Methods and compositions for reduced putrescine methyl transferase and reduced quinolate phosphoribosyl transferase in plants

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 quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase), 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 quinolate phosphoribosyl transferase and a reduced amount of putrescine methyl transferase as compared to a non-transgenic tobacco plant of the same variety grown under similar conditions.  
   
     
     
         75 . The method of  claim 74 , further comprising collecting tobacco leaves from said transgenic tobacco plant, wherein said tobacco leaves have a reduced amount of quinolate phosphoribosyl transferase and a reduced amount of putrescine methyl transferase as compared to the amount of quinolate phosphoribosyl transferase and putrescine methyl transferase that would be present in said tobacco plant in the absence of said nucleic acid sequence.  
     
     
         76 . The method of  claim 74 , further comprising collecting tobacco seed from said transgenic plant, wherein said tobacco seed comprises said nucleic acid sequence in a copy number sufficient to reduce the amount of quinolate phosphoribosyl transferase and putrescine methyl transferase as compared to the amount of quinolate phosphoribosyl transferase and putrescine methyl transferase that would be present in said tobacco plant in the absence of said nucleic acid sequence  
     
     
         77 . The method of  claim 74 , wherein said isolated nucleic acid sequence consists of the nucleotide sequence of SEQ ID NO: 1.  
     
     
         78 . The method of  claim 74 , 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 74 , wherein said introducing step comprises ballistic transformation.  
     
     
         80 . The method of  claim 74 , wherein said introducing step comprises  Agrobacterium  transformation.  
     
     
         81 . The method of  claim 74 , 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 74 , 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 74 , 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 74 , 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 74 , 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.  
     
     
         86 . The method of  claim 74 , 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.  
     
     
         87 . The method of  claim 74 , 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.  
     
     
         88 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines.  
     
     
         89 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0 to 8000 parts per billion (ppb).  
     
     
         90 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-100 ppb.  
     
     
         91 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-300 ppb.  
     
     
         92 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of nicotine.  
     
     
         93 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 30,000 parts per million (ppm).  
     
     
         94 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 20,000 parts per million (ppm).  
     
     
         95 . The method of  claim 74 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 10,000 parts per million (ppm).  
     
     
         96 . The method of  claim 74 , wherein the transgenic tobacco plant has no detectable amounts of one or more tobacco specific nitrosamines.  
     
     
         97 . The method of  claim 74 , wherein the transgenic tobacco plant has no detectable amount of nicotine.  
     
     
         98 . A transgenic tobacco plant produced by the method of  claim 74 .  
     
     
         99 . A tobacco leaf collected from the transgenic tobacco plant of  claim 98 .  
     
     
         100 . A tobacco seed collected from the transgenic tobacco plant of  claim 98 , wherein said tobacco seed comprises said isolated nucleic acid sequence.  
     
     
         101 . A transgenic tobacco plant having a reduced amount of quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase), 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 quinolate phosphoribosyl transferase and the amount of putrescine methyl transferase as compared to the amount of quinolate phosphoribosyl transferase and putrescine methyl transferase that would be present in said plant in the absence of said exogenous nucleic acid sequence.    
     
     
         102 . The transgenic tobacco plant of  claim 101 , wherein said exogenous nucleic acid sequence consists essentially of the nucleotide sequence of SEQ ID NO: 1.  
     
     
         103 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         104 . A tobacco leaf collected from the transgenic tobacco plant of  claim 101 .  
     
     
         105 . A tobacco seed that germinates into the transgenic tobacco plant of claim  101 .  
     
     
         106 . A tobacco seed collected from the transgenic tobacco plant of  claim 101 , wherein said seed comprises said exogenous nucleic acid sequence.  
     
     
         107 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         108 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         109 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         110 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         111 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         112 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         113 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         114 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines.  
     
     
         115 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0 to 8000 parts per billion (ppb).  
     
     
         116 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of tobacco specific nitrosamines in the range of 0-300 ppb.  
     
     
         117 . The transgenic tobacco plant of  claim 101 , 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.  
     
     
         118 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has no detectable amounts of one or more tobacco specific nitrosamines.  
     
     
         119 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of nicotine.  
     
     
         120 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 30,000 parts per million (ppm).  
     
     
         121 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 20,000 parts per million (ppm).  
     
     
         122 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has a reduced amount of nicotine in the range of 1 to 10,000 parts per million (ppm).  
     
     
         123 . The transgenic tobacco plant of  claim 101 , wherein the transgenic tobacco plant has no detectable amount of nicotine.  
     
     
         124 . A tobacco product comprising leaves of the transgenic tobacco plant of  claim 101 .  
     
     
         125 . A blended tobacco product comprising leaves of the transgenic tobacco plant of  claim 101 .  
     
