The role of heterologous chloroplast sequence elements in transgene integration and expression

Abstract

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

Figure 1.

Nucleotide alignment of the psbA 5′ UTR. Intergenic spacers were extracted from complete plastid genomes of 20 species of angiosperm. Colored bases indicate agreements. Colored annotations below the tobacco sequence indicate regulatory elements (from 5′ to 3′): promoter and transcription start, purple; 5′ UTR terminal stem loop region, white; RBS, yellow (RBS3, R…; RBS2, full label; RBS1, R); AU box, green; translational start, blue (D). Identity across taxa is indicated by the histogram shown under the consensus sequence. Complete data from genomic analyses are available at http://chloroplast.cbio.psu.edu.

Figure 2.

Theoretical predictions of secondary structure within the psbA 5′ UTR. The Vienna RNA Websuite tool, RNAfold, was used to produce two-dimensional structures for the psbA 5′ UTR for three representatives each from Solanaceae (top row), Asteraceae (middle row), and Poaceae (bottom row), based on minimum free energy. Unpaired bases upstream and downstream of the predicted stem and loop region are shown as circular, with 5′ and 3′ ends labeled accordingly; this representation is arbitrary and software dependent and is not intended as an indication of structure. ΔG values are given below the species names for each structure. Arrowheads indicate potential ribosome-binding sites (RBS). Small arrows indicate base changes in RBS relative to tobacco; these sites are identical within the families shown. Brackets indicate the AU box.

Figure 3.

Schematic representation of expression cassettes and confirmation of homoplasmy by Southern hybridization. A, Schematic representation of the chloroplast transformation vectors. Ls, L. sativa; Nt, N. tabacum; Prrn, rRNA operon promoter; aadA, aminoglycoside 3′-adenylytransferase gene; TrbcL, 3′ UTR of rbcL; Trps16, 3′ UTR of rps16; g10, 5′ translation control element of bacteriophage T7 gene10; CTB-Pins, coding sequence of CTB subunit fused to human proinsulin; pagA, coding sequence of Bacillus anthracis protective antigen gene; PpsbA, promoter and 5′ UTR of psbA gene; TpsbA, 3′ UTR of psbA gene. B to D, Homoplasmic transformants generated for this study: Ls-Nt-CP (B), Ls-Ls-PA (C), Ls-g10-PA (D). Lane 1, The wild type (B, 4.2 kb; C and D, 3.0 kb); lanes 2 and 3, independent transplastomic lines (B, 6.4 kb; C and D, 7.1 kb). Five micrograms of total DNA was digested completely with AflIII for the Ls-Nt-CP blot and with SmaI for the Ls-Ls-PA and Ls-g10-PA blots.

Figure 4.

Schematic representation of homologous recombination between the tobacco transplastomic genome and the lettuce transformation vector. Total DNA was isolated from the tobacco Nt-Ls-PA transplastomic lines, sequenced using appropriate primers, and aligned with tobacco and lettuce sequences from the National Center for Biotechnology Information database. A unique lettuce intron sequence of the transformation vector is indicated by purple bars. A unique tobacco intron sequence of the plastome is indicated by the green bar. Blank indicates looped out sequence(s). Black bars indicate the exon region.

Figure 5.

Accumulation of foreign protein in transplastomic lettuce and tobacco. A and B, CTB-Pins accumulation estimated by densitometry (A) and PA accumulation estimated by ELISA (B) presented as a function of light and developmental stage. Highest accumulation is shown in the back rows. The order of young, mature, and old is different in A and B because of the accumulation of higher CTB-Pins in older leaves and PA in mature leaves. The bars of the histograms represent means of at least three independent determinations. C, SDS-PAGE stained with Coomassie Brilliant Blue. Lanes 1, 3, and 5, 10, 20, and 30 μg of TLP from Nt-Nt-CP older leaf at 6:00 pm; lanes 2, 4, and 6, corresponding amounts of wild-type protein extract; lane L, molecular mass standards. Arrowheads indicate positions of CTB-Pins (22 kD) and Rubisco (53 kD).

Figure 6.

Northern blotting of total RNA. To examine foreign transcript abundance in transplastomic lines, 2 μg of total RNA (except for Nt-Ls-PA and Nt-Nt-PA) was separated by electrophoresis, blotted to nylon membranes, and probed with radiolabeled CTB-Pins or PA fragment. A, Top panel, autoradiographs; bottom panel; ethidium bromide-stained rRNA. Monocistrons are indicated by one-headed arrows, and dicistrons are indicated by two-headed arrows. B, Plots of integrated density values (IDVs). Broken lines show data points, and solid lines show trend lines. C, Densitometric estimation of signal intensity was used to calculate relative abundance of foreign transcripts in lines with heterologous regulatory elements when compared with those with endogenous elements. Lines with endogenous elements were assigned a value of 1. Mono, CTB-Pins or PA monocistron; Di, aadA and CTB-Pins or PA dicistron; Total, Mono and Di combined values.

Figure 7.

Polysome assay. Suc gradient fractions were separated through 1.2% agarose and transferred for northern blotting with the CTB-Pins probe. Lanes are numbered above A for both A (Ls-Ls-CP) and B (Ls-Nt-CP). Lane T, Total RNA; lane B, blank; lanes 1 to 12, fractions 1 to 12 collected from the bottom of the gradient; lane RNA, RNA standards (in A; bands are 0.5, 1, 2, and 3 kb); lane probe, CTB-Pins probe. C, Lanes 1 to 4 and 5 to 8, fractions 1, 4, 8, and 11 from Ls-Nt-CP and Ls-Ls-CP, respectively. D, Controls. Lanes 1 to 3, Pooled fractions from the wild-type sample; lane 4, blank; lanes 5 to 7, each lane contains two pooled factions from 2 to 7 of puromycin-treated sample (Ls-Ls-CP); lanes 8 to 12, fraction corresponds to lane number. Transcripts identified in fractions 2 to 7 are considered to be polysome associated; fractions 8 to 12 are considered nonpolysomal. Ethidium bromide-stained agarose gels are shown below each blot.

Figure 8.

RNA EMSA competition assay. Stromal proteins isolated from lettuce (A) or tobacco (B) were incubated with radiolabeled endogenous psbA 5′ UTR (lane 1). Competitions included 50× unlabeled endogenous psbA 5′ UTR (lane 2) and 50×, 100×, or 200× unlabeled heterologous psbA UTR (tobacco in A and lettuce in B; lanes 3–5, respectively). Lane 6 shows labeled probe. Brackets indicate free probe, and arrowheads indicate complexes associated with labeled RNA.