The First Introduction of an Exogenous 5ʹ Untranslated Region for Control of Plastid Transgene Expression in Chlamydomonas reinhardtii

The utilization of heterologous 5' untranslated regions (5′UTRs) for expressing foreign proteins in the chloroplast of Chlamydomonas reinhardtii (C. reinhardtii) has posed a persistent challenge over the years. This challenge stems from the lack of a defined and comprehensive set of translational cis-elements responsible for stability, ribosome binding, and translation initiation, which are mediated by trans-acting factors native to C. reinhardtii. In the current study, we aimed to address this bottleneck by employing the 5′UTR from gene 10 of the T7 bacteriophage (T7g10 5'UTR), fused to the promoter of C. reinhardtii small subunit ribosomal RNA (rrnS), to facilitate the translation of a reporter gene, YFP. Using a chimeric construct, the YFP mRNA was efficiently translated utilizing the heterologous T7g10 5′UTR. Furthermore, the accumulation of YFP protein under the control of the T7g10 5′UTR was approximately one third of that observed under the control of the endogenous psaA promoter/5′UTR in the C. reinhardtii chloroplast. The results of computational analyses demonstrated that the T7g10 5ʹUTR sequence shares common elements with the endogenous 5ʹUTRs of the chloroplast genes. Moreover, the findings of the current study highlighted the potential of employing bacteriophage 5′UTRs for the foreign protein accumulation from the chloroplast genome of C. reinhardtii.

Graphical Abstract