MX2007015995A - Oleosin genes and promoters from coffee - Google Patents

Abstract

Oleosin- and steroleosin-encoding polynucleotides from coffee plants are disclosed. Also disclosed are promoter sequences from coffee oleosin genes, and methods for using these polynucleotides and promoters for gene regulation and manipulation of flavor, aroma and other features of coffee beans.

Description

OLEOSIN GENES AND COFFEE PROMOTERS FIELD OF THE INVENTION The present invention relates to the field of agricultural biotechnology. In particular, the invention provides polynucleotides that code for oleosin and steroleosin from coffee plants, promoter sequences for coffee oleosin genes and methods for using these polynucleotides and promoters for gene regulation and manipulation of flavor, aroma and other grain traits of coffee .

BACKGROUND OF THE INVENTION Throughout the specification, several publications are cited, including patents, published applications and academic articles. Each of these publications is included as a reference herein, in its entirety. Appointments not fully established within the specification can be found at the end of the specification. The aroma of coffee and its flavor are key components in consumer preference for coffee varieties and brands. The characteristic aroma and flavor of coffee, emanating from a complex series of chemical reactions, imply taste precursors (Maillard reactions) that occur during the roasting of the beans. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, more than 800 chemicals and biomolecules have been identified that contribute to the flavor and aroma of coffee (Montavon et al, 2003, J. Agrie, Food Chem., 51: 2328-34, Clarke &Vitzthum, 2001). , Coffee: Recent Developments, Blackwell Science). Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor, in order to meet consumer preferences. Both aroma and taste can be imparted artificially in coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (taste). An alternative procedure would be to use molecular biology techniques either to add aroma as for flavor enhancing elements that do not occur naturally in coffee beans, or to improve those elements responsible for the flavor and aroma naturally found in coffee beans. . Genetic enginng is particularly suited to achieve these ends. For example, coffee proteins from different coffee species can be exchanged. In the alternative, the expression of genes that code for coffee proteins of natural origin, which contribute positively to coffee flavor, can be improved. Conversely, the expression of genes that code for coffee proteins of natural origin, which negatively contribute to coffee flavor, can be suppressed. The endogenous proteins of coffee whose expression could be the object of genetic manipulation, and if it should be increased or suppressed to what degree of production of such coffee proteins, have been determined empirically. The US storage protein has been identified as one such candidate coffee protein. (Montavon et al., 2003, J. Agrie, Food Chem. 51: 2335-43). Coffee oleosin, because of its role in oil storage, is another candidate for coffee protein. Coffee oils are known constituents of coffee aroma and flavor. For example, (E) -2-nonenal, and trans-trans-2-4-decadienal are volatile derived lipids, important for coffee aroma (Akiyama et al, 2003; Variyar et al, 2003). Therefore, increasing or decreasing the storage of these oils in the coffee grade should have a measurable effect on the aroma and flavor of the coffee. Oleosins also form lipid bilayers and may contribute to the lipid content as well.

Oleosins have been detected in a range of plant species including rape seed oil (Keddie et al, 1992), African palm oil (NCBI), cotton (Hughes et al, 1993), sunflower (Thoyts et al, 1995). ), peeled grains (Aalen et al, 1994; 1995), rice (Wu et al, 1998), almond (García-Mas et al, 1995), cocoa (Guilloteau et al, 2003) and corn (Qu and Huang, 1990, Lee and Huang, 1994). In vegetable seeds, oil bodies, also called oleosomes, are maintained by oleosins. It is believed that these oil bodies serve as a container for triacylglycerols (TAG) (Tzen et al, 1993). One function of oleosins is to organize the lipid reserves of seeds into small, easily accessible structures (Huang et al, 1996). The seed oil bodies oscillate in their diameter from 0.5 to 2 μM (Tzen et al, 1993), providing a high surface area for the volumetric ratio, which is believed to facilitate the rapid conversion of TAG into free fatty acids through the lipase. , mediated by hydrolysis on the surface of the oily body (Huang et al, 1996). In seeds that contain large amounts of oils, such as rapeseed oil. Oleosins represent 8% -20% of the total protein (Li et al, 2002) and oleosins represent 79% of the proteins associated with the oily bodies of arabidopsis (Jolivet et al, 2004).

