Patent application title: PLANT PROMOTERS AND USES THEREOF
Inventors:
Alain Tissier (Pertuis, FR)
Christophe Sallaud (Montpellier, FR)
Denis Rontein (Greoux Les Bains, FR)
IPC8 Class: AC12N1582FI
USPC Class:
Class name:
Publication date: 2015-08-06
Patent application number: 20150218574
Abstract:
The invention concerns tools, methods and compositions for modifying
plants and/or protein expression in plants. The invention concerns in
particular transcriptional promoters enabling specific expression in the
trichomes, constructs containing said promoters, and their uses for
genetically modifying cells, seeds or plants. The invention also concerns
methods for producing transgenic plants expressing proteins or
metabolites of interest. The invention is generally applicable to any
plant having glandular trichomes, and to the expression of any protein of
industrial interest, in particular therapeutic or phytosanitary.Claims:
1. A composition of matter comprising: a) an isolated nucleic acid
comprising: (i) SEQ ID NO: 2; (ii) a fragment of SEQ ID NO: 2 comprising
at least 100 consecutive nucleotides and having functional
transcriptional promoter activity in glandular trichomes; (iii) a nucleic
acid sequence having at least 80% identity to SEQ ID NO: 2 or a fragment
of SEQ ID NO: 2 comprising at least 100 consecutive nucleotides, said
nucleic acid sequence having functional transcriptional promoter activity
in glandular trichomes; (iv) a sequence displaying at least 90% identity
with a sequence of SEQ ID NO: 2, or a fragment thereof having at least
100 consecutive nucleotides and having functional transcriptional
promoter activity in glandular trichomes; (v) a nucleic acid sequence
selected from SEQ ID NOs: 3 or 4 or a fragment of SEQ ID NOs: 3 or 4
comprising at least 100 consecutive nucleotides and having functional
transcriptional promoter activity in glandular trichomes; (vi) a nucleic
acid sequence selected from SEQ ID NO: 6, 7 or 8; or (vii) a nucleic acid
encoding a plant cell chloroplast transit peptide comprising SEQ ID NO:
10; b) a recombinant expression cassette comprising a gene of interest
operatively linked to a nucleic acid comprising: (i) SEQ ID NO: 2; (ii) a
fragment of SEQ ID NO: 2 comprising at least 100 consecutive nucleotides
and having functional transcriptional promoter activity in glandular
trichomes; (iii) a nucleic acid sequence having at least 80% identity to
SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 comprising at least 100
consecutive nucleotides, said nucleic acid sequence having functional
transcriptional promoter activity in glandular trichomes; (iv) a sequence
displaying at least 90% identity with a sequence of SEQ ID NO: 2, or a
fragment thereof having at least 100 consecutive nucleotides and having
functional transcriptional promoter activity in glandular trichomes; (v)
a nucleic acid sequence selected from SEQ ID NOs: 3 or 4 or a fragment of
SEQ ID NOs: 3 or 4 comprising at least 100 consecutive nucleotides and
having functional transcriptional promoter activity in glandular
trichomes; or (vi) a nucleic acid sequence selected from SEQ ID NOs: 6, 7
or 8; c) an expression vector comprising a gene of interest operatively
linked to a nucleic acid comprising: (i) SEQ ID NO: 2; (ii) a fragment of
SEQ ID NO: 2 comprising at least 100 consecutive nucleotides and having
functional transcriptional promoter activity in glandular trichomes;
(iii) a nucleic acid sequence having at least 80% identity to SEQ ID NO:
2 or a fragment of SEQ ID NO: 2 comprising at least 100 consecutive
nucleotides, said nucleic acid sequence having functional transcriptional
promoter activity in glandular trichomes; (iv) a sequence displaying at
least 90% identity with a sequence of SEQ ID NO: 2, or a fragment thereof
having at least 100 consecutive nucleotides and having functional
transcriptional promoter activity in glandular trichomes; (v) a nucleic
acid sequence selected from SEQ ID NOs: 3 or 4 or a fragment of SEQ ID
NOs: 3 or 4 comprising at least 100 consecutive nucleotides and having
functional transcriptional promoter activity in glandular trichomes; or
(vi) a nucleic acid sequence selected from SEQ ID NOs: 6, 7 or 8; d) a
recombinant cell comprising a recombinant expression cassette, said
recombinant expression cassette comprising a gene of interest operatively
linked to a nucleic acid comprising: (i) SEQ ID No: 2; (ii) a fragment of
SEQ ID NO: 2 comprising at least 100 consecutive nucleotides and having
functional transcriptional promoter activity in glandular trichomes;
(iii) a nucleic acid sequence having at least 80% identity to SEQ ID NO:
2 or a fragment of SEQ ID NO: 2 comprising at least 100 consecutive
nucleotides, said nucleic acid sequence having functional transcriptional
promoter activity in glandular trichomes; (iv) a sequence displaying at
least 90% identity with a sequence of SEQ ID NO: 2, or a fragment thereof
having at least 100 consecutive nucleotides and having functional
transcriptional promoter activity in glandular trichomes; (v) a nucleic
acid sequence selected from SEQ ID NOs: 3 or 4 or a fragment of SEQ ID
NOs: 3 or 4 comprising at least 100 consecutive nucleotides and having
functional transcriptional promoter activity in glandular trichomes; or
(vi) a nucleic acid sequence selected from SEQ ID NOs. 6, 7 or 8; e) a
transgenic plant or transgenic seed comprising a recombinant expression
cassette, said recombinant expression cassette comprising a gene of
interest operatively linked to a nucleic acid comprising: (i) SEQ ID NO:
2; (ii) a fragment of SEQ ID NO: 2 comprising at least 100 consecutive
nucleotides and having functional transcriptional promoter activity in
glandular trichomes; (iii) a nucleic acid sequence having at least 80%
identity to SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 comprising at
least 100 consecutive nucleotides, said nucleic acid sequence having
functional transcriptional promoter activity in glandular trichomes; (iv)
a sequence displaying at least 90% identity with a sequence of SEQ ID NO:
2, or a fragment thereof having at least 100 consecutive nucleotides and
having functional transcriptional promoter activity in glandular
trichomes; (v) a nucleic acid sequence selected from SEQ ID NOs: 3 or 4
or a fragment of SEQ ID NOs: 3 or 4 comprising at least 100 consecutive
nucleotides and having functional transcriptional promoter activity in
glandular trichomes; or (vi) a nucleic acid sequence selected from SEQ ID
NOs: 6, 7 or 8; or f) an isolated plant cell chloroplast transit peptide
comprising SEQ ID NO: 10.
2. The composition of matter according to claim 1, wherein said expression cassette comprises a gene of interest that encodes an enzyme leading to the modification of the composition of the exudate secreted by the trichomes.
3. The composition of matter according to claim 1, wherein said expression cassette comprises a gene of interest that encodes a therapeutic or phytosanitary protein, an enzyme, a resistance protein, a transcriptional activator or a viral genome.
4. The composition of matter according to claim 1, wherein said expression cassette further comprises a nucleic acid encoding SEQ ID NO: 10.
5. The composition of matter according to claim 1, wherein said recombinant cell is a plant cell.
6. The composition of matter according to claim 5, wherein said plant cell is from the family Solanaceae, Asteraceae, Cannabaceae or Lamiaceae.
7. A method for producing a protein in a plant comprising introducing into a plant cell or seed a recombinant cassette comprising a gene of interest that encodes a protein of interest and regenerating a plant from said plant cell or seed that produces said protein of interest, said recombinant expression cassette comprising a gene of interest operatively linked to a nucleic acid comprising: (i) SEQ ID NO: 2; (ii) a fragment of SEQ ID NO: 2 comprising at least 100 consecutive nucleotides and having functional transcriptional promoter activity in glandular trichomes; (iii) a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 comprising at least 100 consecutive nucleotides, said nucleic acid sequence having functional transcriptional promoter activity in glandular trichomes; (iv) a sequence displaying at least 90% identity with a sequence of SEQ ID NO: 2, or a fragment thereof having at least 100 consecutive nucleotides and having functional transcriptional promoter activity in glandular trichomes; (v) a nucleic acid sequence selected from SEQ ID NOs: 3 or 4 or a fragment of SEQ ID NOs: 3 or 4 comprising at least 100 consecutive nucleotides and having functional transcriptional promoter activity in glandular trichomes; or (vi) a nucleic acid sequence selected from SEQ ID NOs: 6, 7 or 8.
