Chimaeric gene coding for a transit peptide and a heterologous polypeptide

• US 5,728,925 A
• Filed: 04/28/1995
• Issued: 03/17/1998
• Est. Priority Date: 12/28/1984
• Status: Expired due to Term

• Current Assignee: Bayer Cropscience NV (Bayer AG)
• Original Assignee: Bayer AG, Plant Genetic Systems NV (Bayer AG)
• Inventors: Van Montagu, Marc, Herrera-Estrella, Luis, Van Den Broeck, Guido, Cashmore, Anthony R., Bohnert, Hans J., Schreier, Peter, Kausch, Albert P., Schell, Jeff, Timko, Michael P.
• Primary Examiner(s): Fox, David T.
• Application Number:
  US08/430,257
• Time in Patent Office: 1,054 Days
• Field of Search: 435/69.7, 435/69.8, 435/70.1, 435/172.3, 435/240.4, 435/320.1, 800/205, 800/250, 536/23.4, 536/24.1
• US Class Current: 800/300
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8221 Transit peptides

Abstract

Chimaeric DNA sequence which encodes: 1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or polypeptide of a plant and 2) a protein or polypeptide that is heterologous to the transit peptide. The chimaeric DNA sequence can be used as a vector for transforming a plant cell so that a chimaeric precursor of the heterologous protein or polypeptide is produced in the cytoplasm of the cell and the chimaeric precursor then transports the heterologous protein or polypeptide in vivo into a chloroplast of the cell.

Claims

1. A plant containing in its nuclear genome a chimaeric DNA sequence, comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and

2) a protein or polypeptide of interest which is different from a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to said plant,

wherein the chimaeric DNA sequence can be expressed in cells of said plant under the control of said promoter, and said protein or polypeptide of interest is translocated into the chloroplasts of said cells of said plant.

2. The plant of claim 1, in which said protein or polypeptide of interest is of bacterial origin.

3. The plant of claim 1 in which said protein or polypeptide of interest is of plant origin.

4. The plant of claim 1 in which said protein or polypeptide of interest confers resistance to an herbicide.

5. The plant of claim 1 in which the first amino acid of said protein or polypeptide of interest is a methionine.

6. The plant of claim 1 in which no more than the first seven amino acids of said protein or polypeptide of interest are encoded by a synthetic nucleotide linker.

7. The plant of claim 1 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein selected from the group consisting of the small subunit of ribulose-1,5-bisphosphate carboxylase, and chlorophyll a/b binding protein.

8. The plant of claim 7 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed, and wheat.

9. The plant of claim 7 in which the transmit peptide has the sequence selected from the group consisting of:

##STR4##

10. The plant of claim 1 in which said promoter is normally associated with the DNA encoding said transit peptide.

11. The plant of claim 10 in which said promoter is a promoter of a gene selected from the group consisting of, the plastocyanin gene, the ferredoxin-NADP+ oxydoreductase gene, the ribulose-1,5-bisphosphate carboxylase gene, and the chlorophyll a/b binding protein gene.

12. The plant of claim 1 in which said promoter is foreign to the DNA encoding said transit peptide.

13. The plant of claim 12 in which said promoter is a promoter of the nopalin synthase gene.

42. A seed of the plant of any one of claims 1 to 4, 7, 14, 15 and 27, comprising said chimaeric DNA sequence.

43. A cell of the plant of any one of claims 1 to 4, 7, 14, 15 and 27, comprising said chimaeric DNA sequence.

14. A plant containing in its nuclear genome a chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and

2) a hybrid protein comprising in sequence;

2.1) a linker, and

2.2) a protein or polypeptide of interest which is different from said chloroplast protein or chloroplast polypeptide

wherein said linker does not significantly alter the biological property of said protein or polypeptide of interest in said hybrid protein, and

b) a promoter upstream of said nucleic acid sequence recognized by polymerases endogenous to a plant,

wherein the chimaeric DNA sequence is expressed in cells of said plant under the control of said promoter, and said hybrid protein is translocated into the chloroplasts of said cells of the plant.

