bZIP transcription factors regulate the conversion of nicotine to nornicotine
阅读说明：本技术 bZIP转录因子调节烟碱向降烟碱的转化 (bZIP transcription factors regulate the conversion of nicotine to nornicotine ) 是由 L·袁 S·K·辛格 S·帕塔奈克 D·M·劳森 于 2018-12-06 设计创作，主要内容包括：本文提供了一种减少烟碱向降烟碱转化的方法。所述方法包括将至少一种碱性区域/亮氨酸拉链(bZIP)型转录因子抑制剂施用于需要其的生物体。本文还提供了一种减少烟碱向降烟碱转化的方法,所述方法包括使烟碱N-脱甲基酶(NND)的启动子上的bZIP型转录因子结合位点突变。本文还提供了一种减少烟碱向降烟碱转化的方法,所述方法包括使植物基因组突变以敲除至少一种bZIP型转录因子。(Provided herein is a method of reducing the conversion of nicotine to nornicotine. The methods comprise administering at least one basic region/leucine zipper (bZIP) -type transcription factor inhibitor to an organism in need thereof. Also provided herein is a method of reducing the conversion of nicotine to nornicotine comprising mutating the bZIP-type transcription factor binding site on the promoter of nicotine N-demethylase (NND). Also provided herein is a method of reducing the conversion of nicotine to nornicotine comprising mutating the genome of a plant to knock out at least one bZIP-type transcription factor.)
1. A method of reducing the conversion of nicotine to nornicotine comprising administering to an organism in need thereof at least one basic region/leucine zipper (bZIP) -type transcription factor inhibitor.
2. The method of claim 1, wherein the at least one bZIP transcription factor inhibitor is selected from the group consisting of a group C bZIP transcription factor inhibitor, a group S bZIP transcription factor inhibitor, and combinations thereof.
3. The method of claim 1 wherein the at least one bZIP transcription factor inhibitor is selected from the group consisting of a NtbZIP1a inhibitor, a NtbZIP1b inhibitor, a NtbZIP2a inhibitor, a NtbZIP2b inhibitor, and combinations thereof.
4. The method of claim 1 wherein the at least one bZIP transcription factor inhibitor comprises a NtbZIP1a inhibitor and a NtbZIP1b inhibitor.
5. The method of claim 1 wherein the at least one bZIP transcription factor inhibitor comprises a NtbZIP2a inhibitor and a NtbZIP2b inhibitor.
6. The method of claim 1 wherein the at least one bZIP transcription factor inhibitor comprises a NtbZIP1a inhibitor, a NtbZIP1b inhibitor, a NtbZIP2a inhibitor, and a NtbZIP2b inhibitor.
7. The method of any one of claims 1 to 6, wherein the at least one bZIP transcription factor inhibitor is selected from the group consisting of antisense oligonucleotides, miRNAs, siRNAs, Locked Nucleic Acid (LNA) nucleotides, or combinations thereof.
8. The method of claim 7 wherein the at least one bZIP transcription factor inhibitor comprises an antisense oligonucleotide to a bZIP transcription factor selected from the group consisting of NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and combinations thereof.
9. A method of reducing the conversion of nicotine to nornicotine, comprising mutating a basic region/leucine zipper (bZIP) type transcription factor binding site on the promoter of nicotine N-demethylase (NND).
10. The method of claim 9, wherein the NND is selected from the group consisting of CYP82E4v1, CYP82E5v2, and CYP82E 10.
11. The method of claim 10, wherein the NND is CYP82E4v 1.
12. The method of claim 11, wherein the bZIP binding site on the CYP82E4v1 promoter is an a/G box having the pre-mutation sequence of TACGTC.
13. The method of claim 12, wherein the mutated binding site has the sequence TGCGTC.
14. The method of claim 13, wherein the mutated binding site is formed by site-directed mutagenesis.
15. A method of reducing the conversion of nicotine to nornicotine, comprising mutating a plant genome to knock out at least one basic region/leucine zipper (bZIP) -type transcription factor.
16. The method of claim 15 wherein the at least one bZIP transcription factor is selected from the group consisting of group C bZIP transcription factors, group S bZIP transcription factors, and combinations thereof.
17. The method of claim 15 wherein the at least one bZIP transcription factor is selected from the group consisting of NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and combinations thereof.
18. The method of claim 15 wherein the at least one bZIP transcription factor is selected from the group consisting of NtbZIP1a and NtbZIP1b, NtbZIP2a and NtbZIP2b, and combinations thereof.
