WO2000070065A1 - PROMOTER OF THIOREDOXINE TaTrxh2 IN WHEAT - Google Patents

Abstract

The invention relates to the cloning of the promoter of thioredoxine TaTrxh2 in wheat and the use thereof in controlling the expression of the gene of interest in transgenic plants or plant cells.

Description

 THIOREDOXIN PROMOTER WHEAT TaTrxh2

The invention is: relating to the cloning and characterization of a wheat thioredoxin promoter.

Thioredcxins are proteins of low molecular weight, which have been demonstrated in a large number of organisms, where they catalyze different oxidation-reduction reactions involving dithiolsulfhydryl exchanges. Their catalytic site includes the conserved sequence: -Trp-Cys-Gly / Pro / Ala- Pro-Cys-. The thioredoxins in oxidized form comprise a disulfide bridge, the reduction of which into -SH groups, by reduced ferredoxin or by NADPH, is catalyzed via a specific system.

In plants, 3 types of thioredoxin have been highlighted: the first 2 (thioredoxins m and f), are ferredoxin-dependent thioredoxins, located in chloroplasts, where they are involved in the regulation of photosynthesis. A third type, called thioredoxin h, has been identified in the cytosol. Thioredoxin h is part of an NADP-dependent thioredoxin system (NTS), where it is associated with NADPH and an enzyme called NADP-thioredoxin reductase (NTR).

Initially, 2 h thioredoxins were extracted and partially purified from the wheat grain (VOGT and FOLLMANN, Biochem. Biophys. Acta 873, 415-418, 1986). Recently, the team of Inventors has isolated and characterized 2 cDNA clones encoding a common wheat thioredoxin h (TaTrxhl), and a durum wheat thioredoxin h (TdTrxhl) (GAUTIER et al., Eur. J. Biochem. 252 , 314-324, 1998). The primary structures deduced from the cDNA clones of the thioredoxins h TaTrxhl and TdTrxhl are very conserved (96% identity between them). They have an N-terminal extension very rich in Ala residues, the analysis of which reveals a putative transmembrane domain of 20 residues. They have strong homologies with cereal thioredoxins h (70 to 80%) and dicotyledonous thioredoxins h (60%).

The thioredoxins h intervene during the germination of the wheat grain, where they participate, at the level of the endosperm, in the mobilization of the reserves necessary for the growth of the embryo. They act in particular: by reducing the disulphide bridges of certain reserve proteins, such as gliadins and glutenins (KOBREHΞL et al., Plant Physiol. 99,

919-924, 1992), which increases their sensitivity to proteolysis;

- by reducing enzymes involved in the mobilization of reserves, or inhibitors of these enzymes, which leads to the activation of the former, and the deactivation of the latter.

It has also been proposed to use thioredoxins h to improve the quality of food, in particular cereal-based; it has in fact been found that they favor the formation of dough during the production of bread (ONG et al. _r Cereal Chem. 70, 113-114, 1993), and that in addition they decrease the allergenicity of certain foods.

The inventors have undertaken the study of the expression of thioredoxins h in cereal seeds, in particular in wheat, in order to provide means of controlling this expression.

In the context of this work, they isolated a gene hereinafter called Ta Trxh2, coding for a common wheat thioredoxin h (Tri ticum aestivum), hereinafter called TaTrxh2, the primary structure of which has 97% similarity to that of thioredoxin h TaTrxhl of common wheat (GAUTIER et al., 1998, publication cited above).

The inventors also isolated the promoter of the Ta Trxh2 gene, and expressed, in rice, the reporter gene gus under the control of this promoter. They thus observed that the expression of the reporter gene was localized exclusively in the grain of rice and more particularly in the starchy endosperm. They also highlighted regions involved in the spatial and temporal regulation of this promoter.

The sequence of the Ta Trxh2 gene and of the 5 'region comprising the promoter are represented in the sequence list in the appendix under the number SEQ ID NO: 1. The subject of the present invention is a promoter consisting of a nucleic acid fragment comprising at least one functional domain specific for the promoter of the Ta Trxh2 gene.

