Modifying nicotine and nitrosamine levels in tobacco

a technology of nitrosamine and nicotine, which is applied in the field of tobacco and tobacco products, can solve the problems of poor taste, fragrance, or smoking properties of products, and achieve the effects of lowering the carcinogenic potential of such products, and reducing the amount of tsnas

Inactive Publication Date: 2005-07-28
VECTOR TOBACCO LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] A further aspect of the present invention involves a method for reducing expression of a quinolate phosphoribosyl transferase gene in a plant cell by growing a plant cell transformed to contain exogenous DNA, where a transcribed strand of the exogenous DNA is complementary to quinolate phosphoribosyl transferase mRNA endogenous to the cell. Transcription of the complementary strand reduces expression of the endogenous quinolate phosphoribosyl gene.
[0019] Yet another aspect of the invention concerns the reduction of the amount of TSNAs, preferably NNN and NNK, and metabolites thereof in humans who smoke, consume or otherwise ingest tobacco. This method is practiced by providing a tobacco product having a reduced amount of TSNAs to said humans, thereby lowering the carcinogenic potential of such product in said humans. By one approach, for example, the carcinogenic potential of side stream or main stream tobacco smoke in a human exposed to said side stream or main stream tobacco smoke is reduced by providing the cured tobacco as described above in a product that undergoes pyrolysis, wherein pyrolysis of said product results in side stream or main stream smoke comprising a reduced amount of TSNAs. Thus, the cured tobacco described above can be used to prepare a tobacco smoking product that produces a reduced amount of TSNAs in the side stream and / or mainstream smoke and thereby reduce the amount of carcinogen in humans who come in contact with tobacco smoke.

Problems solved by technology

Although researchers have developed several approaches to reduce the nicotine content or the nicotine delivery of tobacco products, many techniques result in a product that has poor taste, fragrance, or smoking properties.

Method used

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  • Modifying nicotine and nitrosamine levels in tobacco
  • Modifying nicotine and nitrosamine levels in tobacco
  • Modifying nicotine and nitrosamine levels in tobacco

Examples

Experimental program
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Effect test

example 1

Isolation and Sequencing

[0103] TobRD2 cDNA (Conkling et. al., Plant Phys. 93, 1203 (1990)) was sequenced and is provided herein as SEQ ID NO: 1, and the deduced amino acid sequence as SEQ ID NO: 2. The deduced amino acid sequence was predicted to be a cytosolic protein. Although plant QPTase genes had not yet been reported, comparisons of the NtPT1 amino acid sequence with the GenBank database (FIG. 3) revealed limited sequence similarity to certain bacterial and other proteins; quinolate phosphoribosyl transferase (QPTase) activity has been demonstrated for the S. typhimurium, E. coli. and N. tabacum genes. The NtQPT1-encoded QPTase has similarity to the deduced peptide fragment encoded by an Arabidopsis EST (expression sequence tag) sequence (Genbank Accession number F20096), which may represent part of an Arabidopsis QPTase gene.

example 2

In-Situ Hybridization

[0104] To determine the spatial distribution of TobRD2 mRNA transcripts in the various tissues of the root, in situ hybridizations were performed in untransformed plants. In-situ hybridizations of the antisense strand of TobRD2 to the TobRD2 mRNA in root tissue was done using techniques as described in Meyerowitz, Plant Mol. Biol. Rep. 5: 242 (1987) and Smith et al., Plant Mol. Biol. Rep. 5: 237 (1987). Seven day old tobacco (Nicotania tabacum L.) seedling roots were fixed in phosphate-buffered glutaraldehyde, embedded in Paraplast Plus (Monoject Inc., St. Louis, Mo.) and sectioned at 8 micron thickness to obtain transverse as well as longitudinal sections. Antisense TobRD2 transcripts, synthesized In vitro in the presence of 35S-ATP, were used as probes. The labeled RNA was hydrolyzed by alkaline treatment to yield 100 to 200 base mass average length prior to use.

[0105] Hybridizations were done in 50% formamide for 16 hours at 42° C., with approximately 5×106...

example 3

TobRD2 mRNA Levels in Nic1 and Nic2 Tobacco Mutants and Correlation to Nicotine Levels

[0107] TobRD2 steady-state mRNA levels were examined in Nic1 and Nic2 mutant tobacco plants. Nic1 and Nic2 are known to regulate quinolate phosphoribosyl. transferase activity and putrescence methyl-transferase activity, and are co-dominant regulators of nicotine production. The present results are illustrated in FIGS. 5A and 5B and show that TobRD2 expression is regulated by Nic1 and Nic 2.

[0108] RNA was isolated from the roots of wild-type Burley 21 tobacco plants (Nic1 / Nic1 Nic2 / Nic2), roots of Nic1-Burley 21 (nic1 / nic1 Nic2 / Nic2), roots of Nic2-Burley 21 (Nic1 / Nic1 nic2 / nic2) and roots of Nic1− / Nic2-Burley 21 (nic1 / nic1 nic2 / nic2).

[0109] Four Burley 21 tobacco lines were grown from seed in soil for a month and transferred to hydroponic chambers in aerated nutrient solution in a greenhouse for one month. These lines were isogenic, except for the two low-nicotine loci, and had genotypes of Nic...

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Abstract

The present invention generally relates to tobacco and tobacco products having a reduced amount of nicotine and / or tobacco specific nitrosamines (TSNA). More specifically, several ways to make tobacco plants that have reduced nicotine and TSNA levels have been discovered. Embodiments include tobacco harvested from said tobacco plants, cured tobacco from said tobacco plants, tobacco products made with said cured tobacco and methods of making these compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of and claims priority to U.S. patent application Ser. No. 10 / 729,121, filed Dec. 5, 2003, which is a continuation of and claims priority to International Patent Application No. PCT / US02 / 18040, having international filing date of Jun. 06, 2002, designating the United States of America and published in English, which claims the benefit of priority of U.S. provisional patent application No. 60 / 297,154, filed Jun. 08, 2001. The disclosures of all of the aforementioned applications are hereby expressly incorporated by reference in their entireties.FIELD OF THE INVENTION [0002] The present invention generally relates to tobacco and tobacco products having a reduced amount of nicotine and / or tobacco specific nitrosamines (TSNA). More specifically, several ways to make tobacco plants that have reduced nicotine and TSNA levels have been discovered. Embodiments include tobacco harvested from said tobacco plants...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A24B15/20A24B15/10C07K14/415C12N9/10C12N15/82
CPCA24B15/10A24B15/18A24B15/20C12N15/8243A24B15/245C07K14/415C12N9/1077A24B15/243A61P25/34A61P35/00
Inventor CONKLING, MARK
Owner VECTOR TOBACCO LLC
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