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

Abstract

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 .- 38 . (canceled) 
     
     
         39 . An isolated cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence set forth in SEQ ID NO: 4;   (b) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of (a); and   (c) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequences of (a) or (b), and which encodes a transcription factor that regulates alkaloid biosynthesis.   
     
     
         40 . An expression vector comprising the cDNA molecule of  claim 39  operably linked to one or more control sequences suitable for directing expression in a host cell. 
     
     
         41 . The expression vector of  claim 40 , wherein the host cell is a  Nicotiana  cell. 
     
     
         42 . A  Nicotiana  cell comprising the expression vector of  claim 40 . 
     
     
         43 . The isolated cDNA molecule of  claim 39 , wherein the nucleotide sequence comprises the sequence as set forth in SEQ ID NO: 4. 
     
     
         44 . The isolated cDNA molecule of  claim 39 , wherein the nucleotide sequence comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 4. 
     
     
         45 . A method for increasing alkaloid levels in a population of plants, comprising:
 (a) providing a population of mutated plants;   (b) detecting and selecting a target mutated plant within the population, wherein the target mutated plant has decreased expression of a transcription factor that negatively regulates alkaloid biosynthesis as compared to a control plant, wherein the detection comprises using the cDNA molecule of  claim 39  as a primer or probe; and   (c) selectively breeding the target mutated plant to produce a population of plants having decreased expression of a transcription factor that negatively regulates alkaloid biosynthesis as compared to a population of control plants.   
     
     
         46 . The method of  claim 45 , further comprising overexpressing within the mutated plant at least one of NBB1, A622, quinolate phosphoribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO). 
     
     
         47 . The method of  claim 45 , further comprising overexpressing at least one transcription factor that positively regulates nicotinic alkaloid biosynthesis. 
     
     
         48 . The method of  claim 45 , further comprising suppressing the expression of at least one additional transcription factor that negatively regulates nicotinic alkaloid biosynthesis. 
     
     
         49 . The method of  claim 45 , wherein the plants are  Nicotiana  plants. 
     
     
         50 . The method of  claim 49 , wherein the  Nicotiana  plants are  Nicotiana tabacum  plants. 
     
     
         51 . A genetically engineered alkaloid-producing plant comprising a chimeric nucleic acid construct comprising a nucleic acid molecule encoded by a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence set forth in SEQ ID NO: 4;   (b) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of (a); and   (c) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequences of (a) or (b), and which encodes a transcription factor that regulates alkaloid biosynthesis.   
     
     
         52 . The engineered plant of  claim 51 , wherein the plant is a member of the genus  Nicotiana, Duboisia, Solanum, Anthocercis,  or  Salpiglossis.    
     
     
         53 . The engineered plant of  claim 51 , wherein the plant is a  Nicotiana tabacum  plant. 
     
     
         54 . Seeds from the engineered plant of  claim 51 , wherein the seeds comprise the nucleic acid construct. 
     
     
         55 . A product comprising the engineered plant of  claim 51  or portions thereof, wherein the product has a modified level of an alkaloid as compared to a product comprising a control plant or portions thereof. 
     
     
         56 . The product of  claim 55 , wherein the product is selected from the group consisting of a cigarette, cigarette tobacco, cigar tobacco, a cigar, pipe tobacco, chewing tobacco, snuff, snus, lozenges, reconstituted tobacco, leaf tobacco, shredded tobacco, and cut tobacco. 
     
     
         57 . The engineered plant of  claim 51 , wherein the alkaloid is a nicotinic alkaloid. 
     
     
         58 . The engineered plant of  claim 51 , wherein the nucleotide sequence comprises the nucleotide sequence as set forth in SEQ ID NO: 4.