The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression

    Authors

    T. Ruhlman; D. Verma; N. Samson;H. Daniell

    Comments

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    Abbreviated Journal Title

    Plant Physiol.

    Keywords

    PSBA MESSENGER-RNA; PENTATRICOPEPTIDE REPEAT PROTEIN; 5' UNTRANSLATED; REGION; ESCHERICHIA-COLI RECA; LACTUCA-SATIVA L.; TOBACCO CHLOROPLASTS; SPINACH CHLOROPLAST; HIGHER-PLANTS; 5'-UNTRANSLATED REGION; TRANSLATION; EFFICIENCY; Plant Sciences

    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.

    Journal Title

    Plant Physiology

    Volume

    152

    Issue/Number

    4

    Publication Date

    1-1-2010

    Document Type

    Article

    Language

    English

    First Page

    2088

    Last Page

    2104

    WOS Identifier

    WOS:000276335900029

    ISSN

    0032-0889

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