8. The method according to claim 7, further comprising recovering the protein of interest from exudate at the leaf surface.
9. The method according to claim 7, wherein the plant, plant seed or plant cell is from the family Solanaceae, Asteraceae, Cannabaceae or Lamiaceae.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No. 11/664,153, filed Mar. 29, 2007, now U.S. Pat. No. 8,993,836, which is the the U.S. national stage application of International Patent Application No. PCT/FR2005/002530, filed Oct. 12, 2005, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.
[0002] The invention concerns tools, methods and compositions for modifying plants and/or protein expression in plants. The invention concerns in particular transcriptional promoters enabling specific expression in the trichomes, constructs containing said promoters, and their uses for genetically modifying cells, seeds or plants. The invention also concerns methods for producing transgenic plants expressing proteins or metabolites of interest. The invention is generally applicable to any plant having glandular trichomes, and to the expression of any protein of industrial interest, in particular therapeutic or phytosanitary.
INTRODUCTION
[0003] The surface of the leaves of certain higher plants (Angiosperms) contains organs called trichomes which, depending on their anatomy, are divided into two main types: nonsecreting trichomes on the one hand and secreting trichomes on the other hand. The main function of glandular secreting trichomes is to secrete at the leaf surface resins and essential oils composed of a few major compounds.
[0004] In the tobacco plant, glandular secreting trichomes are composed of a foot made up of several (3-5) linearly arranged cells and a head containing a variable number (2-20) of secretory cells. Said cells primarily secrete sucrose esters and diterpenes. The latter account for up to 60% of the exudate and 10% of the dry weight of the adult plant (Wagner et al., 2004). In cultivated tobacco species (Nicotiana tabacum), the diterpenes produced belong to two separate families, the labdanes and the cembranes. The relative levels of production of the two families depend on the cultivar although cembrane production is generally four to ten times higher than labdane production. In N. sylvestris, a presumed progenitor of N. tabacum, labdanes are absent and a compound from the cembrane family, cembratriene-diol (CBT-diol), by itself accounts for more than 60% of the terpenes produced by the plant.
[0005] The steps leading to the biosynthesis of CBT-diol in tobacco have been partially elucidated and can be broken down into two different parts:
[0006] Biosynthesis of the universal precursor of all diterpenes, geranylgeranyl pyrophosphate (GGPP), via the so-called "Rohmer" pathway (Rohmer et al., 1996), takes place in the chloroplast.
[0007] Biosynthesis of CBT-diol from GGPP. Wang and Wagner (2003) have proposed the following biosynthetic pathway:
[0007] GGPP→CBT-ol→CBT-diol.
[0008] The first cyclization step would be carried out by an enzyme from a large family of enzymes known as terpene synthases (Bohlmann et al., 1998). The diterpene synthase of tobacco would use GGPP as substrate to form CBT-ol. The second step whereby CBT-diol is produced from CBT-ol is a hydroxylation catalyzed by an enzyme from the cytochrome P450 family. Professor G. Wagner's group (University of Kentucky) has used subtractive PCR to identify two N. tabacum candidate genes for each of these steps:
[0009] a sequence displaying high homology with sequences coding for terpene synthases (CYC-2; Genbank No. AF401234. NID: AY495694).
[0010] a sequence coding for a cytochrome P450-type enzyme (CYP71D16, NID: AF166332) (Wang et al., 2001, Wang & Wagner 2003).
[0011] Studies of the extinction of expression of these genes by co-suppression and RNA interference in N. tabacum have shown (i) a decrease in CBT-diol and CBT-ol correlated with a decrease in CYC-2 gene expression, and (ii) an increase in CBT-ol accumulation and a decrease in CBT-diol formation correlated with a decrease in CYC71D16 gene expression in the trichomes. These studies have suggested that (i) the CYC-2 gene codes for the CBT-ol cyclase responsible for CBT-ol synthesis and (ii) the CYP71D16 gene codes for a CBT-ol hydroxylase which converts CBT-ol to CBT-diol. Moreover, a genomic sequence of a gene very similar to CYC-2 mRNA has recently been deposited in the database (CYC-1, NID: AY049090), which suggests the existence of not one but several CBT-ol cyclase genes.
[0012] The expression of proteins in plants and/or the genetic modification of plants in order to confer them with particular properties is of major appeal, in both the phytosanitary and the pharmaceutical field. The availability of tools which can control or regulate the expression of genes in plants is therefore a key factor in the exploitation and development of said systems. In this regard, the development of promoters or other regulatory sequences specific of tissues, particularly of secreting trichomes, would be a very important advantage.
[0013] In this context, G. Wagner's group has shown that a 1852 bp regulatory sequence, located upstream of the ATG of the CYP71D16 gene, specifically directs the expression of the uidA reporter gene in the trichome secretory cells of tobacco (application US 2003/0100050 A1, Wagner et al., 2003). Furthermore, several promoter sequences extracted from different species have been shown to direct the expression of a heterologous gene in tobacco trichomes (Table 1).
[0014] Among said promoters, that of the LTP3 gene, coding for a cotton protein involved in lipid transfer (LTP), is specifically expressed in cotton fiber cells. The regulatory sequence of the gene (1548 bp) has been studied in tobacco. Said sequence specifically directs the expression of the uidA gene in leaf trichomes. The 315 bp sequence located between positions -614 and -300 upstream of the ATG is thought to underlie the promoter's specificity. The promoter of the LTP6 gene would also enable trichome-specific expression in cotton. Based on the literature, however, it would appear that the expression occurs in cells at the foot of the trichome, and not in the secretory cells. Moreover, when said promoters are introduced into tobacco, expression is no longer specific, with in particular a signal in epidermal cells (see Table 1).
[0015] Therefore, today, there is an important need for promoters or expression regulatory sequences, adapted to the expression of genes in plants, and enabling in particular a high and tissue-specific expression, in particular in secreting trichomes.
SUMMARY OF THE INVENTION
[0016] The application describes the identification and characterization of regulatory nucleic acid sequences of plant origin, which make it possible to specifically direct the expression of a gene of interest in the secretory cells of plant trichomes. Said promoters are easy to manipulate, of reasonable size, modulable, can be adapted to the expression of heterologous genes, and can drive expression specifically in certain plant tissues. Thus the application describes promoters, expression cassettes and vectors that can be used for modifying plants and expressing products of interest in plant tissues or cells.
[0017] A first object of the invention more particularly concerns a nucleic acid having functional transcriptional promoter activity in glandular trichomes, characterized in that it comprises a sequence selected in the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 22, a fragment thereof having at least 100 consecutive bases or a functional variant thereof. More particularly, the nucleic acid comprises a sequence selected in the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 22, and a fragment thereof having at least 100 consecutive bases or a sequence displaying at least 80% identity with one of said sequences, and is specific of glandular trichomes, in particular of the secretory cells of glandular trichomes.
[0018] Another object of the invention concerns a plant cell chloroplast transit peptide, comprising the sequence MSQSISPLMFSHFAKFQSNIWRCNTSQLRVIHSSYASFGGRRKERVRRMNRAMDLSS (SEQ ID No. 10) or a functional fragment thereof, as well as a nucleic acid coding for one such peptide.
[0019] The invention also concerns any recombinant expression cassette comprising a gene of interest operatively linked to a nucleic acid such as defined hereinabove; any expression vector comprising a nucleic acid or a cassette such as defined hereinabove; and any modified cell comprising a cassette or a vector such as defined hereinabove. Preferably the cell is a plant cell, in particular from the family of Solanaceae, Asteraceae, Cannabaceae or Lamiaceae.
[0020] Another object of the invention concerns a method for producing a protein in the trichomes of a plant, comprising introducing into said plant a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein.
[0021] Another object of the invention concerns a method for producing a protein in the trichomes of a plant, comprising introducing into a plant cell or seed a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein, and regenerating a plant from said cell or seed.
