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

• US 10,968,459 B2
• Filed: 07/20/2018
• Issued: 04/06/2021
• 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): Collins, Cynthia E
• Application Number:
  US16/041,152
• Publication Number:
  US 20180327766A1
• Time in Patent Office: 991 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 positively negatively regulates alkaloid biosynthesis, such as nicotine biosynthesis. In particular, the present disclosure provides methods for the inhibition of Nicotiana benthamiana auxin response factor 1 (NbTF1) to reduce alkaloid biosynthesis in plants.

Claims

1. A method for reducing nicotinic alkaloid in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is down-regulated by:

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

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

1 or SEQ ID NO;

2;

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

3; 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;

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

(b) producing a Nicotiana plant comprising the plant cell; and

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

2. The method of claim 1, further comprising suppressing within the Nicotiana 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 suppressing the expression of at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis is selected from NbTF4, NbTF5, or NbTF6.

4. The method of claim 1, further comprising overexpressing at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis, wherein the at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis is selected from NbTF3 or NbTF7.