Patent EA-004652-B1 (EA)
Title: QUINOLATE PHOSPHORIBOSYL-TRANSFERASE (QPRTase) OF TOBACCO PLANT ENCODING SAID ENZYME AND METHODS
Inventor: CONKLING MARK A
IPC: C12N15/11
Filed: 1998-06-10
Abstract: 1. An isolated DNA molecule comprising encoding quinolate phosphoribosyl transferase (QPRTase) of a tobacco plant selected from the group consisting of: (a) DNA sequence according to SEQ ID NO:1 given in the List of sequences; (b) DNA encoding (QPRTase) enzyme of a tobacco plant with amino acid having SEQ ID NO:2; (c) DNA sequences which hybridize to isolated DNA of (a) or (b) above under stringent conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 70 degree C; (d) DNA sequences which differ from the DNA of (a), (b) or (c) above due to the degeneracy of the genetic code; and (e) DNA sequences comprising at least 30 nucleotides according to SEQ ID NO:1. 2. An isolated DNA molecule according to claim 1, wherein characterized by SEQ ID NO:1 given in the List of sequences. 3. An isolated DNA molecule according to claim 1, wherein encoding (QPRTase) enzyme of a tobacco plant with amino acid having SEQ ID NO:2. 4. An isolated DNA molecule according to claim 1, which is hybridized to isolated DNA of (a) or (b) under stringent conditions represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 70 degree C. 5. An isolated DNA molecule according to claim 1, which differs from the DNA of (a), (b) or (c) due to the degeneracy of the genetic code. 6. An isolated DNA molecule encoding quinolate phosphoribosyl transferase (QPRTase) of a tobacco plant, comprising at least 30 nucleotides according to SEQ ID NO:1. 7. An isolated DNA molecule encoding quinolate phosphoribosyl transferase (QPRTase) of a tobacco plant, comprising at least 50 nucleotides according to SEQ ID NO:1. 8. An exogenous DNA construct comprising an expression cassette, in the 5' to 3' direction, a promoter operable in a plant cell and a DNA segment according to claim 1 positioned downstream from said promoter and operatively associated therewith. 9. An exogenous DNA construct comprising an expression cassette, in the 5' to 3' direction, a promoter operable in a plant cell and a DNA segment according to claim 1 positioned downstream from said promoter and operatively associated therewith, said DNA segment in antisense orientation. 10. An exogenous DNA construct comprising in the 5' to 3' direction, a promoter operable in a plant cell and a DNA encoding quinolate phosphoribosyl transferase of a tobacco plant, having SEQ ID NO:1, said DNA is operatively associated with said promoter. 11. An exogenous DNA construct comprising in the 5' to 3' direction, a promoter operable in a plant cell and a DNA encoding quinolate phosphoribosyl transferase of a tobacco plant, having SEQ ID NO:1, said DNA DNA segment in antisense orientation and operatively associated with said promoter. 12. An exogenous DNA construct comprising an expression cassette, in the 5' to 3' direction, a promoter operable in a plant cell and a DNA comprising at least 50 nucleotides according to SEQ ID NO:1 1 positioned downstream from said promoter and operatively associated therewith. 13. An exogenous DNA construct according to any one of claims 8-12, which promoter is constitutively active in plant cells. 14. An exogenous DNA construct according to any one of claims 8-12 wherein said promoter is selectively active in plant root tissue cells. 15. An exogenous DNA construct according to any one of claims 8-12 wherein said promoter is selectively active in plant root cortex tissue cells. 16. An exogenous DNA construct according to any one of claims 8-12 wherein said construct further comprises a plasmid. 17. A plant transformation vector comprising DNA construct according to any one of claims 8-12. 18. A plant transformation vector comprising DNA construct according to any one of claims 8-12 wherein the plant transformation vector is an Agrobacterium tumefaciens vector. 19. A transgenic plant comprising DNA construct according to any one of claims 8-12. 20. A transgenic plant cell comprising DNA construct according to any one of claims 8-12. 21. An isolated quinolate phosphoribosyl transferase enzyme of a tobacco plant encoding DNA sequence according to (a), (c) and (d) of claim 1. 22. An isolated quinolate phosphoribosyl transferase enzyme of a tobacco plant having amino acid sequence SEQ ID NO:2. 23. A method of making a transgenic plant cell having reduced quinolate phosphoribosyl transferase (QPRTase) expression, said method comprising: providing a plant cell of a type known to express quinolate phosphoribosyl transferase; transforming said plant cell with said exogenous DNA construct according to any one of claims 8-16 to produce transgenic cells, said plant cell having reduced expression of QPRTase compared to an untransformed cell. 24. A method of claim 23, wherein said plant cell is Nicotiana tabacum. 25. A method according to claim 23, wherein said transforming step is carried out by bombarding said plant cell with microparticles carrying said DNA construct. 