[0022] Another object of the invention concerns a method for producing a plant expressing a recombinant protein, comprising introducing into a plant cell or seed a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein, and regenerating a plant from said cell or seed. Advantageously, said protein is an enzyme whose activity in secretory cells leads to a modification of the composition of the exudate.
[0023] Another advantage consists in that the plant secretes the protein in the glandular trichome, and the protein is recovered from the exudate at the leaf surface.
[0024] Another object of the invention concerns any plant or seed comprising an expression cassette or a vector such as defined hereinabove.
[0025] The invention is also directed at the use of a nucleic acid such as defined hereinabove in order to specifically express a protein in the glandular trichomes of a plant. The invention also relates to kits comprising nucleic acids, vectors, cassettes and/or cells such as described earlier.
[0026] As described in detail hereinbelow, the invention is applicable to the expression of any gene of interest in any plant or plant tissue, preferably higher plants comprising glandular trichomes, for different applications, such as for the production of pharmaceutical or phytosanitary products of interest, or for the production of plants having improved or adapted properties.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present application describes the identification of plant trichome-specific promoters from the CBT-ol synthase genes of tobacco. Starting from the sequence of a cDNA coding for a terpene synthase, i.e. CBT-ol cyclase (CYC-2, NID: AF401234), four genes (named NsTPS-02a, 02b, 03, and 04) displaying strong sequence similarity to CYC-2 were identified from genomic DNA of the species Nicotiana sylvestris, using PCR methods.
[0028] An analysis of expression showed that said genes (in particular NsTPS-02a, 02b, and 03) are expressed in tobacco trichomes.
[0029] The promoter sequences, heretofore unknown, located upstream of the ATG of each of said four genes, have been identified and characterized. Each sequence is approximately 1 kb in size. The sequences are given in SEQ ID Nos. 1 to 4. Alignment of the four sequences reveals that they share more than 93% identity (FIG. 1, Table 2). In addition, said alignment reveals identical regions, most probably involved in the specificity of expression.
[0030] Construction of recombinant expression cassettes using said promoters has demonstrated, in an unexpected manner, an expression profile which is restricted and specific of trichome secretory cells. Such a profile was unexpected in so far as the expression characteristics of terpene synthases have not been documented at all in the prior art. Moreover, the results obtained by the applicants show that even the CYC-1 gene (NID: AY049090) whose genomic sequence was deposited by G. Wagner, is barely expressed in N. tabacum trichromes.
[0031] The present application therefore provides novel promoters, particularly advantageous for the expression of proteins in plants or plant tissues, in particular for specific expression in the cells of glandular trichomes. Said promoters are particularly advantageous since they enable the production of recombinant products directly in the exudate secreted by the leaves, thereby greatly facilitating the recovery of the recombinant products, if need be.
[0032] A particular object of the invention therefore concerns any nucleic acid having functional transcriptional promoter activity in glandular trichomes, characterized in that it comprises a sequence selected in the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 22, a fragment thereof having at least 100 consecutive bases or a functional variant thereof (or of their complementary strand).
[0033] In a particular embodiment, the invention concerns any nucleic acid such as defined hereinabove, characterized in that it comprises a sequence selected in the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 22, a fragment thereof having at least 100 consecutive bases or a sequence displaying at least 80% identity to one of said sequences, and in that it has transcriptional promoter activity specific of glandular trichomes.
[0034] In a specific embodiment of the invention, the nucleic acid comprises a sequence selected in the group consisting of SEQ ID Nos. 1, 2, 3, 4 and 22, preferably the sequence SEQ ID No. 2.
[0035] As illustrated in the examples, the 1065 base pair sequence of the TPS-02b gene promoter (SEQ ID No. 2) was cloned upstream of a uidA reporter gene (the sequence of the TPS02b promoter/uidA gene fusion is given by SEQ ID No. 5). Said construct (cassette No. 1) was inserted into a T-DNA vector (pBI121) containing a kanamycin resistance gene under the control of the nopaline synthase promoter of Agrobacterium tumefasciens (nos). Said T-DNA was introduced into N. sylvestris by genetic transformation using the strain Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1993). UidA gene expression was detected in several transformants. The results in FIG. 2 show that expression was present in secretory cells, but not in the cells constituting the base of the trichomes. Furthermore, no uidA expression was detected in any of the other organs of the plant. These data indicate that the sequence under study can direct the expression of a heterologous gene in the secretory cells of glandular trichomes in a highly specific manner.
[0036] In the spirit of the invention, the term "nucleic acid" shall be understood to mean any molecule based on DNA or RNA. These can be molecules that are synthetic or semi-synthetic, recombinant, optionally amplified or cloned into vectors, chemically modified or containing non-natural bases. Typically they are isolated DNA molecules, synthesized by recombinant methods well known to those skilled in the art.
[0037] In the spirit of the invention, "specific" promoter shall be understood to mean a promoter mainly active in a given tissue or cell group. It shall be understood that a residual expression, generally lower, in other tissues or cells cannot be entirely excluded. For example, residual expression in other tissues or cells does not exceed 30% of the expression observed in glandular trichomes, preferably it does not exceed 20%, 10% or 5%. A particular feature of the invention is based on the ability to construct promoters specific of glandular trichome secretory cells, enabling a modification of the composition of the leaf secretions of the plant, and in particular enabling the expression therein of products of interest.
[0038] A nucleic acid "fragment" according to the invention advantageously refers to a fragment comprising at least 10 consecutive bases, more generally at least 20, 30, 40, 50, 60, 70, 80, 90 or 100. Nucleic acid "fragments" are typically fragments containing 100, 200, 300, 400 or 500 consecutive nucleotides of the sequence or more. Nucleic acid "fragments" are preferably fragments containing at least 100, 200, 300, 400 or 500 consecutive nucleotides of the sequence. Nucleic acid "fragments" can be used alone, or combined with other transcriptional regions, in order to construct chimeric promoters having a specificity for glandular trichomes. A promoter according to the invention is typically a region of the sequence displaying promoter activity.
[0039] FIG. 1 shows that the promoters according to the invention share a very high percentage of sequence identity with each other. In this regard, the analysis of said sequences allowed the inventors to identify parts thereof as conserved motifs necessary for specific expression in trichome secretory cells. Elimination of said motifs leads to a significant reduction in expression or a loss of specificity of expression. Said motifs, three in number, are defined by the following consensus sequences, where the letters K, W and Y represent nucleic acid bases according to the international nomenclature with K=G or T, W=A or T, and Y=C or T:
TABLE-US-00001 Motif 1: 5'-KKTCGTWGCAWT-3' (SEQ ID No. 6) Motif 2: 5'-AGTAATWTYW-3' (SEQ ID No. 7) Motif 3: 5'-TTGTAGCWAW-3' (SEQ ID No. 8)
[0040] In a preferred embodiment, a fragment in the context of the invention comprises at least one sequence selected in the group consisting of SEQ ID No. 6, 7 or 8. Similarly, a particular object of the invention concerns any nucleic acid sequence comprising a sequence selected in the group consisting of SEQ ID No. 6, 7 or 8 or a functional variant thereof. Said nucleic acids are typically recombinant nucleic acids having transcriptional promoter activity. In particular they may be chimeric promoters, that is to say, comprising a sequence selected in the group consisting of SEQ ID No. 6, 7 or 8 or a functional variant thereof, operatively linked to a minimal promoter.
[0041] The term "functional variant" designates any nucleic acid bearing one or more modifications (that is to say for example a mutation, deletion, or addition of one or more bases) with respect to the parent sequences described herein, and conserving an activity, either of a transcriptional promoter, or a signal of expression specificity, or of transit. For example the functional variants can correspond to promoters derived from the corresponding genes of other plant species. For instance, the studies described in the examples herein concern sequences from the species Nicotiana sylvestris, one of the presumed diploid parents of cultivated tobacco, N. tabacum. It is known that the sequences of N. sylvestris and N. tabacum are extremely similar (up to 99% sequence identity between N. tabacum and N. sylvestris sequences), such that the promoters of the corresponding genes of N. tabacum constitute functional variants in the context of the invention, and can be easily prepared by conventional methods (hybridization, amplification, and the like). Other functional variants are nucleic acids, synthetic, recombinant or natural, whose sequence hybridizes, under conditions of high stringency, with one of the sequences SEQ ID No. 1, 2, 3, 4, 22 or a fragment having at least 100 consecutive nucleotides and which has transcriptional promoter activity, in particular specific of glandular trichomes.
