外源FBA对具有碳减排潜力的小球藻光合活性的调控研究

Global warming arising from the increasing level of greenhouse gases (GHGs) in the atmosphere, has received great concern worldwide. CO2 is a primary GHG and contributes largely to the global warming. Thus, developing low carbon technologies for CO2 mitigation has garnered a surge of attention. Chlorella can fix CO2 by photosynthesis and convert it into value-added bioactive compounds such as lipids and proteins. Besides, Chlorella has some unique advantages such as better photosynthetic efficiency, rapid growth and low cost. These strengths make Chlorella-mediated biomitigation a green and economic strategy. However, the inherent relatively low photosynthetic capacity of Chlorella has hampered the practical use of this strategy for CO2 biomitigation applications. The present project aims to engineer the Calvin cycle to enhance the photosynthetic capacity in Chlorella vulgaris (a promising algal for biomitigation), through introducing of a fructose 1,6-bisphosphate aldolase (FBA) derived from Chlamydomonas into its chloroplast. The Venus fluorescent protein gene will be employed as a reporter to evaluate the heterologous expression in C. vulgaris. Then several promoters and transformation methods will be examined, developing a high-efficiency genetic system for C. vulgaris. On this basis, the FBA derived from Chlamydomonas will be introduced into C. vulgaris. Transgenic lines will be grown under different culture conditions and their physiochemical characteristics will therefore be examined. Those with high CO2 assimilation rates will be selected. Then comparative transcriptomic and metabonomic analysis, as well as other methods will be employed to reveal the possible regulative mechanisms of exogenous FBA on the photosynthetic capacity of C. vulgaris under different culture conditions. Overall, the present study will provide deep insights into targeted genetic engineering toward algal trait improvement for CO2 biomitigation uses.

由CO2为主的温室气体导致的全球变暖现象已引起全世界的广泛关注。发展低碳技术、实现碳减排，成为当前研究的热点。小球藻可通过光合作用，将CO2转化成脂质和蛋白质等高附加值产物。同时其具有光合效率较高、生长快和培养成本低等优势，因此通过小球藻固定CO2实现碳减排是一种绿色经济的技术。然而小球藻内在固定CO2的能力仍有限，这制约了该技术的实际应用。本项目旨通过基因工程将衣藻卡尔文循环途径中的关键酶—果糖1,6-二磷酸醛缩酶（FBA）导入到具有良好碳减排潜力的普通小球藻中，以提高其固碳效率。首先以Venus荧光蛋白作为报告基因，通过优化启动子和转化方法，获得高效的小球藻遗传体系。其次将衣藻FBA导入小球藻中，考察在不同生态环境下的生理特性，获得高同化CO2的藻株，并通过比较转录组学和代谢组学等技术揭示外源FBA对藻光合活性的调控机制。此研究将为藻类定向遗传改良以用于生物碳减排提供坚实的理论基础。

由CO2为主的温室气体导致的全球变暖现象已引起全世界的广泛关注。发展低碳技术、实现碳减排，成为当前研究的热点。小球藻可通过光合作用，将CO2转化成脂质和蛋白质等高附加值产物。同时其具有光合效率较高、生长快和培养成本低等优势，因此通过小球藻固定CO2实现碳减排是一种绿色经济的技术。然而小球藻内在固定CO2的能力仍有限，这制约了该技术的实际应用。本项目验证了通过基因改造普通小球藻卡尔文循环中的关键酶进而提高光合效率的可行性。首先，本项目考察了6种不同的普通小球藻（Chlorella vulgaris）藻株的生长状况和CO2固定速率，选择了其中表现最佳的C. vulgaris FACHB-1068作为出发藻株。之后，通过抗生素敏感性实验，以Venus荧光蛋白作为报告基因考察了4种不同启动子的驱动效率，在普通小球藻FACHB-1068中建立了以AphVIII基因作为筛选标记的高效稳定的遗传转化体系。在此基础上，通过将衣藻的果糖1,6-二磷酸醛缩酶（FBA）基因导入到普通小球藻中，有效地提高了藻细胞的生长和光合速率。最后经分子生物学和生理生化分析，表明外源FBA的过表达可能通过促进卡尔文循环中核酮糖1,5-二磷酸的再生和光系统中的能量传递速率从而提高光合效率。本项目可为藻类定向遗传改良以用于生物碳减排提供坚实的理论基础。