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