Regulating alkaloids
View Patent ↗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.
1. A nucleic acid construct comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1; and
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
wherein the nucleotide sequence is operatively linked in sense, antisense, or inverted repeat orientation to a heterologous promoter.
2. A plant cell comprising the nucleic acid construct of claim 1 .
3. A method of producing a reduced-alkaloid plant, comprising genetically engineering N-methylputrescine oxidase (MPO) suppression in the plant, wherein the engineering comprises introducing into a plant cell of the plant the nucleic acid construct of claim 1 .
4. The method of claim 3 , wherein the plant is tobacco.
5. A nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous N-methylputrescine oxidase-1 (MPO1) sequence in sense, antisense, or inverted repeat orientation, and a terminator, wherein the MPO1 sequence is the nucleotide sequence of SEQ ID NO: 1.
6. A plant cell comprising the nucleic acid construct of claim 5 .
7. A method of producing a reduced-alkaloid plant, comprising genetically engineering N-methylputrescine oxidase (MPO) suppression in the plant, wherein the engineering comprises introducing into a plant cell of the plant the nucleic acid construct of claim 5 .
8. The method of claim 7 , wherein the plant is tobacco.
9. A method for reducing an alkaloid in a tobacco plant, comprising
(a) genetically engineering N-methylputrescine oxidase (MPO) suppression in the plant, wherein the MPO has the amino acid sequence set forth in SEQ ID NO: 2; 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.
10. A tobacco plant having genetically engineered suppression of N-methylputrescine oxidase (MPO) and reduced content of nicotine, wherein the MPO is suppressed by a nucleic acid encoding a polypeptide that has the amino acid sequence set forth in SEQ ID NO: 2, or its complementary nucleotide sequence.
11. A tobacco plant comprising a chimeric nucleic acid construct, wherein the construct comprises the nucleic acid construct of claim 1 linked to a heterologous nucleic acid.
12. Transformed progeny of the tobacco plant of claim 10 , wherein the transformed progeny have suppression of the MPO having the amino acid sequence set forth in SEQ ID NO: 2.
13. Transformed progeny of the tobacco plant of claim 11 , wherein the transformed progeny have suppression of the MPO having the amino acid sequence set forth in SEQ ID NO: 2.
14. Transformed seeds from the tobacco plant of claim 10 .
15. A reduced-alkaloid tobacco product comprising a portion of a tobacco plant of claim 10 .
16. The tobacco product of claim 15 , wherein the product is a cigarette.
17. A smoking cessation product comprising a portion of a tobacco plant of claim 10 .
18. A reduced-alkaloid tobacco product produced from genetically engineered tobacco having suppressed N-methylputrescine oxidase (MPO), wherein:
(a) the MPO is suppressed by a nucleic acid encoding a polypeptide that has the amino acid sequence set forth in SEQ ID NO: 2, or its complementary nucleotide sequence, and
(b) the product has a reduced collective amount of N′-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone (NNK), N′-nitrosoanatabine (NAT), and N′-nitrosoanabasine (NAB) as compared to a similar tobacco product prepared from a non-genetically engineered control tobacco plant.