Methods and compositions for tobacco plants with reduced nitrosamines

Abstract

The present application describes isolated nucleic acids that contain a Nic gene product responsive element, and the use thereof in methods of producing transgenic tobacco plants having reduced levels of nicotine and/or TSNA therein, as well as other plants or host cells that contain altered levels of a protein of interest therein due to inclusion of a cis-acting decoy element therein.

1 - 73 . (canceled)  
     
     
         74 ). A method of making a transgenic tobacco plant having a reduced amount of tobacco specific nitrosamines, comprising: 
 (i) introducing into at least one tobacco plant cell an isolated nucleic acid sequence that binds a Nic gene product, wherein the nucleic acid sequence is selected from the group consisting of: 
 (a) an isolated nucleic acid sequence of SEQ ID NO:1 or an active fragment thereof, wherein said fragment comprises 20 to 455 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product;  
 (b) an isolated nucleic acid sequence which is at least 80% identical to the nucleic acid sequence of (a) and binds a Nic gene product; and  
 (c) an isolated nucleic acid that hybridizes under stringent conditions to the complement of the nucleic acid sequence of (a) and binds a Nic gene product;  
 to produce at least one transformed tobacco plant cell, and  
   (ii) regenerating said transformed tobacco plant cell into said transgenic tobacco plant, having a reduced amount of tobacco specific nitrosamines as compared to a non-transgenic tobacco plant of the same variety grown under similar conditions.    
     
     
         75 ). The method of claim  1 , further comprising collecting tobacco leaves from said transgenic tobacco plant, wherein said tobacco leaves have a reduced amount of tobacco specific nitrosamines as compared to the amount of tobacco specific nitrosamines that would be present in said tobacco plant in the absence of said nucleic acid sequence.  
     
     
         76 ). The method of claim  1 , further comprising collecting tobacco seed from said transgenic tobacco plant, wherein said tobacco seed comprises said nucleic acid sequence in a copy number sufficient to reduce the amount of tobacco specific nitrosamines in a tobacco plant produced from said seed as compared to the amount of tobacco specific nitrosamines that would be present in the absence of said nucleic acid sequence.  
     
     
         77 ). The method of claim  1 , wherein said isolated nucleic acid sequence consists of the nucleotide sequence of SEQ ID NO:1.  
     
     
         78 ). The method of claim  1 , wherein said isolated nucleic acid sequence is contained within a recombinant nucleic acid construct, and said recombinant nucleic acid construct does not contain a NtQPT1 coding sequence.  
     
     
         79 ). The method of claim  1 , wherein said introducing step comprises ballistic transformation.  
     
     
         80 ). The method of claim  1 , wherein said introducing step comprises  Agrobacterium  transformation.  
     
     
         81 ). The method of claim  1 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 30-400 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         82 ). The method of claim  1 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 50-350 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         83 ). The method of claim  1 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 100-300 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         84 ). The method of claim  1 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 200-400 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         85 ). The method of claim  1 , wherein the transgenic tobacco plant is a Burley tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0 to 8000 parts per billion (ppb).  
     
     
         86 ). The method of claim  1 , wherein the transgenic tobacco plant is an Oriental tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-300 ppb.  
     
     
         87 ). The method of claim  1 , wherein the transgenic tobacco plant is a Flue-cured tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-100 ppb.  
     
     
         88 ). The method of claim  1 , wherein the transgenic tobacco plant has no detectable amounts of one or more tobacco specific nitrosamines.  
     
     
         89 ). A transgenic tobacco plant produced by the method of claim  1 .  
     
     
         90 ). A tobacco leaf collected from the transgenic tobacco plant of claim  16 .  
     
     
         91 ). A tobacco seed collected from the tobacco plant of claim  16 , wherein said tobacco seed comprises said isolated nucleic acid sequence.  
     
