NUCLEIC ACID SEQUENCES ENCODING TRANSCRIPTION FACTORS REGULATING ALKALOID BIOSYNTHESIS AND THEIR USE IN MODIFYING PLANT METABOLISM

• US 20140298535A1
• Filed: 04/24/2014
• Published: 10/02/2014
• Est. Priority Date: 05/25/2007
• Status: Active Grant

• Current Assignee: National Research Council Canada
• Original Assignee: National Research Council Canada
• Inventors: Page, Jonathan, Todd, Andrea T.
• Granted Patent: US 9,175,302 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: A24B 13/00 Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff reconstituted tobacco products A24B3/14
• CPC Class Codes: A24B 15/10 Chemical features of tobacco products or tobacco substitutes
• CPC Class Codes: A24B 15/12 of reconstituted tobacco
• CPC Class Codes: A24D 1/00 Cigars; Cigarettes
• CPC Class Codes: A24D 1/10 with extinguishers
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Abstract

Plant metabolism and alkaloid levels can be regulated by transcription factors that regulate the nicotinic alkaloid biosynthetic pathway. In one embodiment, the disclosure provides a transcription factor that negatively regulates alkaloid biosynthesis, such as nicotine biosynthesis.

Claims

1-38. -38. (canceled)

39. A method for reducing an alkaloid in a plant, comprising up-regulating a transcription factor that negatively regulates alkaloid biosynthesis, wherein the transcription factor is up-regulated by:

(a) introducing into the plant an expression construct comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of;

(i) the nucleotide sequence set forth in SEQ ID NO;

14 or SEQ ID NO;

15;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;

16; and

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and encodes a transcription factor that negatively regulates alkaloid nicotine biosynthesis; and

(b) growing the plant under conditions whereby the expression construct increases levels of the transcription factor in the plant as compared to a control plant grown under similar conditions.

40. A method for reducing a nicotinic alkaloid in a plant, comprising up-regulating a transcription factor that negatively regulates alkaloid biosynthesis using the method of claim 39, and down-regulating at least one of NBB1, A622, QPT, PMT, and MPO.

46. A method for reducing a nicotinic alkaloid in a plant, comprising up-regulating a transcription factor that negatively regulates alkaloid biosynthesis using the method of claim 39, and down-regulating at least one transcription factor that positively regulates alkaloid biosynthesis.

47. A method for reducing a nicotinic alkaloid in a plant, comprising up-regulating a transcription factor that negatively regulates alkaloid biosynthesis using the method of claim 39, and up-regulating at least one additional transcription factor that negatively regulates alkaloid biosynthesis.

41. A method for increasing an alkaloid in a plant, comprising down-regulating a transcription factor that negatively regulates alkaloid biosynthesis by:

(a) introducing into a plant cell a nucleic acid comprising at least 21 consecutive nucleotides of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of;

(i) the nucleotide sequence set forth in SEQ ID NO;

14 or SEQ ID NO;

15;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;

16; and

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and encodes a transcription factor that negatively regulates biosynthesis, wherein said consecutive nucleotides are in sense orientation, antisense orientation;

or both;

(b) producing a plant comprising said plant cell; and

(c) growing the plant under conditions whereby the nucleotide sequence decreases levels of the transcription factor in the plant as compared to a control plant grown under similar conditions.

45. A method for increasing a nicotinic alkaloid in a plant, comprising down-regulating a transcription factor that negatively regulates alkaloid biosynthesis by the method of claim 41, and up-regulating at least one of NBB1, A622, QPT, PMT, and MPO.

48. A method for increasing a nicotinic alkaloid in a plant, comprising down-regulating a transcription factor that negatively regulates alkaloid biosynthesis using the method of claim 41, and up-regulating at least one transcription factor that positively regulates alkaloid biosynthesis.

49. A method for increasing a nicotinic alkaloid in a plant, comprising down-regulating a transcription factor that negatively regulates alkaloid biosynthesis using the method of claim 41, and down-regulating at least one additional transcription factor that negatively regulates alkaloid biosynthesis.

42. A method for increasing an alkaloid in a plant, comprising down-regulating a transcription factor that negatively regulates alkaloid biosynthesis, wherein the transcription factor is down-regulated by:

(a) introducing into a population of plant cells a reagent for site-directed mutagenesis of a target comprising at least 21 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO;

14; and

(b) detecting and selecting a target mutated plant cell or a plant derived from such a cell, wherein the target mutated plant cell or plant has a mutation in a gene encoding transcription factor positively regulating alkaloid biosynthesis and reduced alkaloid content as compared to a control plant.

43. The method of claim 42, wherein the reagent is a recombinagenic oligonucleobase.

44. The method of claim 42, wherein the reagent is a targeted nuclease.

55. A mutated alkaloid-producing plant having reduced expression of a transcription factor that negatively regulates alkaloid biosynthesis and increased alkaloid content, produced by the method of claim 42.

56. The mutated plant of claim 55, wherein the plant is a member of the genus Nicotiana, Duboisia, Solanum, Anthocercis, or Salpiglessis.

57. The mutated plant of claim 55, wherein the plant is a Nicotiana tabacum plant.

58. A product having an increased level of an alkaloid comprising the mutated plant of claim 55, or fractions or portions thereof.

59. Seeds from the mutated plant of claim 55.

61. The product of claim 58, wherein the product is selected from the group consisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, reconstituted tobacco, leaf tobacco, shredded tobacco, and cut tobacco.

50. A genetically engineered alkaloid-producing plant comprising a chimeric nucleic acid construct comprising a nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence set forth in SEQ ID NO;

14 or SEQ ID NO;

15;

(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;

16;

(c) a nucleotide sequence at least 90% identical to the nucleotide sequences of (a) or (b), and which encodes a transcription factor that regulates alkaloid biosynthesis; and

(d) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequences of (a), (b), and/or (c), and which encodes a transcription factor that regulates alkaloid biosynthesis;

wherein the nucleic acid molecule is linked to a heterologous nucleic acid to form a chimeric nucleic acid construct.

51. The engineered plant of claim 50, wherein the plant is a member of the genus Nicotiana, Duboisia, Solanum, Anthocercis, or Salpiglessis.

52. The engineered plant of claim 50, wherein the plant is a Nicotiana tabacum plant.

53. Seeds from the engineered plant of claim 50, wherein the seeds comprise the nucleic acid construct.

54. A product having a modified level of an alkaloid comprising the engineered plant of claim 50, or fractions or portions thereof.

60. The product of claim 54, wherein the product is selected from the group consisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, reconstituted tobacco, leaf tobacco, shredded tobacco, and cut tobacco.