Down regulation of auxin response factor NbTF7 to increase nicotine in a plant

• US 9,976,153 B2
• Filed: 09/11/2015
• Issued: 05/22/2018
• 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): Worley, Cathy Kingdon
• Application Number:
  US14/852,172
• Publication Number:
  US 20160073675A1
• Time in Patent Office: 984 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. In particular, the present disclosure provides methods for the inhibition of Nicotiana benthamiana auxin response factor (NbTF7) to increase alkaloid biosynthesis in plants.

Claims

1. A method for increasing nicotine in a Nicotiana benthamiana or tabacum plant, comprising

introducing into a Nicotiana benthamiana or tabacum plant cell a vector 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

wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both.

2. The method of claim 1, further comprising overexpressing within the Nicotiana plant at least one of berberine bridge enzyme-like protein a (NBB1), isoflavone reductase homolog A622 (A622), quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).

3. The method of claim 1, further comprising overexpressing at least one transcription factor that positively regulates nicotine biosynthesis.

4. The method of claim 1, further comprising suppressing the expression of at least one transcription factor that negatively regulates nicotine biosynthesis.

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

6. A transgenic Nicotiana benthamiana or tabacum plant produced by the method of claim 1, wherein the plant has an increased nicotine content, as compared to a control plant grown under similar conditions.

7. A product comprising the transgenic Nicotiana benthamiana or tabacum plant of claim 6 or portions thereof, wherein the product has an increased level of nicotine as compared to a product produced from a control plant.

8. The product of claim 7, 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, and reconstituted tobacco.

9. Seeds from the transgenic Nicotiana benthamiana or tabacum plant of claim 6, wherein the seeds comprise the nucleic acid molecule.

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

14.

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

15.

12. The method of claim 2, wherein NBB1 is overexpressed.

13. The method of claim 2, wherein A622 is overexpressed.

14. The method of claim 2, wherein QPT is overexpressed.

15. The method of claim 2, wherein PMT is overexpressed.

16. The method of claim 2, wherein MPO is overexpressed.

17. The transgenic Nicotiana plant of claim 6, wherein the plant is a Nicotiana tabacum plant.