[0042] In a general manner, functional variants are nucleic acids displaying at least 80, 85 or 90% identity with sequences SEQ ID No. 1, 2, 3, 4 or 22, or with the fragments thereof having at least 100 consecutive nucleotides, even more preferably more than 95% identity obtained by blastN sequence alignment software (Altschul et al., 1990).
[0043] The percentage of identity is determined by comparing two sequences which have been optimally aligned. The percentage of identity is calculated by determining the number of positions for which an identical residue appears in both sequences at the same position divided by the total number of positions and multiplied by 100. The optimal alignment of two sequences can be achieved for example with an algorithm to look for local homology (Smith & Watherman, 1981), or equivalent systems known to those skilled in the art.
[0044] The identified genes have also enabled the characterization of a transit peptide, which can target or direct a heterologous protein to the chloroplast compartment of plant cells, particularly trichomes. For instance, it is known that diterpene synthases have a peptide signal sequence at their N-terminal end required for their localization in the chloroplast compartment, the site of biosynthesis of geranylgeranyl pyrophosphate (GGPP) (Trapp and Croteau, 2001). By using the ChloroP program (Emanuelsson et al., 2000, Nielsen et al., 1997), a putative transit peptide of 51 amino acids (AA) was identified from the coding sequence of the TPS-02a gene. Said transit peptide (plus the following 6 AA) was introduced into cassette No. 1, upstream of the uidA gene and downstream from the TPS-02b promoter. In this manner a translational fusion of the transit peptide with the GUS protein was produced. Said construct (cassette No. 2) was inserted into a T-DNA vector (pBIl21) carrying a kanamycin resistance gene under the control of the cauliflower mosaic virus promoter (CaMV 35S). This T-DNA was introduced into N. sylvestris by genetic transformation using the strain Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1993).
[0045] As for cassette No. 1, uidA gene expression in several transformants was only detected in glandular trichome secretory cells. Furthermore, microscopic examination showed that GUS activity was located in plasts, indicating that the GUS protein was correctly targeted to the chloroplast compartment. These data demonstrate that the sequence under study, corresponding to the CBT-ol cyclase transit peptide of N. sylvestris (TPS-02b), directs the localization of a heterologous protein to the chloroplast compartment.
[0046] Another object of the invention therefore concerns a plant cell chloroplast transit peptide, comprising the following amino acid sequence MSQSISPLMFSHFAKFQSNIWRCNTSQLRVIHSSYASFGGRRKERVRRMNRAMDLSS (SEQ ID No. 10) or any fragment or functional variant thereof, as well as a nucleic acid coding for a plant cell chloroplast transit peptide, comprising the nucleotide sequence: "ATGAGTCAATCAATTTCTCCATTAATGTTTTCTCACTTTGCAAAATTTCAGTCGA ATATTTGGAGATGCAATACTTCTCAACTCAGAGTTATACACTCATCATATGCCTC TTTTGGAGGGAGAAGAAAAGAGAGAGTAAGAAGAATGAATCGAGCAATGGATC TTTCTTCA" (SEQ ID No.9), or any fragment or functional variant thereof.
[0047] The invention also relates to any recombinant expression cassette, characterized in that it comprises a gene and/or a promoter of interest operatively linked to a nucleic acid such as defined hereinabove.
[0048] The term expression cassette designates a nucleic acid construct comprising a coding region and a regulatory region, operatively linked. The expression "operatively linked" indicates that the elements are combined in such a way that the expression of the coding sequence (the gene of interest) and/or the targeting of the coded protein are under control of the transcriptional promoter and/or the transit peptide. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance therefrom which is compatible with control of expression. Likewise, the sequence of the transit peptide is generally fused upstream of the sequence of the gene of interest, and in frame with it, and downstream of any promoter. Spacer sequences may be present, between the regulatory elements and the gene, as long as they do not prevent expression and/or targeting.
[0049] The gene of interest can be any nucleic acid (for example a DNA or RNA) containing a region coding for an expression product (for example a mRNA or a protein). In a preferred embodiment, the gene of interest codes for a therapeutic or phytosanitary protein, an enzyme, a resistance protein, a transcriptional activator or a viral genome.
[0050] Another object of the invention relates to any (expression) vector comprising a nucleic acid or a cassette such as defined hereinabove. The vector can be DNA or RNA, circular or not, single- or double-stranded. Typically it is a plasmid, phage, virus, cosmid, artificial chromosome, etc. Advantageously it is a plant vector, that is to say, capable of transforming a plant cell. Examples of plant vectors are described in the literature, among which one can cite in particular the A. tumefaciens T-DNA plasmids pBIN19 (Bevan, 1984), pPZP100 (Hajdukewicz et al., 1994), pCAMBIA series (R. Jefferson, CAMBIA, Australia). The vectors of the invention can additionally comprise an origin of replication and/or a selection gene and/or a plant recombination sequence, etc. The vectors can be constructed by conventional molecular biology methods, well known to those skilled in the art, using for example restriction enzymes, ligation, clonings, replication, etc. Specific examples of vectors according to the invention are provided in the experimental section, and include in particular pLIBRO-01 and pLIBRO-02.
[0051] The gene constructs of the invention can be used to genetically modify plants, and in particular to introduce and express proteins in plant tissues, including whole plants.
[0052] Introduction of the inventive constructs into a plant cell or tissue, including a seed or plant, can be carried out by any method known to those skilled in the art. Plant transgenesis methods are well known in the field, and comprise for example the use of the bacterium Agrobacterium tumefaciens, electroporation, conjugative transfer, gene gun methods, and the like.
[0053] A commonly used method is based on the use of the bacterium Agrobacterium tumefaciens, which mainly consists in introducing the construct of interest (nucleic acid, cassette, vector, etc.) in the bacterium A. tumefaciens, then contacting said bacterium with the leaf disks of the chosen plant. The expression cassette is typically introduced in the bacterium by using as vector the Ti plasmid (or T-DNA), which can be transferred into the bacterium for example by heat shock. Incubation of the transformed bacteria with leaf disks leads to transfer of the Ti plasmid into the genome of the disk cells. The latter can optionally be cultivated in suitable conditions in order to regenerate a transgenic plant, the cells of which comprise the construct of the invention. For further details or variant implementations of the A. tumefaciens transformation method, reference can be made to Horsch et al., 1985 or Hooykaas and Schilperoort, 1992 for example.
[0054] Another method of plant transformation is based on projecting microparticles (typically microbeads) to which gene constructs are attached, directly on plant cells, then culturing said cells in order to regenerate a transgenic plant. The particles which are used are typically gold particles, which are typically projected by means of a particle gun (see in particular Russell et al., In Vitro Cell. Dev. Biol., 1992, 28P, p. 97-105).
[0055] The microinjection method is based primarily on injecting the gene constructs into plant protoplasts or embryos, then cultivating said tissues so as to regenerate whole plants. Other plant transgenesis methods are well known, or other protocols implementing the above methods are described in the prior art (Siemens, J and Schieder, 1996) and can be employed in the invention.
[0056] Once regenerated, the transgenic plants can be tested for expression of the product of interest in the trichomes. This can be done by collecting the leaf exudate and testing for the presence of the product in said exudate, when the product is meant to be secreted. This can also be done by analyzing the presence of the expression product of the gene of interest in the leaves and, more particular, in the trichome cells (for example by analyzing mRNA or genomic DNA with specific primers or probes). Optionally the plants can be selected, crossed, treated, etc. in order to obtain plants displaying improved levels of expression.
[0057] In this regard, another object of the invention concerns a recombinant cell comprising a cassette or a vector such as defined hereinabove. For example it can be a plant cell, in particular from the Solanaceae, Asteraceae, Cannabaceae or Lamiaceae family. The cells can be cultivated in vitro, and used to regenerate tissues or whole plants, in order to produce proteins in culture, or else to study the properties of genes or proteins of interest (for example by functional genomics).