Oleosins cover the surface of these oily bodies (Huang, 1996), where they are thought to help stabilize the lipid body during the desiccation of the seed, by preventing the coalescence of the oils. Related lipids that contain particles are also found in certain specialized cells. For example, the tapetum, a structure involved in the development of pollen; it also has lipid particles similar to a specific oily body, called tapetosomes. These particles similar to the oily body are involved in the provision of functional components required for the development of microspores and pollen (Murphy et al, 1998, Hernandez-Pinzon et al, 1999). n Oleosin proteins are composed of three distinctive domains: a centrally conserved hydrophobic fragment of approximately 72 amino acids, flanked by a highly variable N-terminal carboxylic portion and a C-terminal amphipathic a-helix (Huang, 1996; Li et al, 2002). The lengths of the amino and carboxy portions are very variable, and as a consequence, oleosins can range in size from 14 to 45 kDa (Tai et al, 2002, Kim et al, 2002). The antipathetic amino and carboxy portions allow the protein to reside stably on the surface of the oily bodies (Huang, 1996). The amino acids in the center of the hydrophobic region contain three conserved prolines and a conserved serine that forms the KNOT proline moiety. It is believed that this portion allows the central fragment to be folded into a hydrophobic fork, which anchors the oleosin in the oily central matrix (Huang, 1996). The role of the KNOT portion of proline on protein function was further investigated by Abell et al (1997) who showed that, if the three proline residues were replaced by leucine residues, an oleosin-beta-glucuronidase fusion protein failed in addressing oily bodies in both transient embryonic expression and in stably transformed seeds. Oleosins have been classified as high or low MR isoforms (oleosin H and L) depending on relative molecular masses (Tzen et al, 1990). The sequential analysis showed that the main difference between the H and L oleosins was the insertion of 18 residues in the C-terminal domain of oleosins H (Tai et al, 2002) and Tzen et al (1998) showed that both forms coexist in the oily bodies. In Zea mays, Lee and Huang (1994) identified three genes, OLE1 6, OLE1 7 and OLE18 with molecular weights of 16, 17 and 18 kDa, respectively, which are expressed during seed maturation. The corresponding protein ratios are 2: 1: 1 respectively in the isolated oily bodies (Lee and Huang, 1994; Ting et al, 1996). Lee et al. (1995) classified OLE1 6 as an oleosin L and O E1 1 and OLE18 as oleosins H, indicating that the oily bodies of Zea mays contain equal amounts of oleosins H and L in the oily bodies. In addition, they discovered that the oily bodies of rice embryos contain a similar amount of two different oleosins of molecular masses 18 and 16 kDa, corresponding to the H form and the L form respectively (Tzen et al, 1998; Wu et al, 1998) . Two oleosins were also identified in the Theobroma ca cao seed (Guilloteau et al, 2003). In 15 and 16.1 kDa these proteins represent an L form and a H form, respectively. Kim et al (2002) have characterized the oleosin genes in Arabidopsis in three groups. The first group consists of oleosins expressed specifically in the seeds (S), the second is expressed in the seeds and in the floral microspores (SM) and the final group is expressed in the floret tapetum (T). Of the sixteen oleosin genes identified in the Arabidopsis genome, five genes were specifically expressed in seed maturation, three genes were expressed in the maturation of seeds and floral microspores and eight in the floral tapetum (Kim et al, 2002) . The five specific oleosins of Arabidopsis seeds were also previously classified as three oleosins of form H and two oleosins of form L by Wu et al (1999). Sesame, corn and rice have all been shown to code for three specific seed oleosins (Tai et al, 2002; Ting et al, 1996; Chuang et al, 1996; Wu et al, 1998; Tzen et al, 1998). It is believed that the expression of oleosin will be regulated in development and spatially, mainly at the level of transcription (Keddie et al, 1994). Wu et al (1998) showed that transcripts of two rice oleosins appear seven days after pollination and disappear in mature seeds. A similar result was obtained by Guilloteau et al (2003) who showed that the level of the two cacao oleosin transcripts decreased in the mature seeds. While the gene transcription of oleosin has been studied semi-quantitatively in a variety of seed types, there are no reports in which the transcript levels of most, or all, of the oleosins in a seed type have been determined quantitatively during the development of the seed. Despite the fact that coffee beans have an oil content between 10 and 16%, little is known about the oleosin proteins in coffee. There is a shortage of scientific data regarding the number of coffee oleosins, their protein structure, their levels of expression and distribution through the coffee plant and between coffee species, their oil storage capacities, and the regulation of their expression at the molecular level. Thus, there is a need to identify and characterize proteins, genes, and genetic regulatory elements of coffee oleosin. Such information will make it possible for the oleosin proteins of coffee to be genetically manipulated, with the goal of improving one or several characteristics of coffee, which include the content and stability of the oil, which in turn can affect the parameters of roasting, impacting finally the aroma and taste of coffee. For purposes of increasing or suppressing the production of coffee proteins such as oleosins, it is desirable to have available a group of promoters compatible with the coffee plant. In addition, any genetic manipulation should ideally be located primarily or solely on the coffee bean, and should not adversely affect the reproduction or propagation of the coffee plant. Seed-specific promoters have been described. Examples of such promoters include the 5 'regulatory regions from such genes as cruciferane (US Patent No. 6,501,004), napin (Kridl et al, Seed Sci. Res. 1: 209: 219, 1991), phaseolin (Bustos et al. Plant Cell, 1 (9): 839-853, 1989), soybean trypsin inhibitor (Riggs et al, Plant Cell 1 (6): 609-621, 1989), ACP (Baerson et al, Plant Mol. Biol. , 22 (2): 255-267, 1993), stearoyl-ACP desaturase (Slocombe et al, Plant Physiol. 104 (4) .167-176, 1994), beta-conglycinin soy subunit (P-Gm7S , Chen et al, Proc. Nati, Acad. Sci. 83: 8560-8564, 1986), USP de Vi cia faba (P-Vf.Usp, US Patent Application Serial No. 10 / 429,516). In addition, an L3 oleosin promoter from Zea mays (P-Zm.L3, Hong et al, Plant Mol. Biol., 34 (3): 549-555, 1997) has been described. Seed-specific promoters have found application in plant transformation. For example, groups have used genetic manipulation to modify the level of seed constituents. See, Selvaraj et al, U.S. Patent No. 6,501,004, Peoples et al U.S. Pat. No. 6, 586, 658, Shen et al, U.S. Patent Application. Series No. 10 / 223,646, Shewmaker et al, U.S. Patent Application. Series. No. 10 / 604,708, and Wahlroos et al, U.S. Patent Application. Series No. 10 / 787,393. It should be noted that oleosin promoters have been used successfully in these systems. However, seed-specific promoters, and more specifically, 'oleosin promoters' of coffee, have not yet been used to transform coffee plants. Thus, there is a need for additional, available gene regulatory sequences to control the expression of coffee proteins. In the same context, there is a need to have gene regulatory sequences available to control the expression of oleosins in coffee plants. In addition, there is a need for gene regulatory sequences available to control the expression of coffee proteins in the coffee bean. In this regard, the specific promoters for gene expression in the coffee bean are very attractive candidates, among these promoters are the coffee oleosin promoters.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the present invention offers nucleic acid molecules, isolated from coffee (Coffea spp.), Which have coding sequences that code for oleosins. In certain embodiments, the coding sequences encode oleosins having molecular weights between about 14 kDa and about 19 kDa. In certain embodiments, the coding sequences encode fragments of oleosins, for example, (a) residues 1 to approximately 27, approximately 28 to approximately 109, or approximately 110 to the C terminus of SEQ ID NOS: 8 or 9; (b) residues 1 to approximately 15, approximately 16 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 10; (c) residues 1 to approximately 30, approximately 31 to approximately 114, or approximately 115 to the C-terminus of SEQ ID NO: 11; (d) residues 1 to approximately 18, approximately 19 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 12; or (e) residues 1 to about 40, about 41 to about 115, or about 116 to the C terminus of SEQ ID NO: 13. In certain embodiments, the encoded oleosms have amino acid sequences greater than 80% identical to either SEQ ID NOS: 8-13. Another aspect of the invention offers a nucleic acid molecule isolated from coffee. { Coffea spp. ), which has a coding sequence that encodes a steroleosin. In certain embodiments, the nucleic acid molecule encodes a fragment of a sterolema protein, e.g., residues 1 to about 50, about 50 to about 80, about 81 to about 102, about 103 to about 307, and about 308 to about the carboxy terminus of SEQ ID NO: 14. In other embodiments, the nucleic acid molecule encodes a steroleosin having an amino acid sequence greater than 80% identical to SEQ ID NO: 14. The nucleic acid molecules that code for oleosine or coffee steroleosin described above may be in one of several forms, including (1) a gene having an open reading structure comprising the coding sequence, (2) a mRNA molecule produced by the transcription of that gene , (3) a cDNA molecule produced by reverse transcription of mRNA, or (4) an oligonucleotide between 8 and 100 bases in length , which is complementary to a segment of one of the above forms of the nucleic acid molecule. Other aspects of the invention provide vectors comprising the nucleic acid molecules encoding oleosin or coffee steroleosins, described above. In certain embodiments, the vector is an expression vector, such as a plasmid, cosmid, baculovirus, bacmid, bacterial, yeast or viral vector. In certain embodiments, the vector contains the coding sequence of oleosin or steroleosin, operably linked to a constitutive promoter. In other 4 embodiments, the coding sequence is operably linked to an inducible promoter. In other embodiments, the coding sequence is operably linked to a tissue-specific promoter, which is a seed specific promoter in some embodiments, and a specific coffee seed promoter in particular embodiments. In those embodiments, the specific seed promoter of coffee may be an oleosin gene promoter. Another aspect of the invention provides host cells transformed with a vector of the type described above. The host cells can be plant cells, bacterial cells, fungal cells, insect cells or mammalian cells. In certain embodiments, the host cells are plant cells that can be coffee, tobacco, Arabidopsis, corn, wheat, rice, soybeans, barley, rye., oats, sorghum, alfalfa, clover, cañola, safflower, sunflower, peanut, cocoa, tomatillo, potato, pepper, eggplant, beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, zinnia, and pastures. The invention also provides fertile plants, produced from plant cells. Another aspect of the invention provides a method for modulating the flavor or aroma of coffee beans, which comprises modulating the production of one or more oleosins or steroleosins within coffee seeds. In certain embodiments, the method involves increasing the production of one or more oleosins or steroleosins, such as by increasing the expression of one or more endogenous oleosin or steroleosin genes within the coffee seeds, or by introducing a transgene. which codes for oleosin or steroleosin in the plant. In other embodiments, the method involves decreasing the production of one or more oleosins or steroleosins, such as by introducing a nucleic acid molecule in coffee, which inhibits gene expression of oleosin or steroleosin. Another aspect of the invention provides a promoter isolated from a coffee plant gene encoding an oleosin. In certain embodiments, the promoter is isolated from a gene encoding an oleosin having an amino acid sequence greater than 80% identical to any of SEQ ID NOS: 8-13. In particular embodiments, the promoter contains one or more regulatory sequences, selected from the group consisting of TTAAAT, TGTAAAGT, CAAATG, CATGTG, CATGCAAA, CCATGCA and ATATTTATT. In a specific embodiment, the promoter comprises SEQ ID NO: 15. Another aspect of the invention provides a chimeric gene comprising an oleosin gene promoter, operably linked to one or more coding sequences. Vectors, and host cells and fertile transgenic plants comprising such chimeric genes are also provided. Other features and advantages of the present invention will be understood by reference to the drawings, the detailed description and the following examples.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Optimal alignment of coffee protein sequences. Alignments were generated with a ClustalW program in the Lasergene software package (DNASTAR) and then manually adjusted to optimize the alignment. The location of the knot portion of proline P- (5X) -SP- (3X) -P is indicated, with highly conserved prolines and serines, shown in bold type. The conserved sequences are put in frame. The insertion of form H (see figure 4) is shown in the heavy type box. The access numbers of the aligned oleosin sequences are: CaOLE-I (SEQ ID NO: 8; AY9280S4), CcOLE-I (SEQ ID NO: 9; AY841271), CcOLE-2 (SEQ ID NO: 10; AY841272), CcOLE-3 (SEQ ID NO: 11; AY841273), CcOLE-4 (SEQ ID NO: 12; AY841274) and CcOLE-5 (SEQ ID NO: 13; AY841275).

