Artificial control of transgene expression in Chlamydomonas reinhardtii chloroplast using the lac regulation system from Escherichia coli

Abstract

Systems that can control the expression of a gene both temporally and spatially are important for the study of transgenic plants. Here, we describe an artificial, controllable gene expression system using the lac regulation system from Escherichia coli that we constructed in the Chlamydomonas reinhardtii chloroplast. This system consists of a controllable reporter gene expression cassette and the Lac repressor expression cassette. We created controller promoters by modifying two promoter sequences, rbcL and 16S rRNA, known to be highly active in the C. reinhardtii chloroplast. We inserted a synthetic lac operator sequence in different positions around these promoters, and both repression and induction of transcription were examined using appropriate repressor and inducer molecules. The effect of differing amounts of repressor protein on transcription was also investigated in stable chloroplast transformants. In the case of the modified rbcL promoter, although complete transcription repression was not achieved with the repressor, rapid, full induction was achieved within 1 h. In contrast, although the modified 16S rRNA promoter permitted almost complete repression, full transcription induction was not observed.