US 5,877,402 A · DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein

Abstract

DNA constructs are provided for stable transformation of plastids of multicellular plants and expression of foreign proteins in plastids. The DNA constructs comprise a transforming DNA which is targeted to a pre-determined location on the plastid genome and inserted into the plastid genome by homologous recombination with targeting segments comprising DNA sequences homologous to the pre-determined region of the plastid genome. The transforming DNA contains a non-lethal selectable marker gene which confers a selectable phenotype on cells having plastids in which substantially all of the genomes therein contain the transforming DNA (i.e., homoplasmic cells or tissues). The transforming DNA further comprises at least one insertion site 4 for an additional DNA segment, such as a gene encoding a protein for improving a characteristic of the transformed plant. The non-lethal selectable marker gene is preferably provided as a chimeric gene by assembly from an expression cassette comprising 5' and 3' regulatory segments, preferably derived from plastid genes. A coding segment encoding the non-lethal selectable marker is inserted between the 5' and 3' regulatory segments to form the chimeric gene. The non-lethal selectable marker coding segment preferred in the present invention is the coding region of aadA from bacteria, which encodes aminoglycoside 3"-adenylyltransferase to confer spectinomycin and streptomycin resistance.

Claims34

Dependent claims

A DNA construct for stably transforming plastids of multicellular plants, which comprises a transforming DNA having:

a) a targeting segment comprising a DNA sequence homologous to a pre-determined plastid genomic sequence of a genome within a plastid to be transformed, said targeting segment enabling insertion of said transforming DNA into said plastid genome by homologous recombination with said pre-determined plastid genomic sequence, said insertion not interfering with the normal function of the plastid genome;
b) a chimeric selectable marker gene disposed within said targeting segment, positioned so as not to disrupt said homologous recombination, said chimeric selectable marker gene comprising:
i) a selectable marker coding segment encoding a gene product that confers to cells of said plant a selectable phenotype in the presence of a selection agent which is non-lethal to plant cells containing plastids transformed with said DNA construct but lethal to untransformed plastids;
ii) a 5' regulatory segment of plant chloroplast origin, operably linked to said selectable marker coding segment to promote expression of said selectable marker coding segment in said plastids;
and
iii) a 3' regulatory segment of a plant chloroplast mRNA molecule, operably linked to said selectable marker coding segment to promote stability of mRNA produced during said expression of said selectable marker coding segment in said plastids;
and
c) at least one cloning site adapted for insertion of at least one additional DNA segment, said at least one cloning site being disposed within said targeting segment so as not to interfere with said conferring of said selectable phenotype to said plant cells.

5 more elements

A DNA construct for stably transforming plastids of multicellular plants and expressing at least one gene product within said transformed plastids, which comprises a transforming DNA having:

a) a targeting segment comprising a DNA sequence homologous to a pre-determined plastid genomic sequence of a genome within a plastid to be transformed, said targeting segment enabling insertion of said transforming DNA into said plastid genome by homologous recombination with said pre-determined plastid genomic sequence, said insertion not interfering with the normal function of the plastid genome;
b) a selectable marker gene disposed within said targeting segment, said selectable marker gene conferring to cells of said plant a selectable phenotype in the presence of a selection agent which is non-lethal to plant cells containing plastids transformed with said DNA construct but lethal to untransformed plastids;
and
c) at least one additional expressible DNA segment disposed within said targeting segment, positioned so as not to disrupt said homologous recombination, and so as not to interfere with said conferring of said selectable phenotype to said plant cells, said at least one additional expressible DNA segment containing;
i) at least one coding segment encoding a gene product;
ii) a 5' regulatory segment of plant chloroplast origin, operably linked to said at least one coding segment to promote expression of said coding segment in said plastids;
and
iii) a 3' regulatory segment of a plant chloroplast mRNA molecule operably linked to said at least one coding segment to promote stability of mRNA produced during said expression of said coding segment in said plastids.

5 more elements

Dependencies are inferred from the claim text.

DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein

Life of this patent

DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein

Life of this patent

Abstract

Claims34