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AssigneeAuburn University
InventorHenry Daniell
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The invention provides universal chloroplast integration and expression vectors which are competent to stably transform and integrate genes of interest into chloroplast genome of multiple species of plants. Transformed plants and their progeny are provided. Monocotyledonous and dicotyledonous plants are transformed which have never been transformed heretofore. Plants transformed with a synthetic gene express valuable biodegradable protein-based polymers (PBPs). Transformed plants produce high value molecules. Resistance is provided to agricultural crops against the major classes of chemical herbicides. Herbicide resistance is used as a lethal selectable marker for chloroplast transformation. The transformed plants are capable of expressing in addition to the targeted trait, a desirable, secondary non-targeted trait. Insect resistance is provided to transformed plants, both against insects that are susceptible to Bt toxins and against insects that have developed resistance to Bt toxins.
A stably transformed herbicide resistant target plant species or the progeny thereof, which comprises chloroplast stably transformed with a universal integration and expression vector competent for stable transformation of the chloroplast of different plants species which comprises an expression cassette which comprises a heterologous protein of interest expressed by a heterologous DNA sequence in the chloroplast genome of the target plant species, a selected phenotype other than tolerance to said herbicide, and flanking each side of the expression cassette, flanking DNA sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous protein into the chloroplast genome of the target plant was facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome.
A process for conferring herbicide resistance to a target plant species which comprises introducing into the plant a universal integration and expression vector competent for stably transformed with a universal integration and expression vector competent for stably transforming the chloroplast of different plants species which comprises an expression cassette which comprises, operably joined, a heterologous DNA sequence coding for a protein of interest which confers resistance to a herbicide and control sequences positioned upstream from the 5′ and downstream from the 3′ ends of the coding sequence to provide expression of the coding sequence in the chloroplast of the target plant, a heterologous nucleotide sequence coding for a selectable phenotype other than for is tolerance to said herbicide, and flanking each side of the expression cassette, flanking sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle and the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous coding sequence into the chloroplast genome of the target plant is facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome and growing the transformed plant.
A process of determining chloroplast transformation and expression of a target trait on the basis of a target plant species acquisition of resistance to a selected herbicide due to the transformation of the plant species which comprises introducing into the plant a universal integration and expression vector competent for stably transforming the chloroplast of different plant species which comprises an expression cassette which comprises, operably joined, a heterologous DNA sequence coding for the desired target trait and control sequences positioned upstream from the 5′ and downstream from the 3′ ends of the coding sequence to provide expression of the coding sequence in the chloroplast of a target plant, a heterologous nucleotide sequence coding for a selectable phenotype and flanking each side of the expression cassette, flanking DNA sequences which comprise each one a portion of the intergenic spacer 2 region between the tRNAIle and the tRNAAla genes of the chloroplast genome, which are homologous to a spacer sequence of the target chloroplast genome, which sequence is conserved in the chloroplast genome of different plant species, whereby stable integration of the heterologous coding sequence into the chloroplast genome of the target plant is facilitated through homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome, exposing the plants into which the vector has been introduced to a lethal concentration of the herbicide and selecting the plants which do not die from exposure thereto, thereby having selected the transformed plants which express the desired target trait.
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