Figure 2. Optimal alignment of the steroleosin protein of Coffea canephora, CcSTO-I sequence with the two sequences of the nearest data bank. Access numbers of the aligned oleosin sequence are: CAB39626 for A. thaliana (At) (AtSTOLE-7, SEQ ID NO .: 17), AY841276 for Coffea canephora (SEQ ID NO: 14), and AF498264 for Sesamun indicum (Lin and Tzen, 2004) (SiSTO-B, SEQ ID NO. : 16). The alignments were generated with a ClustalW program in the Lasergene software package (DNASTAR) and then manually adjusted to optimize the alignment. The conserved regions are put in picture. The locations of the potential active site of S- (12X) -Y- (3X) -K conserved and the KNOT portion of proline P- (11X) -P are indicated, with highly conserved residues shown in bold (Lin et ah, 2002 ). The NADPH and sterol binding regions identified by Lin et al. (2002) are also indicated. Figure 3. ClustalW-based phylogeny of the five oleosins of C. canephora and 16 oleosins of Arabidopsis. The protein sequences of each gene were aligned with the ClustalW program of the Lasergene package and then manually adjusted to optimize the alignment. To illustrate the potential evolutionary relationships between the various sequences, the resulting alignment is presented in the form of a phylogenetic tree. The scale represents the branch distance as the number of residual changes between the adjacent ones. The H and L forms of A are indicated. Thaliana The locations of the Arabidopsis sequences are shown as seed / microspore (SM), seed (S) and Tapetum (T). The access numbers of the aligned oleosin sequences are: AAF01542, BAB02690, CAA44225, Q39165, AA022633, AAF69712, BAB02215, AAC42242, NP196368, NP196369, CABS7942, NP196371, NP 196372, NP 196373, NPl 96377 and NP200969 for Arabidopsis SI, S2, S3, S4, S5, SM1, SM2, SM3, TI, T2, T3, T4, TS, T6, T7 and T8 respectively. Figure . Optimal alignment of the region containing the insertion portion of the H form of 18 residues in the C-terminal domain of oleosins. The region containing the site of the insertion of 18 residues of all coffee oleosins was aligned with selected oleosins of other plant species using the ClustalW program with a subsequent manual optimization stage. The conserved residues are put in picture; the residues with the highest conservation are in bold. The access numbers of the aligned oleosin sequences are: AAFO 1542, BAB02690, CAA44225, Q39165 and AA022633, for Arabidopsis seed 1 (SI), S2, S3, S4, and S5 (Kim et al, 2002; Tai et al, 2002) (SEQ ID NOS: 1S-22, respectively); AY928084 for Coffea arabica OLE-I (SEQ ID NO .: 1); P21641, S52030 and S52029 for Mai ze Hl, H2 and L (SEQ ID NOS: 23-25, respectively); U43931, U43930 and BAD23684 for Rice H, Ll and L2 (SEQ ID NOS: 26-28, respectively); U97700 (Chen et al, 1997) AF302807 and AF091840 (Tai et al, 2002) for Sesamum indi cum H2, Hl and L (SEQ ID NOS: 29-31 respectively); AF466102 and AF466103 for T. cacao 16.9 and 15.8 (Guilloteau et al, 2003) (SEQ ID NOS: 32-33, respectively). Figure 5. Expression of oleosin genes of Coffea canephora and Coffea arábi ca in different tissues and during the maturation of the seeds. Transcription levels for A) OLE-I, B) OLE-2, C) OLES, D) OLE-4,?) OLE-5 in various tissues, and in the development of the seed and tissues of the pericarp of coffee beans in different stages, was determined by both conventional (panels inserted above histograms) and by quantitative RT-PCR (histograms). The expression levels were determined in relation to the expression of transcripts of the RPL39 gene expressed constitutively in the same samples. F) shows the RPL39 control transcript in all tissues and samples. SG, small green bean; LG, big grain; YG, yellow grain; RG, mature grain; SP, small green pericarp; LP, large pericarp; YP, yellow pericarp; RP, red pericarp; St, stem; Le, sheet; Fl, flower; Rt, root.

Figure 6. Expression of oleosin and steroleosin genes in coffee. A) Expression of the CSP1 gene coding for the US storage protein in Coffea camphora and Coffea arabica in different tissues and during the maturation of the seed. Reverse transcription was carried out with equivalent amounts of total RNA. SG, small green bean; LG, big grain; YG, yellow grain; RG, mature grain; SP, small green pericarp; LP, large pericarp; YP, yellow pericarp; RP, red pericarp; St, stem; Le, sheet; Fl, flower; Rt, root. B) Expression of steroleosin in various tissues, determined by quantitative PCR. C) Expression of steroleosin in Coffea arabica (T-2308) during germination of the seed. The transcription levels were analyzed in the grain in five different stages of germination. Mature (fully developed grain), TO (after inhibition), 2DAI (two days after inhibition), 5DAI, 30DAI and 60DAI. Figure 7. Levels of transcription of oleosin in Coffea arabica (T-2308) during germination of the seed. The transcription levels were analyzed in the grain in five different stages of germination. TO (after inhibition), 3DAI (three days after inhibition), 5DAI, 30DAI and 60DAI.