The term “promoter” is understood to mean a double-stranded DNA sequence comprising at least the sequences necessary for the initiation of transcription of a gene, optionally associated with cis-control sequences of said transcription; the expression “functional domain specific to a promoter” means a sequence of said promoter comprising one or more DNA motifs involved in the initiation of transcription, or else a double-stranded DNA sequence constituting a regulatory domain comprising one or more of the DNA motifs involved in the cis control of transcription by said promoter. Promoters in accordance with the invention can comprise in particular: a) the nucleic acid fragment represented in the annexed sequence list by the sequence SEQ ID NO: 2, as well as in FIG. 1, and which corresponds to the region 5 'non-coding of the Ta TrxhΣ gene extending from position -1 to position -1111 relative to the ATG initiation codon, or portions of said fragment, in particular:

* the nucleic acid fragment whose sequence extends from position -1 to position -83 relative to the ATG codon of the Ta Trxh2 gene; this fragment includes the sequences involved in the initiation of transcription, and necessary for the basic activity of the promoter;

* nucleic acid fragments comprising functional domains involved in the regulation of the transcription of the Ta Trxh.2 gene, and in particular in its tissue specificity and / or in its expression at different stages of plant development; these are in particular: - the nucleic acid fragment whose sequence extends from position -591 to position -1111 relative to the ATG codon of the Ta Trxh2 gene; this fragment comprises a regulatory domain involved in the inhibition of the expression of the Ta Trxh2 gene in the epithelium of the scutellum; the nucleic acid fragment whose sequence extends from position -228 to position -451 relative to the ATG codon of the TaTrxh2 gene; this fragment comprises a regulatory domain involved in the induction of the expression of the Ta Trxh2 gene at the start of grain maturation; the nucleic acid fragment whose sequence extends from position -451 to -591 relative to the ATG codon of the Ta Trxh2 gene; this fragment comprises a regulatory domain involved in the induction of the expression of the Ta TrxhΣ gene in the epithelium of the scutellum; the nucleic acid fragment whose sequence extends from position -83 to position -228 relative to the ATG codon of the Ta Trxh2 gene; this fragment includes sequences involved in the induction of expression at the starchy endosperm. b) of the nucleic acid fragment constituting the first intron (positions 1232-2203 on the sequence SEQ ID NO: 1) of the Ta Trxh2 gene; this fragment could include a type of regulatory domain enhancer, increasing the level of expression of the Ta Trxh2 gene.

Those skilled in the art can, from the fragments comprising at least one functional domain of the promoter of the Ta Trxh2 gene specified above, more precisely identify the limits of these functional domains, as well as the DNA motifs involved in the function of each of them, by techniques known in themselves, for example by the technique of DNA fingerprints (footprints), by incubating these fragments with nuclear extracts of grain albumen cells, as well as with nuclear extracts of cells in which the promoter of the Ta Trxh2 gene is inactive.

The invention encompasses in particular any promoter obtainable from a nucleic acid fragment comprising at least one functional domain of the promoter of the Ta Trxh2 gene, by conventional techniques of genetic engineering, in particular by mutagenesis and / or genetic recombination . It is thus possible to produce artificial promoters having the level of activity, and the degree of specificity desired.

It is thus possible, for example, to deactivate one or more of the functional regulatory domains located in the 5 ′ non-coding region of the Ta Trxh2 gene, for example by deleting at least one nucleotide or a nucleotide sequence of the motifs d 'DNA involved in the function of the domain (s) concerned. It is also possible to associate the nucleic acid molecules comprising functional domains of the promoter of the Ta Trxh2 gene together, and / or with functional domains originating from promoters other than that of the Ta Trxh2 gene.

According to a preferred embodiment of a promoter in accordance with the invention, it comprises at least the Ta Trxh2 promoter sequences which allow a specific expression in the grain, especially in starchy endosperm.

The invention also includes: expression cassettes, comprising, in addition to a promoter in accordance with the invention, a gene of interest placed under transcriptional control of said promoter, or a site allowing the insertion of said gene of interest; recombinant vectors, resulting from the insertion of a promoter or of an expression cassette in accordance with the invention in a host vector.

The promoters in accordance with the present invention can be used to control the expression of a gene of interest in plant cells, in particular of monocots.

Said gene of interest can for example be either the Ta Trxh2 gene, placed under the control of an artificial promoter, as defined above, derived from the promoter

Ta Trxh2, either a heterologous gene encoding a thioredoxin other than TaTrxh2, or any other protein of interest.

It is possible, for example, to introduce a gene of interest under the control of the promoter of the Ta Trxh2 gene, or of an artificial promoter constructed from the regulatory elements thereof which confer specificity of expression in the cells of albumen. , in order to express said gene of interest only in the endosperm cells of the grain. It is also possible to selectively delete the sequences of the promoter of the Ta Trxh2 gene responsible for the specificity of expression, in order to construct an artificial promoter making it possible to ensure ubiquitous expression of a thioredoxin h, or of another protein d 'interest.

The subject of the invention is also plant cells and transgenic plants, in particular monocots, and in particular cereals, transformed with at least one nucleic acid molecule comprising a promoter according to the invention.

The inventors have thus obtained transgenic rice, in which a heterologous gene has been placed under transcriptional control of the gene promoter.

Ta Trxh2 and observed in these plants a specific expression in the endosperm cells of the grain.

The transformed cells and the transgenic plants in accordance with the invention can also be used as models for studying and / or modifying the expression of different genes in the albumen cells of the grain.

The present invention will be better understood using the additional description which follows, which refers to nonlimiting examples illustrating the cloning and characterization of the Ta Trxh2 gene and its promoter. EXAMPLE 1: ISOLATION AND CHARACTERIZATION OF THE TaTrx GENE Σ

1. - Screening of a wheat genomic DNA bank A genomic DNA bank was produced from DNA extracted from common wheat leaves (Tri ticum aestivum) of the Andain variety. After partial digestion of genomic DNA with Mbol, the medium-sized fragments 15 kb were cloned at the BamH.1 site of the phage EMBL3 SP6 / T7, which was propagated in the host bacterium K802 -K 802 (galK2, gal T22 , HsdR2, (r k-, mk +), mcrA,

+ mcrB, metBl, mrr, supE44).

6

6.10 clones of the genomic DNA library were spread and screened with a 669 bp probe (TRX) containing the entire sequence coding for thioredoxin h of common wheat TaTrxhl (GAUTIER et al., 1998, cited above), and the positive clones were then screened by PCR (polymerase chain reaction) using a pair of primers (THP2 and THM2) derived from the same sequence. One of the selected clones (λ4), which contains a wheat genomic DNA fragment of approximately 10 kb, was digested with PstI, releasing two fragments, one of 1.5 kb and the other of 3, 8 kb, both recognized by the TRX probe. These two fragments were cloned into the vector pLITMUS 29 (BIOLAB) at the PstI restriction site. The two clones obtained are called CTRX3 and CTRX4. The clone CTRX3 corresponds to the 1.5 kb fragment and the clone CTRX4 to the 3.8 kb fragment. Analysis of the nucleotide sequences of clones CTRX4 and CTRX3 shows that they each contain part of the same gene coding for a wheat thioredoxin h, truncated during digestion with PstI.

From the nucleotide sequences of these 2 clones, the inventors chose two primers (THP8 and THM8) making it possible to amplify a thioredoxin h gene over a length of approximately 2.6 kb. The PCR was carried out on the undigested DNA of the λ4 clone, and a fragment of the expected size was cloned in the vector pGEM-T (PROMEGA). The clone obtained contains the Ta Trxh2 gene coding for a common wheat thioredoxin h.

It includes a promoter region of 1111 bp, a coding region of 1447 bp, and a 3 'non-coding region of 131 bp. The coding region of the Ta TrxhΣ gene comprises three exons of 120, 123 and 135 bp separated by two introns, of 972 bp and of 93 bp. The first exon codes for a 40 amino acid polypeptide, the second exon codes for a 41 amino acid polypeptide containing the active site, and the third exon codes for a 45 amino acid polypeptide.

The nucleotide sequence of the Ta Trxh2 gene codes for a common wheat thioredoxin h, named TaTrxh2, of 126 amino acids, of calculated molecular mass 13435 Da and of calculated pi 5.0. The comparison of the sequences of the translation products of the Ta Trxh2 gene and of the Ta Trxhl genes previously described by GAUTIER et al. (1998, publication cited above) and TdTrxhl (thioredoxin h of durum wheat) shows that they are very conserved. Indeed, the peptide sequence of TaTrxh2 has 97% similarity and 94% identity with that of TaTrxhl and 95% similarity and 90% identity with that of TdTrxhl.

The N-terminal domain of TaTrxh2 is shorter than that of TaTrxhl and TdTrxhl. The primary structure of TaTrxh2 does not contain a signal peptide, suggesting that the protein is localized in the cytoplasm. However, it has an N-terminal extension already demonstrated in the primary structure of TaTrxhl and TdTrxhl, which may correspond to a transmembrane domain. Analysis of the N-terminal extension of TaTrxh2 with the RAO ARGOS program (PC / gene, RAO et al., Biochem. Biophys. Acta 869, 197-214, 1986) reveals a putative transmembrane domain between residues 2 and 19. The active site, formed of the following 5 amino acids: WCGPC, is conserved between the 3 h thioredoxins of wheat TaTrxh2, TaTrxhl and TdTrxhl.

The introns have different sizes from those of the wheat thioredoxin h gene introns previously highlighted by ROBERT (1994) indicating that the Ta Trxh2 gene is different from these. The introns of the Ta Trxh2 gene are of type 0 and are limited in 5 'by the GTA sequence and in 3' by the CAG sequence, which correspond to consensus sequences of the intron-exon limits.

The 3 'non-coding region of the Ta Trxh2 gene presents the AATAAA polyadenylation signal common to the genes transcribed by RNA polymerase II. EXAMPLE 2: STRUCTURAL ANALYSIS OF THE TaTrxhΣ GENE PROMOTER

The promoter of the Ta TrxhΣ gene was analyzed to find putative regulatory motifs capable of intervening in the control of expression, and was notably compared to that of the thioredoxin h genes of C. reinhardtii (STEIN et al., Plant Mol. Biol. 28, 487-503, 1995), tobacco (BRUGIDOU et al., Mol. Gen. Genêt 238, 285-293, 1993) and rice (ISHIWATARI et al., 1995), thioredoxin m from C. reinhardtii (STEIN et al., 1995), and murine thioredoxins (MATSUI et al., Gene 152, 165-171, 1995) and human (TONISSEN et al., Gene 102, 221-228, 1992; KAGHAD et al., Gene 140, 6643-6653, 1994). The Ta Trxh2 gene promoter sequence is shown in Figure 1.

The transcription initiation site (represented in FIG. 1 in bold and underlined with a double line) is an adenine located at -65 bp from the ATG. The Ta Trxh2 gene promoter does not contain a consensus sequence corresponding to a TATAn box or a CAAT box at the positions expected for the genes transcribed by RNA polymerase II.

On the other hand, it contains a TATA-like box (AATTTAT, underlined with a double line in FIG. 1) at -105 bp from ATG.

It also contains a GC box (GGGCCGGG, underlined in dotted lines in FIG. 1) located at -84 bp from the ATG of the Ta Trxh2 gene. The GC boxes are recognized by Spl type transcription factors (DYNAN et al. Nature 316, 774-778, 1985), and are involved in the constitutive expression of genes. GC boxes are present in all known promoters of thioredoxin genes. A sequence rich in adenine, interrupted by a residue G (AAAAAAAGAAAAAAAAA, in bold type underlined with a single line in FIG. 1), is located at -227 bp from the ATG of the Ta Trxh2 gene; sequences of this type have also been identified previously in the promoters of the αe thioredoxin h genes of tobacco and rice.

BHLH (CANNTG) sequences, recognized by transcription factors of the helix / loop / helix family, are located at -206 bp and -411 bp from the ATG of the Ta Trxh2 gene; they are shown in Figure 1 in lowercase letters.

Bzip sequences (ACGT, underlined with a single line in FIG. 1) recognized by transcription factors of the leucine zipper family (bZIP), are located at -251 bp and -184 bp in ATG of the Ta Trxh2 gene. The bZIP proteins have been described in the activation of the expression of genes encoding grain reserve proteins. ACGT motifs have also been described in ABRE consensus sequences (ABA-responsive element) of gene promoters whose expression is regulated by abscissic acid (ABA) (MUNDY et al., Proc. Natl. Acad. Sci USA, 87, 1406-1410, 1990).

Two pyrimidine boxes (CCTTTCTCT and TCTTTCTTC, boxed in FIG. 1) are respectively located at -553 bp and -541 bp from the ATG of the Ta Trxh2 gene. The pyrimidine boxes (CCTTTT) are involved in the regulation of expression by giberellic acid, generally in association with GARE (GA-responsive element) (TAACAAA) sequences (HUANG et al., Plant Mol. Biol. 14, 115 -121, 1990), and 02S sequences (opaque-2-binding sequence) or box I (TATCCAT) (GUBLER et al., Plant Cell 4, 1435-1441, 1992; LANAHAN et al., Plant Cell, 4, 203-211, 1992), with which they are organized into a complex called GARC (GA-responsive complex) (BETHKE et al., Bot. 48, 1337-1356, 1997). No GARE or 02S sequence was demonstrated in the 1111 bp upstream of the AT Tr of the Ta Trxh2 gene.

A TGTGTGAGCA motif (in bold type, and underlined with a dotted line in FIG. 1) is located at -403 bp from the ATG of the Ta Trxh2 gene. This motif differs only in the presence of an additional G residue, of the consensus sequence "GCN4-like" (TGTGTGACA) of the "albumen box" involved in the albumen-specific expression of wheat glutenin genes (HAMMOND- KOSACK et al., EMBO J. 12, 545-554, 1993). However, the other motif of the albumen box, called EM (TGTAAAAGT), and the presence of which is also necessary for the albumen-specific expression (ALBANI et al., Plant Cell 9, 171-184, 1997), is not has not been demonstrated in the 1111 bp upstream of the ATG of the Ta TrxhΣ gene.

Trimeric diades CAA and TTG (in italics in FIG. 1) separated by 10 bases, are present at -107 bp and -97 bp respectively from the ATG of the Ta Trxh2 gene. These motifs are associated with a specific expression in the aleurone layer (THOMAS et al., Plant Cell 2, 1171-1180, 1990).

EXAMPLE 3: FUNCTIONAL ANALYSIS OF THE TaTrxh GENE PROMOTER. 2.

The sequence of 1111 bp at 5 ′ of the ATG of the Ta Trxh2 gene, or different fragments of this sequence, were cloned upstream of the coding sequence of the reporter gene gus in the vector pSPORT1-GUS. The vector pSPORTl-GUS (DIGEON, 1997) contains the coding sequence of the gus gene (β-glucuronidase E 'E. coli) and the nos-ter terminator of the nopaline synthase gene, inserted at the EcoRI-HindIII site of the vector pSPORTl ( GIBCO BRL).

The constructions carried out are as follows:

- PI: this construction comprises the entire sequence of 1111 bp upstream of the ATG of the Ta Trxh2 gene; - P2: this construction comprises the sequence of 589 bp upstream of the ATG of the Ta Trxh2 gene;

- P3: this construction comprises the sequence of 481 bp upstream of the ATG of the Ta Trxh2 gene; - P4: this construction comprises the sequence of 228 bp upstream of the ATG of the Ta Trxh2 gene;

- P5: this construction comprises the 83 bp sequence upstream of the ATG of the Ta Trxh2 gene.

The limits of the regions of the promoter of the Ta Trxh2 gene used in the constructions are indicated in FIG. 1.

As a positive control, the vector pUGCl was used (CHAIR et al., 1996), which allows the constitutive and ubiquitous expression of the gus gene under the control of the promoter, the first exon and the first intron of the gene encoding corn ubiquitin.

The vector pSPORTl-GUS was used as a negative control.

These different constructions were transferred by bombardment according to the protocol described by FAUQUET et al. (Proc. Third. Int. Rice Genêt. Symp., Ed. GS Khush, 153-165, 1996), in young embryogenic rice calluses (var. Japonica IRAT 349) derived from the proliferation of the scutellum of the mature embryo . All the constructs tested were co-transferred with the vector pILTAB227 (FAUQUET et al., 1997), which confers resistance to hygromycin and which allows the selection of transformed cells.

A mixture of the vector carrying the construction to be tested, and of the vector pILTAB227 (molar ratio: vector to be tested / pILTAB227 = 4/1) is used to coat microparticles of gold, at a rate of 5 μg of total DNA (3 μg of DNA to be tested + 2 μg of pILTAB227) at a concentration of 1 μg / μl, for 3 mg of a mixture in equal quantity of gold microparticles with diameters 1.0 μ and 1.6 μm, suspended in 50 μl of distilled water.

The bombardment is carried out using a PDS-1000 / He particle gun (PARTICULE DELIVERY SYSTEM, BIORAD).

The bombarded embryogenic calluses are then screened on a selection medium containing hygromycin. Callals resistant to hygromycin are selected and placed on a regeneration medium devoid of hygromycin. The regenerated plants (generation F0) were then transferred to pots, and, after acclimatization in phytotron, are grown in the greenhouse.

