MXPA00000743A - Coffee storage proteins - Google Patents

Abstract

The subject of the present invention is proteins derived from the coffee bean, and DNAs encoding and regulating the expresion of at least one of these proteins.

Description

CAFE RESERVE PROTEINS Description of the Invention The object of the present invention are the proteins derived from the coffee bean, and the DNAs that encode and regulate the expression of at least one of these proteins. It is known that during its growth, numerous plants are able to produce reserve proteins in their embryos, in their tubers and in particular in their seeds. These reserve proteins play an important role, particularly in the amino acid reserve for the germination of the grain. They are also important in the structure and content of amino acids. Some of these proteins have been isolated and, in some cases, have been expressed in host plants. Thus, for example, EP 0,295,959 demonstrates in particular the expression in a host plant of DNA derived from Bertholletia excelsa H.B.K. (Brazil nut) that encodes at least one subunit of the reserve protein known as 2S. In addition, WO 9119801 demonstrates the existence of two reserve proteins derived from Theobroma Cacao, its precursor and its genes that encode these proteins.

However, until now no reserve protein derived from the coffee bean is known, nor any sequence capable of regulating the transcription of these proteins. However, it would be very useful to have sequences of these proteins available, in particular to modify the original production of the reserve proteins in the coffee bean. Furthermore, it would also be very useful to have available a sequence capable of regulating the transcription of these proteins in a way that allows, in particular, the expression of a protein encoded by an interesting gene in the coffee bean. The object of the present invention is to provide an answer to these needs. SUMMARY OF THE INVENTION For this purpose, the present invention relates to any DNA derived from the coffee bean that encodes at least 200 consecutive amino acids of the amino acid sequence SEQ ID NO: 2. The present invention relates to any protein derived from the grain of coffee having at least 20 consecutive amino acids of the amino acid sequence SEQ ID NO: 2. Another object of the present invention relates to all or part of the DNA delimited by nucleotides 1 to 2509 of the nucleic sequence SEQ ID NO: 3, capable of regulating the transcription of the reserve proteins according to the invention, as well as the use of all or part of this DNA to direct the expression of genes of interest in plants, in particular in the coffee tree. The present invention also relates to the use of all or part of the DNA delimited by nucleotides 33 to 1508 of the nucleic sequence SEQ ID N0: 1 or of its complementary strand of at least 10 bp (base pairs) to carry out a PCR (polymerase chain reaction) or as a probe to detect in vitro or inactivate a coffee bean gene encoding a reserve protein in vivo. In addition, the invention relates to any recombinant plant cell capable of expressing a recombinant reserve protein according to the invention. Finally, the present invention relates to any food, cosmetic or pharmaceutical product comprising all or part of the DNA or recombinant proteins according to the invention. Accordingly, the present invention opens up the possibility of using all or part of the DNA according to the invention to modify the original production of the reserve proteins in the coffee bean. It is therefore possible, in particular, to contemplate the superexpression or under-expression of all or part of the DNA according to the invention in the coffee bean. For the purposes of the present invention, "homologous nucleic sequence" means any nucleic sequence that differs from the nucleic sequences according to the invention only in the substitution, deletion and / or insertion of a small number of base pairs. In this context, two nucleic sequences encoding the same protein due to the degeneracy of the genetic code will be considered as homologous in particular. The one that exhibits more than 70% homology with the nucleic sequence according to the invention will also be considered a homologous sequence. In the latter case the homology is determined by the ratio between the number of base pairs of a homologous sequence and that of a nucleic sequence according to the invention. Furthermore, for the purposes of the present invention, a homologous nucleic sequence is also understood as a sequence that hybridizes under astringent conditions, that is, any nucleic sequence capable of hybridizing to the nucleic sequences according to the present invention by means of the Southern-Blot method. (southern transfer), in order to avoid specific hybridizations or hybridizations that are not stable (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold.Spring Harbor Laboratory Press, USA, 1989, chapter 9.31 to 9.51). Finally, for the purposes of the present invention, "homologous amino acid sequence" is understood to mean any amino acid sequence that differs from the amino acid sequences according to the invention only in the substitution, insertion and / or deletion of at least one amino acid. The one that exhibits more than 50% homology with the amino acid sequence according to the invention will also be considered as the homologous sequence. In the latter case, the homology is determined by the ratio between the number of amino acids of a homologous sequence and that of an amino acid sequence according to the invention. In the remainder of the description the sequences SEQ ID NO: refer to the sequences presented in the sequence listing below. The synthetic oligonucleotides SEQ ID NO: 5 to SEQ ID NO: 18 which are mentioned in the description and which are presented in the sequence listing below are provided by Genset S.A., 1 passage Delaunay, 75011 Paris, France. The reserve proteins are only present in the coffee bean and are abundantly expressed in the endosperm. In the mature coffee bean they represent approximately 50% of the total of the proteins and they play an important role in the maturation of the coffee bean. These proteins influence in particular the structure and density of coffee beans as well as their amino acid content. They also play an important role in the reserve of amino acids for the germination of the grain. It is possible to isolate the DNA encoding as well as the DNA that regulates the expression of the coffee grain reserve proteins by carrying out an inverse PCR starting with nucleic primers derived from the nucleic sequences SEQ ID NO: 1 and SEQ ID NO: 3. Those skilled in the art are in fact trained, for example, to choose the primers that are most suitable for carrying out this PCR. For this purpose, a DNA encoding at least 20 consecutive amino acids of the amino acid sequence SEQ ID NO: 2 was isolated from the coffee bean. Preferably this DNA encodes at least one protein derived from the coffee bean selected from the group comprising the protein of reserve aß having the amino acid sequence SEQ ID NO: 2, the segmentation protein a delimited in the amino acid sequence SEQ ID NO: 2 by amino acids 1 to 304, the segmentation protein ß delimited in the amino acid sequence SEQ ID NO: 2 by amino acids 305 to 492, or any nucleic sequences homologous to these sequences. For the sake of the benefit of the present invention, the invention relates to DNA delimited by nucleotides 33 to 1508 of the nucleic sequence SEQ ID NO: 1 encoding the aβ reserve protein, or any nucleic sequence homologous to this sequence. In particular, the invention relates to DNA comprising, at least in the nucleic sequence SEQ ID NO: 1 nucleotides 33 to 944 encoding the cleavage protein a and / or nucleotides 945 to 1508 encoding the β-segmentation protein. The present invention also relates to the use of all or part of the DNA delimited by nucleotides 33 to 1508 of the nucleic sequence SEQ ID NO: that of its complementary strand of at least 10 bp (base pairs) as a primer to carry perform a PCR (polymerase chain reaction) or as a probe to detect in vi tro or modify the expression in vivo of at least one gene of the coffee bean that encodes at least one reserve protein. The DNA according to the present invention can conveniently be used to express at least one recombinant reserve protein derived from the coffee bean in a host plant or microorganism. For this purpose it is possible to clone all or part of the nucleic sequence SEQ ID NO: 1 delimited by nucleotides 33 to 1508 in an expression vector downstream of a promoter, or of a promoter and a signal sequence, and upstream of a terminal while preserving the reading frame, subsequently this vector can be introduced, for example, into a plant, a yeast or a bacterium. Below are specific examples of application. In addition, all or part of the DNA delimited by nucleotides 33 to 1508 of the nucleic sequence SEQ ID NO: 1 can be conveniently used in the coffee bean in a modified form by mutagenesis to modify the original production of reserve proteins in the bean of coffee and therefore modify the organoleptic quality of the coffee bean. The invention also relates to the aβ reserve protein having the amino acid sequence SEQ ID NO: 2, the segmentation protein having the sequence delimited by amino acids 1 to 304 of the amino acid sequence SEQ ID NO: 2, and ß-segmentation protein having the sequence delimited by amino acids 305 to 492 of the amino acid sequence SEQ ID NO: 2, or any amino acid sequence that is homologous thereto.

It was demonstrated that the reserve proteins derived from coffee bean are synthesized in an extensive precursor that is segmented into two proteins, the segmentation protein a and the segmentation protein ß. Segmentation proteins a and β can be recombined in a polymerized form through at least one disulfide bridge. In fact it has been possible to isolate polymerized forms of segmentation proteins a and / or β and / or their homologous sequences in the endosperm of the coffee bean. For this purpose the present invention also relates to the polymerized form of recombinant aβ, a and / or β reserve proteins, as well as their homologous sequences. Another object of the present invention relates to all or part of the DNA delimited by nucleotides 1 to 2509 of the nucleic sequence SEQ ID NO: 3, capable of regulating the expression of the reserve protein having the amino acid sequence SEQ ID NO: 2. The invention also relates to the use of all or part of the DNA delimited by nucleotides 1 to 2509 of the nucleic sequence SEQ ID NO: 3, to allow the expression of the protein in the coffee bean or in a heterologous plant. α-reserve protein encoded by nucleotides 33 to 1508 of the nucleic sequence SEQ ID NO: 1 or of a protein encoded by a gene of interest.

The DNA delimited by nucleotides 1 to 2509 of the nucleic sequence SEQ ID NO: 3 can be conveniently used by fusing it completely or partially with a gene of interest while preserving the reading frame, and then cloning all into an expression vector that It is introduced into coffee, in order to allow the expression of the protein encoded by this gene in the coffee bean. The invention also covers all food, cosmetic or pharmaceutical products comprising all or part of the DNA or recombinant proteins according to the invention. Those skilled in the art are truly able to detect their presence in small amounts by means of oligonucleotide probes or appropriate antibodies. The reserve proteins derived from the coffee bean, the DNA derived from the coffee bean that encodes at least one of these proteins, as well as the DNA capable of regulating its transcription, according to the invention, are characterized in greater detail below with the help of biochemical and molecular analysis. I. Identification of the coffee grain reserve proteins The total proteins are extracted from the mature fruits of Coffea arabica of the Caturra variety.