Figure 8. In silico genomic sequence of the CcOLE-1 gene. The primers used for the genewalker system are underlined in the sequence. Sequence analysis of the CcOLE-1 promoter (pOLE-1, SEQ ID NO .: 15). The deduced and nucleotide protein sequences OLE-1 from C. canephora (SEQ ID NO: 2, SEQ ID NO: 9). An arrow indicates the start site of the transcript. The putative TATA table (==) is displayed. The RY portion is shown by a box. The "portion of endosperm" ("» "" ") is indicated;] _ portion similar to the AT-rich enhancer () and the E-frames (T-o). The sequence access number of the CcOLE-1 promoter ( pOLE-1, SEQ ID NO: 15) deposited in the EMBL / Genebank database is AY841277.The complete transcribed sequence of CcOLE-1 is shown in bold type C. CcOLE-1 amino acids are indicated below the codon first base The start and stop of the codon are indicated in Table 1. A restriction site of HindIII is indicated at position 123 bp from the transcriptional start site Figure 9A-9E Optimal alignment of each Coffea canephora protein sequence with the four sequences from the most similar data bank: Figure 9A) CcOLE-1 (AY841271), Figure 9B) CcOLE-2 (AY841272), Figure 9C) CcOLE-3 (AY841273), Figure 9D) CcOLE-4 (AY841274) and Figure 9E) CcOLE-5 (AY841275) The alignments were generated with the ClustalW program in the Lasergene software package (DNASTAR) and after or they were adjusted manually to optimize the alignment. The location of the knot portion of proline P- (5X) -SP- (3X) -P is indicated as a line above and in the box of the conserved P and S residues. The conserved sequences are put in frame; the highly conserved regions are shown in bold. The access numbers of the aligned oleosin sequences are: AAF69712 and BAB02215 for Arabidopsis seed / microspore 1 (SM1), and SM2 (Kim et al, 2002) (SEQ ID NOs 1.42, respectively); AY928084 for Coffea arabica (Ca) OLE-I (SEQ ID NO.:l); AAO65960 for Corylus avellana (Cav) OLE-L (SEQ ID NO.:38); T10121 for Ci trus sinensis OLE (SEQ ID NO -.36) (Naot et al, 1995); AAL92479 for Olea europaea OLE; Q43804 for Prunus dulcis for PdOLE-1 (SEQ ID NO.:37) (Garcia-Mas et al, 1995); AAG24455, AAG09751, AAG43516 and AAG43517 for Perilla frutescens OLN-Lb, OLN-La, and OLN-Sa (SEQ ID NOS: 39, 40, and 35, respectively); U97700 (Chen et al, 1997); AF302807 and AF091S40 (Tai et al, 2002) for Sesamum indicum H2, Hl and L (SEQ ID NOS: 29-31, respectively). Figure 10. Hydrophobicity profiles for the oleosin family of C. canephora. Hydropathy charts were generated according to the Kyte and Doolittle method (1982) using the appropriate program in the Lasergene software package (DNASTAR). Negative values indicate hydrophobic regions. The site of the knot portion of the proline is shown by an arrow. Figure 10 (F) is a graph of hydrophilicity. Figure 11. Analysis of Southern blot in the gene CcOLE-1. Evaluation of the number of copies of OLE-1 in the genome of C. canephora. The genomic DNA of robus ta was cut with Dral, SspI, Notl, Rsal or HindIII / SspI and Dral / Rsal. The genomic spots were probed with the full-length cDNA labeled with p32 which includes the 3 'and 5' untranslated region for CcOLE-1. The autoradiography presented was exposed for 10 days at -80 ° C. Figure 12. Expression of OLE-1 in the leaves of Coffea arabica (catimor) under drought stress. The transcription levels for OLE-1 were determined by quantitative RT-PCR. The expression levels were determined in relation to the expression of transcripts of the rpl39 gene expressed constitutively in the same samples. The unlabeled bars in each case represent the average transcription levels in the three very watery controls. The levels of transcription in three independent plants stressed by water are shown in bars marked diagonally.

DETAILED DESCRIPTION OF ILLUSTRATIVE MODALITIES Definitions: Various terms in relation to biological molecules and other aspects of the present invention are used throughout the specification and the claims. "Isolated" means altered "by the hand of man" from the natural state. If a composition or substance is present in nature, it has been "isolated" if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living or animal plant is not "isolated", but the same polynucleotide or polypeptide, separated from the coexisting materials of its natural state, is "isolated", as the term is used in the present. "Polynucleotide", also referred to as "Nucleic acid molecule", generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, double and single stranded DNA, DNA which is a mixture of double and single stranded regions, double and single stranded RNA and RN which is a mixture of double and single stranded regions, hybrid molecules that they comprise DNA and RNA that can be single-stranded or, more commonly, double-stranded or a mixture of double-stranded and single-stranded regions. In addition, "polynucleotides" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA that contains one or more modified bases and DNA or RNA with spinal columns modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inopine. A range of modifications to DNA and RNA can be made; thus, "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides as commonly found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "polynucleotides" also encompass relatively short polynucleotides, often referred to as oligonucleotides. "polypeptide" refers to any peptide or protein comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "polypeptide" refers to both short chains commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. The polypeptides may contain amino acids other than the 20 amino acids encoded by gene. "Polypeptides" include amino acid sequences, modified either by natural processes, such as post-translational processing or by chemical modification techniques that are well known in the art. Such modifications are described perfectly in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polynucleotide, which includes the peptide backbone, the amino acid side chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or in varying degrees, at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. The polypeptides can be branched as a result of ubiquitination, and these can be cyclic, with or without branching. The branched cyclic, branched and cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent coupling of flavin, covalent coupling of a heme moiety, coupling, covalent of a nucleotide or nucleotide derivative, covalent coupling of a lipid-derived lipid, covalent coupling of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition by transfer RNA, from amino acids to proteins such as arginylation, and ubiquitination. See, for example, Proteins-Structure and Molecular Properties, 2nd Edition, T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for Protein Modifications and Nonprotein Cofactors," Meth Enzymol (1990) 182: 626-646 and Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging," Ann NY Acad Sci (1992) 663: 48- 62"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains the essential properties. A common variant of a polynucleotide differs in the nucleotide sequence of another reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide modified by the reference polynucleotide. The nucleotide changes can result in substitutions, additions, deletions, fusions and truncations of the amino acids in the polypeptide encoded by the reference sequence, as described above. A typical variant of a polypeptide differs in the amino acid sequences of another reference polypeptide. Generally, the differences are limited, so that the sequences of the reference polypeptide and the variant are very similar globally and, in many regions, identical. A reference variant and polypeptide may differ in amino acid sequence by one or more substitutions, additions or deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide can be of natural origin, such as an allelic variant, or it can be a variant that is known to appear naturally. Variants of polynucleotide and polypeptides of non-natural origin can be made by mutagenesis techniques or by direct synthesis. In relation to mutant plants, the terms "null mutant" or "mutant with loss of function" are used to designate an organism or genomic DNA sequence with a mutation, which causes a gene product to be non-functional or very absent. Such mutations may occur in the coding and / or regulatory regions of the gene, and may be changes of individual residues, or insertions or deletions of nucleic acid regions. These mutations can also occur in the coding and regulatory regions of other genes, which by themselves can regulate or control an encoded gene and / or protein, to cause the protein to be non-functional or very absent. The term "substantially the same" refers to amino acid or nucleic acid sequences having sequential variations that do not materially affect the nature of the protein (e.g., the structure, stability characteristics, substrate specificity and / or biological activity of the protein ). With particular reference to the nucleic acid sequence, the term "substantially equal" is intended to refer to the coding region and to conserved sequences that govern expression, and refers primarily to degenerate codons encoding the same amino acid, or alternating codons encoding conservative substituted amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially equal" refers in general to conservative substitutions and / or variations in regions of the polypeptide, not involved in the determination of the structure or function. The terms "identical percent" and "similar percent" are also used herein in comparisons between amino acid and nucleic acid sequences. When referring to amino acid sequences, "identity" or "identical percent" refers to the percent of the amino acids of the subject amino acid sequences that have been adjusted to identical amino acids in the amino acid sequences compared by a test program of "Similar percent" sequences refer to the percent of the amino acids in the amino acid target sequence that has been adjusted to identical or conserved amino acids. Conserved amino acids are those that differ in structure, but are similar in physical properties such as the exchange of one for another that would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor, Biol. 119: 205). When referring to nucleic acid molecules, "identical percent" refers to the percent of nucleotides in the target nucleic acid sequence, which have been adjusted to identical nucleotides by a sequence analysis program.

