莱茵衣藻,一种单细胞绿藻,是绿色生物技术和研究细胞器功能和生物发生的突出模型,如叶绿体和纤毛。然而,reinhardtii中来自核基因组的外源基因的稳定表达面临着一些限制,包括低表达水平和由于基因组位置效应而导致的克隆之间的显着差异,表观遗传沉默,和耗时的程序。我们开发了一种强大的瞬时表达系统来克服这些限制。我们证明了体外转录的mRNA通过电穿孔有效进入无壁细胞和酶解壁的野生型细胞。内源性或外源性元件可以促进mRNA在莱茵衣原体中的有效瞬时表达,包括PsaD的5'UTR和衍生自嗜血杆菌的充分表征的Kozak序列。在优化的系统中,在转化后120h检测到mRNA表达,在转化后4h左右出现峰值。荧光标记蛋白成功瞬时表达,使细胞器标记和蛋白质亚细胞定位的实时测定。值得注意的是,瞬时表达的IFT46补偿了IFT46-1突变表型,表明细胞内IFT46的正确蛋白质折叠和功能。此外,我们证明了我们的系统使用双分子荧光互补研究活细胞中蛋白质-蛋白质相互作用的可行性。总之,建立的瞬时表达系统为研究蛋白质定位提供了强大的工具,函数,以及在相对较短的时间内进行C.reinhardtii的相互作用,这将大大促进基因功能的研究,基因组结构,以及在C.reinhardtii和其他藻类中进行绿色生物制造。
Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles\' function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via
electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5\' UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120h with a peak around 4h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46-1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.