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

• US 9,988,640 B2
• Filed: 04/07/2015
• Issued: 06/05/2018
• 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): Abraham, Amjad
• Assistant Examiner(s): Fan, Weihua
• Application Number:
  US14/681,073
• Publication Number:
  US 20150203859A1
• Time in Patent Office: 1,155 Days
• 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 genetically engineered, reduced nicotinic alkaloid-producing Nicotiana plant comprising reduced expression of a NbTF5 transcription factor as compared to a control plant, wherein the amino acid sequence of said NbTF5 transcription factor is set forth in SEQ ID NO:

10, wherein the Nicotiana plant comprises a recombinant nucleic acid construct selected from the group consisting of;

(a) a polynucleotide sequence in sense orientation or antisense orientation that suppresses expression of the transcription factor, and

(b) a short interfering RNA molecule comprising a polynucleotide sequence that suppresses expression of the transcription factor,

wherein the polynucleotide sequence of (a) and (b) comprises at least 21 consecutive nucleotides of a sequence selected from the group consisting of;

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

8 or SEQ ID NO;

9; and

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

10;

wherein the polynucleotide sequence in (a) and (b) is operably linked to one or more heterologous sequences.

2. The genetically engineered plant of claim 1, wherein the plant is a decreased-nicotinic alkaloid Nicotiana tabacum plant.

3. The genetically engineered Nicotiana plant of claim 1, wherein the recombinant nucleic acid construct further comprises one or more of the NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT) and N-methylputrescine oxidase (MPO) genes, wherein the expression of the one or more genes is suppressed as compared to a control plant.

4. Seeds from the genetically engineered Nicotiana plant of claim 1, wherein the seeds comprise the recombinant nucleic acid construct.

5. A product having a reduced level of a nicotinic alkaloid comprising the genetically engineered Nicotiana plant of claim 1, or portions thereof.

6. The product of claim 5, wherein the tobacco product is selected from the group consisting of a reduced-nicotine cigarette, a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, and reconstituted tobacco.

7. A method of producing the genetically engineered reduced nicotinic alkaloid-producing Nicotiana plant of claim 1, wherein the method of reducing the nicotinic alkaloid comprises down-regulating the NbTF5 transcription factor, wherein the transcription factor is down-regulated by:

a) introducing into the Nicotiana plant a nucleotide sequence comprising at least 21 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

8, SEQ ID NO;

9, and a sequence that encodes the amino acid sequence set forth in SEQ ID NO;

10, wherein the consecutive nucleotides are in either sense or antisense orientation; and

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

8. A method of producing the genetically engineered reduced nicotinic alkaloid-producing Nicotiana plant of claim 1, wherein the method of reducing the nicotinic alkaloid comprises down-regulating the NbTF5 transcription factor, wherein the NbTF5 transcription factor is down-regulated by introducing into a plant cell of the Nicotiana plant a nucleic acid construct that expresses the short interfering RNA molecule of claim 1 that suppresses expression of a gene product encoded by SEQ ID NO:

8 or SEQ ID NO;

9.

9. A method of producing the genetically engineered reduced nicotinic alkaloid-producing Nicotiana plant of claim 1, wherein the method of reducing the nicotinic alkaloid comprises down-regulating the NbTF5 transcription factor, wherein the NbTF5 transcription factor is down-regulated by introducing into a plant cell of the Nicotiana plant a nucleic acid construct comprising, in the 5′

to 3′

direction, (a) a promoter operably linked to a heterologous nucleic acid encoding SEQ ID NO;

10 in the sense or antisense orientation, and (b) a terminator.