HERBICIDE TOLERANT RICE PLANTS AND METHODS FOR IDENTIFYING SAME

• US 20080289060A1
• Filed: 08/24/2007
• Published: 11/20/2008
• Est. Priority Date: 08/24/2006
• Status: Active Grant

• Current Assignee: Bayer Cropscience NV (Bayer AG)
• Original Assignee: Bayer Bioscience NV (Bayer AG)
• Inventors: JOHNSON, KIRK, DE BEUCKELEER, MARC, MICHIELS, FRANK
• Granted Patent: US 7,928,295 B2
• Time in Patent Office: Days
• US Class Current: 800/260
• CPC Class Codes: C12N 15/8277 Phosphinotricin

Abstract

The invention provides specific transgenic rice plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the rice genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.

Claims

1. A method for identifying elite event GAT-OS3 in biological samples comprising detecting a GAT-OS3 specific region with a specific primer or probe that specifically recognizes the 5′

or 3′

flanking region of GAT-OS3.

2. The method of claim 1, wherein said method comprises amplifying a DNA fragment of between 50 and 500 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′

flanking region of GAT-OS3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or the 3′

flanking region of GAT-OS3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, the other primer of said primers recognizing a sequence within the foreign DNA of GAT-OS3 comprising the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

3. The method of claim 2, wherein said primer recognizing the 5′

flanking region of GAT-OS3 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or said primer recognizing the 3′

flanking region of GAT-OS3 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, and said primer recognizing a sequence within the foreign DNA of GAT-OS3 consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

4. The method of claim 2, wherein said primer recognizing the 5′

flanking region of GAT-OS3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or said primer recognizing the 3′

flanking region of GAT-OS3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, and said primer recognizing a sequence within the foreign DNA of GAT-OS3 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

5. The method of claim 4, wherein said primer recognizing the 5′

flanking region comprises the sequence of SEQ ID NO;

3 and said primer recognizing a sequence within the foreign DNA comprises the sequence of SEQ ID NO;

4.

6. The method of claim 5, wherein said method comprises amplifying a fragment of about 278 bp.

16. The method of claim 1, further comprising hybridizing a nucleic acid from said biological samples with a specific probe for GAT-OS3.

17. The method of claim 16, wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of GAT-OS3 and the sequence of the foreign DNA contiguous therewith.

18. The method of claim 17, wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:

1 from nucleotide 583 to 624 or SEQ ID NO;

2 from nucleotide 52 to 93, or the complement of said sequences.

7. A kit for identifying elite event GAT-OS3 in biological samples, said kit comprising one primer recognizing the 5′

flanking region of GAT-OS3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or one primer recognizing the 3′

flanking region of GAT-OS3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, and one primer recognizing a sequence within the foreign DNA of GAT-OS3, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

8. The kit of claim 7, wherein said primer recognizing the 5′

flanking region of GAT-OS3 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or said primer recognizing the 3′

flanking region of GAT-OS3 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, and said primer recognizing a sequence within the foreign DNA of GAT-OS3 consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

9. The kit of claim 7, wherein said primer recognizing the 5′

flanking region of GAT-OS3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or said primer recognizing the 3′

flanking region of GAT-OS3 comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, and said primer recognizing a sequence within the foreign DNA of GAT-OS3 comprises at its 3′

end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

10. The kit of claim 7, including a primer comprising the sequence of SEQ ID NO:

3 and a primer comprising of the sequence of SEQ ID NO;

4.

11. A primer comprising a sequence which, under optimized detection conditions, specifically recognizes a sequence within the 5′

or 3′

flanking region of GAT-OS3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 and said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607.

12. The primer of claim 11, wherein said primer consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO:

1 from nucleotide 1 to nucleotide 603 or a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607.

13. The primer of claim 11, wherein said primer comprises at its extreme 3′

end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607.

14. A primer comprising at its extreme 3′

end the sequence of SEQ ID NO;

3.

15. A primer comprising at its extreme 3′

end the sequence of SEQ ID NO;

4.

19. A kit for identifying elite event GAT-OS3 in biological samples, said kit comprising a specific probe, capable of hybridizing specifically to a specific region of GAT-OS3.

20. The kit of claim 19, wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of GAT-OS3 and the sequence of the foreign DNA contiguous therewith.

21. The kit of claim 20, wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:

1 from nucleotide 583 to 624 or SEQ ID NO;

2 from nucleotide 52 to 93, or the complement of said sequences.

22. A specific probe for identifying elite event GAT-OS3 in a biological sample.

23. The probe of claim 22, wherein said probe comprises a nucleotide sequence comprising at least 80% sequence identity with a sequence comprising part of the 5′

flanking sequence or the 3′

flanking sequence of GAT-OS3 and the sequence of the foreign DNA contiguous therewith, or the complement thereof.