The expression of the gus gene was sought in the vegetative organs and in the seeds of rice of the generations T0 and Tl. Only the fertile plants and having a normal phenotype, were retained for the analysis. The integration of the transgene in the plants analyzed was verified by PCR 'and Southern blot. The detection of β-glucuronidase activity was carried out by histochemical test, detecting the blue coloration resulting from the hydrolysis of 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid (X-GLU ), and its quantification was carried out by fluorometric test, by measuring 4-methylumbelliferone (MU) formed from 4-methylumbellirefyl β-glucuronic acid, according to the protocols described by JEFFERSON et al. (Plant Mol. Biol. Report 5, 387-405, 1987). 1. Expression of the gus gene in the vegetative organs The analysis was carried out on the roots, stubble, and leaves.

In the case of untransformed rice plants, or those transformed with the vector pSPORTl-GUS, the histochemical analysis does not reveal any GUS activity, and the fluorimetric measurements show only very little or no activity. In the case of rice plants transformed with the vector pUGCI, a high GUS activity (greater than 500 pmol MU / min / mg of protein) is observed in all the vegetative organs tested. In the case of rice plants transformed with the constructs PI, P2, P3, P4 or P5, no coloration is observed in the vegetative organs incubated in the presence of X-GLU, and the GUS activity measured by fluorometry is not not significantly different from those measured for plants transformed with the vector pSPORTl-GUS or untransformed plants. These results show that the promoter of the Ta Trxh2 gene does not allow the expression of the gus gene in the vegetative organs. 2. Expression of the gus gene in grains

Histochemical analysis

Whole grain

The rice grains, taken at 35 JAF (days after fertilization) were cut longitudinally and then incubated in the presence of X-GLU.

No coloration is detected in the rice grains transformed with the vector pSPORTl-GUS.

On the contrary, an intense coloration of the whole grain is detected for the rice grains transformed with the vector pUGCI.

In the case of rice grains transformed with the constructions PI, P2, P3 or P4, a blue coloration is detected in the albumen of the grains, but not in the embryo (cotyledon axis and scutellum), the envelopes or the spikelets. At the albumen level, this coloration appears in particular at the periphery of the embryo, above the epithelium of the scutellum, and in a median zone of the albumen over the entire length of the grain. This coloration is less intense, and appears less quickly than that observed in rice grains transformed with pUGCI. The intensity of the coloring seems to vary depending on the construction (PI, P2, P3 or P4) concerned. The highest intensity is observed in rice grains transformed with the P2 construction, and the lowest in those processed with the P construction. These results are confirmed by the analysis of the Tl grains, where the coloration appears more quickly than in the T0 grains and is more intense.

In the case of rice grains transformed with the P5 construction, no coloration is detected, either in the embryo, in the albumen, or in the husks. At the level of the different grain fabrics

To precisely determine the location of the expression of the gus gene under the control of the promoter of the Ta Trxh2 gene, histological sections were made and observed under a light microscope. These observations show that, for the constructs PI, P2, P3 or P4, the labeling is localized in a small number of starchy endosperm cells located at the periphery of the embryo and in the central zone of the starchy endosphere. The cells of the embryo, the aleurone layer and the envelopes are not marked. For construction P5, no marking is visible. Fluorimetric analysis

GUS activity was measured on the one hand on embryos and on the other hand on the endosperm of rice grains.

GUS activity is zero or very weak in rice grains transformed with the vector pSPORTl-

GUS, as in unprocessed rice grains. On the other hand, it is very strong (> 500 pmol / MU / min / mg of protein) in the embryo and the endosperm of rice grains transformed with the vector pUGCI.

The GUS activity measured in the embryos of rice grains transformed with the constructs PI, P2 P3, P4 or P5 is not significantly different from that measured in non-transformed rice grains or of rice transformed with the vector pSPORTl- GUS.

On the other hand, the activity measured in the endosperm of rice grains transformed with the constructions PI, P2, P3 or P4, is 25 to 40 times higher than that measured in the endosperm of untreated or transformed rice grains with the vector pSPORTl-GUS. It varies from 40 pmol / MU / min / mg of protein for the rice grains transformed with the construction P2, to 25 pmol / MU / min / mg of protein for those transformed with the construction P4. For construction P5, no GUS activity is detected in the grains.