19. The method of any one of claims 9 to 18, further comprising administering at least one bZIP-type transcription factor inhibitor to an organism in need thereof.
20. The method of claim 19 wherein the at least one bZIP transcription factor inhibitor is selected from the group consisting of a NtbZIP1a inhibitor, a NtbZIP1b inhibitor, a NtbZIP2a inhibitor, a NtbZIP2b inhibitor, and combinations thereof.
Technical Field
The present invention relates to articles and methods for modulating the conversion of nicotine (nicotinine) to nornicotine (nornicotine). In particular, the presently disclosed subject matter relates to transcription factors for modulating the conversion of nicotine to nornicotine and methods of use thereof.
Background
Common tobacco (Nicotiana tabacum, common tobacaco) is a natural heterotetraploid that originated approximately 20 million years ago. The maternal S genome is from the ancestor of nicotiana americana (n.sylvestris) and the paternal T genome is from the relatives of nicotiana villosa (n.tominosiformis). Nicotine is the major alkaloid accumulated in most cultivated tobacco varieties. Over the past decades, significant progress has been made in the isolation and characterization of structural genes in the nicotine biosynthetic pathway. Jasmonic Acid (JA) is the major inducer of nicotine biosynthesis. JA-responsive Transcription Factors (TF) belong to two major families, APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH), and are known to induce expression of genes encoding key enzymes in the nicotine biosynthetic pathway.
Nicotine and other tropane alkaloids (such as hyoscyamine and scopolamine) are synthesized in the root and transported to the leaves through the xylem. Various transporters have been isolated and their role in alkaloid transport and vacuolar uptake (vacuolar sequencing) in plants has been characterized. In tobacco, many transporters belonging to the multidrug and toxic compound efflux (MATE) family, including MATE1/2 and jasmonate-induced alkaloid transporter (JAT1/2), are involved in nicotine transport and vacuolar uptake. However, studies of the TF involved in the regulation of these transporters are not thorough.
In addition to nicotine, tobacco plants accumulate three other pyridine alkaloids, nornicotine, neonicotine, and dehydroneonicotine. Nornicotine is demethylnicotine (free of methyl groups) derived from nicotine by an enzymatic process. Nornicotine is also a precursor to N-nitrosonornicotine (NNN) that is produced during curing and processing of tobacco materials. More specifically, during post-harvest processing, nornicotine chemically reacts with nitrosating agents to form NNNs. Since NNNs belong to a class of carcinogens associated with smoking, known as Tobacco Specific Nitrosamines (TSNAs), there is a strong need to reduce TSNAs in tobacco products.
There are two possible ways to reduce TSNA. One is to reduce the overall nicotine content; the other is to eliminate the conversion of nicotine to nornicotine. The conversion of nicotine to nornicotine is catalyzed by nicotine N-demethylase (NND), a small family of cytochrome P450 enzymes. In the conversion of nicotine to nornicotine in tobacco, three NND genes have been identified: CYP82E4v1 (from hairy tobacco), CYP82E5v2 (from hairy tobacco) and CYP82E10 (from american tobacco). CYP82E4v1(E4) plays a major role in nicotine conversion in senescent leaves to nornicotine, whereas CYP82E10(E10) expression is reported to be in the root and CYP82E5(E5) plays a role in both root and leaf. However, to date, no Transcription Factor (TF) involved in regulating the conversion of nicotine to nornicotine (i.e., the transcriptional regulator of the E4, 5, and 10 genes) has been identified. Thus, although significant progress has been made in the biochemical and molecular characterization of these nicotine transporters and enzymes involved in nornicotine biosynthesis, the underlying molecular mechanisms regulating these genes remain to be elucidated.
Thus, there is a need for articles and methods for modulating the conversion of nicotine to nornicotine.
Disclosure of Invention
The presently disclosed subject matter fulfills some or all of the above-described needs, as will become apparent to those of ordinary skill in the art after studying the information provided in this document.
Brief Description of Drawings
The subject matter of the present disclosure will be better understood and features, aspects, and advantages other than those described above will become apparent when consideration is given to the following detailed description. Such detailed description makes reference to the following drawings, in which:
FIG. 1 shows a graph illustrating a hierarchical cluster analysis of transcriptome data for eight different tobacco tissues. Each tissue forms a different cluster based on expression.
Figure 2 shows a graph illustrating the distribution of different TF families in tobacco and its progenitors.
FIG. 3 shows a graph illustrating the analysis of co-expression of TF genes and structural genes in the nicotine biosynthetic pathway in different tissues. TF and structural genes are divided into 8 different modules (color-coded sidebars) based on their expression. The black module contains most of the structural genes of the nicotine biosynthetic pathway. YF, flower buds; MF, mature flowers; YL, young leaf; ML, mature leaf; SL, senescent leaves.