"Identity" and "similarity" can be easily calculated by known methods. The nucleic acid sequences and the amino acid sequences can be compared using computer programs that align the similar sequences of the amino acids or nucleic acids and thus define the differences. In preferred methodologies, the BLAST programs (NCBI) and parameters used herein are employed, and the DNAstar system (Madison, Wl) is used to align sequential fragments of genomic DNA sequences. However, equivalent alignments and similarity / identity assessments can be obtained through the use of standard alignment software. For example, version 9.1 of the Wisconsin GCG package, available from Genetics Computer Group in Madison, Wisconsin, and the default parameters used (gap creation penalty or gap = 12, space extension penalty = 4) for that program, it can also be used to compare the identity and similarity of the sequences. "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain and humanized antibodies, as well as antibody fragments (e.g., Fab, Fab ', F (ab') 2 and F), which include the products of a Fab or another immunoglobulin expression library. With respect to antibodies, the term "immunologically specific" or "specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but that do not substantially recognize and bind to other molecules in a sample containing a protein. mixed population of antigenic biological molecules. Selection assays for determining the binding specificity of an antibody are well known and are routinely practiced in the art. For a comprehensive description of such trials, see Harlow et al. (Eds.), ANTIBODIES TO LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). ). More preferably, the preparation comprises at least 75% by weight, and more preferably 90-99% by weight, of the compound of interest. Purity is measured by methods appropriate for the compound of interest (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). With respect to the single-stranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to allow such hybridization under predetermined conditions, generally used in the art (sometimes referred to as "substantially complementary"). In particular, the term refers to the hybridization of an oligonucleotide with a substantially complementary sequence, contained within a single-stranded DNA or RNA molecule, for the substantial exclusion of oligonucleotide hybridization with single-stranded, non-sequence nucleic acids. complementary A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information, necessary to produce a gene product, when the sequence is expressed. The coding sequence may comprise untranslated sequences (e.g., introns or 5 'or 3' untranslated regions) within translated regions, or may lack such untranslated sequences (e.g., as in cDNA). "Intron" refers to polynucleotide sequences in a nucleic acid, which do not encode information related to protein synthesis. Such sequences are transcribed into mRNA, but are deleted before translation of the mRNA into a protein.

The term "operably linked" or "operably inserted" means that the regulatory sequences, necessary for the expression of the coding sequence are placed in a nucleic acid molecule at the appropriate positions with respect to the coding sequence, to make possible the expression of the coding sequence. By way of example, a promoter is operably linked to a coding sequence when the promoter is capable of controlling the transcription or expression of that coding sequence. The coding sequences can be operably linked to promoters or regulatory sequences in an antisense sense or orientation. The term "operably linked" is sometimes applied to the arrangement of other transcription control elements (eg, "enhancers" in an expression vector.) Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers , polyadenylation signals, terminators, and the like, which provide for the expression of a coding sequence in a host cell The terms "promoter", "promoter region" or "promoter sequence" refer generally to the transcriptional regulatory regions of a host. gene, which can be found on the 5 'or 3' side of the coding region, or within the coding region, or within introns.Commonly, a promoter is a DNA regulatory region, capable of binding RNA polymerase in a cell and initiate transcription of a downstream coding sequence (3 'direction.) The common 5' promoter sequence is linked at its end 3 'by the transcription initiation site and extends upstream (5' direction) f to include the minimum number of bases or elements necessary to initiate the transcription, at levels detectable above the antecedent. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensual sequences) responsible for the RNA-polymerase binding. A "vector" is a replicon, such as plasmid, phage, cosmid, or virus, to which another segment of nucleic acid can be operably inserted, to cause replication or segment expression. The term "nucleic acid construction" or "DNA construction" is sometimes used to refer to a sequence or coding sequences operably linked to appropriate regulatory sequences and inserted into a vector to transform a cell. This term can be used interchangeably with the term "transformation DNA" or "transgene". Such a nucleic acid construct can contain a coding sequence for a gene product of interest, together with a selectable marker gene and / or a reporter gene. A "marker gene" or "selectable marker gene" is a gene whose encoded gene product confers a characteristic that makes it possible for a cell containing the gene to be selected from other cells that do not contain the gene. The vectors used for the manipulation by genetic engineering commonly contain one or several selectable marker genes. Types of selectable marker genes include (1) antibiotic resistance genes, (2) pesticide tolerance or resistance genes, and (3) metabolic or auxotrophic marker genes that make it possible for transformed cells to synthesize an essential component, usually an amino acid , which the cells can not produce otherwise. A "reporter gene" is also a type of marker gene. It commonly encodes a gene product that is probable or detectable by standard laboratory means (eg, enzymatic activity, fluorescence). The term "express," "expressed," or "expression" of a gene refers to the biosynthesis of a gene product.