These results show that the region (-1111 bp to -83 bp) of the Ta Trxh2 gene promoter allows expression of the gus gene only in starchy endosperm cells, and that only the deletion leaving only 83 bp in upstream of ATG has deleted the sequences responsible for spatial expression, some of which are probably located in the promoter region between -228 bp and -83 bp.

The GCN4-like motif identified during the analysis of the structure of the Ta Trxh2 gene promoter is therefore apparently not solely responsible for the tissue specificity of the expression; in fact, despite its deletion in constructs P3 and P4, the expression of the gus gene remains specific to the endosperm of the grain.

Two sequences: AACAAATCC, and AACAAAGTG (shown in bold in Figure 1), are present at -51 bp and -381 bp, respectively, relative to the ATG of the Ta Trxh2 gene. These sequences have a similarity with AACA motifs (AACAAACTCTATC) recently highlighted in the promoters of 6 genes coding for rice glutelins, and intervening in the albumen-specific expression of these genes.

EXAMPLE 4 EVOLUTION OF THE EXPRESSION OF THE GUS GENE DURING THE DEVELOPMENT OF GRAINS OF TRANSGENIC RICE

The expression of the gus gene was followed during the maturation and germination of rice grains transformed with the constructs PI, P2, P3, P4 or P5. 1. During maturation Three stages were analyzed: 10 JAF, 25 JAF and 35 JAF. The expression of the gus gene was evaluated, either by histochemical localization of the GUS activity, or by detection of the transcripts by Northern blot.

GUS activity Histochemical analysis shows that for the three stages of maturation studied, 10, 25, and 35 JAF, GUS activity is always detected in the starchy endosperm of rice grains transformed with the constructions Pi, P2, P3 or P4 . On the other hand, for the P5 construction, no GUS activity is detected.

At 10 JAF, GUS activity is detected in the starchy endosperm, at the periphery of the embryo. At 25 JAF, the GUS activity progresses towards the middle zone of the starchy endosperm. At 35 JAF, GUS activity is detected on the entire surface of the starchy endosperm.

The intensity of the coloring varies with the nature of the construction and the stage of maturation, in particular in the case of construction P4, for which the coloration is very difficult to detect at the start of maturation.

These results are confirmed by more detailed observations at the level of each tissue of the grain, which show that:

- At 10 JAF: for the PI, P2 or P3 constructs, the GUS activity is very strong in the cells of starchy endosperm at the periphery of the embryo, and it is not detected in cells in the middle area of starchy endosperm. For the P4 construction, the GUS activity in the cells of the starchy endosperm is very low, or even not detectable. In addition, in rice grains transformed with the P2 construct, GUS activity is also detected in the cells of the epithelium of the scutellum.

- At 25 JAF: for the constructions PI, P2 or P3, the GUS activity decreased in the cells of starchy endosperm at the periphery of the embryo and increases in those of the central zone of starchy endosperm; for rice grains transformed with the construction P2, no more GUS activity is detected in the cells of the epithelium of the scutellum. In rice grains transformed with the P4 construct, GUS activity increased in starchy endosperm cells located at the periphery of the embryo and in the central area. - At 35 JAF: the GUS activity is much weaker than at 25 JAF in all the starchy endosperm cells of rice grains transformed with PI, P2 or P3; in rice grains transformed with the P4 construct, GUS activity is detected in cells in the middle zone of starchy endosperm.

For construction P5, no GUS activity is detected whatever the stage of maturation or the tissue of the grain analyzed.

Whatever the construction used, no GUS activity is detected during maturation, in the cells of the embryonic axis, the aleurone layer, or the husks of the grains.

Detection of transcripts of the gus gene

The presence of the transcripts of the gus gene was sought in the total RNAs extracted, at the various stages of maturation, from the rice grains transformed with constructions PI, P2, P3, P4 or P5. Detection was carried out by Northern blot, using the P3 + GUS probe. This 2.6 kb probe comprises the sequence of 481 bp upstream of the ATG of the Ta Trxh2 gene, the coding sequence of the gus gene and the terminator of the nos gene.

For each of the constructions PI, P2, P3, P4 or P5, this probe makes it possible to detect the presence of transcripts whose expected size is between 1.9 and 2.4 kb, depending on the construction. The presence of these transcripts varies according to the construction used for the transformation, and the stage of maturation.