FIG. 4 shows a graph illustrating the expression of NtbZIP1a/b and NND in different tobacco tissues.
FIG. 5 shows an image illustrating the bZIP family in tobacco. NtbZIP1a and NtbZIP1b are denoted by a.
FIG. 6 shows an image comparing the nucleotide and amino acid sequences of NtbZIP1a and 1 b.
FIG. 7 shows a graph demonstrating that transient overexpression of NtbZIP1a in tobacco leaves induces the expression of CYP82E4v1, CYP82E5v2 and CYP82E 10.
Fig. 8 shows a graph illustrating that NtbZIP1a significantly activates the CYP82E4v1 and CYP82E10 promoters in tobacco cells.
FIG. 9 shows a diagram illustrating that NtbZIP1a activates the CYP82E4v1 promoter in tobacco cells by binding to the A/G cassette.
Fig. 10 shows a graph illustrating that NtbZIP1a and b significantly activate the CYP82E4v1 promoter in tobacco cells.
FIG. 11 shows a graph illustrating the toping down-regulation of expression of NtbZIP and NND in tobacco plants.
FIGS. 12A-C show graphs and images illustrating overexpression of NtbZIP1a in tobacco plants. (A) Genomic DNA PCR and cDNA PCR of control and three transgenic lines (
Fig. 13 shows an image illustrating the alignment of the amino acid sequences of NtbZIP2a and 2 b.
FIG. 14 shows a graph illustrating that NtbZIP2a and b have a similar expression pattern in tobacco tissue.
Fig. 15 shows a diagram illustrating the synergy of NtbZIP2 with NtbZIP1 to activate the E4 promoter in tobacco cells.
FIGS. 16A-B show images illustrating protein-protein interactions of NtbZIP1 and NtbZIP2 using a yeast two-hybrid assay. (A) A yeast two-hybrid assay showing protein-protein interactions between NtbZIP1 and
While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the following appended claims.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, including the methods and materials described below.
Following long-standing patent law convention, the terms "a", "an" and "the" as used in this application, including the claims, mean "one or more". Thus, for example, reference to "a cell" includes a plurality of cells, and so on.
The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (such as reaction conditions), and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term "about," when referring to a value or amount of mass, weight, time, volume, concentration, percentage, or the like, is intended to encompass variations of, in some embodiments, ± 50% with respect to the specified amount, in some embodiments, ± 40% with respect to the specified amount, in some embodiments, ± 30% with respect to the specified amount, in some embodiments, ± 20% with respect to the specified amount, in some embodiments, ± 10% with respect to the specified amount, in some embodiments, ± 5% with respect to the specified amount, in some embodiments, ± 1% with respect to the specified amount, in some embodiments, ± 0.5% with respect to the specified amount, and in some embodiments, ± 0.1% with respect to the specified amount, as such variations are suitable for carrying out the disclosed methods.
As used herein, a range can be expressed as from "about" one particular value, and/or to "about" another particular value. It will also be understood that a number of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.
All combinations of method or process steps used herein can be performed in any order, unless otherwise indicated by the context in which the referenced combination is made or clearly contradicted.
Detailed Description
The details of one or more embodiments of the presently disclosed subject matter are set forth in this document. Modifications to the embodiments described in this document, as well as other embodiments, will be apparent to persons of ordinary skill in the art upon study of the information provided in this document. The information provided herein, and in particular the specific details of the described exemplary embodiments, is provided primarily for clarity of understanding and thus no unnecessary limitations are to be understood therefrom. In case of conflict, the present specification, including definitions, will control.
The presently disclosed subject matter relates to articles and methods for modulating the conversion of nicotine to nornicotine. In some embodiments, the preparation comprises one or more Transcription Factor (TF) inhibitors. In one embodiment, for example, the article of manufacture comprises one or more inhibitors of basic region/leucine zipper (bZIP) -type transcription factors. In another embodiment, the bZIP-type transcription factor is derived from tobacco. In another embodiment, the inventors have identified 133 bZIP-type transcription factors in tobacco, divided into ten subgroups: A. b, C, D, E, F, G, H and S, suitable bZIP-type transcription factors include at least one of the 27 bZIPs in subgroup S, at least one of the 6 bZIPs in subgroup C, other bZIP 63 homologs, or combinations thereof. In certain embodiments, the subgroup S bZIP transcription factors include, but are not limited to, NtbZIP1a (SEQ ID NOS: 1 and 2), NtbZIP1b (SEQ ID NOS: 3 and 4), or combinations thereof; and/or subgroup C bZIP transcription factors include, but are not limited to, NtbZIP2a (SEQ ID NO: 5), NtbZIP2b (SEQ ID NO: 6), or combinations thereof.
