Translation control elements for high-level protein expression in the plastids of higher plants and methods of use thereof
AssigneeRutgers, The State University of New Jersey
InventorsPal Maliga, Hiroshi Kuroda, Muhammad Khan
Life of this patent
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Abstract
DNA constructs containing translational control elements are provided. These 5′ regulatory segments facilitate high level expression of transgenes introduced into the plastids of higher plants.
Claims28
A recombinant DNA construct for expressing at least one heterologous protein in the plastids of higher plants, said construct comprising a chimeric 5′ regulatory region which includes a promoter element, a leader sequence and a downstream box element operably linked to a coding region of said at least one heterologous protein, said chimeric regulatory region enhancing translational efficiency of an mRNA molecule encoded by said DNA construct, relative to constructs lacking said chimeric regulatory region.
A plasmid for transforming the plastids of higher plants, said plasmid being selected from the group consisting of pHK30(B), pHK31(B), pHK60, pHK32(B), pHK33(B), pHK34(A), pHK35(A), pHK64(A), pHK36(A), pHK37(A), pHK38(A), pHK39(A), pHK40(A), pHK41(A), pHK42(A), pHK43(A), pMSK56, pMSK57, pMSK45, pMSK48, pMSK49, and pMSK35, pMSK53 and pMSK54.
A method for producing transplastomic monocots, comprising:
- a) obtaining embryogenic cells;
- b) exposing said cells to a heterologous DNA molecule under conditions whereby said DNA enters the plastids of said cells, said heterologous DNA molecule encoding at least one exogenous protein, said at least one exogenous protein encoding a selectable marker;
- c) applying a selection agent to said cells to facilitate sorting of untransformed plastids from transformed plastids, said cells containing transformed plastids surviving and dividing in the presence of said selection agent;
- d) transferring said surviving cells to selective media to promote shoot regeneration and growth;
- and
- e) rooting said shoots, thereby producing transplastomic monocot plants.
A method for producing transplastomic rice plants, said method comprising:
- a) obtaining embryogenic calli;
- b) inducing proliferation of calli on modified CIM medium;
- c) obtaining embryogenic cell suspensions of said proliferating calli in liquid AA medium;
- d) bombarding said embryogenic cells with microprojectiles coated with plasmid DNA;
- e) tranferring said bombarded cells to selective liquid AA medium;
- f) transferring said cells surviving in AA medium to selective RRM regeneration medium for a time period sufficient for green shoots to appear;
- and
- g) rooting said shoots in a selective MS salt medium.
A method for containing transgenes in transformed plants, comprising:
- a) determining the codon usage in said plant to be transformed and in microbes found in association with said plant;
- and
- b) genetically engineering said transgene sequence via the introduction of rare codons to abrogate expression of said transgene in said plant associated microbe.
Dependencies are inferred from the claim text.