For rice transformed with the PI construction, the transcripts are detected at the 3 stages of maturation with a maximum at mid-maturation.

For rice transformed with construction P2, the transcripts are detected at the start of maturation.

For rice transformed with construction P3, the transcripts are detected at the beginning and at the end of the grain maturation.

For rice processed with construction P4, the transcripts are detected at mid-ripening and at the end of the grain's ripening. For rice processed with construction

P5, no transcript of the gus gene is detected whatever the maturation stage analyzed.

2. During germination

For the study of the expression of the gus gene during germination, the GUS activity was analyzed by histochemistry in rice grains transformed with the constructs PI, P2, P3, P4 or P5.

For each construction, 10 grains were put to germinate in the dark and collected at different times after imbibition: 0, 12, 24, 48 and 72 hours. GUS activity is detected in the starchy endosperm of rice grains transformed with the constructs PI, P2, P3 or P4 regardless of the germination stage. On the other hand, for rice grains transformed with the P5 construction, no GUS activity is detected.

The study of the accumulation of transcripts of the gus gene in the grains transformed with the constructs PI, P2, P3 or P4 shows that these are not accumulated during germination whatever the construction.

These results indicate that the promoter (1111 bp upstream of the ATG) of the Ta Trxh2 gene does not allow the expression of the gus gene during germination. The GUS activity detected in the seed kernels is certainly a residual activity due to the very high stability of β-glucoronidase in the grain.

Conclusion

Analysis of the expression of the gus gene under the control of the promoter of the Ta Trxh2 gene during the development of rice grains transformed with the constructs PI, P2, P3, P4 or P5 shows an effect of the deletions of the promoter of the Ta Trxh2 gene on the temporal and spatial expression of the gus gene in the grains of processed rice.

The PI, P2, P3 constructs allow expression of the gus gene earlier than the P4 construct during maturation. Construction P5 does not allow expression of the gus gene since no transcript is detected. Indeed, transcripts of the gus gene are detected at 10 JAF for the 3 constructions PI, P2, P3, and only 25 JAF for the construction P4. This suggests that the differences in expression level previously reported between the constructs P2 and P4, probably result from a delay in the expression of the gus gene under the control of the P4 promoter, rather than of a lower level of expression. The Ta Trxh2 promoter region between -1111 bp and - 228 bp certainly contains regulatory sequences which allow expression of the gus gene in the early stages of maturation.

With regard to spatial expression, the region of the Ta Trxh2 gene promoter between -1111 bp and -591 bp probably contains a sequence inhibiting the expression of the gene in the epithelium of the scutellum. Indeed, when it is deleted (construction P2) the expression of the gus gene is observed in this tissue. Conversely, the region between -591 bp and -451 bp would contain a sequence activating expression in the epithelium of the scutellum, because when it is deleted (construction P3) there is no more expression of the gus gene in this tissue.

The results show that during the maturation of the rice grains, the promoter of the TaTrxh2 gene allows expression of the gus gene specific for starchy endosperm. This expression is detected in a limited number of cells distributed in a central area of the endosperm and at the periphery of the embryo.

Claims

1) Promoter characterized in that it consists of a nucleic acid fragment comprising at least one functional domain specific for the promoter of the Ta Trxh2 gene. 2) Promoter according to claim 1, characterized in that said nucleic acid fragment is chosen from the group consisting of: the nucleic acid fragment whose sequence extends from position -1 to position -1111 relative to at the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -1 to position -83 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -451 to position -591 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -591 to position -1111 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -228 to position -451 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -451 to -591 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence extends from position -83 to position -228 relative to the ATG codon of the Ta Trxh2 gene; the nucleic acid fragment whose sequence is that of the first intron of the Ta Trxh2 gene. 3) Expression cassette characterized in that it comprises a promoter according to any one of claims 1 or 2. 4) Recombinant vector characterized in that it comprises at least one promoter according to any one of claims 1 or 2. 5) Plant cell transformed by at least one promoter according to any one of claims 1 or 2. 6) Transgenic plant transformed by at least one promoter according to any one of claims 1 or 2. 7) transgenic plant according to claim 6, characterized in that it is a monocotyledon. 8) Use of a promoter according to any one of claims 1 or 2 to control the expression of a gene of interest in a plant cell.