Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation - PubMed (original) (raw)

Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation

Tania Quesada-Vargas et al. Plant Physiol. 2005 Jul.

Abstract

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

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Figures

Figure 1.

Figure 1.

Transcriptional and translational analysis of the _Cry_2Aa2 operon. A, Schematic representation of the _orf_1-orf2-_cry_2Aa2 operon in transgenic lines, including the aad_A gene and the upstream P_rrn promoter (P); upstream native chloroplast 16S ribosomal RNA gene with its respective promoter (Prrn) and the _trn_I and _trn_A are shown. Arrows represent expected transcripts and their respective sizes. B, RNA hybridized with the _cry_2A probe, loaded as follows: wt, wild-type control; lanes 1 to 3, _cry_2Aa2 operon transgenic lines. Transcripts of the _cry_2Aa2 operon are indicated by lowercase letters and correspond to the transcripts depicted in A. C, Relative heterologous transcript abundance within each line hybridized with the _cry_2A probe. D, Transcript analysis showing RNA hybridization with the _aad_A probe, loaded as follows: wt, wild-type control; lanes 1 to 3, _cry_2Aa2 operon transgenic lines. Transcripts of the _cry_2Aa2 operon are of sizes as described for the _cry_2Aa2 probe; f is _aad_A/_orf_1/orf2 tricistron, 2.5 knt. E, Heterologous transcript quantification for samples hybridized with the _aad_A probe. F, RNA hybridization using the orf1,2 probe. Samples were loaded in the same order as in D and predicted transcript sizes correspond to those observed in D. G, Relative transcript abundance within each transgenic line obtained by hybridization with the _orf_1,2 probe. H, Western-blot analysis using the _Cry_2Aa2 antibody. wt, Wild-type control; lanes 1 and 2, _cry_2Aa2 operon transgenic lines; lane 3, positive control (_Cry_2Aa2 protein). The expected polypeptide of 65 kD is shown in both transgenic plants and the positive control. I, Western-blot analysis using the _ORF_2 antibody. wt, Wild-type control; lanes 1 and 2, cry2Aa2 operon transgenic lines; lane 3, positive control (_ORF_2 protein). The expected polypeptide of 45 kD is shown in both transgenic plants and the positive control.

Figure 2.

Figure 2.

Polysome fractionation assays of the _cry_2Aa2 operon. A, RNA hybridized with the _cry_2A probe after fractionation through a Suc gradient. WT, Wild-type control; T, total RNA sample; lanes 1 to 12, RNA collected from the different fractions of the gradient. Lower fractions correspond to the bottom of the Suc gradient (polysomal fractions). P. _cry_2Aa2 probe. C, Transcript “c” (_aad_A-_orf_1-_orf_2-_cry_2A polycistron) described in Figure 1A. B, Same RNA blot after stripping and rehybridizing with _orf_1,2 probe. Lane P is omitted because no _orf_1,2 probe was loaded. C, Puromycin release and wild-type controls. Cry2Aa2 samples were treated with puromycin before loading onto Suc gradients, whereas an additional wild-type sample was loaded onto Suc gradients and used as a negative control. RNA was hybridized with the _aad_A probe. The gel was loaded as follows: WT, wild-type RNA; T, total RNA; 1 to 11, RNA collected from the different fractions of the Suc gradient and hybridized with the _aad_A probe. Lanes 12 to 16, Wild-type RNA from fractions 2, 4, 6, 8, and 10 collected from the Suc gradient. P, _aad_A probe. c, transcript “c” (_aad_A-_orf_1-_orf_2-_cry_2A polycistron) described in Figure 1A.

Figure 3.

Figure 3.

Transcriptional and translational analysis of the hsa operons. A, Schematic representation of the hsa operons (rbs-hsa, 5′UTR-hsa, _orf_1-_orf_2-hsa) in transgenic lines, including the aad_A gene and upstream P_rrn promoter (P); upstream native chloroplast 16S ribosomal RNA gene and promoter (Prrn) as well as _trn_I/_trn_A genes are shown. Arrows represent expected transcripts and their respective sizes. B, RNA hybridization with the hsa probe. wt, Wild type; lanes 1 to 3, rbs-hsa transgenic lines; lanes 4 to 6, 5′UTR-hsa transgenic lines; lanes 7 to 9, _orf_1,2-hsa transgenic lines. Lowercase letters correspond to the transcripts predicted in A. C, Relative abundance of the transcripts obtained with the hsa probe. D, mRNA transcripts hybridized with the aad_A probe and loaded in the same order as in B. Transcripts a to i corresponded to the same transcripts observed in B; k corresponds to the 16_rrn/hsa polycistron (6.9 knt). E, Quantification of relative heterologous transcript abundance obtained with the _aad_A probe. F, mRNA transcripts of wild-type (wt) and _orf_1,2-hsa transgenic lines (lanes 1–3) hybridized with the _orf_1,2 probe. G, Relative abundance obtained for the transcripts detected with the _orf_1,2 probe. H, Western-blot analysis using the HSA antibody. wt, Wild-type control. Lanes 1 and 2, RBS-hsa transgenic lines; lanes 3 and 4, 5′UTR-hsa transgenic lines; lanes 5 and 6, _orf_1,2-hsa transgenic lines; lane 7, positive control (HSA protein). Lane marked with (--) was left blank. All samples presented 66-kD and 132-kD peptides, corresponding to the size of the HSA protein and its dimeric form, respectively. I, Western-blot analysis using the ORF2 antibody. Lanes 1 and 2, _orf_1,2-hsa transgenic lines; lane 3, wild-type control; lane 4, positive control (ORF protein). Shown are 45-kD ORF2 and 90-kD dimer.

Figure 4.

Figure 4.