For this purpose, the maternal tissues of coffee beans that are rapidly crushed in liquid nitrogen are separated and then reduced to a powder according to the method of Damerval et al. (Electoforésis 7, 52-54, 1986). The coffee proteins are then extracted from 10 mg of this powder which is dissolved in 100 μl of solution containing 3% w / v of CHAPS, 8.5 M of urea, 0.15% w / v of DTT and 3% v / v of support ampholyte pH 3-10. The mixture is then subjected to centrifugation at 13,000 g for 5 minutes, and the supernatant containing the total proteins of the coffee beans is recovered. A one-dimensional electrophoresis is carried out in this supernatant based on a pH gradient using, for example, the Multiphore system (Pharmacia Biotech AB, Bjdrkgatan 30, 75182 Upsula, Sweden). For this purpose, 50 μl / electrophoretic gel is deposited. To separate the total proteins according to their molecular weights, a second SDS-PAGE electrophoresis is then carried out on the gels derived from the first electrophoresis, using, for example, a Bio-Rad device (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, California 94547 USA) under standard conditions, according to the Laemmli method (Nature, 277, 680-688, 1970). For this purpose, the gels derived from the one-dimensional electrophoresis are equilibrated with 5 ml / gel Tris regulator containing 6 M urea, 30% v / v of glycerin, 2% w / v of SDS, 2% w / v of DTT and 2.5% w / v of iodoacetamide, are placed on the gels of the second SDS-PAGE electrophoresis, and the migration of the proteins are carried out in a Bio-Rad equipment at 40 A and at a temperature of 12 ° C for 9 hours, for example. The gels produced in this manner are subjected to silver staining by the method Bjellqvist et al. (Electrophoresis, 14, 1357-1365, 1993). The images are then analyzed with the help of a scanner (Scanner XRS 12CX, X-Ray Scanner Corporation, 4030 Spencer Street, Torrance, California 90503 USA) and, for example, with the help of the Bio Image program (Bio Image, 777 East Eisenhower Parkway, Suite 950, Ann Arbor, Michigan 48108, USA). Proteins separated by two-dimensional electrophoresis are transferred to PVDF membranes in a CAPS regulator with the aid of a Bio-Rad Transblot Cell cell (bio-Rad, USA), maintained at 420 mA and at a temperature of 4 ° C for 1 hour 30 minutes , and then they are stained with coomassie blue according to the instructions of Applied Biosystems (Applies Biosystems Inc., 850 Lincoln Center Drive, Foster City, California 94404 USA).

After transfer, the membranes are dried at room temperature before storage at 18 ° C in plastic bags. The microsequencing of the N-terminal sequences of the protein transfers is carried out with the aid of a Beckmann LF 3000 sequencer and the Beckmann Gold high performance liquid chromatography system (Beckmann Instruments Inc., 250 Harbor Boulevard, Box 3100 , Fullerton, California, 92634 USA). For this the protein transfers are cut separating them from the membrane and subjected to digestion of trypsin at pH 8.3 in 50 μl of digestive regulator containing 10% v / v of trypsin, 100 mM of Tris HCl, 1% v / v of Triton RTX and 10% v / v of acetonitrile. The peptides are then separated by high performance liquid chromatography on a C18 column (Merck KGAA, Frankfurter Strasse 250, 64923 Darnstadt, DE) using a water / acetonitrile gradient containing 0.05% TFA, the peptide fractions are concentrated and rediluted in 30% acetonitrile and 0.01% TFA, and sequenced as described above. The two-dimensional electrophoretic profile under denaturing conditions of the endosperm of mature grains of C. arabica shows 4 groups of proteins that are represented in particular, proteins which have an apparent molecular weight of the order of 70, 56, 32 and 23 kDa (kiloDaltons). It can be observed that 2 proteins from the group of 23 kDa proteins as well as 2 proteins from the group at 70 kDa have an N terminal sequence that is identical to the N terminal sequence of the β-segmentation protein. In addition, it was demonstrated that 3 proteins from the 32 kDa protein group and 1 protein from the 56 kDa protein group have an N-terminal sequence identical to the N-terminal sequence of the segmentation protein a. It was also possible to establish 7 internal sequences of 5 to 15 amino acids of one of the proteins at 32 kDa. In addition, with the help of the SwissProt database (Genetics Computer Group Inc., University Research Park, 575 Science Drive, Madison, Wisconsin 53711 USA) and using the FASTA program (Pearson and Lipman, Proc. Nati. Acad. Sci. USA) , 85, 2444-2448, 1988) it was possible to demonstrate the fact that the N-terminal sequences of the proteins of 23, 32, 56 and 70 kDa and the internal sequences of the 32 kDa protein have a high homology with the sequences of the reserve proteins of certain plant species, such as for example Glycine max glycines, 12s proteins of Arabidopsis thaliana, Brassica napus cruciferin, Oryza sativa glutelins and Curcubi maxim protein lis. In light of these results it has been possible to develop the following hypotheses about the structure of the reserve proteins derived from coffee bean. The 56 kDa protein group represents an extensive precursor, the mature aß reserve protein comprising two domains, the a domain and the beta domain. The two-dimensional electrophoretic profile also demonstrates the existence of the segmentation protein a, present in severe isoforms at 32 kDa and that of the ß protein, present in several isoforms at 23 kDa. Therefore, as with the aβ reserve protein, segmentation proteins a and β can exist in several isoforms. Finally, the 70 kDa protein group represents the trimeric form of the β segmentation protein. In addition, the existence of a fragment of the 13 kDa segmentation protein in the two-dimensional electrophoretic profile has been demonstrated. II. Estimation of the amount of reserve proteins contained in the coffee bean and the specificity of the expression of the reserve proteins derived from the coffee bean. The amount of reserve proteins contained in the coffee bean is calculated in percent relative to the integrated total density of the two-dimensional electrophoretic profile. For this purpose, the integrated intensity of the protein transfers representing the reserve protein aß is measured, the segmentation protein a, the segmentation protein ß, the trimeric form of the segmentation protein ß and the fragment of the segmentation protein a. It is accepted that the total integrated intensity of the two-dimensional electrophoretic profile is equivalent to 100%. In this way, a value of 50% of reserve proteins contained in the coffee bean is obtained. In addition, the expression of the reserve proteins of the coffee bean in other tissues of the coffee bean than those of the endosperm is also verified by two-dimensional electrophoresis. It is therefore possible to demonstrate the fact that the reserve proteins are only synthesized in a large amount in the endosperm and in a much smaller proportion in the embryo of the coffee bean. III. Isolation and translation in vi tro of the polyA + messenger RNAs of the total RNAs of the coffee bean Total RNAs are extracted from the coffee beans harvested 4 to 40 weeks after flowering. For this purpose, the maternal tissues of the coffee beans that are rapidly milled in liquid nitrogen are separated before being reduced to powder. This powder is then resuspended in 8 ml of buffer at pH 8 containing 100 mM of Tris-HCl, 0.1% w / v SDS and 0.5% v / v of β-mercaptoethanol, homogenized with a volume of phenol saturated with 100 mM Tris-HCl, pH 8, and then centrifuged at 12,000 g for 10 minutes at 4 ° C to extract the aqueous phase, which is centrifuged (i) once more with an equivalent volume of phenol, (ii) two times with an equivalent volume of phenol: chloroform (1: 1) and (iii) twice with an equivalent volume of chloroform. The total nucleic acids are then precipitated for 1 hour at -20 ° C by the addition to the aqueous phase of 1/10 of the volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes of ethanol- The whole is then subjected to centrifuged to 12,000 g for 30 minutes at 4 ° C, and the pellet is absorbed in 10 ml of H20 before re-precipitating the nucleic acids again in the presence of LiCl (final 2 M) and ethanol (2.5 volumes).

After centrifugation the pellet of total RNAs is absorbed in 1 ml of H20 and digested for 1 hour at 37 ° C with DNAse RQ1 (Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711 USA) to remove any traces of DNA , and the total RNAs are then deproteinized by treatment with phenol and with chloroform before precipitating them in the presence of sodium acetate as described above. The total RNAs are then absorbed in 500 μl of H20 and quantified by an electrophoretic assay at 260 nm. Its quality is analyzed by electrophoresis with agarose gel in the presence of formaldehyde and by in viral translation. For this purpose the polyA + messenger RNAs (mRNA) are then purified from 500 μg of total RNAs using the Oligotex dT purification system (Qiagen INC., 9600 De Soto Avenue, Chatsworth, California 91311 USA), and then the quality of the mRNAs through their ability to synthesize proteins in vi tro. For this, translational experiments with 1 μg of mRNA are carried out in the presence of a rabbit reticulocyte lysate (Promega, USA) and then the proteins synthesized in this way are marked by the incorporation of 35S-methionine (Amersham International foot , Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, UK). The labeled proteins are then separated by two-dimensional electrophoresis as described above. After fixing in a mixture of acetic acid / ethanol (40/10) the gels are incubated in the presence of Amplify (Amersham, UK), dried under vacuum and exposed to -80 ° C against an autoradiographic film. On the one hand, the results of the translations in vi tro with RNAs extracted from grains from 4 to 40 weeks of age after flowering demonstrate the presence of numerous proteins with molecular weights of between 1 and 100 kDa. On the other hand, the results of the translations in vi tro with the RNAs extracted from grains harvested between 16 and 30 weeks after flowering show the presence in large quantities of proteins corresponding to the aβ form of the reserve proteins. On the other hand, no product of the translation that corresponds in size to segmentation proteins a and ß is observed. This result confirms the aforementioned hypothesis according to which these two segmentation proteins are effectively derived from the in vivo segmentation of the extended aβ precursor. To isolate the cDNA from these reserve proteins, two libraries were formed in the manner described below.

IV. Construction and screening of cDNA libraries The synthesis of cDNA, necessary for the construction of libraries, is carried out according to the recommendations provided in the kit (reagents) "Riboclone cDNA synthesis system M-MLV (H- ) (Promega, USA) using the mRNA extracted from coffee beans harvested 16 and 30 weeks after flowering. The efficiency of this reaction is monitored by the addition of [alpha-32P] dCTP during the synthesis of the two strands of DNA. After migration in an alkaline agarose gel (Sambrook et al., Molecular Cloning - A Laboratory Manual, 1989), it is estimated that the size of the new cDNA synthesized varies from 0.2 to more than 4.3 kb. The quantifications, with the help of the DNA Dipstick Kit (DNA dipstick kit) (Invitrogen BV, De Schelp 12, 9351 NV Leek, The Netherlands) show that about 100 ng of cDNA of 1 μg of mRNA are synthesized. The new synthesized cDNA (s) are treated below according to the recommendations provided in the RiboClone EcoRI Adaptator Ligation System kit (Promega, USA) and ligated (n) within the pBluescript II SK (+) plasmid (Stratagene, 11011 North Torrey Pines Road, La Jolla, California 92037, USA) previously digested with the restriction enzyme EcoRI and dephosphorylated by treatment with calcine intestine alkaline phosphatase. The entirety of this ligated mixture is used to convert the E. coli XLl-Blue MRF 'strain (Stratagene, USA). Bacteria containing recombinant vectors are selected on plates with LB medium (Luria-Bertani) containing 12.5 μl / ml tetracycline, 20 μg / ml ampicillin, 80 μg / ml methicillin and in the presence of IPTG and X-Gal (Sambrook et al., 1989). They are then cultured in petri dishes to obtain about 300 clones per plate. These clones are transferred to nylon filters and then treated according to the recommendations provided by Boehringer Mannheim (Boehringer Mannheim GmbH, Biochemica, Postfach 310120, Mannheim 31, DE). In addition, the sequence of the amino acids from 325 to 330 of the sequence SEQ ID NO: 2 is chosen in the amino acid sequence of the β-segmentation protein because it allows designating an oligonucleotide probe that is relatively only slightly degenerate, the OLIGO probe. 1, having the nucleic sequence SEQ ID NO: 4 which is marked at its 5 'end by the addition of the digoxigenin radical (Genosys Biotechnologies Inc., 162A Science Park, Milton Road, Cambridge CB4 4BR, UK).

The filters are prehybridized at 65 ° C for 4 hours in the hybridizing solution defined in the protocol of the 3 'end labeling kit of the oligonucleotide DIG (Boehringer Mannheim, DE), and the hybridization is carried out at 37 ° C for 10 hours. hours in the presence of the 0LIG01 probe (10 pmol / ml final). After hybridization the filters are washed in the presence of tetramethylammonium chloride according to the protocol defined by Wood et al., (Proc Nati, Acad, Sci., USA, 82, 1585-1588, 1985) and then submitted to immunological detection in the presence of CSPD (Tropix, 47 Wiggins Avenue, Bedford, Massachussetts 01730 USA) according to the recommendations provided by Boehringer Mannheim (DIG luminescent detection kit (kit)). A positive clone that hosts the recombinant vector, which we call "pCSPl" for the remainder of the description, is selected from the cDNA library scan carried out 16 weeks after flowering. This vector contains a cDNA cloned in the EcoRI site of the vector pBluescript II SK (+), which is sequenced according to the protocol of the "T7 sequencing kit" (Pharmacia, Sweden) in the presence of [alpha-35S] dATP. This cDNA comprises the last 819 nucleotides of the sequence SEQ ID NO: 1, and consequently is unable to encode the aβ reserve protein.

To isolate the cDNA encoding the entire aβ reserve protein, a novel nucleic probe called SOI is synthesized in the remainder of the description. For this purpose a PCR is carried out (US patent 4,683,195 and US patent 4,683,202) using the synthetic oligonucleotide OLIGO 2 having the nucleic sequence SEQ ID NO: 5, and the synthetic oligonucleotide OLIGO 3 having the nucleic sequence SEQ ID NO: 6. The PCR reaction is carried out in the presence of 0.1 ng of the pCSPl vector in a final volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl 2, 0.1 mg / ml of gelatin, 0.2 mM of each dNTP, 0.25 μM of each oligonucleotide (OLIGO 2 and OLIGO * 3), and 3 units of Taq DNA polymerase (Stratagene, USA). The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C-30 s, 42 ° C-30 s, 72 ° C-2 min) followed by a final extension at 72 ° C for 7 minutes. The fragment obtained after amplification is purified in a Microcon 100 cartridge (Amicon INC, 72 Cherry Hill Drive, Beverly, Massachusetts 01915 USA) and 50 ng of this fragment are labeled by random primer extension with 50 μCi of [alpha -32P] dCTP according to the Megaprime kit (Amersham, UK). In addition, the nylon filters used during screening with the OLIGO 1 probe are dehybridized by two 15 minute washes at 37 ° C in the presence of 0.2 N-NaOH-01% SDS (w / v) and then prehybridized for 4 hours at 65 ° C in a solution containing dxSSC, lxDenhart (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% BSA fraction IV) and 50 μg / ml of denatured salmon sperm DNA. They are then hybridized for 10 hours at 65 ° C in the same solution with the total labeled SOI probe, and then washed three times for 30 minutes at 65 ° C in the presence of, successively, 2xSSC-0.1% SDS, lxSSC -0.1% SDS and 0.1xSSC-0.1% SDS. Accordingly, a positive clone harboring the recombinant vector, which we will call "pCSP2", is selected for the remainder of the description from the cDNA library scan carried out 30 weeks after flowering. This vector contains the sequence SEQ ID NO: 1 of 1706 bp, which corresponds to the cDNA encoding the totality of the aβ reserve protein, having as sequence amino acid sequence SEQ ID NO: 2 and a theoretical molecular weight of 54999 Da. A search of the SwissProt databank with SEQ ID NO: 2 confirms that this coffee protein belongs to the family of plant type reserve proteins. The precursor cleavage site is located between amino acids 304 and 305 of the amino acid sequence SEQ ID NO: 2, as was observed for all the other plant proteins of type lis (Borroto and Dure, Plant Mol. Biol. 8, 113-131, 1987). This is also confirmed by the N-terminal segmentation of the segmentation protein ß described above. Consequently, the cleavage protein a corresponds to the first 304 amino acids of the amino acid sequence SEQ ID NO: 2, whereas the segmentation protein β corresponds to the last 188 amino acids of this sequence. The theoretical molecular weights of a and ß are respectively 34125 Da and 20892 Da and agree with those described in the above under "Identification of the coffee grain reserve proteins". The N-terminal sequences of the segmentation proteins a and β analyzed in the foregoing are in the amino acid sequence SEQ ID NO: 2 with the exception of a few amino acids. These differences are probably explained by the existence of several isoforms of these proteins that can differ from each other by a few amino acids (Shirsat, Developmental Regulation of Plant Gene Expression, Grierson Ed., Blackie, Chapman and Hall, NY, 153-181, 1991). V. Expression of the gene encoding the aβ reserve protein during the development of the Arabica Coffea grain The expression of the gene encoding the aβ reserve protein in the coffee beans harvested at various stages of development is verified (a 9, 12, 16, 30 and 35 weeks after flowering). For this purpose, 10 μg of Total RNAs of these coffee beans for 15 minutes at 65 ° C in IxMOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7), in the presence of formamide (50%) and formaldehyde (0.66 M final). They are then separated by electrophoresis for 6 hours at 2.5 V / cm in the presence of the lxMOPS buffer on a 1.2% agarose gel containing 2.2 M formaldehyde as the final concentration. After migration the RNAs are stained with ethidium bromide (BET) according to Sambrook et al. 1989, which allows to standardize the amounts deposited in a gel by the fluorescence intensities of ribosomal RNAs 16S and 23S. The total RNAs are then transferred and fixed to a positively charged nylon membrane, according to the recommendations provided by Boehringer Mannheim (Boehringer Mannheim, DE). The prehybridization and the hybridization are carried out according to the conditions described in the foregoing in chapter IV.

The mRNAs that encode the aβ reserve protein are completely absent from the grains harvested up to 9 weeks after flowering. They begin to be detected very weakly in the grains harvested at 12 weeks after flowering, and they are very abundant in grains harvested from 16 to 30 weeks after flowering, when again they are represented very weakly in coffee beans. mature (35 weeks after flowering). In all cases, the SOI probe hybridizes only with a class of mRNA whose estimated size of about 1.8 kb is close to that of the nucleic sequence SEQ ID NO: 1. The kinetics of accumulation of mRNAs is similar to that of observed for most of the genes for reserve proteins (Shirsat, 1991). According to the tissue tests carried out during the maturation of coffee beans, it is observed that the increase in the amount of mRNA between 12 and 16 weeks after flowering occurs at the same time as the absorption of the perisperm by the endosperm. In comparison with the analyzes carried out previously by means of two-dimensional electrophoresis on the accumulation of proteins, during the maturation of the grain, a perfect superposition of the kinetics of accumulation of the mRNAs with that of the reserve proteins is observed. In the mature stage, the persistence of the reserve proteins in the absence of their corresponding messenger RNAs is explained by a high stability of these proteins in vivo. According to these observations, and as has been demonstrated in other plant species (Shirsat, 1991), it seems that the expression of the gene encoding the aβ reserve protein is essentially controlled by a promoter, a sequence capable of regulating the transcription of the gene that is expressed specifically in the endosperm of the coffee bean. SAW. Isolation of the promoter of the gene encoding the aß protein of Coffea arabica The promoter of the gene encoding the aß protein of Coffea arabica is isolated by several inverse PCRs according to the method of Ochman et al. (Geneties 120, 621-623, 1988). For this purpose, the nuclear DNA of coffee is isolated from young leaves of C. arabica, Caturra variety, according to the protocol described by Rogers and Bendich (Plant Mol. Biol. Manuel, Gelvin, Schilperoort and Verma Eds. Kluwer Academic Publishers Dordrecht, The Netherlands, A6, 1-11, 1993). 0.5 to 1 μg of this DNA is digested with several restriction enzymes, such as, for example, Dral, HincII and Ndel, and then treated with phenol: chloroform (1: 1) and precipitated for 12 hours at -20 °. C in the presence of 0.3M final sodium acetate and ethanol (2.5 volumes). After centrifugation at 10,000 g for 15 minutes at 4 ° C, the DNA is absorbed in about 500 μl of binding buffer containing 30 mM Tris-HCl, pH 7.8, 10 mM MgCl 2, 10 mM DTT and 0.5 mM rATP, for a final DNA concentration of approximately 1 to 2 ng / μl. The ligation is carried out for 12 hours at 14 ° C in the presence of DNA ligase T4 at 0.02 Weiss u / μl, and then the self-ligated genomic DNA is precipitated as described above and absorbed in 20 μl of H20 before quantification with the DNA dipstick kit (Invitrogen, The Netherlands). a) Reverse PCR reaction No. 1 This first reaction is carried out using the synthetic oligonucleotide SO10 having the nucleic sequence SEQ ID NO: 7, and the synthetic oligonucleotide SOll having the nucleic sequence SEQ ID NO: 8. This PCR reaction Inverse is carried out in the presence of 50 ng of bound genomic DNA in a final volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 0.1 mg / ml gelatin , 0.2 M of each dNTP, 0.25 μM of each oligonucleotide (SO10 and SOll), and 3 units of Taq DNA polymerase (Stratagene, USA). The reaction mixture is then covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C-30 s, 56 ° C-30 s, 72 ° C-2 min) followed by a final extension cycle at 72 ° C for 7 minutes. The amplified DNA fragments are then analyzed by molecular hybridization (J. Southern, Mol. Biol.98, 503-517, 1975), separated by electrophoresis on 1% agarose gel stained with ethidium bromide and then transferred. in the presence of 0.4 N NaOH for 12 hours to positively charged nylon membrane (Boehringer Mannheim, DE). After transfer, the membrane is baked for 15 minutes at 120 ° C and then prehybridized at 65 ° C for 4 hours in the hybridization solution defined in the "3-end labeling kit of the DIG oligonucleotide" (Boehringer Mannheim , FROM) . The membrane is then hybridized at 37 ° C for 10 hours in the presence of the synthetic oligonucleotide S012 (10 pmol / ml) having the nucleic sequence SEQ ID NO: 9 and marked at its 5 'end with a digoxigenin radical. After hybridization the filters are washed in the presence of tetramethylammonium chloride according to the protocol defined by Wood et al., 1985, and then subjected to immunological detection in the presence of CSPD (Tropix, USA) according to the recommendations provided. in the DIG luminescent detection kit (Boehringer Mannheim, DE). After autoradiography, the presence of a DNA fragment of approximately 1.7 kb is detected, derived from the inverse PCR reaction to the genomic DNA initially digested with the restriction enzyme HincII that binds the S012 probe. This DNA is then cloned into the vector pCR-Script (SK +) (Stratagene, USA). For this purpose, 10 μl of the inverse PCR reaction is mixed with 100 μl of sterile water, and then the mixture is centrifuged for 10 minutes at 3000 g in a Microcon 100 cartridge (Amicon, USA). 3 μl of the DNA purified in this way are treated in the presence of native Pfu DNA polymerase (Stratagene, USA) in order to convert their cohesive ends to truncated ends. This reaction is carried out in a final volume of 10 μl containing 10 mM KCl, 6 mM (NH4) 2 SO4, 20 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 2 mM MgCl2. , 1 mM of each dNTP, 10 μg / ml of BSA, and the reaction mixture is covered with 50 μl of mineral oil, incubated for 30 minutes at 72 ° C, and then 1 μl of this reaction mixture is added to the reaction mixture. used directly in the linkage reaction with the vector pCR-Script SK (+).

