Compositions and methods to stack multiple nucleotide sequences of interest in the genome of a plant

• US 20040083500A1
• Filed: 08/12/2003
• Published: 04/29/2004
• Est. Priority Date: 11/18/1997
• Status: Active Grant

• Current Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Original Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Inventors: Baszczynski, Christopher L., Bowen, Benjamin A., Tagliani, Laura A., Peterson, David J.
• Granted Patent: US 9,222,098 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

Methods and compositions for the stacking of multiple nucleotide sequences at precise locations in the genome of a plant or plant cell are provided. Specifically, transfer cassettes comprising nucleotide sequences of interest flanked by non-identical recombination sites are introduced into a plant comprising a target site. The target site contains at least a set of non-identical recombination sites corresponding to those on the transfer cassette. Exchange of the nucleotide sequences flanked by the recombination sites is effected by a recombinase. The transfer cassettes and target sites are designed so as to allow for the stacking or ordering of nucleotide sequences at precise locations in the plant genome.

Claims

1. A method to combine multiple transfer cassettes at one location in a genome of a plant cell, said method comprising:

a) stably incorporating into the genome of the plant cell, a target site comprising at least a first and a second non-identical recombination site;

b) introducing into the plant cell a first transfer cassette comprising in the following order at least the first, a third, and the second non-identical recombination sites, wherein the first and the third non-identical recombination sites of the first transfer cassette flank a first nucleotide sequence of interest;

c) providing a first recombinase that recognizes and implements recombination at the first and the second non-identical recombination sites;

d) introducing into the plant cell a second transfer cassette comprising at least the second and the third non-identical recombination sites, wherein the second and the third non-identical recombination sites of the second transfer cassette flank a second nucleotide sequence of interest wherein, introducing said first or said second transfer cassette comprises sexual crossing; and

,

e) providing a second recombinase that recognizes and implements recombination at the second and the third non-identical recombination site.

2. The method of claim 1 wherein, said second transfer cassette further comprises in the following order the third, a fourth, and the second non-identical recombination sites, wherein the third and the fourth non-identical recombination sites flank the second nucleotide sequence of interest.

3. The method of claim 2 further comprising:

a) introducing into the plant cell a third transfer cassette comprising in the following order at least the fourth, a fifth, and the second non-identical recombination sites, wherein the fourth and the fifth non-identical recombination sites of the third transfer cassette flank a third nucleotide sequence of interest and wherein introducing said third transfer cassette comprises sexual crossing; and

,

b) providing a third recombinase that recognizes and implements recombination at the fourth and the second non-identical recombination sites.

4. The method of claim 1, wherein said first, said second, or said third non-identical recombination site is selected from the group consisting of an FRT site, a mutant FRT site, a LOX site, and a mutant LOX site.

5. The method of claim 4, wherein said mutant FRT sites comprises FRT 5 (SEQ ID NO:

3), FRT 6 (SEQ ID NO;

4), or FRT 7 (SEQ ID NO;

5).

6. The method of claim 1, wherein said first or said second recombinase is FLP or Cre.

7. The method of claim 6, wherein said FLP recombinase has been synthesized using maize preferred codons.

8. The method of claim 6, wherein said Cre recombinase has been synthesized using maize preferred codons.

9. The method of claim 1, wherein said plant cell is from a monocot.

10. The method of claim 9, wherein said monocot is maize.

11. The method of claim 1, wherein said plant cell is from a dicot.

12. The method of claim 11, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

13. The method of claim 1, wherein said plant cell is in plant.

14. The method of claim 1, wherein providing said first, said second, or said first and said second recombinase comprises genetically transforming said plant cell with one or more expression cassettes comprising a nucleotide sequence encoding said first or said second recombinase.