The process involves the transcription of the gene into mRNA and then the translation of the mRNA into one or more polypeptides, and encompasses all post-translational modifications of natural origin. "Endogenous" refers to any constituent, for example, a gene or nucleic acid, or polypeptide, which can be found naturally within the specified organism. A "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the largest molecule by nature. Thus, when the heterologous region comprises a gene, the gene will usually be flanked by DNA that does not flank the geonomic DNA in the genome of the source organism. In another example, a heterologous region is a construct in which the coding sequence itself is not found by nature (eg, a cDNA in which the genomic coding sequence contains introns, or synthetic sequences having codons, different from the native gene) . Allelic variations or mutational events of natural origin do not give rise to a heterologous region of DNA as defined herein. The term "DNA construction", as defined above, is also used to refer to a heterologous region, particularly one constructed to be used in the transformation of a cell. A cell has been "transformed" or "transfected" by exogenous or heterologous DNA, when such DNA is introduced into the cell. The transforming DNA may or may not be integrated (covalently linked) in the cell genome. In prokaryotes, yeasts and mammalian cells, for example, the transforming DNA can be maintained in an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome, so that it is inherited by daughter cells through the replication of chromosomes. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of developing stably in vitro for many generations. "Grain", "seed", refers to a plant unit in flowering reproduction, capable of developing in another plant of this type. As used herein, especially with respect to coffee plants, the terms are used synonymously and indistinctly. As used herein, the term "plant" includes reference to whole plants, plant organs, leaves, stems, shoots, roots, seeds, pollen, plant cells, organelles of the plant cell and progeny Of the same. Parts of transgenic plants will be within the scope of the invention to include, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, seeds, pollen, fruits, leaves or roots that are originate in transgenic plants or their progeny. Description: In one of its aspects, the present invention provides coffee nucleic acid molecules, which encode a range of oleosins, as well as a steroleosin. Representative examples of nucleic acid molecule encoding oleosin and steroleosin were identified from the databases of more than 47,000 expressed sequence markers (ESTs) from several Coffea canephora cDNA libraries (robusta) with RNA isolated from young leaves. and the grain and tissue of the pericarp of grains harvested at different stages of development. Overlapping ESTs were identified and "put in clusters" in unigenes (contigs) comprising complete coding sequences. Unigene sequences were scored by performing a BLAST search of each individual sequence against the non-redundant protein database NCBI (National Center for Biotechnology Information). The open reading structures of five of the unigenes expressed during grain development were scored as glycine-rich coding proteins, determined to be oleosins. A sixth open reading structure was identified by BLAST analysis of the databases with a known sequence of steroleosin. The ESTs representing the full-length cDNA for each oleosin or steroleosin unit were isolated and sequenced. A full-length cDNA for one of the oleosins (OLE-1) was also isolated and sequenced. These cDNAs are referred to herein as CaOLE-1 (SEQ ID NO: 1) and CcOLE-1 (SEQ ID NO.:2), CcOLE-2 (SEQ ID NO: 3), CcOLE-3 (SEQ ID NO: 4), CcOLE-4 (SEQ ID NO: 5), CcOLE-5 (SEQ ID NO: 6) and CcSTO-1 (SEQ ID NO: 7). The ESTs that form the oleosin or steroleosin unigenes, came from libraries obtained from grain at either 30 and 46 weeks after fertilization.

The deduced amino acid sequences of CaOLE-1 and CcOLE-1 up to CcOLE-5, were established herein as SEQ Numbers: 8-13, have molecular masses of 15.7, 14.1, 18.6, 15.3 and 17.9 kDa respectively. Each of the proteins contains a hydrophobic region of 81, 73, 80, 72 and 75 amino acids, respectively with the KNOT signature portion containing three conserved prolines and a serine conserved at its center. The amino acid sequence, deduced from Ce STO-1, set forth herein as SEQ ID NO: 14, has a molecular mass of 40.5 kDa, with a KNOT portion of proline within the N-terminal domain. Close orthologs of the five oleosins and coffee steroleosin have been identified in Arabidopsis and other plants with well characterized oily bodies, such as sesame, rice and corn. The quantitative expression analysis indicates that there may be at least two types of expression patterns for the seed (S) and floral microespore (SM) steroleosins; it was discovered that one group of genes has a higher level of expression at the beginning of the oleosin gene expression, while it was discovered that the other group shows greater expression slightly after the development of the grain. As evidenced by the data set out in greater detail in the examples, there appear to be significant differences in the levels and distribution of the oleosin transcripts in two coffee species, C. arabica and C. canephora (robusta), with the grain of C. arábi ca containing a greater amount of oil, which has a higher overall level of oleosin transcripts in relation to the expression of a constitutively expressed ribosomal protein. This observed variation in the global level of oleosin proteins between two coffee species, can provide a basis for coffee handling, through genetic techniques or traditional breeding, to influence the commercially important characteristics of coffee, such as oil content and profile, size and structure of oily bodies, formation of volatile lipid-derived materials, (E) -2-nonenal, and trans-trans-2-4-decadienal during coffee roasting, and generation of "foam" during the extraction of espresso coffee. Another aspect of the invention provides promoter sequences and related elements that control the expression of oleosin genes in coffee. As described in more detail in the examples, a promoter sequence, pOLE-1 (contained in SEQ ID Number 15), of one of these genes was identified by the transit of the primer assisted by PCR. The pOLE-1 promoter was shown to contain several seed regulatory elements, as shown in Figure 8 and described in the examples. Using this promoter linked to the GUS reporter gene, it was determined that this promoter is specific for the seeds, cotyledons, hypocotyledons and first true leaves of the developing seeds. The expression of the gene also showed that it is induced by water stress. Although the polynucleotides encoding oleosins and steroleosin from Coffea canephora are described and exemplified herein, it is intended that this invention encompass nucleic acids and encoded proteins from other Coffea species that are sufficiently similar to be used interchangeably with the polynucleotides and proteins of C. canephora for the purposes described above. Accordingly, when the terms "oleosin" or "steroleosin" are used herein, they are intended to encompass all Coffea oleosins or steroleosins having the general physical, biochemical, and functional characteristics described herein, and the polynucleotides that are incorporated herein by reference. encode Considered in terms of their sequences, polynucleotides encoding oleosin and steroleosin of the invention include allelic variants and natural mutants of SEQ ID NOS: 1-7, which are probably found in different varieties of C arabica or C canephora, and homologs of SEQ ID NOS: 1-7 that are probably found in different coffee species. Because such variants and homologs are expected to have certain differences in nucleotide and amino acid sequence, this invention provides isolated oleosin or steroleosin-encoding nucleic acid molecules, which encode respective polypeptides having at least about 80% (and, with order increasing preference, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% and 99%) of identity with any of SEQ ID NOS: 8-14, and comprises a nucleotide sequence having equivalent identity ranges to any of SEQ ID NOS: 1-7 . Because the natural sequence variation probably exists between oleosins and steroleosins, and the genes that encode them in different varieties of coffee and species, someone skilled in the art would expect to find this level of variation, while still retaining the properties unique of the polypeptides and polynucleotides of the present invention. Such expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of variations of amino acid sequences, conservative, which do not appreciably alter the nature of the encoded protein. Accordingly, such variants and homologs are considered practically the same as others and are included within the scope of the present invention. Several domains or fragments of the oleosin and steroleosin coffee genes and proteins are also considered within the scope of the invention. For example, the hydrophilic or antipathetic amino and carboxy-terminal domains of the oleosin polypeptides (for example, the N-terminus of about 10-40 residues and the C-terminus of about 30-50 residues, and the corresponding coding polynucleotides can be used to distinguish an oleosin protein or oleosin-encoding gene from another.The conserved hydrophobic core domains and corresponding coding polynucleotides can be useful for identifying oleosin orthologs from other species or genera.Also, the minor conserved portions of the polypeptide are of steroleosin (eg, residues 1 to about 50, about 81 to about 102, and about 308 of the carboxy terminus) and the corresponding coding polynucleotides can be distinguished from steroleosins closely related to each other, while conserved portions (eg residues) 50 up to about 80, and about 103 to about 307) can be used to identify less closely related orthologs. The preserved hydrophobic core domains will find particular utility for the subject recombinant proteins for oil bodies of the plant, which includes coffee, as described in US Patent No. 6,137,032. US Pat. No. 6,137,032 also describes the association of recombinant proteins comprising a hydrophobic oleosin domain of coffee with oily bodies (any of the natural or artificially constructed "body-like" structures formed using, for example, a vegetable oil ) can be used to facilitate the purification of such recombinant proteins (van Rooijen &; Moloney, 1995, Bio / Technology 13: 72-77). As mentioned, the inventors have demonstrated that oleosin gene expression is seed-specific and in coffee storage, it is also inducible by stress due to drought. Accordingly, gene regulatory sequences, associated with oleosin genes, are of practical utility and are considered within the scope of the present invention. The OLE-1 promoter of C. canephora is exemplified herein. The region upstream of the OLE-1 genomic sequence of C. canephora is set forth herein as SEQ ID NO: 15, and contains part or all of an exemplary promoter of the invention, although other portions of the promoter may be found in other sites in the gene, as explained in the definition of "promoter" set forth herein. However, promoters and other gene regulatory sequences of oleosin and steroleosin from any coffee species can be obtained by the same methods described below, and can be used according to the present invention. The promoters and regulatory elements that govern the tissue specificity and the temporal specificity of the gene expression of oleosin and steroleosin can be used to have an advantage in order to alter or modify the oily body profile of several coffee species, among other utilities. . The following sections establish the general procedures involved in the practice of the present invention. To the extent that specific materials are mentioned, it is only for the purpose of illustration, and it is not intended to limit the invention. Unless otherwise specified, general biochemical and molecular biological procedures, such as those set forth in Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory (1989) or Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons (2005) are used.