The entirety of this ligation mixture (10 μl) is used to transform E.coli XLl-Blue MRF 'strain (Stratagene, USA). The bacteria containing the recombinant vectors are selected on plates with LB medium containing 20 μg / ml ampicillin, 80 μg / ml methicillin and in the presence of IPTG and X-Gal (Sambrook et al., 1989). At the end of the transformation, about 100 clones are obtained which are transferred to a nylon filter and analyzed by means of molecular colony hybridization (Grunstein and Hogness, Proc. Nati, Acad. Sci. USA 72, 3961-3965, 1975) with the S012 probe according to the conditions described above. This screening allows to isolate a positive clone that hosts the recombinant vector pCSPP1. This vector contains the genomic DNA fragment detected by autoradiography that is cloned in the Sfrl site of the pCR-Script vector (SK +). This DNA is sequenced according to the protocol defined by Pharmacia (T7 sequencing kit) in the presence of [alpha-35S] dATP. It comprises the last 1717 base pairs of the nucleic sequence SEQ ID NO: 3, flanked at each end by an HincII restriction site. It contains 750 base pairs upstream of the codon to initiate translation of the gene encoding the aβ reserve protein and the first 968 base pairs of this nuclear gene. Given the fact that this gene belongs to a multigene family it will be called CSP1 from now on. This partial sequence of the CSP1 gene shows the presence of two introns of identical size (111 bp), located respectively between nucleotides 2811-2921 for the first, and nucleotides 3239-3349 for the second nucleic sequence SEQ ID NO: 3. These two introns have smaller sizes than those observed, for example, in Arabidopsis thaliana, but on the other hand they are located in the same positions as those observed in this plant (Pang et al., Plant, Mol. Biol. 11, 805- 820). b) Reverse PCR reaction No. 2: first scan To obtain the nucleic sequences located upstream of the HincII site (position 1763 of the nucleic sequence SEQ ID NO: 3) another reverse PCR reaction is carried out using this time the synthetic oligonucleotides S016 and S017 deduced from the sequence already cloned into the plasmid pCSPP1 and having respectively the nucleic sequences SEQ ID NO: 10 and SEQ ID NO: 11. This reverse PCR reaction is carried out under conditions identical to those described for the PCR reaction No. 1, except for the following parameters: the ligand of the oligonucleotides was carried out at 57 ° C and 35 cycles of polymerization were carried out.

As defined above, the DNA fragments amplified by this PCR are analyzed by molecular hybridization after being separated on an electrophoresis gel, and transferred to a nylon membrane. This membrane is then prehybridized for 4 hours at 65 ° C in a solution containing ßxSSC, lxDenhart (0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2% BSA fraction IV) and 50 μg / ml of denatured salmon sperm DNA, and then it is hybridized for 10 hours at 65 ° C in the same solution with the SO1016 probe. This probe is in fact synthesized by PCR using the synthetic oligonucleotides SO10 and S016 described above, in the presence of 0.1 ng of the vector pCSPPl, in a final volume of 50 μl containing 50 mM KCl, 10 mM Tris- HCl, pH 8.8, 1.5 mM MgCl2, 0.1 mg / ml gelatin, 0.2 mM of each dNTP, 0.25 μM of each oligonucleotide (SO10 and S016), and 3 units of Taq DNA polymerase (Stratagene, USA). The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C - 30 s, 46 ° C - 30 s, 72 ° C - 2 min) followed by a final cycle extension to 72 ° C for 7 minutes. The fragment obtained after amplification (698 bp) is purified in a Microcon 100 cartridge (Amicon, USA), and 50 ng of this fragment is labeled by random primer extension with 50 μCi of [alpha-32P] dCTP from according to the protocol of the Megaprime kit (Amersham, UK). After hybridization the membrane is washed three times for 30 minutes at 65 ° C in the successive presence of 2xSSC-0.1% SDS, lxSSC-0.1% SDS and 0.1xSSC-0.1% SDS and analyzed by autoradiography to detect a DNA fragment of approximately 1 kb that binds the SO1016 probe. This DNA derived from the reverse PCR reaction in the genomic DNA initially digested with the restriction enzyme Ndel is then treated with Pfu DNA polymerase and then ligated to the pCR-Script vector (SK +) as described above. This linkage is then used to transform the E. coli XLl-Blue MRF 'strain and the transformants are selected and analyzed by molecular hybridization with the SO1016 probe according to the conditions described in the foregoing. This screening allows to isolate a positive clone that hosts the vector pCSPP2. As expected, this vector results from cloning to the Sfrl site of the pCR-Script vector (SK +) of the DNA fragment previously identified by hybridization. The latter, which corresponds to the DNA segment between nucleotides 1514 and 2523 of the nucleic sequence SEQ ID NO: 3, flanked at each end by a restriction site Ndel and consequently additionally contains 250 bp upstream of the fragment of Genomic DNA cloned into the vector pCSPP1 is sequenced. c) Reverse PCR reaction No. 2: second screening To clone nucleotides 1 to 1513 of the nucleic sequence SEQ ID NO: 3, another molecular hybridization is carried out on the DNA fragments derived from the reverse PCR reaction No. 2. For This purpose is derived from the probe used, designated SO1720, of the nuclear DNA sequence of coffee cloned into the vector pCSPP2 and synthesized by PCR using the oligonucleotide S017 described above and the SO20 olucleotide having the nucleic sequence SEQ ID NO. : 12. This reaction is carried out in the presence of 0.1 ng of the vector pCSPP2 under conditions identical to those used for the synthesis of the SO1016 probe, except for the temperature for the ligation of the oligonucleotides, which is 50 ° C. The fragment obtained after the amplification (262 bp) is labeled as described above and is used as a probe to verify the reverse PCR reactions No. 2. The nylon membrane used during the screening of the products of the Reverse PCR reaction No. 2 with probe S01016 is inhibited by two 15 minute washes at 37 ° C in the presence of 0.2 N NaOH - 0.1% SDS (w / v), then prehybridized and hybridized as described in what precedes with the SO1720 probe. At the end of this hybridization a DNA fragment of approximately 1.9 kb derived from the reverse PCR reaction No. 2 is detected in the genomic DNA initially digested with the restriction enzyme Dral. As described above, this DNA is then treated with Pfu DNA polymerase, ligated into the pCR-Script vector (SK +) and all linkage is used to transform the E. coli XLl-Blue MRF 'strain. The transformants are then selected and screened by molecular hybridization with the SO1720 probe. It is thus possible to isolate a positive clone harboring the vector pCSPP3 resulting from cloning to the Sfrl site of the pCR-Script vector (SK +) of the DNA fragment previously identified by hybridization. The latter, which corresponds to the DNA segment between nucleotides 1 and 1886 of the nucleic sequence SEQ ID NO: 3, flanked at each end by a Dral restriction site, is sequenced. Accordingly it additionally contains 1513 base pairs upstream of the cloned genomic DNA fragment within the pCSPP2 vector. d) Cloning of genomic DNA fragments Reverse PCR experiments form chimeric linear molecules by combining non-contiguous DNA fragments in the genome with one another (Ochman et al., 1988). further, measurements of the mutation frequency show that Pfu DNA polymerase is approximately twelve times more accurate than Taq DNA polymerase, which reduces the chances of point mutations during PCR amplifications (Lundberg et al., Gene 108, 1- 6, 1991). For these reasons, a PCR reaction is carried out on the native genomic DNA of C. arabica, Caturra variety, in the presence of the Pfu DNA polymerase. This reaction is carried out in the presence of 10 ng of the genomic DNA, in a final volume of 50 μl containing 50 mM KCl, 6 mM (NH4) 2 SO4, 20 M Tris-HCl, pH 8.0, 0.1% of Triton X-100, 2 mM MgCl2, 10 μg / ml of BSA, 0.2 mM of each dNTP, 0.25 μM of the SOlO and SO20 oligonucleotides described above, and 3 units of Puf DNA polymerase. Oligonucleotide SOlO is located in the backbone of the nucleic sequence SEQ ID NO: 3, between nucleotides 2512 and 2534, while oligonucleotide SO20 is located in the sense strand of the nucleic sequence SEQ ID NO: 3 between nucleotides 1565 and 1584. The reaction mixture is then covered with 50 μl of mineral oil and incubated for 45 cycles (94 ° C - 30 s, 50 ° C - 30 s, 72 ° C - 3 min) followed of a final extension cycle at 72 ° C for 7 minutes.

After this PCR a single fragment is obtained which is cloned into the vector pCR-Script (SK +) to give the vector pCSPP4. Sequencing demonstrates that this genomic DNA fragment corresponds to the sequence between oligonucleotides SOlO and SO20. The DNA amplified during this PCR reaction is then used for the construction of the vectors, as explained in the following: VII. Construction of the genetic transformation vectors necessary for the functional analysis of the promoter of the gene encoding the aβ reserve protein of Coffea arabica The sequences located upstream of the translation initiation site, placed at 2510 of the nucleic sequence SEQ ID NO : 3 are analyzed in order to verify their ability to regulate the expression of the uidA reporter gene in the grains of the transformed plants. For this purpose, several constructions are made in the binary transformation vector pBHOl (Clontech Laboratories Inc., 1020 East Meadow Circle, Palo Alto, California 94303-4320 USA). This vector contains the reporter gene uidA that encodes the β-glucuronidase (GUS) and the bacterial gene nptll, which encodes the neomycin phosphotransferase. The latter confers resistance to kanamycin in transformed plants. These two genes are flanked by the right and left ends of the T-DNA of the plasmid pTiT37 of Agrobacterium tumefaciens (Bevan, Nucí, Acids Res. 12, 8711-8721, 1984) that define the region of the DNA capable of being transferred within the genome. of plants infected with this bacterium. The vector pBHOl is digested with the restriction enzyme Ba HI and dephosphorylated by treatment with calf alkaline phosphatase (Promega, USA) according to the protocol defined by the supplier. Next, the DNA fragments of different sizes that are obtained by PCR in the presence of the pCSPP4 vector, of Pfu DNA polymerase and of two synthetic oligonucleotides each containing at their 5 'end the nucleic sequence SEQ ID are cloned into the PBI101 vector. NO: 13. This sequence comprises a BamHI restriction site that allows the cloning of the PCR products into the pBHOl vector linearized with the same enzyme. On the one hand a synthetic oligonucleotide capable of binding to the promoter is used, and on the other hand the oligonucleotide BAGUS having the nucleic sequence SEQ ID NO: 14. The use of the latter allows, after digestion of the PCR products with the restriction enzyme BamHI, obtain a translational fusion between the first 5 amino acids of the reserve protein aß of the coffee bean and the N-terminal end of the β-glucuronidase. a) Construction of pCSPPS The PCR reaction is carried out with 5 ng of the plasmid pCSPP4, in a volume of 50 μl containing 50 mM KCl, 6 mM (NH4) 2S04, 20 mM Tris-HCl, pH 8 , 0.1% Triton X-100, 2 mM MgCl2, 10 μg / ml BSA, 0.25 mM oligonucleotide UP210 having the nucleic sequence SEQ ID NO: 15, and BAGUS, which has the nucleic sequence SEQ ID NO: 14 , and 3 units of Puf DNA polymerase. The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C - 30 s, 55 ° C - 30 s, 72 ° C - 2 min) followed by a final cycle extension to 72 ° C for 7 minutes. The PCR fragment of approximately 950 bp is purified in a Microcon 100 cartridge (Amicon, USA) and digested for 12 hours at 37 ° C with BamHI (Promega, USA) and ligated into the linearized vector pBHOl in the presence of T4 DNA ligase (Promega, USA) according to the recommendations provided by the provider. The E. coli XLl-Blue MRF1 strain is then transformed with the entire ligation mixture. The plasmids are extracted independently of several transformants and sequenced to determine the orientation of the PCR fragment in the binary vector. Accordingly, this analysis allows the plasmid pCSPP5 to be selected. b) Construction of pCSPPd The construction of this vector is carried out as described for the vector pCSPP5, except for the fact that the oligonucleotide UP210 is replaced with the oligonucleotide UP211 having the nucleic sequence SEQ ID NR: 16. The cloning of the PCR product (approximately 700 bp), correctly oriented in the vector pBHOl gives the vector pCSPP6 . c) Construction of pCSPP7 The construction of this vector is carried out as described for the vector pCSPP5, except for the fact that the oligonucleotide UP210 is replaced with the oligonucleotide UP212 having the nucleic sequence SEQ ID NR: 17. The cloning of the PCR product (450 bp), correctly oriented in the vector pBHO1 gives the vector pCSPP7. c) Construction of pCSPPd The construction of this vector is carried out as described for the vector pCSPP5, except for the fact that the oligonucleotide UP210 is replaced with the oligonucleotide UP213 having the nucleic sequence SEQ ID NR: 18. The cloning of the PCR product (250 bp), correctly oriented in the pBHOl vector gives the vector pCSPPd. VIII. Transformation of Agrobacterium tumefaciens The vectors described above (pCSPP5-8), as well as plasmids pBIlOl and pBI121 (Clontech) are introduced independently into the unarmed C58pMP910 strain of Agrobacterium tumefaciens (Koncz and Schell, Mol.Gen.Genet., 204, 383 -396, 1986) according to the direct transformation method described by An et al. (Plant Mol, Biol. Manuel, Gelvin, Schilperoort and Verma Eds, Kluwer Academic Publishers Dordrecht, The Netherlands, A3, 1-19, 1993). Agrobacterium clones are selected for each transformation. recombinant tumefaciens in LB medium supplemented with kanamycin (50 μg / ml) and rifamycin (50 μg / ml). To verify the structure of the plasmids introduced in Agrobacterium tumefaciens, they are extracted using the rapid mini-repair technique described by An et al. (1993) and analyzed by restriction mapping after reverse transformation in the XLl-Blue MRF 'strain of E. coli. In the plasmid pBIlOl, the uidA gene is silent because it has no promoter. In contrast, this same gene is expressed in plants transformed with the vector pBI121 because it is under the control of the constituent promoter CaMV 35S (Jefferson et al., J. EMBO, 6, 3901-3907, 1987). These two plasmids were respectively used as negative and positive controls for the expression of the uidA reporter gene.

IX. Transformation and regeneration of Nicotiana tabacum The transformation of Nicotiana tabacum variety XHFD8 is carried out with the bearers previously described (pCSPP5-8, pBHOl and pBI121), according to the protocol described by Horsch et al. (Plant Mol, Biol. Manuel, Gelvin, Schilperoort and Verma Eds, Kluwer Academic Publishers Dordrecht, The Netherlands, A5, 1-9, 1993). For this purpose, leaf discs of seedlings that were germinated in vitro for about 2 minutes are incubated with a stationary phase culture transformed from Agrobacterium tumefaciens diluted in a 0.9% NaCl solution to obtain an OD measurement at 600 nm of between 0.2 and 0.3. They are then dried on 3 MM paper (Whatmann), incubated without selection pressure in a culture chamber on medium MS-stem (MS salts 4.3 g / 1, sucrose 30 g / 1, agar 8 g / 1, myoinositol 100 mg / 1, thiamine 10 mg / 1, nicotinic acid 1 mg / 1, pyridoxine 1 mg / 1, naphthaleneacetic acid (NAA) 0.1 mg / 1, benzyladenine (BA) 1 mg / 1) (Murashige and Skoog, Physiol. 15, 473-497, 1962). After 3 days the discs are transferred on MS medium supplemented with kanamycin (100 μg / ml) and with cefotaxime (400 μg / ml) in order to multiply the transformed cells to obtain calli. These discs are then subcultured each week on fresh "MS-stem" medium. After 21 to 28 days the buds that germinate are cut from the calli and subcultured in standard MS medium, that is in MS medium free of phytohormones, supplemented with kanamycin (100 μg / ml) and with cefotaxime (200 μg / ml). After rooting in a Petri dish, the seedlings are transplanted into pots in a substrate composed of peat and compost (fermented residues) and then grown in a greenhouse at a temperature of 25 ° C and with a photoperiod of 16 hours. For each transformation experiment 30 seedlings are selected (R0 generation). All these plants turned out to be morphologically normal and fertile. They were individualized and gave seeds (Rl generation). X. Analysis of the genomic DNA of tobacco plants transformed with Agrobacterium tumefaciens The genomic DNA of transgenic tobacco plants is extracted from the leaves according to the protocol described by Rogers and Bendich (Plant Mol. Biol. Manuel, Gelvin, Schilperoort and Verma Eds. , Kluwer Academic Publishers Dordrecht, The Netherlands, A6, 1-11, 1993) and then analyzed by PCR and by molecular hybridization, according to the Southern blot technique. The PCR reactions are carried out with 10 ng of DNA in a final volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 0.1 mg / ml gelatin, 0.2 M of each dNTP, 3 units of Taq DNA polymerase, 0.25 μM of the oligonucleotide BI104 having the nucleic sequence SEQ ID NO: 19 described in the sequence listing below, and 0.25 μM of the oligonucleotide BI105, which has the nucleic sequence SEQ ID NO : 20 described in the sequence listing below. Oligonucleotide BI104 is located 27 bp downstream of the BamHI site of plasmid pBHOl, and oligonucleotide BI105 is located 73 bp upstream of the Ba HI site of plasmid pBHOl. PCR reactions are carried out for 30 cycles (94 ° C - 30 s, 54 ° C - 30 s, 72 ° C - 2 min) followed by a 7 minute cycle at 72 ° C (final extension). The amplified DNA fragments of transgenic tobacco plants transformed with the pBHOl plasmids (negative control), pBI121 (positive control), pCSPP5, pCSPPβ, pCSPP7 and pCSPP8 have molecular weights of about 280 bp, 1030 bp, 1230 bp, 980 bp, 730 bp and 430 bp respectively. In all cases it is concluded that the fragment initially cloned upstream of the uidA reporter gene is intact. 10 μg of DNA from tobacco plants transformed with Agrobacterium tumefaciens are digested with BamHI. Then the restriction fragments that are obtained are separated by electrophoresis on agarose gel (1%) and the DNA is transferred to a nylon filter., before hybridizing it independently with a uidA probe and an nptll probe. The uidA probe is synthesized by PCR using the synthetic oligonucleotide GMP1 having the sequence SEQ ID NO: 21 described in the sequence listing below and the synthetic oligonucleotide GMP2 having the sequence SEQ ID NO: 22 described in the sequence listing Then, in the presence of 0.1 ng of the vector pBHOl in a final volume of 50 μl containing 50 mM of KCl, 10 mM of Tris-HCl, pH 8.8, 1.5 mM of MgCl2, 0.1 mg / ml of gelatin, 0.2 mM of each dNTP, 0.25 μM of each oligonucleotide and 3 units of Taq DNA polymerase (Stratagene, USA). The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C - 30 s, 46 ° C - 30 s, 72 ° C - 2 min) followed by a cycle at 72 ° C for 7 minutes.