ELISA analysis of the _orf_1-_orf_2-hsa transgenic line. Total soluble protein content of young, mature, and old leaf extracts of the _orf_1-_orf_2-hsa transgenic lines determined by ELISA analyses. Transgenic plants were subjected to the following light conditions: 4, 8, and 16 hours of light, as well as total darkness.

Figure 5.

Figure 5.

Transcriptional and translational analysis of the _tps_1 operon. A, Schematic representation of the _tps_1 operon in transgenic lines, including the aad_A gene and upstream P_rrn promoter (P). Upstream native chloroplast 16S ribosomal RNA gene and promoter (Prrn) as well as _trn_I/_trn_A genes are shown. Arrows represent expected transcripts and their respective sizes. B, Northern-blot analysis obtained by hybridization with the _tps_1 probe, loaded as follows: wt, wild-type control; lanes 1 to 3, _tps_1 transgenic lines. Transcripts of the _tps_1 operon correspond to those depicted in A, indicated with lowercase letters. C, Relative transcript abundance per transgenic line, obtained with the _tps_1 probe. D, RNA transcripts hybridized with the _aad_A probe, loaded as follows: wt, wild-type control; lanes 1 to 3, _tps_1 transgenic lines. Transcript bands obtained for the _tps_1 operon are of sizes as described for _tps_1 probe (B). D, Relative abundance of transcripts in each sample after hybridization with the _aad_A probe. E, Western-blot analysis using the TPS1 antibody. Lane 1, Positive control (TPS1 protein); lane 2, wild-type control; lane 3, _tps_1 transgenic line. A polypeptide of 65 kD was observed in the transgenic clone, corresponding to the expected size of the TPS1 protein, as observed in the positive control.

Figure 6.

Figure 6.

Transcriptional and translational analysis of the CTB operons. A, Schematic representation of the 5′UTR-_ctb_-gfp and RBS-ctb operon in transgenic lines, including the aad_A gene and the upstream P_rrn promoter (P); upstream native chloroplast 16S ribosomal RNA gene with its respective promoter (Prrn) and the _trn_I and _trn_A are also shown. Arrows represent expected transcripts and their respective sizes. B, Northern-blot analysis showing RNA hybridized with the CTB probe. Samples were loaded as follows: wt, wild-type control; lanes 1 to3, 5′UTR-_ctb_-gfp transgenic lines; lanes 4 to 6, _rbs_-ctb transgenic lines. The transcripts and respective sizes correspond to those indicated in A with lowercase letters. C, Relative transcript abundance, within each line, of the transcripts shown in B. D, RNA hybridization using the _aad_A probe, and loaded according to the following: M, molecular weight marker; wt, wild-type control; lanes 1 to 3, RBS-ctb transgenic lines; lanes 4 to 6, 5′UTR-_ctb_-gfp transgenic lines. Lanes marked with (--) were left blank. The transcripts observed correspond to the same as in B. E, Relative transcript abundance, per line, for the transcripts shown in C. F, Western-blot analysis of the RBS-CTB transgenic lines using anti-CTB antibody. Lanes 1 to 3, transgenic clones; lane 4, wild-type control; lane 5, positive control (CTB protein). CTB from transgenic lines is in trimeric form. E, Western-blot analysis of the 5′UTR-_ctb_-gfp transgenic lines using the CTB antibody. Lane 1, Wild-type control; lanes 2 to 5, transgenic lines; lane 6, positive control (CTB protein).

Figure 7.

Figure 7.

Transcription of heterologous operons using the _psb_A 3′UTR probe. A, Northern-blot analysis and corresponding quantification of transcripts obtained from different HSA transgenic lines described in Figure 3, as well as of the native _psb_A transcripts. The RNA gels were loaded as follows: wt, wild-type; lanes 1 to 3, RBS-HSA transgenic lines; lanes 4 to 6, 5′UTR-HSA transgenic lines; lanes 7 to 9, ORF-1,2-HSA transgenic lines; P, _psb_A 3′UTR probe. Lowercase letters correspond to the same transcripts predicted in Figure 3A. Transcript abundance was normalized against the wild-type _psb_A, to which a value of 1 was assigned. B, Northern-blot analysis and corresponding transcript quantification of the _cry_2Aa2 operon. Gel loading was as follows: wt, wild-type RNA; lanes 1 to 3, Cry2Aa2 transgenic lines; P, _psb_A 3′UTR probe. Lowercase letters correspond to transcripts predicted in Figure 1A. Native _psb_A transcript is indicated. Transcript abundance was normalized against the wild-type _psb_A, to which a value of 1 was assigned. The low transcript abundance of lane 1 is due to partial RNA degradation in the sample. C, RNA blot and transcript quantification of the transgenic TPS1 lines. The RNA gel was loaded as follows: wt, wild type; lanes 1 to 3, TPS1 transgenic lines; P, _psb_A 3′UTR probe. Lowercase letters correspond to transcript sizes shown in Figure 5A. Native _psb_A transcript is indicated. Transcript abundance was normalized against the wild-type _psb_A, showing a value of 1. D, Northern-blot analysis of the RBS-CTB and 5′UTR-CTB-GFP transgenic lines. Samples were loaded as follows: wt, wild type; lanes 1 to 3, RBS-CTB transgenic lines; lanes 4 to 6, 5′UTR-CTB-GFP transgenic lines; P, _psb_A 3′UTR probe. Lowercase letters correspond to transcripts shown in Figure 6A. Transcripts a* and b* are similar in size to the native _psb_A and therefore they cannot be distinguished from the native transcript. Because such transcripts were shown to be very abundant in Figure 6B, and because of increase in transcript abundance in comparison to the wild-type _psb_A transcript, it is assumed that such transcripts are present. Transcript abundance was normalized against the wild-type _psb_A, to which a value of 1 was assigned.

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