[0058] Another object of the invention also concerns a plant or seed comprising an expression cassette or a vector such as defined hereinabove.
[0059] The invention further concerns a method for producing a protein in a plant, in particular in the glandular trichome of a plant, comprising introducing into said plant a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein.
[0060] Another aspect of the invention concerns a method for producing a protein in a plant, in particular in the glandular trichome of a plant, comprising introducing into a plant cell or seed a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein, and regenerating a plant from said cell or seed.
[0061] The invention also concerns any method for producing a plant expressing a recombinant protein, comprising introducing into a plant cell or seed a cassette or a vector such as defined hereinabove, comprising a gene coding for said protein, and regenerating a plant from said cell or seed.
[0062] Advantageously, the recombinant protein expressed in the trichome leads to the production by the secretory cells of a molecule secreted in the leaf surface exudate and/or to a modification of the exudate composition.
[0063] Advantageously, the plant secretes the protein in the glandular trichome, and the protein is recovered in the exudate at the leaf surface.
[0064] The invention can thus be used to modify plants, plant cells or tissues, in order to make them express different products of interest.
[0065] The invention can be used in particular to express products of interest specifically in the secretory cells of glandular trichomes of higher plants (in particular Angiosperms). The invention can be applied in particular to any plants from families having glandular trichomes, for example Asteraceae, Solanaceae, Cannabaceae and Lamiaceae. The invention is particularly adapted to plants from the Solanaceae family, such as for example the genuses Solanum, Lycopersicon, Capsicum, Petunia, Datura, Atropa, etc., and to Nicotianae, for example Nicotiana sylvestris and N. tabacum.
[0066] The product of interest can be any recombinant protein, including peptides, enzymes, antibodies, and the like. In particular it can be a protein having a biological activity of industrial interest, for example medical or phytosanitary. It can also be a protein intended to confer particular properties to the plant (in particular a modification of the composition of the secretion (exudate)), such as resistance to pathogens (destructive insects, fungi, bacteria, viruses, etc.), improved growth, a modified metabolite content or a modified synthetic pathway, etc.
[0067] The invention can also be used in order to express, in a plant cell or a plant, a transcriptional activator. In this case, the transcriptional activator produced will enable control of the expression of a protein of interest placed under control of a promoter responding to said activator. Such a binary system, using two cassettes (present on a same vector or on different vectors) makes it possible to amplify the specific expression obtained with the aid of the invention. One such example is a system comprising a first cassette containing the gene coding for a transcriptional activator (for example GAL4) under the control of the trichome-specific promoter, and another cassette comprising the gene coding for the protein of interest under the control of elements known to be regulated by the transcription factor (Ga14).
[0068] Another amplification system consists in the use of a viral RNA vector. In fact, some plant RNA viruses code for an RNA-dependent RNA polymerase which allows amplification of the transcripts of a given gene. In this embodiment, the gene of interest is cloned downstream from the promoter of said RNA polymerase, in place of the open reading frame coding for the viral coat protein. The virus is itself placed under the control of the specific promoter. Expression of the virus is therefore restricted to trichome secretory cells, thereby allowing selective amplification of expression of the gene in said cells.
[0069] Other aspects and advantages of the invention will become apparent in the following examples, which are given for purposes of illustration and not by way of limitation.
LEGENDS OF FIGURES
[0070] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.
[0071] FIG. 1: Alignment of promoter sequences of the invention: The sequences of the four TPS promoters (NsTPS02a, SEQ ID NO:1; NsTPS02b, SEQ ID NO:2; NsTPS03, SEQ ID NO:3; and NsTPS04, SEQ ID NO:4) approximately 1 kb in length were aligned with the AlignX alignment program (VectorNTI suite, INFORMAXINC). Motifs required for specific trichome activity are shaded in gray.
[0072] FIGS. 2A-2D: Visualization under a microscope (A and B) and a low-power stereo microscope (B and D) of trichome glandular cells of different organs after histochemical staining of the tissues with X-Gluc. UidA reporter gene expression, stained in blue, is detected only in trichome glandular cells. The cells at the base of the trichome are not stained. Staining was carried out on two tobacco lines transformed with the pLIBRO-01 construct. A and C: leaf trichomes, B: stalk trichomes, D: sepal trichomes.
EXAMPLES
1. Sequence Isolation and Characterization
[0073] Genomic DNA was extracted from tobacco leaves (N. sylvestris or N. tabacum) with the aid of a Qiagen® commercial kit (DNeasy Plant Maxi Kit). The promoter regions of the different TPS genes (TPS02a, 02b, 03, and 04) were cloned by using the "adapter-anchor" PCR method (Siebert et al., 1995). Briefly, genomic DNA was digested separately with different restriction enzymes generating blunt ends (DraI, SspI, NaeI) and ligated to an adapter composed of the following primers:
ADPR1 5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGGAGGT (SEQ ID No. 14) and ADPR2 5'-ACCTCCCC (SEQ ID No. 15). The TPS-02 gene promoter region was then identified by successively carrying out two PCR reactions (PCR1 followed by PCR2) with primers complementary to the adapter (AP1 and AP2) and different primers of the published CYC-2 gene coding sequence (Genbank No. AF401234, NID: AY495694). To identify promoter sequences of approximately 1 kb, the PCR1 reaction was carried out with the AP1 primer (5'-GGATCCTAATACGACTCACTATAGGGC, SEQ ID No. 16) and the external primer 2A04 (5'-AGAGGCATATGATGAGTGTATAACTC, SEQ ID No. 17). An aliquot of the PCR1 reaction was used to carry out the PCR2 reaction with the AP2 primer (5'-CTATAGGGCTCGAGCGGC, SEQ ID No. 18) and the internal primer 2A02 (5'-ACCTCCAAATATTCGACTGAAATTT, SEQ ID No. 19). The fragments obtained by this method ranged in size from 200 to 1000 bp. In order to isolate the largest sequences, the same process was repeated on the newly identified sequences. For example, to identify the 1.7 kb promoter sequence corresponding to the TPS-02b gene, the PCR1 reaction was carried out on the same genomic material and in the same PCR conditions but using the external primer 2G07 (5'-GTCATGTGCTTGAAGTTACCATATC, SEQ ID No. 20) and the internal primer 2G08 (5'-GTTACCATATCAATATTATTTACCAT, SEQ ID No. 21). The PCR amplifications were all carried out in the same standard PCR conditions with 0.25 unit Taq polymerase (supplier: Eppendorf) in 25 μl of reaction mixture in the following conditions: 1 cycle at 94° C. 2 min, then 35 cycles at 94° C. 30 sec, 68° C. 30 sec and 1 cycle at 72° C. 5 min using 50 ng of genomic DNA.
[0074] To clone the TPS-02b promoter (PromTPS02b) in the binary vector, another PCR amplification was carried out on genomic DNA using the sense primer, 3A04 (sequence 5'-CTAAGCTTAATTTATTTTTGTAAAACTTC, SEQ ID No. 11) which has a 5' HindIII site and the antisense primer, 3A03 (sequence 5'-ATGGATCCCTCTTTCTCTCGCCAAACGAGT, SEQ ID No. 12) which has a 5' BamH1 site. To clone the TPS-02b promoter comprising the transit peptide (promTPS02b-TP), the sense primer 3A04 was used with the antisense primer 4C01 (5'-TGGATCCTGAAGAAAGATCCATTGCTCGA, SEQ ID No. 13), comprising a 5' HindIII site. To clone the TPS-02b--1.7 kb promoter (PromTPS02b--1.7, SEQ ID No. 22), the sense primer 4E07 which has a 5' SacI site (5' TGAGCTCAAAGAGGTGAAACCTAATCTAGTATGCAA 3', SEQ ID No. 23) was used with the primer 3A03. The amplifications were carried out with Taq polymerase (Eppendorf®) in the following conditions: 1 cycle at 94° C. 2 min, 35 cycles at 94° C. 30 sec, 57° C. 30 sec, 72° C. 1 min and 1 cycle at 72° C. 5 min using 50 ng of genomic DNA in a 25 μl reaction. The PCR products were purified on a Qiaquick column (Qiagen) and cloned into the pGEM-T vector (Promega®) to yield plasmids pPromTPS02b and pPromTPS02b-TP, respectively. The clones were sequenced, and the sequences of the 1 kb promoter regions are represented by SEQ ID Nos. 1 to 4. Alignment of the sequences (FIG. 1) revealed 93% identity or higher. Alignment with the 1.7 kb sequences gave sequence identities greater than 90%.