The nptll probe is synthesized by PCR using the synthetic oligonucleotide NPTII-1 having the sequence SEQ ID NO: 23 described in the sequence listing below and the synthetic oligonucleotide NPTII-2 having the sequence SEQ ID NO: 24 described in FIG. sequence listing below, in the presence of 0.1 ng of the pBHOl vector in a final volume of 50 μl containing 50 mM KCl, 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl2, 0.1 mg / ml gelatin , 0.2 mM of each dNTP, 0.25 μM of each oligonucleotide and 3 units of Taq DNA polymerase (Stratagene, USA). The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C - 30 s, 46 ° C - 30 s, 72 ° C - 2 min) followed by a cycle at 72 ° C for 7 minutes. These two probes are purified and then labeled as described in the above in the VI test. The hybridization profiles obtained for each probe are then compared to select the tobacco plants transformed with Agrobacterium tumefaciens that integrated into their genome a single ungrouped copy of the T-DNA. The selection of these plants is also confirmed by the results of the analysis of the segregation of the kanamycin resistance character after in vitro germination on standard MS medium of the Rl seeds of these plants. In fact, in this case a segregation of 3 / 4-1 / 4 of the character of resistance to kanamycin is observed, which is compatible with the integration of T-DNA in a single place of the nuclear DNA. XI. Study of the performance characteristics of the coffee promoter and its derivatives in transgenic tobacco plants This study is carried out in plants of the RO generation and in mature seeds of the Rl generation. Therefore, measurements of GUS activity are carried out on leaves and seeds according to the method described by Jefferson et al. (1987) using as substrate MUG (methyl umbelliferyl glucuronide) and measuring by fluorometry the appearance of MU (metilumbelliferone). For this purpose, the leaf explants (10 mg) and seeds (approximately 40) are crushed in the presence of sterile sand in 300 μl of extraction buffer (50 mM Na2HP04, pH 7.0, 10 mM EDTA, 10 mM ß -mercaptoethanol). The cell debris is removed by centrifugation for 15 minutes at 4 ° C and the soluble proteins of the supernatant are quantified according to the Bradford method (Anal. Biochem. 72, 248-254, 1976) according to the protocol defined by Bio-Rad (USA). ) and using BSA as a standard. Measurements of GUS activity are carried out in microtiter plates incubated at 37 ° C using 1 μg of soluble proteins in 150 μl reaction regulator (extraction buffer with 1 mM MUG). Fluorescence measurements, expressed in pmol MU / min / mg of protein, are carried out at an excitation wavelength of 365 n and at an emission wavelength of 455 nm (Fluoroskanll, Labsystem). The results of the GUS activity measurements presented in FIG. 1 below demonstrate that no enzymatic activity is observed in the leaves and seeds of the plants containing the T-DNA of the pBHOl plasmid. For the other transformation experiments, the differences in GUS activities observed between each of the transgenic plants transformed with the same generic construction can be explained by a positional effect that results from the random integration of T-DNA into the genome (Jones et al., J. EMBO, 4, 2411-2418, 1985). Plants containing the pBI121 construct have a glucuronidase activity of between 1500 and 20,000 pmol MU / min / mg protein. For these same plants no significant differences were observed between the measurements of specific GUS activities carried out using seeds and leaves. These observations confirm the constitutive nature of the CaMV 35S promoter in plants (Odell et al., Nature 313, 810-812, 1985).s of the results shows that the GUS activities measured in the leaves of the plants that independently contain the T-DNAs of the plasmid constructs pCSPP5, pCSPP6, pCSPP7 and pCSPPd are respectively 60, 60, 30 and 12 times higher than the activity Average GUS measured in the seeds of the plants transformed with the pBI121 plasmid. It is also observed that the maximum expression of the uidA gene is obtained with the vectors pCSPP5 and pCSPPd, reaching on average 465 nmol MU / min / mg of protein. From this observation it can be concluded that the DNA fragment between nucleotides 1572 (5 'end of the sequence SEQ ID NO: 15) and 1815 (5' end of the sequence SEQ ID NO: 16) of the sequence SEQ ID NO: 3 does not contain any sequence that is critical to the operation of the coffee promoter. The more substantial deletions made in the promoter (corresponding to the vectors pCSPP7 and pCSPPd) result in a reduction in the level of expression of the uidA reporter gene, which is higher in the more substantial the deletion. On the other hand, these deletions in no case lead to a loss of the specificity of the expression of the promoter, since in all the transgenic plants that were analyzed the uidA reporter gene continues to be specifically expressed in the seeds.

