常指能进行光合作用的特殊真核微生物，在生物能源应用方面前景广阔。微藻油脂研究已经取得了不少成果，但仍然面临着许多挑战。如何进一步提高油脂含量，是微藻能源生物技术领域里的一大难点。这需要深入了解微藻油脂代谢的调控机制。最新研究显示表观遗传可能参与了微藻非生物胁迫下的油脂代谢调控。我们的前期工作表明，莱茵衣藻组蛋白去甲基化酶CrHDM1的突变导致甘油三酯显著下降，证明表观遗传参与了微藻油脂代谢调控。本项目以莱茵衣藻CrHDM1为重点对象，构建突变株、回补株、标签融合蛋白（GFP、TAP、HA等）过量表达株为研究材料，1）通过高通量RNA测序分析CrHDM1在甘油三酯代谢中的基因调控网络，2）通过高通量染色质免疫沉淀测序和荧光定量PCR分析靶基因，3）通过串联亲和纯化系统和免疫共沉淀技术分析互作蛋白，结合脂质分析阐明其调控微藻油脂代谢的机制，进而为提高微藻油脂产量提供理论基础和新的方法途径。

微藻常指能进行光合作用的特殊真核微生物，在生物能源应用方面前景广阔。微藻油脂研究已经取得了不少成果，但仍然面临着许多挑战。如何进一步提高油脂含量，是微藻能源生物技术领域里的一大难点。这需要深入了解微藻油脂代谢的调控机制。本项目利用模式藻莱茵衣藻及其实验体系，综合采用分子生物学、生物化学、细胞生物学、遗传学、组学、生物信息学等现代生物学研究方法和技术，拟研究参与油脂代谢的重要因子的功能和基因调控网络，并探索转录调控和表观调控微藻脂质代谢的分子机理。通过该项目的实施，我们解析了组蛋白去甲基化酶（CrHDM1）的底物特异性、靶基因和调控碳代谢的机制；探究了bZIP转录因子（CrbZIP2）在莱茵衣藻脂质代谢中的调控作用和可能的机制；阐释了MYB转录因子（CrMYB1）在莱茵衣藻脂质代谢中的调控机制并探讨了它的应用潜力；揭示了长链脂酰CoA合成酶（CrLACS）成员在莱茵衣藻脂质代谢应对环境变化中的作用机制。综合来讲，我们的工作不仅有助于阐明微藻油脂代谢的转录调控和表观调控的机理，也为今后通过基因工程手段提高微藻油脂含量和产能的实现提供了理论基础和新的方法途径。