2. Construction of Transformation Vectors and Cassettes
[0075] The CaMV 35S promoter located upstream of the uidA gene in the binary vector pBI121 (AF485783) was removed by HindIII and BamH1 enzymatic digestion and purified on an agarose gel on a Qiaquick column (Qiagen®). The PromTPS02b and PromTPS02b-TP promoter sequences were purified on a Qiaquick column (Qiagen®) after HindIII and BamH1 digestion of plasmids pPromTPS02b and pPromTPS02b-TP. The promoter sequences were inserted into the HindIII and BamH1 sites of the pBI121 vector by replacing the CaMV 35S promoter thereby creating a transcriptional fusion with the uidA gene, to yield constructs respectively named pLIBRO-18 and pLIBRO-19. The two binary vectors pLIBRO-01 and 02 were introduced into Agrobacterium strain LBA4404 by electroporation.
[0076] The promTPS02b--1.7 promoter was cloned into the EcoR1 site of the vector pCAMBIA1391Z (NID: AF234312) to yield vector pLIBRO-32.
3. Genetic Transformation
[0077] Transgenic tobacco lines (N. sylvestris) carrying the pLIBRO-01, pLIBRO-02 and pLIBRO-32 constructs were obtained by genetic transformation with the corresponding Agrobacterium strains by using the leaf disk method (Horsch et al., 1985). Transformants were selected with 100 mg/1 kanamycin sulfate and Agrobacterium was eliminated with 250 mg/l carbenicillin.
4. Analysis of uidA Gene Expression
[0078] UidA gene expression was detected by the histochemical GUS assay as described by Jefferson et al. (1987). A low power stereo microscope was used to visualize the staining.
5. Demonstration of Specific Expression in Trichromes
[0079] UidA gene expression was measured in several transgenic tobacco lines (N. sylvestris) carrying the pLIBRO-01 and pLIBRO-32 construct. The results for the pLIBRO-01 construct are presented in FIG. 2 (see legend above).
[0080] The data show that expression occurred in secretory cells, but not in the cells forming the base of the trichomes. Furthermore, no uidA gene expression was detected in other organs of the plant. These results demonstrate that the test sequence directs the expression of a heterologous gene in secretory cells of glandular trichomes in a highly specific manner. Identical results were obtained with the pLIBRO-32 construct (data not shown).
6. Demonstration of Chloroplast Targeting
[0081] UidA gene was measured in several transgenic tobacco lines (N. sylvestris) carrying the pLIBRO-02 construct.
[0082] As for cassette No. 1, uidA gene expression in several transformants was detected only in glandular trichome secretory cells. Furthermore, under the microscope, GUS activity was found to be localized in chloroplast cells, indicating that the GUS protein was correctly targeted to the chloroplast compartment. These results show that the transit peptide sequence of the invention targets a heterologous protein to the chloroplast compartment.
TABLE-US-00002 TABLE 1 Gene Plant abbreviation Name of gene Plant Promoter (bp) transformed Expression References LTP3 Lipid transfer protein Cotton 1548 Tobacco Trichomes, peripheral Liu et al., 2000, 1143 epidermis of leaves and BBA, 1487:106111 614 vascular tissues LTP6 Lipid transfer protein Cotton 447 Tobacco Trichomes and stomate guard Hsu et al., 1999, Plant 272 cells Science, 143:6370 wax9D Lipid transfer protein Brassica 972 Tobacco Epidermis of leaf, stems and Pyee and oleracea flowers, petals, sepals, ovules, Kolattukudy, 1995, and trichomes Plant J. 7:4559 LTP1 Lipid transfer protein Arabidopsis 1149 Arabidopsis Epidermal cells from various Thoma et al., 1994, tissues Plant Physiol. 105 3545 CYC71D16 CBT-ol hydroxylase Tobacco 1852 Tobacco Trichomes Wang et al., 2002, J. Exp. Bot. 1891-1897
TABLE-US-00003 TABLE 2 NsTPS02a- NsTPS02b- NsTPS03- NsTPS04- Prom1kb Prom1kb Prom1kb Prom1Kb NsTPS02a-Prom1kb 100 96 93 94 NsTPS02b-Prom1kb 100 93 93 NsTPS03-Prom1kb 100 93 NsTPS04-Prom1Kb 100
REFERENCES
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[0084] Bevan M (1984) Nucl. Acids Res. 12:8711-8721.
[0085] Bohlmann J, Meyer-Gauen G, Croteau R (1998) Proc. Natl. Acad. Sci. USA 95:4126-4133.
[0086] Emanuelsson O, Nielsen H, Brunak S and Von Heijne G (2000) J. Mol. Biol. 300:1005-1016.
[0087] Jefferson R A. (1987) Plant Mol. Biol. Rep. 5:387-405.
[0088] Hajdukiewicz P, Svab Z, Maliga P (1994) Plant Mol. Biol. 25:989-994.
[0089] Nielsen H, Engelbrecht J, Brunak S, and Von Heijne G (1997) Prot. Eng. 10:1-6.
[0090] Hoekema A, Hirsch P R, Hooykaas P J J and Schilperoort R A (1983) Nature 303:179-180.
[0091] Hooykaas P J J and Schilperoort R A (1992) Plant Mol. Biol. 19:15-38.
[0092] Horsch R B, Fry J, Hoffmann N L, Wallroth M, Eichholtz D, Rogers S G, and Fraley R T (1985) Science 227:1229-1231.
[0093] Rohmer M, Seeman M, Horbach S, Bringer-Meryer S, and Sahm H (1996) J. Am. Chem. Soc. 118:2564-2566.
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[0102] Wang E and Wagner G J (2003) Planta 216:686-691.
