Polyphenol oxidase cDNA

• US 6,160,204 A
• Filed: 06/07/1995
• Issued: 12/12/2000
• Est. Priority Date: 01/31/1992
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Steffens, John C.
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US08/481,190
• Time in Patent Office: 2,015 Days
• Field of Search: 800/205, 800/284, 800/286, 800/298, 800/317.2, 800/317.3, 800/317.4, 536/23.6, 435/69.1, 435/172.3, 435/240.4, 435/240.49, 435/320.1, 435/419, 435/468, 435/252.2, 435/252.3
• US Class Current: 800/284
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 9/0059 Catechol oxidase (1.10.3.1), i.e. tyrosinase
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12Y 110/03001 Catechol oxidase (1.10.3.1), i.e. tyrosinase
• CPC Class Codes: C12Y 114/18001 Tyrosinase (1.14.18.1)

Abstract

The present invention describes the cloning and sequencing of plant Polyphenol Oxidase (PPO) cDNAs which has subsequently permitted their use to genetically transform plants to achieve a variety of desired phenotypes.

Claims

1. An isolated nucleic acid molecule encoding a plant polyphenol oxidase.

2. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is deoxyribonucleic acid.

3. The isolated nucleic acid molecule of claim 2 wherein said deoxyribonucleic acid is cDNA.

4. The isolated nucleic acid molecule of claim 3 wherein said cDNA has a nucleotide sequence as shown in SEQ NO:

1.

5. The isolated nucleic acid molecule of claim 3 wherein said cDNA has a nucleotide sequence as shown in SEQ ID NO:

5.

6. The isolated nucleic acid molecule of claim 3 wherein said cDNA has a nucleotide sequence as shown in SEQ ID NO:

14.

7. The isolated nucleic acid molecule of claim 3 wherein said cDNA has a nucleotide sequence as shown in SEQ ID NO:

17.

8. The isolated nucleic acid molecule of claim 1 wherein said plant polyphenol oxidase is a tomato polyphenol oxidase.

9. The isolated nucleic acid molecule of claim 1 wherein said plant polyphenol oxidase is a potato polyphenol oxidase.

10. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:

4.

11. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:

8.

12. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:

16.

13. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:

19.

14. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is ribonucleic acid.

15. The isolated nucleic acid molecule of claim 14 wherein said ribonucleic acid is mRNA.

16. An isolated antisense nucleic acid molecule complementary to the mRNA of claim 15.

17. The isolated antisense nucleic acid molecule of claim 16 wherein said nucleic acid comprises ribonucleic acid.

18. The isolated antisense nucleic acid molecule of claim 16 wherein said nucleic acid comprises single-stranded deoxyribonucleic acid.

19. The isolated antisense nucleic acid molecule of claim 16 wherein said plant polyphenol oxidase is a tomato polyphenol oxidase.

20. The isolated antisense nucleic acid molecule of claim 16 wherein said plant polyphenol oxidase is a potato polyphenol oxidase.

21. A recombinant cell transformed with the isolated antisense nucleic acid molecule of claim 16.

22. The recombinant cell of claim 21 wherein said recombinant cell is Agrobacterium tumefaciens.

23. An expression vector comprising the isolated antisense nucleic acid molecule of claim 16.

24. The expression vector of claim 23 wherein said expression vector is a plasmid.

25. A cell comprising the expression vector of claim 23.

26. The cell of claim 25 wherein said cell is Agrobacterium tumefaciens.

27. A transgenic plant transformed with the isolated antisense nucleic acid molecule of claim 16.

28. The transgenic plant of claim 27 wherein said plant is a tomato plant.

29. The transgenic plant of claim 27 wherein said plant is a potato plant.

30. The transgenic plant of claim 27 wherein said plant is a tobacco plant.

31. A method of decreasing polyphenol oxidase production in a plant, said method comprising:

selecting a plant with a first level of polyphenol oxidase production; and

introducing the isolated antisense nucleic acid molecule of claim 16 into said plant, wherein said isolated antisense nucleic acid molecule blocks translation of said mRNA so as to decrease expression of polyphenol oxidase, resulting in a second level of polyphenol oxidase production in said plant, said second level being less than said first level.

32. The method of claim 31 wherein said second level is about zero.

33. The method of claim 31 wherein said plant is a tomato plant.

34. The method of claim 31 wherein said plant is a potato plant.

35. The method of claim 31 wherein said plant is a tobacco plant.

36. A recombinant cell transformed with the isolated nucleic acid molecule of claim 1.

37. The recombinant cell of claim 36 wherein said recombinant cell is Agrobacterium tumefaciens.

38. An expression vector comprising the isolated nucleic acid molecule of claim 1.

39. The expression vector of claim 38 wherein said expression vector is a plasmid.

40. A cell comprising the expression vector of claim 38.

41. The cell of claim 40 wherein said cell is Agrobacterium tumefaciens.

42. A transgenic plant transformed with the isolated nucleic acid molecule of claim 1.

43. The transgenic plant of claim 42 wherein said plant is a tomato plant.

44. The transgenic plant of claim 42 wherein said plant is a potato plant.

45. The transgenic plant of claim 42 wherein said plant is a tobacco plant.

46. A method of increasing polyphenol oxidase production in a plant, said method comprising:

selecting a plant with a first level of polyphenol oxidase production; and

introducing the nucleic acid molecule of claim 1 into said plant, wherein when said nucleic acid molecule is expressed and polyphenol oxidase is produced, resulting in a second level of polyphenol oxidase production in said plant, said second level being greater than said first level.

47. The method of claim 46 wherein said first level is about zero.

48. The method of claim 46 wherein said plant is a tomato plant.

49. The method of claim 46 wherein said plant is a potato plant.

50. The method of claim 46 wherein said plant is a tobacco plant.

51. A method of obtaining DNA encoding a plant polyphenol oxidase, said method comprising:

selecting a DNA molecule encoding a plant polyphenol oxidase, said DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

5, SEQ ID NO;

14, and SEQ ID NO;

17;

designing an oligonucleotide probe for a plant polyphenol oxidase based on the nucleotide sequence of the selected DNA molecule;

probing a genomic or cDNA library of a plant with the oligonucleotide probe; and

obtaining clones from said library that are recognized by said oligonucleotide probe, so as to obtain DNA encoding a plant polyphenol oxidase.