Nucleic acid encoding N-methylputrescine oxidase and uses thereof
The gene encoding N-methylputrescine oxidase (MPO) and constructs comprising such DNA are provided, including methods of regulating MPO expression independently or with other alkaloid biosynthesis genes to modulate alkaloid production in plants and host cells. MPO genes or fragments thereof are useful for reducing pyrrolidine or tropane alkaloid production in plants, for increasing pyrrolidine or tropane alkaloid production in plants, and for producing an MPO enzyme in host cells.
1. A reduced alkaloid Nicotiana plant produced by a method selected from:
(i) genetically engineering down-regulation of N-methylputrescine oxidase (MPO) expression relative to a control plant, wherein the genetic engineering comprises introducing into the Nicotiana plant a nucleic acid construct comprising at least 21 consecutive nucleotides of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;
(b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3; and
(c) a nucleotide sequence that is at least 95% identical to the nucleotide sequences of (a) or (b);
wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both; or
(ii) detecting a target mutated plant within a population of mutated plants, wherein said target mutated plant has a mutation within an MPO gene and decreased expression of or decreased activity of MPO enzyme compared to a control plant, wherein the detecting comprises using primers developed from SEQ ID NO: 1 or SEQ ID NO: 2 to PCR amplify regions of the MPO gene from mutated plants in the population of mutated plants, identifying mismatches between the amplified regions and corresponding regions in wild-type MPO gene that lead to the decreased expression of MPO gene or decreased activity of MPO enzyme and identifying the mutated plant that contains the mismatches, and selectively breeding the target mutated plant, thereby producing the reduced alkaloid Nicotiana plant; and
wherein the reduced alkaloid Nicotiana plant comprises the nucleic acid construct or the mutation within the MPO gene and decreased expression or decreased activity of MPO enzyme as compared to a control plant.
2. A reduced alkaloid product produced from the Nicotiana plant of claim 1 .
3. The reduced alkaloid Nicotiana plant of claim 1 , wherein said alkaloid is a pyrrolidine alkaloid.
4. The reduced alkaloid Nicotiana plant of claim 1 , wherein the reduced alkaloid is nicotine.
5. The reduced alkaloid product of claim 2 , wherein the reduced alkaloid is nicotine.
6. The reduced alkaloid product of claim 5 , wherein said product is selected from the group consisting of smoking cessation products, cigarettes, cigarette tobacco, cigars, cigar tobacco, pipe tobacco, chewing tobacco, snus, tobacco lozenges, food products, food ingredients, feed products, feed ingredients, nutritional supplements, and biofuels.