Sequence CWU
1
1
2311068DNANicotiana sylvestris 1aatttatttt cataaaactt ctctaatttt
tgaacaaaat cttatattga ttttttaatc 60aaagccaaaa tatttattta actatgaaaa
ttttttaaca actaatttat tatggtaaat 120aatattgata tggtaacttc aagcacatga
caaaaattat aactaactgc agaagtttac 180tgtctctctg aatcttgtgg ctatgtcatt
ctatcataac aaatacttgt agctaatacg 240ccaacgatgt tctcgatttc atataatttg
aattttaaaa tagcttttaa atttaatatt 300tatttcaaat cattattgtg actaacatgt
tataaccgca gtaatatttg gagatgcaat 360acttatattt agctacaaaa ttttattgta
tcataataag tttgtagcta ttaagttagt 420ttttgccaca aatttttata attgaagcaa
aaatacctat tcaactacaa tattttgtat 480cgagtaatat tttgtgacta gaagattaat
attattacag taatttcaga cgtgtggcaa 540aaactcataa ttatctacaa aatattgtcg
tagcaataat tttttatatc tattaatcca 600attattgcta catgctttta taacttgagg
caaaaatatc tatttagcta taacattttg 660ttagaagtaa tttttgtgac tataaagttg
ttattgctac agtaatttca aatgcgtggc 720aaaaaaaata cgattagcta cgaaatttta
ttgtagcaat aaatttgtag ctatttgggt 780aatattgcta cgacagttag caattatagc
aaaaatgcta aatcagcttt gtcgatttaa 840ttttgtagct aattttttta tgaatttgta
aatagctatg aaattttaat ttttgtggct 900attgttaggt attagccaca tatagctaag
aatttgtagc tatatataca taatgttgta 960gtggcaaatt ctaacattgt aagcttggct
gccttttttt ttttttggct acaaaactct 1020aaagtaaagg aactagaaaa ctcgtttggc
gagagaaaga gagagatg 106821060DNANicotiana sylvestris
2aatttatttt tgtaaaactt ctctaatttt tggacaaact cttatattga ttttttaatc
60aaagccaaaa tatttattta actatgaaaa aattttaaca actaatttat tatggtaaat
120aatattgata tggtaacttc aagcacatga caaaaattat aactaactgc agaagtttat
180tgtctctctg aatcttgtgg ctatatcata acaaatactt gtagctaata agccaacgat
240gttctcggtt tcatataatt tgaattttaa aatagttttt aaatttaata tttatttcaa
300atcattattg tggctaacat gttataatcg cagtaatatt tggagatgca atacttatat
360ttagctacaa aattttattg tatcagaata agtttgtagc tattaagtta gtttttgcca
420caaattttta taattgaagc aaaaatactt attcagctac agtattttgt atcgagtaat
480attttgtgac tagaagatta atattgctac agtaatttca gacgtgtggc aaaaactcat
540aattagctac aaaatattgt cgtagcaata attttttata tctattaatg caattattac
600tacatgcttt tataacttga ggcaaaaata tctaatagct ataacatttt gttagaagta
660atttttgtgg ctataaaatt ggtattgcta cagtaatttc aaatgcgtgg caaaaaaata
720cgattaacta cgaaatttta ttgtagcaat aactttgtag ctatttgggt aatattgcta
780cgacagttag caattatagc aaaaatgcta aatcagcttt gtcaatttaa ttttgtagct
840aattttttta tgaaattgta aatagctatg aaattttaat ttttgtggct attgttaggt
900attagccaca tatagctaag aatttgtagc tatatataca taatgttgta gtggcaaatt
960ctaacattgt acgcttggct gccctttttt ttttttttgg ctacaaaact ctaaagtaaa
1020ggaactagaa aactcgtttg gcgagagaaa gagagagatg
106031060DNANicotiana sylvestris 3aatttatttt cgtaaaattt ctctaatttg
gacaaactct tatattgatt tttttaatca 60aagccaaaat atttatttaa ctatgaaaat
tttttaacaa ctaatttatt atggtaaata 120atattgatat ggtaacttca agcacatgat
aaaaattata actaactgca gaagtttact 180gtctctttga atcttgtggt tatatcattc
tatcataaca aatacttgta gctaataagc 240caacgatgtt ctcggtttca tataatttga
attttaaaat agtttttaaa tttaatattt 300atttcaaatt attattgtgg ctaacatgtt
ataaccgcag taatatttgg agatgcaata 360cttatattta gcttgaaaat tttattgtat
cagaacaagt ttgtagctat taagttagtt 420tttgccacaa atttttataa ttgaagcaaa
aatacctatt cagctacagt attttgtatc 480gagtaatatt ttgtgactag aagattaata
ttgctacagt aatttcagac gtgtggcaaa 540aactcataat tagctacaaa atattgtcgt
agcaataatt ttttatatct attaatccaa 600ttattgctac atgcttttat aacttgaggc
aaaaatatct atttagctat aacattttat 660taaaagtaat ttttgtggct ataaagttgt
tattgctaca gtaatttcaa atgcgtggca 720aaaaaaatac gattagctac gaaattttat
tgtagcaata aatttgtagc tatttgggta 780atattgctac gacagttagc aattatagca
aaaatgctaa attagctttg ttaatttaat 840tttgtagcta aactttttta tgaaatttta
atttttgtgg ctattgatag gtattagcta 900caattttcat atatgtagct aagaatttgt
agctatatat acataatgtt gtagtggcaa 960attctaacat tgtacgcttg gctgcccttt
ttttttggct acaaaactct aaagtaaagg 1020aactagaaaa ctcgtttggc gagagaaaga
gagagagatg 106041087DNANicotiana sylvestris
4aatttatttt cgtaaaattt ctctaatttt tggacaaact cttatattgg ttttttaatc
60aaagccaaaa tatttattta actatgaaat tttgttgaac aactaattta ttatggtaaa
120taatattgat atggtaactt caagcacatg acaaaaatta taactaactg cagaagttta
180ctgtctctct gaatcttgtg gctatatcat tctatataac aaatacttgt agctaataag
240ccaacgatgt tctcggtttc atataatttg aattttaaaa tagtttttaa atttaatatt
300tatttcaaat cattattgtg gctagcatgt tataaccgca gtaatatttg gagatgcaat
360acttatattt agctacaaaa ttttattgta tcagaataag tttgtaacta ttaagttagt
420ttttgccaca aatttttata attgaagcaa aaatacctat tcagctacga tattttgtat
480cgagtaatat tttgtgacta gaagattaat attgctagag taatttcaga cgtgtggcaa
540aaactcataa ttagctacaa aatattgtcg tagcaataat tgtttatatc tattaatcca
600attattgcta tatgctttta taacttgagg caaaaatatt tatttagcta taacattttg
660ttagaagtaa tttttgtggc tataaagttg ttattgctac ggtaatttca aatgcgtggc
720aaacaaatac gattagctac gaaattttat tgtagcaata aatttgtagc tatttgggta
780atattgctac gacagttagc aattatagca aaaatgctaa attagctttg tcaatttaat
840tttttagcta aattttttta taaaattgta aatagccatg aaattttaat ttttgtggct
900attgttaggt attagccaca attttcatat atgtatctaa gaatttgtag ctatatatac
960ataatgttgt agtggcaaat tctaacattg taagcttagc tgcccttttt tttttttttt
1020tttggctaca aaactctaaa gtaaaggaac tagaaaactc gtttggcgag agaaagaggg
1080atccatg
108752896DNAArtificial sequenceTPS02b promoter/uidA gene fusion
5aagcttaatt tatttttgta aaacttctct aatttttgga caaactctta tattgatttt
60ttaatcaaag ccaaaatatt tatttaacta tgaaaaaatt ttaacaacta atttattatg
120gtaaataata ttgatatggt aacttcaagc acatgacaaa aattataact aactgcagaa
180gtttattgtc tctctgaatc ttgtggctat atcataacaa atacttgtag ctaataagcc
240aacgatgttc tcggtttcat ataatttgag ttttaaaata gtttttaaat ttaatattta
300tttcaaatca ttattgtggc taacatgtta taatcgcagt aatatttgga ggtgcaatac
360ttatatttag ctacaaaatt ttattgtatc agaataagtt tgtagccatt aagttagttt
420tcgccacaaa tttttataat tgaagcaaaa atacttattc agctacagca ttttgtatcg
480agtaatattt cgtgactaga agattaatat tgctacagta atttcagacg tgtggcaaaa
540actcataatt agctacaaaa tattgtcgta gcaataattt tttatatcta ttaatgcaat
600tattactaca tgcttttata acttgaggca aaaatatcta atagctataa cattttgtta
660gaagtaattt ttgtggctat aaaattggta ttgctacagt aatttcaaat gcgtggcaaa
720aaaatacgat taactacgaa attttattgt agcaataact ttgtagctat ttgggtaata
780ttgctacgac agttagcaat tatagcaaaa atgctaaatc agctttgtca atctaatttt
840gtagctaatt tttttatgaa attgtaaata gctatgaaat tttaattttt gtggctattg
900ttaggtatta gccacatata gctaagaatt tgtagctata tatacataat gttgtagtgg