Measurements of GUS activity show that the DNA sequence of the coffee between nucleotides 1572 and 2524 of the sequence SEQ ID NO: 3 described in the sequence listing below effectively contains a promoter that behaves as a very strong promoter in the seeds of tobacco compared to the 35S promoter of CaMV. It is also observed that this same DNA sequence, as well as the deletions derived from it, contain the necessary and sufficient information to direct the expression of the uidA reporter gene in the seeds of transgenic tobacco plants at a level that in all cases is higher than the one that confers the reference promoter CaMV35S. XII. Expression of the reserve protein lis of coffee in Escherichia coli To overexpress and purify the protein lis of the coffee in Escherichia coli an amplification of the DNA sequence between nucleotides 108 and 1517 of the sequence SEQ ID NO: 1 is carried out with the aid of the oligonucleotide TAG_1_having the sequence SEQ ID NO: 25, and the oligonucleotide TAG_2_having the sequence SEQID NO: 26. These two sequences are described in the sequence listing below. These two oligonucleotides allow introducing the unique EcoRI and PstI sites in the amplified coffee sequence by PCR. They also allow to amplify the DNA sequence of the coffee that encodes the coffee reserve protein but that suffers from its cellular passage sequence called "signal peptide", which is between amino acids 1 and 26 of the sequence SEQ ID NO: 2. strategy was followed to limit the toxic effects due to an overexpression in Escherichia coli of proteins that contain very hydrophobic sequences. This reaction is carried out in the presence of 50 ng of the pCSP2 vector in a final volume of 100 μl containing 1.5 units of Pwo DNA polymerase (Boehringer Mannheim), 10 μl of 10X Pwo DNA polymerase buffer (Boehringer Mannheim), 0.1 mM of each dNTP and 2 nM of each oligonucleotide, TAG_1_and TAG_2_. The reaction mixture is covered with 50 μl of mineral oil and incubated for 30 cycles (94 ° C - 30 s, 40 ° C - 60 s, 72 ° C - 2 min) followed by a final cycle extension to 72 ° C for 7 minutes. Next, 30 μl of the PCR mixture is digested with the restriction enzymes EcoRI and PstI according to the recommendations provided by Promega (USA). The DNA fragment of the coffee (1400 bp) amplified by PCR is separated by electrophoresis on a 0.8% agarose gel and purified according to the recommendations provided in the QIAquick Gel Extraction kit (Qiagen Inc. 9600 De Soto Avenue, Chatsworth , CA91311, USES) . It is then ligated to the expression vector pQE31 (Qiagen, USA) previously digested with the enzymes EcoRI and PstI and dephosphorylated by an alkaline phosphatase treatment of calf intestine. The use of the expression vector pQE31 allows introducing 6 histidines (6 His tag) in phase with the N-terminus of the reserve protein lis of the coffee, which then facilitates the purification of this recombinant protein after passing over a column of Ni-NTA resin containing Ni2 + ions (Hochuli et al., J. Chromatography, 411, 177-184, 1987). Linkage roving is used to transform competent cells of the M15 strain (pREP4) of Escherichia coli according to the recommendations provided by Qiagen (USA), and the recombinant bacteria are selected on plates with LB medium containing 25 μg / ml of kanamycin and 100 μg / ml ampicillin. To verify the expression of the reserve protein lis of coffee in Escherichia coli, the bacteria are then cultured in 50 ml of liquid LB medium supplemented with the antibiotics as indicated above until an OD at 600 nm = 1. Induction is carried out by adding IPTG to a final concentration of 1 mM to the culture medium, and culture samples are collected every 30 minutes. Bacteria are lysed and soluble proteins are extracted from Escherichia coli under denaturing conditions. These proteins are then separated on a Ni-NTA resin column following the protocol defined by QIAGEN (QIAexpress system). The fractions of proteins eluted successively are then analyzed by electrophoresis SDS-PAGE. Therefore, it is demonstrated that the only protein capable of binding to the Ni-NTA column corresponds to the recombinant protein lis of coffee. This protein is expressed in Escherichia coli with an approximate molecular weight of 55 kDa, which agrees with that observed in coffee beans for the reserve protein in its precursor form, and this taking into account the modifications of the protein sequence which were carried out during the construction of the expression vector.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: SOCIETE DES PRODUITS NESTLE (B) STREET: AVENUE NESTLE 55 (C) CITY: VEVEY (D) STATE OR PROVINCE: VAUD (E) COUNTRY: SWITZERLAND (F) POSTAL CODE: 1600 (G) TELEPHONE: 021/924 34 20 (H) TELEFAX: 021/924 28 80 (ii) TITLE OF THE INVENTION: COFFEE PROTEINS (iii) SEQUENCE NUMBER: 26 (iv) FORM COMPUTER READING (A) TYPE OF MEDIUM: Soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) LOGICAL SUPPORT: Patentln Relay # 1.0, Version # 1.30 (EPO) ) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1706 base pairs (B) TYPE: nucleotide (C) HEBRATION: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (A) DENOMINATION / KEY: CDS (B) LOCATION: 33..1508 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: AAACACACTA CACTCTCCTC TTGTTTCAGA GA ATG GCT CAC TCT CAT ATG ATT 53 Met Ala His Ser His Met He 1 5 TCT CTT TCC TTG TAC GTT CTT TTG TTC CTC GGC TGT TTG GCT CAA CTA 101 Ser Leu Ser Leu Tyr Val Leu Leu Phe Leu Gli Cys Leu Wing Gln Leu 10 15 20 GGG AGA CCA CAG CCA AGG CTC AGG GGT AAA ACT CAG TGC GAT ATT CAG 149 Gly Arg Pro Gln Pro Arg Leu Arg Gly Lys Thr Gln Cys Asp He Gln 25 30 35 AAG CTT AAT GCA CAA GAA CCA TCC TTC AGG TCC CCA TCA GAG GCT 197 Lys Leu Asn Ala Gln Glu Pro Ser Phe Arg Phe Pro Ser Glu Ala Gly 40 45 50 55 TTA ACT GAA TTC TGG GAT TCT AAT AAT CCA GAA TTT GGG TGC GCT GGT 245 Leu Thr Glu Phe Trp Asp Ser Asn Asn Pro Glu Phe Gly Sis Wing Gly 60 65 70 GTG GAA TTT GAG CGT AAC ACT GTC CA A CCT AAG GGC CTT CGT TTG CCT 293 Val Glu Phe Glu Arg Asn Thr Val Gln Pro Lys Gly Leu Arg Leu Pro CAT TAC TCT AAC GTG CCT AAA TTC GTC TAC GTT GTC GAA GGT ACC GGT 341 His Tyr Ser Asn Val Pro Lys Phe Val Tyr Val Val Glu Gly Thr Gly 90 95 100 GTT CAA GGC ACT GTG ATC CCT CGT TGT GCT GAA ACA TTT GAA TCC CAG 389 Val Gln Gly Thr Val He Pro Gly Cys Ala Glu Thr Phe Glu Ser Gln 105 110 115 GGT GAA TCA TTT TGG GGT GGT CAG GAA CAG CCG GGC AAA GGG CAA GAA 437 Gly Glu Ser Phe Trp Gly Gly Gln Glu Gln Pro Gly Lys Gly Gln Glu 120 125 130 135 GGC CAA GAG CA GGT TCC AAA GGT GGT CAG GAA GGG CGA AGG CAA AGG 485 Gly Gln Glu Gln Gly Ser Lys Gly Gly Gln Glu Gly Arg Arg Gln Arg 140 145 150 TTT CCA GAC CGC CAT CAG AAG CTC AGA AGG TTC CAA AAA GGA GAT GTC 533 Phe Pro Asp Arg His Gln Lys Leu Arg Arg Phe Gln Lys Gly Asp Val i 155 160 165 CTT ATA TTG CTT CCT GGT TTC ACT CAG TGG ACA TAT AAT GAT GGA GAT 581 Leu He Leu Leu Pro Gly Phe Thr Gln Trp Thr Tyr Asn Asp Gly Asp 170 175 180 GTT CCA CTT GTC ACT GTC GCT CTT CTT GAT GTT GCC AAT GAG GCT AAT 629 Val Pro Leu Val Thr Val Wing Leu Leu Asp Val Wing Asn Glu Wing Asn 185 190 195 CAG CTT GAT TTG CAG TCC AGG AAA TTT TTC CTA GCC GGA AAC CCG CAÁ 677 Gln Leu Asp Leu Gln Being Arg Lys Phe Phe Leu Wing Gly Asn Pro Gln 200 205 210 215 CAG GGT GGT GGA AAG GAA GGC CAT CAG GGC CAG CAG CAG CAG CAT AGA 725 Gln Gly Gly Gly Lys Glu Gly His Gln Gly Gln Gln Gln Gln His Arg 220 225 230 AAC ATC TTC TCA GGA TTT GAT GAC CAÁ CTT TTG GCT GAT GCT TTC AAT 773 Asn He Phe Ser Gly Phe Asp Asp Gln Leu Leu Wing Asp Wing Phe Asn 235 240 245 GTT GAC CTC AAA ATA CAG AAA TTG AAG GGT CCG AAA GAT CAG AGG 821 Val Asp Leu Lys He He Gln Lys Leu Lys Gly Pro Lys Asp Gln Arg 25o 255 260 GGT AGC ACA GTC CGA GCT GAA AAA CTT CAA CTG TTC CTG CCT GAA TAT 869 Gly Ser Thr Val Arg Ala Glu Lys Leu Gln Leu Phe Leu Pro Glu Tyr 265 270 275 AGT GAG CAÁ GTG CAÁ CAÁ CA CA CA CAG CA CA CA CA CA CA CA CA CA CA CA CA 9 CA CA CA CA CA CA CA GA CA CA CA CA CA CA CA 917 TGC 965 Gly Val Gly Arg Gly Trp Arg Ser Asn Gly Leu Glu Glu Thr Leu Cys 300 305 310 ACG GTG AAG CTT AGT GAA AAC ATT GGC CTC CCC CAA GAG GCT GAT GTA 1013 Thr Val Lys Leu Ser Glu Asn He Gly Leu Pro Gln Glu Wing Asp Val 315 320 325 TTC AAT CCT CGT GCT GGC CGC ATT ACC ACT GTT AAT AGC CAA AAG ATT 1061 Phe Asn Pro Arg Wing Gly Arg He Thr Thr Val Asn Ser Gln Lys He 330 335 340 CCT ATC CTC AGC AGC CTC CAA CTT AGT GCA GAA AGA GGA TC CTC TAC 1109 Pro He Leu Ser Ser Leu Gln Leu Ser Wing Glu Arg Gly Phe Leu Tyr 345 350 355 AGC AAT GCC ATT TT GCA CCA CAC TGG AAT ATC AAT GCA CAT AAT GCC 1157 Ser Asn Wing He Phe Pro Wing His Trp Asn He Asn Wing His Asn Ala 360 365 370 375 CTG TAT GTG ATT AGA GGA AAT GCA AGA ATT CAG GTG GTG GAT CAC AAA 1205 Leu Tyr Val He Arg Gly Asn Wing Arg He Gln Val Val Asp His Lys 380 385 390 GGA AAC AAA GTT TT GAC GAT GAA GTA AAA CAG GGT CAG CTA ATA ATT 1253 Gly As Lys Val Phe Asp Asp Glu Val Lys Gln Gly Gln Leu He He 395 400 405 GTG CCA CA TAC TTT GCT GTG ATC AAG AAA GCT GGA AAC CA GGA TTT 1301 Val Pro Gln Tyr Phe Wing Val He Lys Lys Wing Gly Asn Gln Gly Phe 410 415 420 GAG TAC GTT GCA TC AAG ACG AAC GAC AAT GCC ATG ATT AAC CCA CTT 1349 Glu Tyr Val Wing Phe Lys Thr Asn Asn Asn Wing Met He Asn Pro Leu 425 430 435 GTT GGA AGA CTT TCG GCA TT CGA GCA ATT CCT GAG GAA GTT TTG AGG 1397 Val Gly Arg Leu Ser Ala Phe Arg Ala He Pro Glu Val Glu Leu Arg 440 445 450 455 AGC CT TC CAÁ ATT TCC AGC GAG GAA GCT GAG GAA TTG AAG TAT GGA 1445 Be Ser Phe Gln Be Ser Be Glu Glu Wing Glu Glu Leu Lys Tyr Gly 460 465 470 AGA CAG GAG CGT TTG CTT TTG AGT GAG CAG TCT CAG CAG GGG AAA AAG 1493 Arg Gln Glu Arg Leu Leu Leu Ser Glu Gln Ser Gln Gln Gly Lys Lys 475 480 485 AGT AGA TGC TTG AGC TAATTATGTA AAAATATCG TATATTAGTC CATGCATAGT 1548 Cys Ser Leu Ser Arg 490 CTACCAACTA TATGTGTGAA TCTAATTCCA AAATAAAATG GTCAATGGAT GTAAAGACAT1608 GGCAATCCAA GCCTTACTAC TGGCGTTGAT TGCGAGAAGT TTGATGTTTG GTGACCATGA1668 GTCAATAATA AACTATGATA ATTAATGTAA AATTTTCC 1706 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 492 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FROM THE SEQUENCE: SEQ ID NO: 2: Met Ala His Ser His Met He Ser Leu Ser Leu Tyr Val Leu Leu Phe 1 5 10 15 Leu Gly Cis Leu Wing Gln Leu Gly Arg Pro Gln Pro Arg Leu Arg Gly 20 25 30 Lys Thr Gln Cys Asp lie Gln Lys Leu Asn Wing Gln Glu Pro Ser Phe 40 45 Arg Phe Pro Ser Glu Wing Gly Leu Thr Glu Phe Trp Asp Ser Asn Asn Pro Glu Phe Gly Cys Wing Gly Val Glu Phe Glu Arg Asn Thr Val Gln 65 70 75 80 Pro Lys Gly Leu Arg Leu Pro His Tyr Ser Asn Val Pro Lys Phe Val 85 90 95 Tyr Val Val Glu Gly Thr Gly Val Gln Gly Thr Val He Pro Gly Cys 100 105 110 Wing Glu Thr Phe Glu Ser Gln Gly Glu Ser Phe Trp Gly Gly Gln Glu 115 120 125 Gln Pro Gly Lys Gly Gln Glu Gly Gln Glu Gln Gly Ser Lys Gly Gly 130 135 140 Gln Glu Gly Arg Arg Gln Arg Phe Pro Asp Arg Hís Gln Lys Leu Arg 145 150 155 160 Arg Phe Gln Lys Gly Asp Val Leu He Leu Leu Pro Gly Phe Thr Gln 165 170 175 Trp Thr Tyr Asn Asp Gly Asp Val Pro Leu Val Thr Val Wing Leu Leu 180 185 190 Asp Val Wing Asn Glu Wing Asn Gln Leu Asp Leu Gln Being Arg Lys Phe 195 200 205 Phe Leu Wing Gly Asn Pro Gln Gln Gly Gly Gly Lys Glu Gly His Gln 210 215 220 Gly Gln Gln Gln Gln His Arg Asn He Phe Ser Gly Phe Asp Asp Gln 225 230 235 240 Leu Leu Ala Asp Ala Phe Asn Val Asp Leu Lys He He Gln Lys Leu 245 250 255 Lys Gly Pro Lys Asp Gln Arg Gly Ser Thr Val Arg Ala Glu Lys Leu 260 265 270 Gln Leu Phe Leu Pro Glu Tys Ser Glu Gln Val Gln Gln Pro Gln Gln 275 280 285 Gln Gln Gln Gln Gln Gln His Gly Val Gly Arg Gly Trp Arg Ser Asn 290 295 300 Gly Leu Glu Glu Thr Leu Cys Thr Val Lys Leu Ser Glu Asn He Gly 305 310 315 320 Leu Pro Gln Glu Wing Asp Val Phe Asn Pro Arg Wing Gly Arg He Thr 325 330 335 Thr Val Asn Ser Gln Lys He Pro He Leu Ser Ser Leu Gln Leu Ser 340 345 350 Wing Glu Arg Gly Phe Leu Tyr Ser Asn Wing He Phe Wing Pro His Trp 355 360 365 Asn He Asn Wing His Asn Wing Leu Tyr Val He Arg Gly Asn Wing Arg 370 375 380 He Gln Val Val Asp His Lys Gly Asn Lys Val Phe Asp Asp Glu Val 385 390 395 400 Lys Gln Gly Gln Leu He He Val Val Gln Tyr Phe Ala Val He Lys 405 410 415 Lys Wing Gly Asn Gln Gly Phe Glu Tyr Val Wing Phe Lys Thr Asn Asp 420 425 430 Asn Wing Met He Asn Pro Leu Val Gly Arg Leu Ser Wing Phe Arg Wing 435 440 445 He Pro Glu Glu Val Leu Arg Ser Ser Phe Gln He Ser Ser Glu Glu 450 455 460 Wing Lys Tyr Gly Arg Gln Glu Arg Leu Leu Leu Ser Glu 465 470 475 480 Gln Ser Gln Gln Gly Lys Lys Arg Ser Cys Leu Ser 485 490 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3477 base pairs (B) TYPE: nucleotide (C) HEBRATION: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) DENOMINATION / KEY: promoter (B) LOCATION: 1..2509 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TTTAAAAGTT TGGTAGAAAA TTTGAGATAT TTGACGTCTA CGAGGCCAGA TATCAATTTG 60 CTGTTGGTGT GATTAACAAG TTTATGAAAA ATCCACGTCA GTCACATTTG TAGGCGGTTA 120 AGAAGATTTT GAAATATATT GAAAGTACTC ACAGTGTTGG CATTTTTTAT TCAGAAAATT 180 ATCCAGTTGA ATGGTTTGGC TACTGATAGT TATTGAGCAG GTGATACAAT AGAGAAGAAG 240 AGTACTTCAA GTTATGCATT TTTTATTGGT TCTGACGTAT TTTCCTCGAG TTCAAAGAAA 300 CAACAGGTGA TTGCATTGTT TACAGCAGAA GCAGAGTATA TTGCAGCGGC TAATAGTGCT 360 AATCAAGTTT TGTGGTTACG TTGCATGTTT GGTATTCTAC AATACAAGCA GGTTGATCTT TA 420 ACGAAAATTT ATTGTGATAG AGTCAGCT ATTGAATTGT CCAAGAATTT AGTACTTCAT 480 GGATGTAACA AGCATATTGG CATCAAATAT CACTTCACAC GTGAGTTGGT TCGAGAGAGA 540 GAGAGAGGTT GAAATTGATT ATTGCAGAAT TAAATAGTAA GTGGCTGACA TTTTCACCAA 600 GACATTGAAG ATAGAGATTT TTGTCAAGTT GAAGAATATG TTAGGCATGT CCAAGTTAGA 660 GGAGATTCGT TTAATGGAGG CAATATAGAA ACACAAACCA AGCCTTTATT ATTTGTTTAT 720 GCTGTCATGT GGGATTGGTA GTAGTATTGT TGGTTGGTAG GGTGGTCACA TGGGATTGAA 780 TTTCCTATGA CTAGTAGAGT TAGTAATAGA AGTTAGCCGC CAAGGGGTTT TGATGTGTAG_840_CTGTTGCGTC CGTCTTTTTT AGCCTTAAGA AGAAGTAGTC ACCTCCGTTG TGTTCTGCAT 900 GGTGTAGCAG AGCCTTGTTA TGATTAATAG AAAATTTTCC TTTGCCTCAA TATCGTTTTT 960 TTTTTTTATT GTTTCTGTGG GTTTTGTGTA TTTATCAATT TGGGTCCACC ACTTTTTCCA1020 ACCATGATCT TAAGCATATC AGCTCACTTC CACTCTATTT CTTTACCATG ATTTTAAGTA1080 CAATAATTTC CTAAAAAACC AAAAAAGAAT CATATCTATA AATTTTGAGA AAAGCATATA1140 TACTGCTAAC ATGATTCTAC GTATAATAGT CGATTATTAA AAATTATTAA TTACATATTT1200 TTGACATAAC CATCGGTGTA CCAAAAGCAC TAATGATTAC AACACTAAAC ACGCAAAGTT1260 GAGTAATTGA AACTGAAATT ACACATAGAC AAAAACTCAA CTAAACAATG TTAGAATGGA1320 ATAGATTAGA GAACCATTGA ATGATCTAAC TCTGGAACTG GGGTTAAGAC AGTCTTCCCA1380 AGCAACTTTT TTTGTCCATG ATTTGGCTAT CATATCACTA TCTTGAAATT TGTTCAGACA1440 CACTGTGGGA GGCTGGAATC AATAGCTTGG ACTTGGATCA TTTATAGAAG CTGATGATCA1500 TTATTGCTCA ACATATGAAT TTGATACAAA TGTCACTGGA ATCAACTTCG TACTTTTTTT1560 TTTTCCTCTT TTCTTTTGGA GTACAAGCCT ACCTACAAGG GGAAGGATAG AGGAAATGCA1620 TAGAGGGAGA TTTAACCTCT ACCCAAGCGG CAGATACAAT GGGTCACGAT ACAGCTGGTT1680 TATTGATGTA TTACAGCGGA AAACGATGTA GATGAGCAAC CTTTTCAAAG AACATAAGTC1740 AAAATCATAG ATGTAAAGCA GTCAACTGAG TCTGTGGCAA TTGTTAGACG TAAAACTCTA1800 TTCCATGTCA TTATTAGGTT TCTTGCTCTA TCTTTTAGTT TGATCCAACA TGGATTGGCT1860 GTCTTTTGTT TGCTAATAAA GATTTTAAAT CATGGAATTT CCCTGTAGAA TGCCTTTAAT1 20 TACATGCCAC TAGACTAGAA ACGGTAATTG TTTAACAGAT ATTTATTCCA GGCATTGAAA1980 TTATGAACTG CAACAGTCAT TTGCCTAGAA GTGTAAACCA ATTGTCTTCA ATAAAGGTGA2040 ATAAAAATCG ATGAAGATAG ATAGGTGCTA GAAACTTAAA AGCAGAAGAT GATAGGTGTG2100 ATGTAATACG CAGCAGTAGT GATCATCTTT CCATATCACA TCTTGAAAGA TCCCAAGAT G2160 AATGTGTGTT TGATTTGGGG TTTGATTCAT CAAAAGCCAT CGTAGCAGAT AATGCACCTT2220 ACCATGCCAT TGCTAAAGTA CAAAAATTTC ATGCAAATAC AAACACAAAA GATTGAACAA2280 TACATGTCAG AAACTCTATG CCACCAAGGC TTACACATCA TCTTTGGTGT AAAGAAGTGT2340 TCATCTTCAT CAGCCATGCA CAAGACTGAG TAGCCAAGTG TAAAATGAAA ATTTTGACGT2400 GTCGATTCCT CATCTTCCAT TACATGTTAT AAAAGGAGCC ATTTCCAAGC TCTAATCGCC2 60 GCATCCCCTC ACCACAAAAA CACACTACAC TCTCCTCTGT TGTCAGAGAA TGGCTCACTC2520 TCATATGATT CTCTTTCCT TGTACGTTCT TTTGTTCCTC GGCTGTTTGG CTCAACTAGG2580 GAGACCACAG CCAAGGCTCA GGGGTAAAAC TCAGTGCGAT ATTCAGAAGC TTAATGCACA2640 AGAACCATCC TTCAGGTTCC CATCAGAGGC TGGTTTAACT GAATTCTGGG ATTCTAATAA2700 TCCAGAATTT GGGTGCGCTG GTGTGGAATT TGAGCGTAAC ACTGTCCAAC CTAAGGGCCT2760 TCGTTTGCCT CATTACTCTA ACGTGCCTAA ATTCGTCTAC GTTGTCGAAG GCAGTTTCAT2820 TTCCCATCCT TTCCATTATT TCTGGAGTTT TTTTTCTATT TTCTTCTTAA TCATCGTATT2880 ATTCATTTTC TTCATGATTT AATCATTTTG GCATAATGCA GGTACCGGTG TTCAAGGCAC2940 TGTGATCCCT GGTTGTGCTG AAACATTTGA ATCCCAGGGT GAATCATTTT GGGGTGGTCA3000 GGAACAGCCG GGCA AAGGGC AAGAAGGCCA AGAGCAAGGT TCCAAAGGTG GTCAGGAAGG3060 GCGAAGGCAA AGGTTTCCAG ACCGCCATCA GAAGCTCAGA AGGTTCCAAA AAGGAGATGT3120 CCTTATATTG CTTCCTGGTT TCACTCAGTG GACATATAAT GATGGAGATG TTCCACTTGT3180 CACTGTCACA CTTCTTGATG TTGCCAATGA CGTGAATCAG CTTGATTTGC AGTCCAGGGT3240 AAGAAAACTT TCAATCCAAA CTTGCCAAGT ATTAATCAAA AAATAATCTC TTTCTGGGCA3300 TATTTTATTG CGGTACCATC TTAATAAAAA AAAAATTTTA TACTTTCAGA AATTTTTCCT3360 AGCCGGAAAC CCGCAACAGG GTGGTGGAAA GGAAGGCCAT CAAGGCCAGC AGCAGCAGCA3420 TAGAAACATC TTCTCAGGAT TTGATGACCA CTTTTGGCTG ATGCTTTCAA TGTTGAC 3477 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 17 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "nucleotide" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: GCNGAYGTNT TYAAYCC (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE (A) ) LENGTH: 17 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: AAACATTGGC CTCCCCC (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCAAACATCA AACTTCTCG (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) ) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GAGAAATCAT ATGAGAGTGA GCC (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 23 pairs of base (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: TTCTTTTGTT CCTCGGCTGT TTG (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 17 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: GTGAGCCATT CTCTGAC (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 23 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: AGTTTGATCC AACATGGATT GGC (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS D E THE SEQUENCE (A) LENGTH: 24 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO "(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GCAAGAAACC TAATAATGAC ATGG (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE : nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ií) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 12: CCTCTTTTCT TTTGGAGTAC (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 12 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: CGCGGATCGG CG (2) INFORMATION FOR THE SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO : 14: CGCGGATCCG CGATGAGAGT GAGCCATTCT CTG (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 35 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15 CGCGGATCCG CGCCTCTTTT CTTTTGGAGT ACAAG (2) INFORMATION FOR SEQ ID NO : 16: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: CGCGGATCCG CGTAGGTTTC TTGCTCTATC TTTTAG (2) INFORMATION FOR A SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other acid nucleic (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: CGCGGATCCG CGGTGCTAGA AACTTAAAAG CAGAAG (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE ( A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: CGCGGATCCG CGACAAAAGA TTGAACAATA CATGTC (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 30 base pairs (B) TYPE: nucleotide ( C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: TTTGATTTCA CGGGTTGGGG TTTCTACAGG (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) HEBRATION : simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: GGCTCGTATG TTGTGTGGAA TTGTGAGCGG (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: ATGTTACGTC CTGTAGAA (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE (A) ) LENGTH: 18 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) D ESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GCAAAGTCCC GCTAGTGC (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 17 pairs base (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: CTGGATCGTT TCGCATG (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 16 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEO IDO" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: CCAGAGTCCC GCTCAG (2) INFORMATION FOR SEQ ID NO : 25: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLEC ULA: another nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDO" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25 ACTAGGGGAT CCACAGCCAA GGCTCAGGGG (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) HEBRATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "OLIGONUCLEOTIDE" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2 (GTACTCTGCA GACATAATTA GCTCAAGCAA CTTCCC

