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.