960caaattctaa cattgtacgc ttggctgccc tttttttttt ttttggctac aaaactctaa
1020agtaaaggaa ctagaaaact cgtttggcga gagaaagagg gatcccccgg gtggtcagtc
1080ccttatgtta cgtcctgtag aaaccccaac ccgtgaaatc aaaaaactcg acggcctgtg
1140ggcattcagt ctggatcgcg aaaactgtgg aattgatcag cgttggtggg aaagcgcgtt
1200acaagaaagc cgggcaattg ctgtgccagg cagttttaac gatcagttcg ccgatgcaga
1260tattcgtaat tatgcgggca acgtctggta tcagcgcgaa gtctttatac cgaaaggttg
1320ggcaggccag cgtatcgtgc tgcgtttcga tgcggtcact cattacggca aagtgtgggt
1380caataatcag gaagtgatgg agcatcaggg cggctatacg ccatttgaag ccgatgtcac
1440gccgtatgtt attgccggga aaagtgtacg tatcaccgtt tgtgtgaaca acgaactgaa
1500ctggcagact atcccgccgg gaatggtgat taccgacgaa aacggcaaga aaaagcagtc
1560ttacttccat gatttcttta actatgccgg aatccatcgc agcgtaatgc tctacaccac
1620gccgaacacc tgggtggacg atatcaccgt ggtgacgcat gtcgcgcaag actgtaacca
1680cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc agcgttgaac tgcgtgatgc
1740ggatcaacag gtggttgcaa ctggacaagg cactagcggg actttgcaag tggtgaatcc
1800gcacctctgg caaccgggtg aaggttatct ctatgaactg tgcgtcacag ccaaaagcca
1860gacagagtgt gatatctacc cgcttcgcgt cggcatccgg tcagtggcag tgaagggcga
1920acagttcctg attaaccaca aaccgttcta ctttactggc tttggtcgtc atgaagatgc
1980ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg cacgaccacg cattaatgga
2040ctggattggg gccaactcct accgtacctc gcattaccct tacgctgaag agatgctcga
2100ctgggcagat gaacatggca tcgtggtgat tgatgaaact gctgctgtcg gctttaacct
2160ctctttaggc attggtttcg aagcgggcaa caagccgaaa gaactgtaca gcgaagaggc
2220agtcaacggg gaaactcagc aagcgcactt acaggcgatt aaagagctga tagcgcgtga
2280caaaaaccac ccaagcgtgg tgatgtggag tattgccaac gaaccggata cccgtccgca
2340aggtgcacgg gaatatttcg cgccactggc ggaagcaacg cgtaaactcg acccgacgcg
2400tccgatcacc tgcgtcaatg taatgttctg cgacgctcac accgatacca tcagcgatct
2460ctttgatgtg ctgtgcctga accgttatta cggatggtat gtccaaagcg gcgatttgga
2520aacggcagag aaggtactgg aaaaagaact tctggcctgg caggagaaac tgcatcagcc
2580gattatcatc accgaatacg gcgtggatac gttagccggg ctgcactcaa tgtacaccga
2640catgtggagt gaagagtatc agtgtgcatg gctggatatg tatcaccgcg tctttgatcg
2700cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc gattttgcga cctcgcaagg
2760catattgcgc gttggcggta acaagaaagg gatcttcact cgcgaccgca aaccgaagtc
2820ggcggctttt ctgctgcaaa aacgctggac tggcatgaac ttcggtgaaa aaccgcagca
2880gggaggcaaa caatga
2896612DNAArtificial sequenceMotif 1 6kktcgtwgca wt
12710DNAArtificial sequenceMotif 2
7agtaatwtyw
10810DNAArtificial sequenceMotif 3 8ttgtagcwaw
109171DNANicotiana
sylvestrisCDS(1)..(171) 9atg agt caa tca att tct cca tta atg ttt tct cac
ttt gca aaa ttt 48Met Ser Gln Ser Ile Ser Pro Leu Met Phe Ser His
Phe Ala Lys Phe 1 5 10
15 cag tcg aat att tgg aga tgc aat act tct caa ctc aga
gtt ata cac 96Gln Ser Asn Ile Trp Arg Cys Asn Thr Ser Gln Leu Arg
Val Ile His 20 25
30 tca tca tat gcc tct ttt gga ggg aga aga aaa gag aga
gta aga aga 144Ser Ser Tyr Ala Ser Phe Gly Gly Arg Arg Lys Glu Arg
Val Arg Arg 35 40 45
atg aat cga gca atg gat ctt tct tca
171Met Asn Arg Ala Met Asp Leu Ser Ser
50 55
1057PRTNicotiana sylvestris 10Met Ser Gln Ser Ile Ser Pro Leu
Met Phe Ser His Phe Ala Lys Phe 1 5 10
15 Gln Ser Asn Ile Trp Arg Cys Asn Thr Ser Gln Leu Arg
Val Ile His 20 25 30
Ser Ser Tyr Ala Ser Phe Gly Gly Arg Arg Lys Glu Arg Val Arg Arg
35 40 45 Met Asn Arg Ala
Met Asp Leu Ser Ser 50 55 1129DNAArtificial
sequenceprimer 11ctaagcttaa tttatttttg taaaacttc
291230DNAartificial sequenceprimer 12atggatccct ctttctctcg
ccaaacgagt 301329DNAArtificial
sequenceprimer 13tggatcctga agaaagatcc attgctcga
291444DNAartificial sequenceprimer 14ctaatacgac tcactatagg
gctcgagcgg ccgcccgggg aggt 44158DNAartificial
sequenceprimer 15acctcccc
81627DNAartificial sequenceprimer 16ggatcctaat acgactcact
atagggc 271726DNAartificial
sequenceprimer 17agaggcatat gatgagtgta taactc
261818DNAartificial sequenceprimer 18ctatagggct cgagcggc
181925DNAartificial
sequenceprimer 19acctccaaat attcgactga aattt
252025DNAartificial sequenceprimer 20gtcatgtgct tgaagttacc
atatc 252126DNAartificial
sequenceprimer 21gttaccatat caatattatt taccat
26221796DNANicotiana sylvestris 22gaattcacta gtgatttgag
ctcaaagagg tgaaacctaa tctagtatgc aaaccatgtt 60aaattctcaa ttgttttgat
agataatgag ttttctgata attaataaat tattagataa 120ttaaaggacc aaatttatat
gacttttgtt ttttatcatc ttgatcatat atacaatgta 180atggatacaa gcttatagtt
gtataaattc tatataatta gttattcata cattaattag 240atatattcaa ttgttcttta
taaatataat tcaaacctga aagcaatact tattttgtaa 300gaattgcaat attgttattt
tgttatggac ttaaatatta accatgttat aatcttaagt 360ttatattatt agaaaaactt
agtttttgaa agactaatat gaacattagt acttatttca 420aaaataagcg cttagatata
tgaaattact ttaagtactt atttaaaata attaagtacc 480acacatacat acatatctct
acaaactgtt aaagttttct atatgagtac ttattttaaa 540ataagagcat aaatataata
aattatgtta aattcttatt taaaataata aaggaccaaa 600catgcataaa ataaagtatg
agcttaataa gtcaagaagc taattgataa gcattgatgc 660caaatgcact tactaactgt
tctatattgt aggaaaaatc taacttttat attaaaaatt 720tattttcata aaacttccct
aatttttgaa caaaatctta tattgatttt ttaatcaaag 780ccaaaatatt tatttaacta
tgaaaatttt ttaacaacta atttattatg gtaaataata 840ttgatatggt aactttcagc
acatgacaaa aattataact aactgcagaa gtttactgtc 900tctctgaatc ttgtggctat
gtcattctat cataacaaat acttgtagct aatacgccaa 960cgatgttctc gatttcatat
aatttgaatt ttaaaatagc ttttaaattt aatatttatt 1020tcaaatcatt attgtgacta
acatgttata accgcagtaa tatttggaga tgcaatactt 1080atatttagct acaaaatttt
attgtatcat aataagtttg tagctattaa gttagttttt 1140gccacaaatt tttataattg
aagcaaaaat acctattcaa ctacaatatt ttgtatcgag 1200taatattttg tgactagaag
attaatatta ttacagtaat ttctgacgtg tggcaaaaac 1260tcataattat ctacaaaata
ttgtcgtagc aataattttt tatatctatt aatccaatta 1320ttgctacatg cttttataac
ttgaggcaaa aatatctatt tagctataac attttgttag 1380aagtaatttt tgtgactata
aagttgttat tgctacagta atttcaaatg cgtggcaaaa 1440aaaatacgat tagctacgaa
attttattgt agcaataaat ttgtagctat ttgggtaata 1500ttgctacgac agttagcaat
tatagcaaaa atgctaaatc agctttgtcg atttaatttt 1560gtagctaatt tttttatgaa
tttgtaaata gctatgaaat tttaattttt gtggctattg 1620ttaggtatta gccacatata
gctaagaatt tgtagctata tatacataat gttgtagtgg 1680caaattctaa cattgtaagc
ttggctgcct tttttttttt ttgggctaca aaactctaaa 1740gtaaaggaac tagaaaactc
gtttggcgag agaaagaggg atccataatc gaattc 17962336DNAartificial
sequenceprimer 23tgagctcaaa gaggtgaaac ctaatctagt atgcaa
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