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 . A method for down-regulating a NbTF1 transcription factor in a  Nicotiana  plant, wherein the transcription factor is down-regulated by the method comprising:
 (a) introducing into a plant a nucleic acid comprising at least 21 consecutive nucleotides of a cDNA molecule comprising a nucleotide sequence selected from the group consisting of:
 (i) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3; and 
 (iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis;
 wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both; and 
 
   (b) growing the  Nicotiana  plant under conditions whereby the nucleotide sequence decreases levels of the transcription factor in the plant as compared to a control Nicotiana plant grown under similar conditions.   
     
     
         40 . The method of  claim 39 , further comprising down-regulating at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO). 
     
     
         41 . The method of  claim 39 , further comprising overexpressing within the  Nicotiana  plant at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis. 
     
     
         42 . The method of  claim 39 , further comprising suppressing the expression of at least one transcription factor that positively regulates nicotinic alkaloid biosynthesis. 
     
     
         43 . The method of  claim 39 , wherein the plant is  Nicotiana tabacum.    
     
     
         44 . A  Nicotiana  plant produced by the method of  claim 39 , wherein the plant comprises the nucleic acid. 
     
     
         45 . A product comprising the plant of  claim 44 , or portions thereof. 
     
     
         46 . The product of  claim 45 , 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, and reconstituted tobacco. 
     
     
         47 . Seeds from the plant of  claim 44 , wherein the seeds comprise the expression construct. 
     
     
         48 . A method for up-regulating a NbTF1 transcription factor in a  Nicotiana  plant, wherein the transcription factor is up-regulated by the method comprising:
 (a) introducing into the plant an expression construct comprising a nucleotide sequence selected from the group consisting of:
 the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and 
 (ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3; and 
   (b) growing the plant under conditions whereby the expression construct increases levels of the transcription factor in the plant as compared to a control plant grown under similar conditions.   
     
     
         49 . The method of  claim 48 , further comprising up-regulating at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO). 
     
     
         50 . The method of  claim 48 , further comprising overexpressing within the  Nicotiana  plant at least one transcription factor that positively regulates nicotinic alkaloid biosynthesis. 
     
     
         51 . The method of  claim 48 , further comprising suppressing the expression of at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis. 
     
     
         52 . The method of  claim 48 , wherein the plant is  Nicotiana tabacum.    
     
     
         53 . A  Nicotiana  plant produced by the method of  claim 48 , wherein the plant comprises the expression construct. 
     
     
         54 . A product comprising the plant of  claim 53 , or portions thereof. 
     
     
         55 . The product of  claim 54 , 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, and reconstituted tobacco. 
     
     
         56 . Seeds from the plant of  claim 53 , wherein the seeds comprise the expression construct.