Nucleic acid molecules, proteins and antibodies: The nucleic acid molecules of the invention can be prepared by two general methods: (1) they can be synthesized from appropriate nucleotide triphosphates, or (2) they can be isolated from biological sources. Both methods use protocols well known in the art. The availability of nucleotide sequence information, such as the cDNA having SEQ ID NOS: 1-7 or the regulatory sequence of SEQ ID NO: 15, makes possible the preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides can be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resulting construct can be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to size limitations, inherent in current synthetic oligonucleotide methods. Thus, for example, a long, double-stranded molecule can be synthesized as several smaller fragments of appropriate complementarity. The complementary segments thus produced can be annealed in such a way that each segment possesses cohesive terms suitable for the coupling of an adjacent segment. Adjacent segments can be ligated by quenching the cohesive ends in the presence of DNA ligase to construct a full-length double stranded molecule. A synthetic DNA molecule thus constructed can then be cloned and amplified into an appropriate vector. In accordance with the present invention, nucleic acids having the sequence homology of appropriate level, share with all the coding and / or regulatory regions of the polynucleotides encoding oleosin or steroleosin, can be identified using hybridization and washing conditions of appropriate rigor. It will be appreciated by those skilled in the art that the aforementioned strategy, when applied to genomic sequences, in addition to making it possible to isolate the oleosin or steroleosin coding sequences, also makes it possible to isolate promoters and other gene regulatory sequences, associated with the oleosin or steroleosin genes, although the regulatory sequences themselves can not share sufficient homology to allow for adequate hybridization.

As a typical illustration, hybridizations can be performed, according to the method of Sambrook et al, using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg / ml sperm DNA of fragmented denatured salmon, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42 ° C for at least six hours. After hybridization, the filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37 ° C in 2X SSC and 0.1% SDS; (4) 2 hours at 45-55 ° C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes. A common formula to calculate the conditions of rigor, required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al, 1989): Tm = 81.5 ° C + 16.6 Log [Na +] + 0.41 (% G + C) - 0.63 (% formamide) -600 / # bp in duplex As an illustration of the above formula, using [Na +] = [0.368] and 50% formamide, with a GC content of 42% and an average probe size of 200 bases, the Tm is 57 ° C. The Tm of a DNA duplex decreases by 1-1.5 ° C with each 1% decrease in homology. In this way, targets with more than approximately 75% sequential identity would be observed using a hybridization temperature of 42 ° C. In one embodiment, the hybridization is at 37 ° C and the final wash is at 42 ° C; in another embodiment, the hybridization is at 42 ° C and the final wash is at 50 ° C; and in yet another embodiment, the hybridization is at 42 ° C and the final wash is at 65 ° C, with the hybridization and the previous wash solutions. High stringency conditions include hybridization at 42 ° C in the above hybridization solution and a final wash at 65 ° C in 0. IX SSC and 0.1% SDS for 10 minutes. The nucleic acids of the present invention can be maintained as DNA in any convenient cloning vector. In a preferred embodiment, the clones are maintained in cloning vector / plasmid expression, such as pGEM-T (Promega Biotech, Madison, Wl), pBluescript (Stratagene, La Jolla, CA), pCR4-TOPO (Invitrogen, Carlsbad, CA) or pET28a + (Novagen, Madison, Wl), all of them can be propagated in a host cell E. proper coli. The nucleic acid molecules of the invention include cDNA, geonomic DNA, RNA, and fragments thereof, which may be single or double stranded or even triple stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA), which have sequences capable of hybridizing to at least one sequence of a nucleic acid molecule of the present invention. Such oligonucleotides are useful as probes for detecting genes encoding oleosin or steroleosin mRNA in plant tissue test samples, for example, by PCR amplification, or for the positive or negative regulation of the expression of genes encoding oleosin or steroleosin on or before translation of mRNA into proteins. The methods in which oligonucleotides or polynucleotides encoding oleosin or steroleosin can be used as probes for such assays include, without restriction: (1) in situ hybridization; (2) Southern hybridization; (3) northern hybridization; and (4) classified amplification reactions such as polymerase chain reactions (PCR) (including RT-PCR) and ligase chain reaction (LCR). The polypeptides encoded by nucleic acids of the invention can be prepared in a variety of ways, according to known methods. If produced in situ, the polypeptides can be purified from appropriate sources, e.g., seeds, pericarps, or other parts of the plant. Alternatively, the availability of isolated nucleic acid molecules encoding the polypeptides makes it possible to produce the proteins using in vitro expression methods known in the art. For example, a cDNA or gene can be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by translation without cells into an appropriate cell-free translation system, such as reticulocytes. of wheat germ or rabbit. In vitro transcription and translation systems are commercially available, for example, from Promega Biotech, Madison, Wl, BRL, Rockville, MD or Invitrogen, Carlsbad, CA. According to a preferred embodiment, large quantities of oleosin or steroleosin polypeptides can be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of the DNA molecule, such as cDNAs having SEQ ID NOS: 1-7, can be inserted into a plasmid vector, adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or in a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements, necessary for the expression of the DNA in the host cell, positioned in such a way as to allow the expression of the DNA in the host cell. Such regulatory elements, required for expression, include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Oleosins or steroleosins produced by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. In a preferred embodiment, a commercially available expression / secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If the expression / secretion vectors are not used, an alternative method involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that specifically bind to the recombinant protein. Such methods are commonly used by practicing experts. The oleosins and steroleosins of the invention, prepared by the aforementioned methods, can be analyzed according to standard procedures. The oleosins and steroleosins purified from coffee or recombinantly produced, can be used to generate monoclonal or polyclonal antibodies, fragments or derivatives of antibodies as defined herein, according to known methods. In addition to producing antibodies to the total recombinant protein, if protein analyzes or Southern and cloning analyzes (see below) indicate that the cloned genes belong to a family of multiple genes, then member-specific antibodies are produced for synthetic peptides corresponding to the non-conserved regions, for example, the N- or C-terminal regions, of the protein can be generated. The kits comprising an antibody of the invention for any of the purposes described herein are also included within the scope of the invention. In general, such equipment includes a control antigen for which the antibody is immunospecific. Oleosms and sterolemas purified from coffee or produced recombinantly, can also be used as emulsifiers or, making use of their inherent capacity to stabilize small drops of oil inside coffee bean cells, can be used as encapsulating agents for soluble molecules in oil. Utilizing these properties, oleosins and coffee sterolemas will have practical utility in the food industry to prepare standard food emulsions, including without restriction cheese, yogurt, ice cream, margarine, mayonnaise, salad dressing or bakery products. These will also be useful in a cosmetic industry for producing soap, skin creams, toothpastes, lipsticks and facial makeup, and the like.

