Genetic engineering of plant chloroplasts
AssigneeAuburn University
InventorsHenry Daniell, Bruce A. McFadden
Life of this patent
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Abstract
Novel chimeric constructions and methods for their use are provided for expression of exogenous genes in a plant chloroplast. Particularly, expression is achieved by the use of a chloroplast or bacterial 5' untranslated region in the expression cassette. The expression cassette may be integrated into the chloroplast genome by the use of chloroplast DNA flanking sequences, or may replicate autonomously if provided with a chloroplast origin of replication. Plants and cells containing the transformed chloroplasts are also provided. The constructs may be used with both monocotyledenous and dicotyledenous chloroplasts.
Claims37
A DNA construct comprising:
- as operably joined components, at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, a transcriptional initiation region functional in a plant chloroplast, a heterologous DNA sequence encoding a polypeptide of interest, wherein transcription of said DNA sequence is regulated by said initiation region, and a transcriptional termination region.
A chloroplast expression vector comprising:
- as operably joined components, at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, a transcriptional initiation region functional in a chloroplast, a DNA sequence comprising one or more cloning site(s) and a transcriptional termination region.
A replication vector comprising:
- as operably joined components, a DNA fragment comprising at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, a transcriptional initiation region functional in a plant chloroplast, a DNA sequence comprising one or more cloning site(s), and a transcriptional termination region.
A plant chloroplast comprising:
- a DNA construct comprising, as operably joined components, at least one chloroplast replication origin functional in a plant chloroplast, a transcriptional initiation region functional in said chloroplast, a heterologous DNA sequence encoding a polypeptide of interest, wherein transcription of said DNA sequence is regulated by said initiation region, and a transcriptional termination region, wherein at least one of said components is heterologous to said chloroplast.
A plant chloroplast comprising:
- a chloroplast expression vector comprising, as operably joined components, (1) a DNA fragment comprising at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, (2) a transcriptional initiation region functional in said chloroplast, (3) a DNA sequence encoding, a polypeptide of interest, and (4) a transcriptional termination region, wherein at least one of said components is heterologous to said chloroplast.
A method for introducing heterologous DNA into a chloroplast, said method comprising:
- bombarding a plant cell with a chloroplast expression vector adsorbed to a microprojectile, said chloroplast expression vector comprising, as operably joined components, (1) at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, (2) a transcriptional initiation region functional in said plant chloroplast, (3) a heterologous DNA sequence encoding a polypeptide of interest, and (4) a transcriptional termination region whereby said heterologous DNA is introduced into chloroplast in said plant cell.
A method for specifically modifying the phenotype of plant cell chloroplast distinct from other plant cell organelles, said the method comprising:
- bombarding a plant cell with a chloroplast expression vector adsorbed to a microprojectile, said vector comprising, as operably joined components, (1) a DNA fragment comprising at least one chloroplast replication origin functional in a plant chloroplast from other than a yeast, (2) a transcriptional initiation region from a chloroplast gene, (3) a heterologous DNA sequence encoding a polypeptide of interest, and (4) a transcriptional termination region, wherein said components are functional in said chloroplast, and
- growing said plant cell whereby the phenotype of said plant chloroplast is modified as a result of expression of said DNA sequence.
A method for specifically modifying the genotype of plant cell chloroplast distinct from other plant cell organelles, said method comprising:
- bombarding a plant cell with a chloroplast expression vector absorbed to a microprojectile, said vector comprising, as operably joined components, (1) a DNA fragment comprising at least one chloroplast replication origin functional in a plant chloroplast, (2) a transcriptional initiation region from a chloroplast gene, (3) a DNA sequence encoding a polypeptide of interest, and (4) a transcriptional termination region, wherein said components are functional in said chloroplast and at least one of said components is heterologous to said plant cell,
- whereby a plurality of chloroplast in said plant cell are transformed and the genotype of said chloroplast is modified as a result of said introducing of said chloroplast expression vector.
Dependencies are inferred from the claim text.