24. The probe of claim 23, wherein said probe comprises a nucleotide sequence comprising at least 80% sequence identity with SEQ ID NO:

1 from nucleotide 583 to 624 or SEQ ID NO;

2 from nucleotide 52 to 93, or the complement of said sequences.

25. A specific probe for identifying elite event GAT-OS3 in a biological sample, said probe comprising a nucleotide sequence being essentially similar to SEQ ID NO:

1 from nucleotide 583 to 624 or SEQ ID NO;

2 from nucleotide 52 to 93, or the complement of said sequences.

26. A method for confirming seed purity comprising detecting a GAT-OS3 specific region with a specific primer or probe that specifically recognizes the 5′

or 3′

flanking region of GAT-OS3, in seed samples.

27. A method for screening seeds for the presence of GAT-OS3 comprising detecting a GAT-OS3 specific region with a specific primer or probe that specifically recognizes the 5′

or 3′

flanking region of GAT-OS3, in samples of seed lots.

28. A method for determining the zygosity status of a plant, plant material or seed comprising elite event GAT-OS3 comprising amplifying DNA fragments of between 50 and 500 bp from a nucleic acid present in a biological sample using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plant DNA of GAT-OS3, wherein said pre-insertion plant DNA of GAT-OS3 is the 5′

flanking region of GAT-OS3, said 5′

flanking region comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 1 to nucleotide 603, or the 3′

flanking region of GAT-OS3, said 3′

flanking region comprising the nucleotide sequence of the complement of SEQ ID NO;

2 from nucleotide 73 to nucleotide 607, or the pre-insertion plant DNA of GAT-OS3 comprising the nucleotide sequence of SEQ ID NO;

23 or the complement thereof, and wherein the third of said primers recognizing a sequence within the foreign DNA of GAT-OS3 comprising the nucleotide sequence of the complement of SEQ ID NO;

1 from nucleotide 604 to nucleotide 743 or the nucleotide sequence of SEQ ID NO;

2 from nucleotide 1 to nucleotide 72.

29. The method according to claim 28, wherein a first primer comprises the nucleotide sequence of SEQ ID NO:

3, a second primer comprises the sequence of SEQ ID NO;

7 and said third primer comprises the sequence of SEQ ID NO;

4.

30. A method of detecting the presence of elite event GAT-OS3 in a biological sample through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:

target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence comprising;

a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 604 to nucleotide 620 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO;

2 from nucleotide 56 to nucleotide 72 or its complement;

b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID NO;

1 from nucleotide 587 to nucleotide 603 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID NO;

2 from nucleotide 73 to nucleotide 89 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;

c) cleaving only the labeled probe within the probe;

target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;

d) recycling of the target nucleic acid sequence by repeating steps (a) to (c); and

e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.

31. A transgenic rice plant, or seed, cells, parts or progeny thereof, each comprising elite event GAT-OS3 in its genome, reference seed comprising said event having being deposited at the ATCC under accession number PTA-2600.

32. The transgenic rice plant, seed, cells, parts or progeny of claim 31, the genomic DNA of which, when analyzed using PCR with two primers comprising the nucleotide sequence of SEQ ID NO:

3 and SEQ ID NO;

4, respectively, yields a DNA fragment of about 278 bp.

38. A method for producing a rice plant or seed comprising elite event GAT-OS3 comprising crossing a plant of claim 31 with another rice plant, and planting the seed obtained from said cross.

40. Genomic DNA produced from a rice plant, plant cell or seed of claim 31.

33. Seed comprising elite event GAT-OS3 deposited at the ATCC under accession number PTA-2600 or derivatives therefrom.

34. A rice plant or a part thereof, or seed therefrom obtained from the seed of claim 33.

35. A rice plant, or seed, cells or tissues thereof, each comprising elite event GAT-OS3 in its genome, obtained by propagation of and/or breeding with a rice plant grown from the seed deposited at the ATCC under accession number PTA-2600.

36. A rice seed comprising elite event GAT-OS3, reference seed comprising said event having been deposited at the ATCC under accession number PTA-2600.

37. A transgenic rice plant, cell or tissue, comprising elite event GAT-OS3, produced from the seed of claim 36.

39. Rice genomic DNA comprising elite event GAT-OS3.

41. An isolated nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID NO:

1 from nucleotide 593 to nucleotide 614 or SEQ ID NO;

2 from nucleotide 62 to 83, or the complement of said sequences.

42. The isolated nucleic acid molecule of claim 41, comprising a nucleotide sequence essentially similar to SEQ ID NO:

1 from nucleotide 583 to nucleotide 624 or SEQ ID NO;

2 from nucleotide 52 to 93, or the complement of said sequences.