15. A method to combine multiple transfer cassettes at one location in a genome of a plant cell, said method comprising:

a) stably incorporating into the genome of the plant cell, a target site comprising in the following order a first, a second, and a third non-identical recombination site;

b) introducing into the plant cell a first transfer cassette comprising the first and the second non-identical recombination sites, wherein the first and the second non-identical recombination sites of the first transfer cassette flank a first nucleotide sequence of interest;

c) providing a first recombinase that recognizes and implements recombination at the first and the second non-identical recombination sites;

d) introducing into the plant cell a second transfer cassette comprising at least the second and the third non-identical recombination sites, wherein the second and the third non-identical recombination sites of the second transfer cassette flank a second nucleotide sequence of interest and wherein introducing said first transfer cassette or said second transfer cassette comprises sexual crossing; and

,

e) providing a second recombinase that recognizes and implements recombination at the second and the third non-identical recombination sites.

16. The method of claim 15, wherein said second transfer cassette further comprises in the following order the second, a fourth, and the third non-identical recombination sites, wherein the second and the fourth non-identical recombination sites flank the second nucleotide sequence of interest.

17. The method of claim 16 further comprising:

a) introducing into the plant cell a third transfer cassette comprising in the following order at least the fourth, a fifth, and the third non-identical recombination sites, wherein the fourth and the fifth non-identical recombination sites of the third transfer cassette flank a third nucleotide sequence of interest, wherein introducing the third transfer cassette comprises sexual crossing; and

,

b) providing a third recombinase that recognizes and implements recombination at the fourth and the third non-identical recombination sites.

18. The method of claim 15, wherein said first, said second, or said third non-identical recombination site is selected from the group consisting of an FRT site, a mutant FRT site, a LOX site, and a mutant LOX site.

19. The method of claim 18, wherein said mutant FRT sites comprises FRT 5 (SEQ ID NO:

3), FRT 6 (SEQ ID NO;

4), or FRT 7 (SEQ ID NO;

5).

20. The method of claim 15, wherein said first or said second recombinase is FLP or Cre.

21. The method of claim 20, wherein said FLP recombinase has been synthesized using maize preferred codons.

22. The method of claim 20, wherein said Cre recombinase has been synthesized using maize preferred codons.

23. The method of claim 15, wherein said plant cell is from a monocot.

24. The method of claim 23, wherein said monocot is maize.

25. The method of claim 15, wherein said plant cell is from a dicot.

26. The method of claim 25, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

27. The method of claim 15, wherein said plant cell is in a plant.

28. The method of claim 15, wherein providing said first, said second, or said first and said second recombinase comprises genetically transforming said plant cell one or more expression cassettes comprising a nucleotide sequence encoding said first or said second recombinase.

29. A plant cell having stably incorporated into its genome at least a first and a second transfer cassette, wherein

said first transfer cassette comprises a first nucleotide sequence of interest flanked by a first and a second recombination site, wherein said first and said second recombination sites are non-identical;

said second transfer cassette comprises a second nucleotide sequence of interest flanked by the second recombination site and a third recombination site, wherein said third recombination site is non-identical to said first and said second recombination site; and

,

said second recombination site is shared between the first and the second transfer cassette, such that the genome of the plant cell comprises in the following order the first recombination site, the first nucleotide sequence of interest, the second recombination site, the second nucleotide sequence of interest, and the third recombination site.

30. The plant cell of claim 29, wherein said first, said second, or said third recombination site is selected from the group consisting of a FRT site, a mutant FRT site, a LOX site and a mutant LOX site.

31. The plant cell of claim 30, wherein the first, the second, or the third recombination site is selected from the group consisting of a FRT site and a mutant FRT site.

32. The plant cell of claim 31, wherein the mutant FRT site is selected from the group consisting of FRT 5 (SEQ ID NO:

3), FRT 6 (SEQ ID NO;

4), and FRT 7 (SEQ ID NO;

5).

33. The plant cell of claim 29, wherein said plant cell is within a plant.

34. The plant cell of claims 29, wherein said plant cell is within a seed.