Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism

• US 9,732,350 B2
• Filed: 10/09/2015
• Issued: 08/15/2017
• Est. Priority Date: 05/25/2007
• Status: Active Grant

• Current Assignee: 22nd Century Limited, LLC (22nd Century Group, Inc.)
• Original Assignee: 22nd Century Limited, LLC (22nd Century Group, Inc.)
• Inventors: Page, Jonathan, Todd, Andrea T.
• Primary Examiner(s): O HARA, EILEEN B
• Application Number:
  US14/879,456
• Publication Number:
  US 20160201074A1
• Time in Patent Office: 676 Days
• Field of Search: None
• 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. A method for increasing a nicotinic alkaloid in a Nicotiana plant, comprising up-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is up-regulated by:

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

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

11 or SEQ ID NO;

12;

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

13; and

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid 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.

2. The method of claim 1, further comprising up-regulating within the plant at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).

3. The method of claim 1, further comprising up-regulating at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.

4. The method of claim 1, further comprising down-regulating at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis.

5. The method of claim 1, wherein the plant is a Nicotiana tabacum plant.

6. The method of claim 1, wherein the expression construct comprises the nucleotide sequence set forth in SEQ ID NO:

11.

7. The method of claim 1, wherein the expression construct comprises the nucleotide sequence set forth in SEQ ID NO:

12.

8. The method of claim 1, wherein the expression construct comprises a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:

13.

9. The method of claim 1, wherein the expression construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:

11, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

10. The method of claim 1, wherein the expression construct comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:

12, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.

11. The method of claim 1, wherein the expression construct comprises a nucleotide sequence that is at least 90% identical to a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:

13, and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis.