RbcS2启动子是叶绿体中二氧化碳固定核心酶RuBisCO小亚基的启动子，稳定表达效率高，广泛应用于微藻外源基因表达。基因组RbcS2在细胞中表达水平稳定，前期研究发现RbcS2启动子与叶绿体跨膜信号肽（cTP）串联使用，外源表达产物呈现生长偶联现象，并成功实现莱茵衣藻油脂与生长同步积累。本项目以野生型莱茵衣藻CC-137为对象，1）借助高通量克隆、培养与表型筛选技术，以荧光蛋白为模式基因，以已知核编码叶绿体蛋白跨膜肽库为基础，构建筛选优化嵌合RbcS2-cTP表达系统；2）对筛选获得的生长偶联型表达系统进行转录组、蛋白组测定，结合相关分析和互作网络分析，辅以RNAi验证，对调控其表达变化的机制进行研究，揭示生长偶联型RbcS2-cTP系统的表达调控机理。本研究将丰富真核藻类表达调控的基础理论，提供新型表达元件和策略，在工程微藻新性状理性设计、微藻合成生物学技术开发方面具有重要意义。

RbcS2 promoter is the promoter of the small subunit (SSU) of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the enzyme responding for the CO2 fixation in the chloroplast. The expression of SSU has been proved high and relatively stable during the cultivation, and RbcS2 and its modifications have been developed as a widely-used promoter driving the exogenous gene expression in microalgae, especially in Chlamydomonas reinhardtii. It has been noted that, the RbcS2 promoter in exogenous genes is not as stable as in RuBisCO SSU while the mechanism is unclear. In our previous work, a growth-coupled expression of exogenous gene was found while RbcS2 was combined with a chloroplast transit peptide (cTP). Basing on it, a C. reinhardtii CC-137 strain with Saccharomyces cerevisiae phospholipid:diacylglycerol acyl transferase (ScPDAT) expressed in the chimeric RbcS2-cTP expression system achieved a 22% increase in total fatty acid and 32% increase in TAG at the end of the exponential growth phase than wild strain, i.e. the simultaneous production of lipids with growth. It brings us the opportunity to understand the regulation of exogenous RbcS2 promoter as well as design new strategy for using microalgae cell as the cell factory..In this project, the wild type of CC-137 will be used as the host cell to work on: 1) the construction, screen, and optimization of growth-coupled RbcS2-cTP expression system, basing on the chimeras of RbcS2, the known cTP and other elements from original RuBisCO SSU, and through the high throughput automatic clone, cultivation and screen platform, with a fluorescence gene as the screen probe; 2) the utilization of transcriptome, proteome, interaction analysis, together with RNAi verification, to investigate the mechanisms underlying. .The goal of this project is to construct and understand a novel expression system for microalgal host cell. The result will enrich the knowledge for the exogenous RuBisCO promoter regulation, and will provide new bio information and materials for the future studies on the rational design of engineered strains, the synthetic biology based on microalgae and the new regulation technologies in the practice of large scale cultivation of microalgae.