     
         126 . The blended tobacco product of  claim 125 , comprising 10 mg or less of nicotine.  
     
     
         127 . The blended tobacco product of  claim 125 , comprising 7.0 mg or less of nicotine.  
     
     
         128 . The blended tobacco product of  claim 125 , comprising 5.0 mg or less of nicotine.  
     
     
         129 . The blended tobacco product of  claim 125 , comprising 2.0 mg or less of nicotine.  
     
     
         130 . The blended tobacco product of  claim 125 , comprising 1.0 mg of less of nicotine.  
     
     
         131 . The blended tobacco product of  claim 125 , comprising no detectable nicotine.  
     
     
         132 . The blended tobacco product of  claim 125 , comprising no detectable TSNAs.  
     
     
         133 . The blended tobacco product of  claim 125 , comprising 25% reduced nicotine/TSNA tobacco and 75% conventional tobacco.  
     
     
         134 . The blended tobacco product of  claim 125 , comprising 50% reduced nicotine/TSNA tobacco and 50% conventional tobacco.  
     
     
         135 . The blended tobacco product of  claim 125 , comprising 75% reduced nicotine/TSNA tobacco and 25% conventional tobacco.  
     
     
         136 . A cured tobacco comprising leaves of the transgenic tobacco plant of  claim 101 .  
     
     
         137 . The cured tobacco of  claim 136 , wherein the amount of TSNAs is in the range of 0 to 8000 parts per billion (ppb).  
     
     
         138 . The cured tobacco of  claim 136 , wherein the amount of TSNAs is in the range of 0 to 300 ppb.  
     
     
         139 . The cured tobacco of  claim 136 , wherein the amount of TSNAs is in the range of 0 to 100 ppb.  
     
     
         140 . The cured tobacco of  claim 136 , wherein the amount of one or more TSNAs is not detectable.  
     
     
         141 . The cured tobacco of  claim 136 , wherein the cured tobacco is selected from the group consisting of Burley, Flue and Oriental.  
     
     
         142 . The cured tobacco of  claim 141 , wherein the cured tobacco is Burley.  
     
     
         143 . The cured tobacco of  claim 141 , wherein the cured tobacco is Oriental.  
     
     
         144 . The cured tobacco of claim  68 , wherein the cured tobacco is Flue.  
     
     
         145 . The cured tobacco of  claim 141 , wherein the amount of nicotine is between 0 and 30,000 parts per million (ppm).  
     
     
         146 . The cured tobacco of  claim 136 , wherein the amount of nicotine is between 0 and 20,000 ppm.  
     
     
         147 . The cured tobacco of  claim 136 , wherein the amount of nicotine is between 0 and 10, 000 ppm.  
     
     
         148 . A tobacco product comprising the cured tobacco of  claim 136 .  
     
     
         149 . A method of making a tobacco product, comprising providing the cured tobacco of  claim 136  and preparing said tobacco product from said cured tobacco.  
     
     
         150 . A method of reducing the amount of a TSNA or a TSNA metabolite in a human that uses tobacco, comprising providing said human the tobacco product of  claim 148 .  
     
     
         151 . A blended tobacco product comprising the cured tobacco of  claim 136 .  
     
     
         152 . A method of making a blended tobacco product, comprising providing the cured tobacco of  claim 136  and preparing said blended tobacco product from said cured tobacco.  
     
     
         153 . A method of reducing the carcinogenic potential of a tobacco product, comprising providing the cured tobacco of  claim 136  and preparing a tobacco product from said cured tobacco, whereby the carcinogenic potential of said tobacco product is thereby reduced.  
     
     
         154 . A method of making a blended reduced nicotine tobacco, comprising: 
 a). providing a first tobacco;    b) providing a second tobacco produced from a genetically modified tobacco plant comprising a reduced level of quinolate phosphoribosyl transferase (QPTase) and a reduced level of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety; and    c) blending said first tobacco with said second tobacco so as to obtain said reduced nicotine tobacco.    
     
     
         155 . A tobacco product comprising the blended reduced nicotine tobacco produced by the method of  claim 154 .  
     
     
         156 . A method of making a blended reduced TSNA tobacco, comprising: 
 a) providing a first tobacco;    b) providing a second tobacco produced from a genetically modified tobacco plant comprising a reduced level of quinolate phosphoribosyl transferase (QPTase) and a reduced level of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety; and    c) blending said first tobacco with said second tobacco so as to obtain said reduced TSNA tobacco.    
     
     
         157 . A tobacco product comprising the blended reduced TSNA tobacco produced by the method of  claim 156 .  
     
