Tobacco specific nitrosamine reduction in plants

A plant and plant cell technology, applied in the fields of tobacco, plant peptides, tobacco treatment, etc.

Active Publication Date: 2016-01-13
PHILIP MORRIS PROD SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a reduction in NNN levels can be obtained, there is more than one TSNA reported to be a carcinogen, which remains preserved in the modified plants

Method used

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  • Tobacco specific nitrosamine reduction in plants
  • Tobacco specific nitrosamine reduction in plants
  • Tobacco specific nitrosamine reduction in plants

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0261] Example 1: Identification of NtCLCe-s sequence

[0262] To identify NtCLCe-s, the associated transcript (NtCLCe-s: NCBI_43350-v4ctg-in) was detected in common tobacco leaves by RT-PCR analysis and the presence of potentially matching EST contigs. Data from an Affymetrix custom-made tobacco exon array (sequence probes from NtPMIa1g22230e1-st) were used to confirm that NtCLCe-s are equally expressed in roots, green leaves and senescent leaves of Nicotiana vulgaris. In addition, it was found that cold stress and strong cadmium stress did not affect the expression level of NtCLCe-s, thus suggesting that NtCLCe-s is constitutively expressed in tobacco root and leaf organs. Constitutive NtCLCe expression may be linked to the maintenance of its essential cellular role in plastids, which are putatively linked to the nitrogen assimilation pathway. According to the WoLFPSORT software, NtCLCe-s are highly predicted to be plastid membrane proteins. RNAseq studies confirm the pr...

example 2

[0263] Example 2: Identification of NtCLCe-t sequence

[0264] To identify NtCLCe-t, associated transcripts were detected in common tobacco leaves by RT-PCR analysis and the presence of corresponding EST contigs. RNAseq studies confirm the presence of transcripts in its progenitor Nicotiana tomentosa, thus suggesting that expression of NtCLCe-t copies may be lost in N. Row.

example 3

[0265] Example 3: Expression of NtCLCe-s or NtCLCe-t in Common Tobacco Leaves

[0266] Both CLC-Nt2-s and CLC-Nt2-t genes are expressed in common tobacco leaves, as indicated by the presence of both transcripts in common tobacco leaves (customized tobacco exon array study validated by RT-PCR ) and corresponding EST contigs (CLC-Nt2-s: MIRA_20760-v4ctg-in; CLC-Nt2-t: NCBI_56794-v4ctg-in). In addition, RNAseq studies confirmed the presence of corresponding transcripts in two ancestral species, N. americanum and N. tomentosa.

[0267] When looking more closely at the transcriptome data from the tobacco exon array using the probes NtPMIa1g19904e2-st and NtPMIa1g50210e2-st specific for CLC-Nt2-t and CLC-Nt2-s, respectively , it can be seen that the two copies are differentially expressed in common tobacco. CLC-Nt2-s is weakly expressed in Burley root (TN90), and CLC-Nt2-t is sensitive to circadian rhythm. Both genes were expressed in smoked tobacco roots and leaves and were in...

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Abstract

In one aspect, there is provided a mutant, non-naturally occurring or transgenic plant cell comprising: (i) a polynucleotide comprising, consisting or consisting essentially of a sequence encoding a member of the CLC family of chloride channels and having at least 60% sequence identity to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:10 or SEQ ID NO:11; (ii) a polypeptide encoded by the polynucleotide set forth in (i); (iii) a polypeptide comprising, consisting or consisting essentially of a sequence encoding a member of the CLC family of chloride channels and having at least 60% sequence identity to SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:12 or SEQ ID NO:13 or SEQ ID NO:14; or (iv) a construct, vector or expression vector comprising the isolated polynucleotide set forth in (i); and wherein the expression or activity of the polynucleotide or the polypeptide is modulated as compared to a control plant and wherein the nitrate levels in the mutant, non-naturally occurring or transgenic plant containing the mutant, non-naturally occurring or transgenic plant cell are modulated as compared to the control plant containing the control plant cell.

Description

technical field [0001] The present invention discloses novel polynucleotide sequences of genes encoding members of the CLC chloride channel family from Nicotiana, as well as variants, homologues, fragments and mutants thereof. The present invention also discloses polynucleotide sequences and variants, homologues, fragments and mutants thereof. The present invention also discloses the modification of the expression of one or more of these genes or the activity of the protein encoded thereby to regulate the level of tobacco-specific nitrosamines (TSNA) in the plant or its component parts. Background technique [0002] Tobacco-specific nitrosamines (TSNAs) are mainly formed during tobacco leaf curing and processing. Tobacco curing is the process of physical and biochemical changes that bring out the aroma and flavor of each tobacco variety. The amount of TSNA in cured tobacco leaves is thought to depend on the accumulation of nitrite, which accumulates during the death of pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/12A24B15/10
CPCA24B15/18A24B15/245C07K14/415C12N15/8218C12N15/8243A24B3/00Y02A40/146A01H5/00
Inventor L·博韦P·坎帕尼
Owner PHILIP MORRIS PROD SA
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