The one or more transcription factor inhibitors include, but are not limited to, antisense oligonucleotides, mirnas, sirnas, Locked Nucleic Acid (LNA) nucleotides, or combinations thereof. In some embodiments, these inhibitors provide RNAi-mediated knock-down/silencing of bZIP-type transcription factors. As will be appreciated by those skilled in the art, the particular sequence/structure of a transcription factor inhibitor is based on the sequence of a particular transcription factor. Thus, as will also be understood by those of skill in the art, antisense oligonucleotides and/or LNAs may be formed by any suitable method using the bZIP-type transcription factor sequences provided herein. For example, in one embodiment, the inhibitor comprises an antisense oligonucleotide having 100% sequence homology to a complementary bZIP-type transcription factor. In another embodiment, the transcription factor inhibitor comprises an antisense oligonucleotide having 100% sequence homology to a bZIP-type transcription factor that is complementary to NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2 b. In such embodiments, the transcription factor inhibitor provides RNAi-mediated knockdown/silencing of NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2b in tobacco.
In some embodiments, provided herein is a method of modulating the conversion of nicotine to nornicotine in a tobacco plant or other nicotine-containing organism. In one embodiment, the method comprises administering one or more bZIP inhibitors disclosed herein to a nicotine-containing organism. Administration of these one or more bZIP inhibitors reduces or eliminates the conversion of nicotine to nornicotine. One or more inhibitors may be administered as a single type of bZIP transcription factor or as a combination of bZIP transcription factors. For example, in one embodiment, the method comprises administering one or more inhibitors of a sbzip-type transcription factor or an inhibitor of a cbzip-type transcription factor. In another embodiment, the method comprises administering one or more inhibitors of S bZIP type transcription factors and one or more transcription factors of C bZIP type transcription factors. In another embodiment, the method comprises administering to the organism one or more inhibitors of NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2 b. In certain embodiments, inhibition of both the S bZIP-type transcription factor and the C bZIP-type transcription factor has a synergistic effect on reducing or eliminating the conversion of nicotine to nornicotine.
The methods disclosed herein comprise administering a single type of inhibitor or any suitable combination of inhibitors, which may be the same or different for each bZIP transcription factor inhibited. For example, in one embodiment, the method comprises administering an antisense oligonucleotide to NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2 b. In another embodiment, the method comprises administering an antisense oligonucleotide to a bZIP transcription factor (such as NtbZIP1a) and LNA nucleotides to another bZIP transcription factor (such as NtbZIP1 b). As will be appreciated by those skilled in the art, while discussed above with respect to certain combinations of bZIP transcription factors and transcription factor inhibitors, the present disclosure is not so limited and may include any other suitable combination of TF and TF inhibitors.
Additionally or alternatively, the methods may include bZIP-type transcription factor knock-out and/or mutation of a bZIP-type transcription factor at a binding site on the promoter of nicotine N-demethylase (NND). For example, in one embodiment, the method comprises editing the plant genome to knock out NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2 b. Genome editing can be performed by any suitable method, such as but not limited to CRISPR/Cas 9-mediated genome editing. In another embodiment, the method comprises mutating a bZIP binding element in the E4 promoter, referred to as the a/G cassette (TACGTC), to TGCGTC by site-directed mutagenesis. Although discussed above with respect to particular mutations in the E4 promoter, as will be understood by those of skill in the art, the present disclosure is not so limited and includes any other mutations in the E4, E5, and/or E10 promoters to reduce or eliminate activation of the corresponding NND by the bZIP-type transcription factor.
Administration of the TF inhibitors, TF knockouts, and/or binding site mutations disclosed herein reduce or eliminate NND activation by bZIP-type transcription factors, which reduces or eliminates the conversion of nicotine to nornicotine. In contrast to prior preparations including E4, E5, and E10 mutants, the preparations disclosed herein control the expression of E4, E5, and E10 to reduce or eliminate the conversion of nicotine to nornicotine. By reducing or eliminating the conversion of nicotine to nornicotine, the articles and methods disclosed herein reduce the deleterious effects of products that typically contain carcinogenic nornicotine, such as, but not limited to, tobacco products.
The presently disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include a compilation of data representing data collected at various times during development and experimentation relating to the subject matter of the present disclosure.
Examples