     
         92 ). A transgenic tobacco plant having a reduced amount of tobacco specific nitrosamines therein, wherein said transgenic tobacco plant comprises cells comprising an exogenous nucleic acid sequence that binds a Nic gene product, wherein said exogenous nucleic acid sequence is selected from the group consisting of: 
 (a) an isolated nucleic acid sequence of SEQ ID NO:1 or an active fragment thereof, wherein said fragment comprises 20 to 455 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product;    (b) an isolated nucleic acid sequence which is at least 80% identical to the nucleic acid sequence of (a) and binds a Nic gene product; and    (c) an isolated nucleic acid that hybridizes under stringent conditions to the complement of the nucleic acid sequence of (a) and binds a Nic gene product, and wherein said exogenous nucleic acid is present in said cells in a copy number sufficient to reduce the amount of tobacco specific nitrosamines in said tobacco plant as compared to the amount of tobacco specific nitrosamines that would be present in said plant in the absence of said exogenous nucleic acid sequence.    
     
     
         93 ). The transgenic tobacco plant of claim  19 , wherein said exogenous nucleic acid sequence consists essentially of the nucleotide sequence of SEQ ID NO:1.  
     
     
         94 ). The transgenic tobacco plant of claim  19 , wherein said exogenous nucleic acid sequence is contained within a recombinant nucleic acid construct, wherein said recombinant nucleic acid construct does not contain a NtQPT1 coding sequence.  
     
     
         95 ). A tobacco leaf collected from the transgenic tobacco plant of claim  19 .  
     
     
         96 ). A tobacco seed that germinates into the transgenic tobacco plant of claim  19 .  
     
     
         97 ). A tobacco seed collected from the transgenic tobacco plant of claim  19 , wherein said seed comprises said exogenous nucleic acid sequence.  
     
     
         98 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 30-400 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         99 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 50-350 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         100 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 100-300 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         101 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising 200-400 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         102 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising at least 20 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         103 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising at least 40 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         104 ). The transgenic tobacco plant of claim  19 , wherein said isolated nucleic acid sequence of SEQ ID NO:1 is a fragment comprising at least 60 consecutive nucleotides of SEQ ID NO:1 and binds a Nic gene product.  
     
     
         105 ). The transgenic tobacco plant of claim  1 , wherein the transgenic tobacco plant is a Burley tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0 to 8000 parts per billion (ppb).  
     
     
         106 ). The transgenic tobacco plant of claim  19 , wherein the transgenic tobacco plant is an Oriental tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-300 ppb.  
     
     
         107 ). The transgenic tobacco plant of claim  19 , wherein the transgenic tobacco plant is a Flue-cured tobacco plant and the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-100 ppb.  
     
     
         108 ). The transgenic tobacco plant of claim  19 , wherein the transgenic tobacco plant has no detectable amounts of one or more tobacco specific nitrosamines.  
     
     
         109 ). A blended tobacco product comprising leaves of the transgenic tobacco plant of claim  19 .  
     
     
         110 ). A tobacco product comprising leaves of the transgenic tobacco plant of claim  19 .  
     
     
         111 ). The tobacco product of claim  37 , selected from the group consisting of pipe tobacco, cigar tobacco, cigarette tobacco, cigars, cigarettes, chewing tobacco, leaf tobacco, shredded tobacco and cut tobacco.  
     
     
         112 ). The transgenic tobacco plant of claim  19 , further comprising a transgene encoding a heterologous protein of interest.  
     
     
         113 ). A method of producing a heterologous protein of interest in a plant, comprising: 
 a) growing the transgenic tobacco plant of claim  39 ; and    b) collecting the heterologous protein of interest from said transgenic plant.    
     
     
         114 ). The transgenic tobacco plant of claim  39 , wherein the heterologous protein of interest is a  Bacillus thuringiensis  insecticidal protein.  
     
     
         115 ). The method of claim  40 , wherein the heterologous protein of interest is a  Bacillus thuringiensis  insecticidal protein.