15. The plant of claim 14, wherein said linker is a N-terminal part of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof.

16. The plant of claim 15 in which said protein or polypeptide of interest is of bacterial origin.

17. The plant of claim 15 in which said protein or polypeptide of interest is of plant origin.

18. The plant of claim 15 in which said protein or polypeptide of interest confers resistance to an herbicide.

19. The plant of claim 15, in which said N-terminal part is encoded by a DNA sequence that comprises an intron.

20. The plant of claim 15 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein selected from the group consistinf of:

the small subunit of ribulose-1,5-bisphosphate carboxylase, and chlorophyll a/b binding protein.

21. The plant of claim 15 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of:

soybean, pea, duckweed, and wheat.

22. The plant of claim 21 in which the transit peptide has the sequence selected from the group consisting of:

##STR5##

23. The plant of claim 15 in which said promoter is normally associated with the DNA encoding said transit peptide.

24. The plant of claim 23 in which said promoter is a promoter of a gene selected from the group consisting of:

the plastocyanin gene, the ferredoxin-NADP+oxydoreductase gene, the ribulose-1,5-bisphosphate carboxylase gene, and the chlorophyll a/b binding protein gene.

25. The plant of claim 15 in which said promoter is foreign to the DNA encoding said transit peptide.

26. The plant of claim 25 in which said promoter is a promoter of the nopaline synthase gene.

27. A plant containing in its nuclear genome a chimaeric DNA sequence comprising:

a) a nucleic acid sequence coding for a chimaeric protein or polypeptide comprising in sequence;

1) a transit peptide of a cytoplasmic precursor of a chloroplast protein or chloroplast polypeptide of a plant species, and,

2) a fusion protein comprising in sequence,

2.1) a N-terminal part consistinf of no more than the first 22 N-terminal amino acids of a mature chloroplast protein or chloroplast polypeptide derived from a natural cytoplasmic precursor thereof, and

2.2) a protein or polypeptide of interest which is different from said chloroplast protein or polypeptide and,

b) a promoter upstream of said nucleic acid sequence recognized by polymerases encodenous to said plant,

wherein the chimaeric DNA sequence is expressed in cells of said plant under the control of said promoter, and said fusion protein is translocated into the chloroplasts of said cells of said plant.

28. The plant of claim 27 in which said protein or polypeptide of interest is of bacterial origin.

29. The plant of claim 27 in which said protein or polypeptide of interest is of plant origin.

30. The plant of claim 27 in which said protein or polypeptide of interest confers resistance to an herbicide.

31. The plant of claim 27 in which said N-terminal part is encoded by a DNA sequence that comprises an intron.

32. The plant of claim 27 in which said N-terminal part is from the small subunit of the ribulose-1,5-bisphosphate carboxylase of Pisum sativum.

33. The plant of claim 27 in which said N-terminal part consists of no more than the first five N-terminal amino acids of said chloroplast protein or polypeptide.

34. The plant of claim 33 in which said N-terminal part has the sequence M-Q-V-W-P.

35. The plant of claim 27 in which the transit peptide is from a cytoplasmic precursor of a chloroplast protein selected from the group consisting of:

the small subunit of ribulose-1,5-bisphosphate carboxylase, and chlorophyll a/b binding proteins.

36. The plant of claim 35 in which the transit peptide is from a cytoplasmic precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase of a plant selected from the group consisting of soybean, pea, duckweed, and wheat.

37. The plant of claim 36 in which the transit peptide has the sequence selected from the group consisting of:

##STR6##

38. The plant of claim 27 in which said promoter is normally associated with the DNA encoding said transit peptide.

39. The plant of claim 38 in which said promoter is a promoter of a gene selected from the group consisting of:

the plastocyanin gene, the ferredoxin-NADP+ oxydoreductase gene, the ribulose-1,5-bisphosphate carboxylase gene, and the chlorophyll a/b binding protein gene.

40. The plant of claim 27 in which said promoter is foreign to the DNA encoding said transit peptide.

41. The plant of claim 40 in which said promoter is a promoter of the nopalin synthase gene.