26. A method according to claim 23, wherein said transforming step is carried out by infecting said plant cell with an Agrobacterium tumefaciens containing a Ti plasmid carrying said DNA construct. 27. The method according to claim 23, wherein said plant cells express endogeneous said quinolate phosphoribosyl transferase messenger RNA (QPRT rnRNA), wherein exogenous DNA construct is complementary to said quinolate phosphoribosyl transferase messenger RNA (QPRT rnRNA) expressed in said plant cell in a region selected from: (a) the 5'-untranslated sequence of said QPRT mRNA; (b) the 3'-untranslated sequence of said QPRT mRNA; and (c) the translated region of said QPRT mRNA. 28. The method according to claim 23, wherein said plant cells express endogeneous said quinolate phosphoribosyl transferase messenger RNA (QPRT rnRNA), wherein said exogenous DNA construct is complementary to at least 200 nucleotides of said quinolate phosphoribosyl transferase messenger RNA expressed in said plant cell. 29. A method of producing a transgenic plant having decreased QPRTase expression comprising producing plant cells according to any one of claims 23 and 24 and regeneration a transgenic plant therefrom. 30. A method of producing transgenic tobacco seeds, comprising growing a transgenic plant according to claim 19 which is a tobacco plant to obtain seeds from said transgenic tobacco plant. 31. A transgenic plant of the species Nicotiana having reduced quinolate phosphoribosyl transferase (QPRTase) expression relative to a nontransformed control plant according to claim 29. 32. A progeny of a transgenic plant according to claim 31. 33. Seeds of a transgenic plant of the species Nicotiana having reduced quinolate phosphoribosyl transferase (QPRTase) expression relative to a nontransformed control plant, wherein said transgenic plant is a plant according to claim 31 or a progeny thereof according to claim 32. 34. A plurality of plants according to claim 31 planted together in an agricultural field. 35. A method for reducing expression of a quinolate phosphoribosyl transferase gene in a plant cell, said method comprising plant cell transformation of exogenous DNA construct according to any one of claims 8-16, wherein a transcribed strand of said exogenous DNA is complementary to quinolate phosphoribosyl transferase mRNA endogenous to said cell, whereby transcription of said complementary strand reduces expression of said quinolate phosphoribosyl gene. 36. A method of producing a plant of the species Nicotiana having decreased levels of nicotine in leaves of said tobacco plant, said method comprising growing a plant of the species Nicotiana, or progeny plants thereof, wherein said plant comprises cells containing transformation of exogenous DNA construct according to any one of claims 8-16, wherein said exogenous DNA construct is complementary to said endogeneous said quinolate phosphoribosyl transferase messenger RNA (QPRT rnRNA) in said cells. 37. A method of making a transgenic plant cell having increased quinolate phosphoribosyl transferase (QPRTase) expression, said method comprising: providing a plant cell of a type known to express quinolate phosphoribosyl transferase; transforming said plant cell with said exogenous DNA construct according to any one of claims 8-16 to produce transgenic cells, said plant cell having increased expression of QPRTase compared to an untransformed cell. 38. A transgenic plant of the species Nicotiana having increased quinolate phosphoribosyl transferase (QPRTase) expression relative to a nontransformed control plant, wherein said transgenic plant comprises plant cells of exogenous DNA construct transformation according to any one of claims 8-16, and having increased expression of QPRTase compared to untransformed cells. 39. A progeny of a transgenic plant according to claim 38. 40. A method for increasing expression of a quinolate phosphoribosyl transferase gene in a plant cell, said method comprising exogenous DNA construct plant cell transformation according to any one of claims 8-16. 41. A method according to claim 40, wherein said plant cells is obtained by a method comprising integrating said exogenous DNA construct into the genome of a host plant cell. 42. A method of producing a plant of the species Nicotiana having increased levels of nicotine in leaves of said tobacco plant, said method comprising growing a plant of the species Nicotiana, or progeny plants thereof, wherein said plant comprises cells containing transformation of exogenous DNA construct according to any one of claims 8-16. 43. A transgenic tobacco plant of the species Nicotiana comprising exogenous DNA construct according to claim 12. 44. A tobacco product obtained from transgenic tobacco plant comprising exogenous DNA construct according to any one of claims 8-16. 45. A tobacco product obtained from transgenic tobacco plant comprising exogenous DNA construct according to claim 12. 46. A method of producing a tobacco product having decreased levels of nicotine comprising making a