【摘要】 全球能源危机和环境污染问题日益严峻，开发可持续、清洁的可再生能源成为科学研究的重点方向。微藻因其高效的光合作用能力、快速生长速率及丰富的脂质含量，是生产生物柴油的理想原料。莱茵衣藻(Chlamydomonas reinhardtii)作为单细胞真核绿藻的模式生物，具有遗传背景清晰、操作便捷等优势，是研究微藻脂质代谢的理想对象。氮胁迫可诱导微藻脂质积累，但其分子机制尚未完全阐明。本研究旨在利用CRISPR干扰(CRISPR interference, CRISPRi)系统靶向调控氮代谢关键基因，模拟氮胁迫环境，探究其对莱茵衣藻脂质积累的影响，为微藻油脂的高效生产提供新的技术策略。以莱茵衣藻(Chlamydomonas reinhardtii) FACHB-2220为研究对象，通过构建CRISPRi系统，抑制硝酸还原酶基因(CrNIT1)和亚硝酸还原酶基因(CrNII1)的表达。通过测定细胞生长、脂质含量及关键基因表达水平，分析抑制氮代谢对脂质积累的影响。CrNIT1基因表达抑制藻株ΔNIT1-4的CrNIT1基因表达量为野生型的10.27%,CrNII1基因沉默组(ΔNII组)中ΔNII1-4的CrNII1基因表达量为野生型的16.02%，表明CRISPRi系统可有效抑制目标基因转录。在氮充足条件下，ΔNIT1-4和ΔNII1-4的细胞密度仅分别为野生型的33.7%和40.2%，但总脂含量分别达干重的34.41%和33.45%，显著高于野生型。本研究通过CRISPRi系统靶向抑制氮代谢关键基因，成功模拟了氮胁迫效应，显著提高了莱茵衣藻的油脂积累效率。本研究阐明了氮代谢与脂质合成之间的调控关系，为微藻生物能源的产业化应用提供了理论基础和技术支撑。更多还原

【Abstract】 The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference(CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene(CrNIT1) and the nitrite reductase gene(CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type(WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the Cr NII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.更多还原