# US 11,306,321 B2 · Transcription factors that regulate nicotine biosynthesis in tobacco

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- Number: US 11,306,321 B2
- Document ID: US11306321B2
- Status: Granted
- Kind: B2
- Application number: 16/915,206
- Filed: 2020-06-29
- Published: 2020-12-03
- Granted: 2022-04-19
- Earliest claimed: 2013-03-01
- Assignees: North Carolina State University
- Applicants: North Carolina State University
- Inventors: Rongda Qu; Bingwu Wang
- Web page: https://patentgrep.com/patent/US11306321B2

## CPC

- C12N 15/8243: {involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine}
- C07K 14/415: from plants
- C12Q 1/6895: for plants, fungi or algae
- C12Q 2600/156: Polymorphic or mutational markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention provides nucleic acids encoding transcription factors and methods of using these nucleic acids to modulate nicotine production in plants and to produce plants having modulated nicotine production.

## Claims

### Claim 1

An isolated nucleic acid molecule comprising a cDNA sequence selected from the group consisting of:
(a) the nucleotide sequence of SEQ ID NO: 7;
(b) a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 7; and
(c) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 8.

### Claim 2 (depends on 1)

A nucleic acid construct comprising, in the 5′ to 3′ direction, a heterologous promoter operable in a plant cell and, positioned downstream from the promoter and operatively associated therewith, the nucleic acid molecule of claim 1.

### Claim 3 (depends on 2)

The nucleic acid construct of claim 2, wherein the promoter is a constitutive promoter, a tissue preferred promoter, or a tissue specific promoter.

### Claim 4 (depends on 1)

A vector comprising the nucleic acid molecule of claim 1.

### Claim 5 (depends on 4)

A transgenic Nicotiana tabacum plant cell or bacterial cell comprising the vector of claim 4.

### Claim 6 (depends on 5)

A transgenic Nicotiana tabacum plant comprising the plant cell of claim 5.

### Claim 7 (depends on 6)

A seed of the transgenic plant of claim 6 or a progeny plant produced from the seed, wherein the seed and progeny plant comprise in their genomes the vector.

### Claim 8 (depends on 6)

A crop comprising a plurality of transgenic Nicotiana tabacum plants according to claim 6 or a transgenic progeny thereof, wherein the plurality of transgenic plants is planted together in an agricultural field, and the transgenic N. tabacum plants and transgenic progeny thereof comprise in their genomes the vector.

### Claim 9 (depends on 6)

A tobacco product produced from the transgenic Nicotiana tabacum plant of claim 6, wherein the tobacco product comprises the vector.

### Claim 10 (depends on 9)

The tobacco product of claim 9, wherein the tobacco product is selected from the group consisting of a smoking cessation product, a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.

### Claim 11

A method of producing a transgenic Nicotiana tabacum plant having decreased alkaloid content, comprising:
(a) introducing into a Nicotiana tabacum plant cell a nucleic acid construct comprising at least 21 consecutive nucleotides of a cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence of SEQ ID NO: 7;
(ii) a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 7; and
(iii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 8;
wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both; and
(b) regenerating the transgenic Nicotiana tabacum plant cell to produce a transgenic Nicotiana tabacum plant comprising the nucleic acid construct.

### Claim 12 (depends on 11)

The method of claim 11, further comprising suppressing within the plant cell at least one of NBB1, A622, putrescine N-methyltransferase (PMT), quinolinic acid phosphoribosyltransferase (QPT), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), and N-methylputrescine oxidase (MPO).

### Claim 13 (depends on 11)

A transgenic Nicotiana tabacum plant produced by the method of claim 11, wherein the transgenic N. tabacum plant comprises a decreased alkaloid content as compared to a non-transgenic control plant grown under similar conditions.

### Claim 14 (depends on 13)

A crop comprising a plurality of the transgenic Nicotiana tabacum plants of claim 13 or a transgenic progeny plant thereof, wherein the plurality of transgenic plants is planted together in an agricultural field.

### Claim 15 (depends on 13)

A tobacco product comprising the transgenic Nicotiana tabacum plant of claim 13, and having a decreased level of alkaloids as compared to a control product comprising the non-transgenic control plant.

### Claim 16 (depends on 15)

The tobacco product of claim 15, wherein the tobacco product is selected from the group consisting of a smoking cessation product, a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.

### Claim 17

A tobacco plant regenerated from a tobacco plant cell comprising a mutant allele of a gene encoding a transcription factor that modulates nicotine biosynthesis, wherein the tobacco plant cell is produced by a method comprising:
(a) introducing into a population of tobacco plant cells a reagent for site-directed mutagenesis comprising an oligonucleotide having a targeting sequence with at least 95% sequence identity to SEQ ID NO: 7, or a complement thereof, thereby producing the tobacco plant cell comprising the mutant allele of the gene encoding the transcription factor that modulates nicotine biosynthesis; and
(b) regenerating a tobacco plant from the tobacco plant cell of step (a),
thereby resulting in a tobacco plant comprising tobacco plant cells comprising the mutant allele of the gene encoding the transcription factor that modulates nicotine biosynthesis.

### Claim 18 (depends on 17)

Seeds from or a progeny plant of the tobacco plant of claim 17, wherein the seeds or progeny plant comprise in their genomes the mutant allele.

### Claim 19 (depends on 17)

A crop comprising a plurality of the tobacco plants of claim 17 or a progeny thereof, wherein the plurality of tobacco plants or progeny thereof comprise in their genomes the mutant allele.

### Claim 20 (depends on 17)

A tobacco product produced from the plant of claim 17, wherein the product comprises the mutant allele, and wherein the tobacco product is selected from the group consisting of a smoking cessation product, a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, leaf tobacco, shredded tobacco, cut tobacco, and any combination thereof.