Characterization of the 16S–23S internal transcribed spacer among 34 higher plants: suitability for interspecific plastid transformation

Abstract

Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S-23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S-23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified approximately 2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3' terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S-23S region as a target for interspecific transformation, and describe a 'primer-to-plastid' system to rapidly generate species-specific flanking regions for transformation vectors.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…Using the same procedure, in the latter region we could detect 23 fragments of identical sequence (from 45 to 237 bp long, average length = 92.7) between potato and tobacco, for a total of 1120 MEPS, a value approximately equal to 40% of that estimated using the homologous potato sequence. A similar reduction in efficiency should have been obtained comparing the homologous vs. the homeologous transformations, assuming conservation of cellular permissiveness towards homeologous recombination at different divergence levels (McNutt et al 2007). Integration of transgene sequences in the recipient plastome has been recently shown to occur by a two-step process with recombination taking place at the ends of the targeting regions (Klaus et al 2004;Sinagawa-Garcia et al 2009).…”

“…The expected efficiency of interspecific plastome recombination between tobacco and various other species was recently estimated, after alignment of the rrn16-rrn23 internal transcribed spacer, considering the number of MEPS (Minimal Efficient Processing Segments), i.e. the number of blocks of sequences identical in the two partners and sufficiently large to allow the initiation of recombination by RecA (Vulić et al 1997;McNutt et al 2007). Based on a MEPS length in higher plant plastids equal to 45 bp, a 19% efficiency, in comparison with that achievable by homologous combinations, was estimated for divergence levels similar to that shown by the rbcL-accD regions in potato and tobacco (McNutt et al 2007).…”

“…the number of blocks of sequences identical in the two partners and sufficiently large to allow the initiation of recombination by RecA (Vulić et al 1997;McNutt et al 2007). Based on a MEPS length in higher plant plastids equal to 45 bp, a 19% efficiency, in comparison with that achievable by homologous combinations, was estimated for divergence levels similar to that shown by the rbcL-accD regions in potato and tobacco (McNutt et al 2007). Using the same procedure, in the latter region we could detect 23 fragments of identical sequence (from 45 to 237 bp long, average length = 92.7) between potato and tobacco, for a total of 1120 MEPS, a value approximately equal to 40% of that estimated using the homologous potato sequence.…”

Spontaneous spectinomycin resistance mutations detected after biolistic transformation of Daucus carota L.

“…The gene rrn16, contained in the rrn operon of the chloroplast genome, is highly conserved, which is reflected in its nearly 100 % homology of both the nucleotide sequence and secondary structure within the higher plants (McNutt et al 2007). …”

Short Communication Characterization of chloroplast region rrn16-rrn23S from the tropical timber tree Cedrela odorata L. and de novo construction of a transplastomic expression vector suitable for Meliaceae trees and other economically important crops

“…This region was chosen because it is useful for the development of plastid transformation vectors (McNutt et al, 2007). Moreover, this region is close to the replication origins oriA and oriB responsible for the high copy number cpDNA, thus increasing the probability of reaching homoplasty after a few regeneration steps (Guda et al, 2000).…”