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

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.

Efficient translation destabilizes transcripts in chloroplasts of Chlamydomonas reinhardtii.

We previously reported that high level of reporter gene transcript does not confer high amount of reporter protein accumulation in Chlamydomonas reinhardtii chloroplast transformants. Here, to further clarify the correlation between the level of transcript and protein accumulation, we generated the beta-glucuronidase (GUS) reporter gene (uidA) constructs with different potential for translation efficiency of the GUS protein by incorporating different 5' and 3'-untranslated regions of chloroplast genes into each construct. The relationship between mRNA stability and translation efficiency of the GUS reporter gene in each construct were then studied in C. reinhardtii stable chloroplast transformants. We found that sequences of the two nucleotides immediately upstream of the initial codon were important for translation efficiency and that transformants showing high GUS activity accumulated lower level of uidA transcripts than the transformants with low GUS activity. Moreover, accumulation and half-lives of these chimeric-uidA transcripts were increased to the same level in the presence of translation inhibitor. The accumulation and/or half-lives of several endogenous chloroplast transcripts were also increased by such inhibitor. Collectively, our results indicate that efficient translation destabilizes transcripts in chloroplasts of C. reinhardtii, and that there is an apparent negative correlation between protein accumulation and mRNA stability.

Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii.

An exogenous gene, placed between the 5'-upstream regions of the Chlamydomonas reinhardtii chloroplast genes, rbcL or psbA, and the 3'-end of the rbcL gene, do not have the same expression pattern as endogenous genes in the C. reinhardtii chloroplast. Here, we chose four chloroplast genes, rbcL, psbA, psbD and atpA, and examine the effects of chloroplast gene coding regions on gene expression in C. reinhardtii. We constructed chimeric genes composed of the promoter, 5'- and 3'-untranslated regions, varying lengths of protein coding regions of the chloroplast genes, and the bacterial beta-glucuronidase (GUS) gene (uidA) as a reporter gene, and introduced into chloroplast genomes. The transformants, which contained the rbcL-uidA and psbA-uidA chimeric genes fused to the coding region of each gene, showed high expression of uidA mRNA as compared with the previously generated transformants, RG and PG, in which uidA was only fused to the promoter and 5'-UTR of each gene. The difference in the accumulation of uidA transcripts among the transformants was the result of different rates of transcription. This result indicates that the coding region is necessary for sufficient expression of rbcL and psbA. On the other hand, the psbD and atpA coding region portions did not affect chimeric gene expression.

Structural and functional characterization of a transcription-enhancing sequence element in the rbcL gene of the Chlamydomonas chloroplast genome.

The structure and function of a transcription-enhancing sequence element in the coding region of the Chlamydomonas reinhardtii rbcL gene was analyzed in Chlamydomonas chloroplast transformants in vivo. The enhancer sequence is contained within a DNA segment extending from position +108 to position +143, relative to the start site of rbcL gene transcription. The sequence remains functional when inverted or when placed 34 bp closer to or 87 bp further downstream of the basic rbcL promoter. However, it does not function from a site about 250 bp downstream of its original location. Besides promoting transcription initiation from the rbcL promoter, the element is able to augment transcription from the promoter of the Chlamydomonas chloroplast atpB gene, but has an inhibitory effect on transcription from the promoter of the chloroplast ribosomal RNA genes. The results suggest that the enhancer-like sequence acts upon transcription initiation in a position-specific and promoter type-specific manner.