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
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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 Nicl and Nic2 Tobacco Mutants and Correlation to Nicotine Levels
[0107] TobRD2 steady-state mRNA levels were examined in Nicl 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 Nicl and Nic 2.
[0108] RNA was isolated from the roots of wild-type Burley 21 tobacco plants (Nicl / Nicl Nic2 / Nic2), roots of Nicl-Burley 21 (nicl / nicl Nic2 / Nic2), roots of Nic2-Burley 21 (Nicl / Nicl nic2 / nic2) and roots of Nic1-Nic2-Burley 21 (nicl / nicl 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 Nicl...
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