Claims (12)

1. CLAIMS 1. DNA derived from coffee bean that encodes at least 20 consecutive amino acids of the amino acid sequence SEQ ID NO: 2.
2. 2. DNA according to claim 1, which encodes at least one protein that is selected from the group comprising the protein of reserve aß having the amino acid sequence SEQ ID NO: 2, the segmentation protein a, delimited in the amino acid sequence SEQ ID NO: 2 by amino acids 1 to 304, and the segmentation protein β, delimited in the amino acid sequence SEQ ID NO: 2 for amino acids 305 to 492.
3. 3. DNA according to claim 1, whose sequence is delimited by nucleotides 33 to 1508, 33 to 944 and / or 945 to 1508 of the nucleic sequence SEQ ID NO: 1, or any nucleic sequence homologous to these sequences.
4. 4. Recombinant recombinant proteins derived from coffee bean having at least 20 consecutive amino acids of the amino acid sequence SEQ ID NO: 2.
5. 5. Reserve proteins according to claim 4 having the amino acid sequence SEQ ID NO: 2 of the reserve protein aß, the sequence delimited by amino acids 1 to 304 of the amino acid sequence SEQ ID NO: 2, corresponding to the reserve protein a, the sequence delimited by amino acids 305 to 492 of the amino acid sequence SEQ ID NO: 2, corresponding to the stock protein ß, or any amino acid sequences homologous to these sequences.
6. 6. Proteins according to claim 5, characterized in that they are polymerized, independently or with each other.
7. 7. All or part of the DNA delimited by nucleotides 1 to 2509 of the nucleic sequence SEQ ID NO: 3, capable of regulating the transcription of the reserve proteins according to claim 5.
8. 8. Use of all or part of the DNA according to the claim 7 to direct the expression of a gene of interest in a plant.
9. 9. Use of all or part of the DNA delimited by nucleotides 33 to 1508 of the nucleic sequence SEQ ID NO: 1 or of its complementary strand of at least 10 bp as a primer to carry out a PCR or as a probe to detect in vitro or modify in vivo at least one coffee bean gene that encodes at least one reserve protein.
10. 10. Recombinant cells of plants capable of expressing a recombinant reserve protein according to claim 5.
11. 11. Plants or seeds that are constituted by plant cells according to claim 10.
12. 12. A food, cosmetic or pharmaceutical composition comprising a DNA according to one of claims 1 to 3, or a recombinant protein according to one of claims 4 to 6.