     
         158 . A method of making a reduced nicotine tobacco product with a desired amount of nicotine, comprising: 
 a) providing a first tobacco, wherein said first tobacco has a measured amount of nicotine;    b) providing a second tobacco produced from a genetically modified tobacco plant comprising a reduced level of quinolate phosphoribosyl transferase (QPTase) and a reduced level of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety, wherein said second tobacco has a measured amount of nicotine; and    c) blending said first tobacco with said second tobacco so as to produce a reduced nicotine tobacco product with a desired amount of nicotine.    
     
     
         159 . The method of  claim 158 , wherein said reduced nicotine tobacco product is a blended cigarette.  
     
     
         160 . The method of  claim 159 , wherein said blended cigarette comprises 10 mg nicotine or less.  
     
     
         161 . The method of  claim 159 , wherein said blended cigarette comprises 7.0 mg nicotine or less.  
     
     
         162 . The method of  claim 159 , wherein said blended cigarette comprises 5.0 mg nicotine or less.  
     
     
         163 . The method of  claim 159 , wherein said blended cigarette comprises 2.0 mg nicotine or less.  
     
     
         164 . The method of  claim 159 , wherein said blended cigarette comprises 1.0 mg nicotine or less.  
     
     
         165 . A tobacco product comprising the blended reduced nicotine tobacco produced by the method of  claim 159 .  
     
     
         166 . A method of making a reduced TSNA tobacco product with a desired amount of TSNA comprising: 
 a) providing a first tobacco, wherein said first tobacco has a measured amount of TSNA;    b) providing a second tobacco produced from a genetically modified tobacco plant comprising a reduced amount of quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety, wherein said tobacco plant has a measured amount of TSNA; and    c) blending said first tobacco with said second tobacco so as to produce a reduced TSNA tobacco product with a desired amount of TSNA.    
     
     
         167 . The method of  claim 166 , wherein said reduced TSNA tobacco product is a blended cigarette.  
     
     
         168 . The method of  claim 167 , wherein said blended cigarette comprises no detectable amount of TSNAs.  
     
     
         169 . A tobacco product comprising the blended reduced TSNA tobacco produced by the method of  claim 166 .  
     
     
         170 . A method of reducing the nicotine consumption of a tobacco user, comprising: 
 a) providing said tobacco user a first tobacco product comprising tobacco produced from a modified tobacco plant comprising a reduced amount of quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety; and    b) providing said tobacco user a second tobacco product comprising tobacco produced from a modified tobacco plant comprising a reduced level of QPTase and a reduced amount of PMTase, as compared to an unmodified tobacco plant of the same variety, wherein said second tobacco product comprises less nicotine than said first tobacco product; and    c) providing said tobacco user with additional tobacco products comprising tobacco produced from a modified tobacco plant comprising a reduced amount of QPTase and a reduced amount of PMTase, as compared to an unmodified tobacco plant of the same variety, wherein said subsequent tobacco products comprise sequentially reduced amounts of nicotine, starting with a third product that comprises less nicotine than said first or second tobacco product.    
     
     
         171 . A method of reducing the TSNA consumption of a tobacco user, comprising: 
 a) providing said tobacco user a first tobacco product comprising tobacco produced from a modified tobacco plant comprising a reduced amount of quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase), as compared to an unmodified tobacco plant of the same variety; and    b) providing said tobacco user a second tobacco product comprising tobacco produced from a modified tobacco plant comprising a reduced amount of QPTase and a reduced amount of PMTase, as compared to an unmodified tobacco plant of the same variety, wherein said second tobacco product comprises less TSNA than said first tobacco product; and    c) providing said tobacco user with additional tobacco products comprising tobacco produced from a modified tobacco plant comprising a reduced amount of QPTase and a reduced amount of PMTase, as compared to an unmodified tobacco plant of the same variety, wherein said subsequent tobacco products comprise sequentially reduced amounts of TSNA, starting with a third product that comprises less TSNA than said first or second tobacco product.    
     
     
         172 . A cured tobacco comprising a genetic modification and a reduced amount of quinolate phosphoribosyl transferase (QPTase) and a reduced amount of putrescine methyl transferase (PMTase).  
     
     
         173 . The cured tobacco of  claim 172 , wherein the tobacco comprises an exogenous nucleic acid comprising the nucleotide sequence of SEQ ID NO: I or an active fragment thereof.  
     
     
         174 . A tobacco product comprising the cured tobacco of  claim 172 .  
     
     
         175 . A method of making a tobacco product, comprising providing the cured tobacco of  claim 172  and preparing said tobacco product from said cured tobacco.  
     
     
         176 . The tobacco product of  claim 174 , wherein the tobacco product is selected from the group consisting of cigarettes, cigars, pipe tobacco, chewing tobacco, snuff, tobacco, tobacco-containing gum and lozenges.  
     
     
         177 . A tobacco-use cessation kit comprising the tobacco product of  claim 174.