REGULATING ALKALOIDS
First Claim
Patent Images
1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
- (a) a nucleotide sequence set forth in SEQ ID NO;
1;
(b) a nucleotide sequence set forth in SEQ ID NO;
3;
(c) a nucleotide sequence comprising at least 15 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO;
1 or 3;
(d) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;
2 or 4; and
(e) a nucleotide sequence that encodes a polypeptide having an amino acid sequence with at least 80% similarity to at least one of SEQ ID NO;
2 and 4 and that has MPO activity.
3 Assignments
0 Petitions
Accused Products
Abstract
MPO1 and MPO2 can be regulated for either decreasing or increasing alkaloid levels in plants, in particular in Nicotiana plants. In particular, suppressing or overexpressing one or more of MPO1 and MPO2 may be used to decrease or increase nicotine and nicotinic alkaloid levels in tobacco plants. Suppression or overexpression of one or more of MPO1 and MPO2 may be used in combination with modification of expression of other genes encoding enzymes on the nicotinic alkaloid biosynthetic pathway such as A622, NBB1, PMT, and QPT.
16 Citations
36 Claims
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1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
-
(a) a nucleotide sequence set forth in SEQ ID NO;
1;(b) a nucleotide sequence set forth in SEQ ID NO;
3;(c) a nucleotide sequence comprising at least 15 consecutive nucleotides of the nucleotide sequence set forth in SEQ ID NO;
1 or 3;(d) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO;
2 or 4; and(e) a nucleotide sequence that encodes a polypeptide having an amino acid sequence with at least 80% similarity to at least one of SEQ ID NO;
2 and 4 and that has MPO activity. - View Dependent Claims (3, 10, 11)
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2. A nucleic acid construct, comprising, in the 5′
- to 3′
direction, a promoter operably linked to a heterologous nucleic acid encoding at least a portion of MPO1 or MPO2 in sense, antisense, or inverted repeat orientation, and a terminator. - View Dependent Claims (4, 6, 7)
- to 3′
-
5. A mutational vector comprising a sequence of oligonucleotides targeting a region comprising 15 consecutive nucleotides of SEQ ID NO:
- 1 or 3.
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8. A method for reducing an alkaloid in a tobacco plant, comprising
(a) genetically engineering MPO suppression in said plant; - and
(b) suppressing at least one additional nicotine biosynthesis enzyme selected from the group consisting of aspartate oxidase, quinolinate synthase, quinolate phosphoribosyl transferase, ornithine decarboxylase, putrescine N-methyltransferase, A622, and NBB1.
- and
- 9. A tobacco plant having genetically engineered suppression of MPO and reduced content of nicotine.
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14. A reduced-alkaloid tobacco product produced from genetically engineered tobacco having suppressed MPO, wherein said product has a reduced collective amount of N′
- -nitrosonornicotine (NNN), 4-methylnitrosoamino-I-(3-pyridyl)-1-butanone (NNK), N′
-nitrosoanatabine (NAT) and N′
-nitrosoanabasine (NAB) compared to a similar tobacco product prepared from a non-genetically engineered control tobacco plant.
- -nitrosonornicotine (NNN), 4-methylnitrosoamino-I-(3-pyridyl)-1-butanone (NNK), N′
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17. An MPO1 polypeptide having the amino acid sequence of SEQ ID NO:
- 2
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18. An MPO2 polypeptide having the amino acid sequence of SEQ ID NO:
- 4
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19. An isolated MPO enzyme encoded by a nucleic acid sequence selected from the group consisting of:
-
(a) a nucleotide sequence set forth in SEQ ID NO;
1;(b) a nucleotide sequence set forth in SEQ ID NO;
3;(c) nucleic acid sequences which hybridize to at least one of SEQ ID NO;
1 and SEQ ID NO;
3 under moderate stringency or high stringency conditions and encode an MPO enzyme; and(d) nucleic acid sequences which differ from the nucleic acid sequence of (a), (b), or (c) above due to the degeneracy of the genetic code and encode an MPO enzyme.
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20. A method of producing an MPO enzyme, comprising
(a) introducing an isolated nucleic molecule encoding at least one of MPO1 and MPO2 into a cell; - and
(b) growing said cell under conditions such that MPO enzyme is produced. - View Dependent Claims (21)
- and
- 22. A method for increasing alkaloids in a plant, comprising overexpressing MPO relative to a control plant.
- 24. A method for increasing nicotine in a Nicotiana plant, comprising overexpressing at least one of MPO1 and MPO2 relative to a control plant.
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31. A method for increasing nicotine and yield in a Nicotiana plant, comprising:
-
(a) introducing into a Nicotiana plant a construct comprising, in the 5′
to 3′
direction, a promoter operably linked to a heterologous nucleic acid encoding an enzyme that increases yield;(b) regenerating transgenic Nicotiana plants from said plant; and (c) selecting a transgenic Nicotiana plant having increased nicotine content and increased yield relative to a control plant. - View Dependent Claims (32)
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33. A method for increasing the ratio of nicotine to total alkaloids in a tobacco plant, comprising:
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(a) overexpressing at last one of MPO1 and MPO2, (b) overexpressing PMT, and (c) suppressing QPT. - View Dependent Claims (34, 35)
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Specification