Vectors, cells, tissues and plants: Vectors and equipment for producing transgenic host cells containing a polynucleotide or oligonucleotide coding for oleosin or steroleosin, or homologue, analog or variant thereof, are also provided in accordance with the present invention. antisense or sense orientation, or reporter gene and other constructs under the control of the oleosin or steroleosin promoter and other regulatory sequences. Suitable host cells include, without restriction, plant cells, bacterial cells, yeast and other fungal cells, insect cells and mammalian cells. Vectors for transforming a wide variety of these host cells are well known to those skilled in the art. These include, without restriction, plasmids, phagemids, cosmids, baculoviruses, bacmides, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Commonly, the kits for producing transgenic host cells will contain one or several appropriate vectors and instructions for producing the transgenic cells using the vector. The kits may also include one or more additional components, such as culture media to grow the cells, reagents to effect cell transformation and reagents to test the transgenic cells for gene expression to name a few. The present invention includes transgenic plants comprising one or more copies of a gene encoding oleosin or steroleosin, or nucleic acid sequences that inhibit the production or function of endogenous oleosins or steroleosins of a plant. This is achieved by transforming the plant cells with a transgene comprising part or all of an oleosin or steroleosin coding sequence, or mutant, antisense or variant thereof, including RNA, controlled by any native or recombinant regulatory sequence, such as it is described later. Coffee species that are presently preferred for processing the transgenic plants described herein, include, without limitation, C. abeokutae, C. arábi ca, C. arnoldiana, C. aruwemiensis, C. bengalensis, C. canephora, C. congensis C. dewevrei, C. excelsa, C. engenioides, and C. heterocalyx, C. kapaka ta, C. kha síana, C. liberi ca, C. moloundou, C. rasemosa, C. salva trix, C. sessi flora, C. stenophylla, C. travencorensi s, C. wigh tiana and C. zanguebariae. Plants of any species are also included in the invention; these include, without restriction, tobacco, Arabidopsis and other "laboratory-friendly" species, cereal crops such as corn, wheat, rice, soybeans, barley, rye, oats, sorghum, alfalfa, clover and the like, oil-producing plants such as cañola, safflower, sunflower, peanut, cocoa and the like, vegetable crops such as tomato, tomatillo, potato, pepper, eggplant, beet, carrot, cucumber, lettuce, pea and similar, horticultural plants such as aster, begonia, chrysanthemum, delphinium , petunia, zinnia, grass and grasses and the like. Transgenic plants can be generated using standard plant transformation methods, known to those skilled in the art. These include without restriction, Agrobacterium um vectors, protoplast treatment with polyethylene glycol, delivery of biolitic DNA, UV laser micro beam, gemini virus vectors or other plant viral vectors, treatment of protoplasts with calcium phosphate, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microspheres covered with the transforming DNA, agitation of cell suspension in solution with silicone fibers covered with transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, for example, Methods for Plant Molecular Biology (Weissbach &; Weissbach, eds. , 1988); Methods in Plant Molecular Biology (Schuler &Zielinski, eds., 25 1989); Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in Plant Molecular Biology - A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem &Varner, eds., 1994). The transformation method depends on the plant that is to be transformed. Frequently Agrobacterium um vectors are used to transform dicotyledonous species. Agrobacterium um binary vectors include, without restriction, BIN19 and derivatives thereof, the pBI vector series, and binary vectors pGA482, pGA492, pLH7000 (Access to GenBank AY234330) and some other appropriate vectors pCAMBIA (derived from pPZP vectors built by Ha dukiewicz, Svab &Maliga, (1994) Plant Mol Biol 25: 989-994, available from CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or through the worldwide network at CAMBIA.org). For the transformation of monocotyledonous species, the biolistic bombardment with particles covered with transforming DNA and silicone fibers covered with transforming DNA are often useful for nuclear transformation. Alternatively, the "superbinary" vectors of Agroba cteri um have been successfully used for the transformation of rice, maize and various other monocotyledonous species. DNA constructs for transforming a selected plant comprise a coding sequence of interest, operably linked to appropriate 5 'regulatory sequences (e.g., translational regulatory and promoter sequences) and 3' regulatory sequences (e.g., terminators). In a preferred embodiment, an oleosin or steroleosin coding sequence is used under the control of its natural 5 'and 3' regulatory elements. In other embodiments, the oleosin and steroleosin coding and regulatory sequences are interchanged (eg, the Ce coding sequence OLE-1 operably linked to the CcOLE-2 promoter) to alter the oil profile of the transformed plant seed for a phenotypic improvement, for example, flavor, aroma or other characteristic. In an alternative embodiment, the coding region of the gene is placed under a potent constitutive promoter, such as the 35S promoter of the cauliflower mosaic virus (CaMV) or the 35S promoter of the scrapie mosaic virus. Other constitutive promoters contemplated for use in the present invention include, without restriction: promoters of T-DNA manpina synthetase, nopaline synthase and octopine synthase. In other embodiments, a strong monocotyledonous promoter is used, for example, the corn ubiquitin promoter, the rice actin promoter or the rice tubulin promoter (Jeon et al., Plant Physiology, 123: 1005-14, 2000). ). Transgenic plants expressing oleosin or steroleosin coding sequences under an inducible promoter are also contemplated within the scope of the present invention. Inducible plant promoters include the repressor-controlled promoter / tetracycline operator, the heat shock gene promoters, stress-induced promoters (e.g., wounds), gene promoters responsible for defense (e.g., phenylalanine-ammonia-lyase genes) , wound-induced gene promoters (e.g. hydroxyproline-rich cell wall protein genes), chemically inducible gene promoters (e.g., nitrate-reductase genes, glucanase genes, chitinase genes, etc.) and dark inducible gene promoters (for example, asparagine-synthetase gene), to name a few. Developmental-specific and tissue-specific promoters are also contemplated for use in the present invention, in addition to the seed-specific oleosin promoters of the invention. Non-limiting examples of other seed-specific promoters include Ciml (message induced by cytokinin), CZ19B1 (19 kDa corn zein), maize (myoinositol-1-phosphate synthase), and celA (cellulose synthase) (US Application Serial Number 09 / 377,648), bean beta-phaseolin, napin-beta-conglycinin , soy lecithin, cruciferin, 15 kDa corn zein, 22 kDa zein, 27 kDa zein, g-zein, waxy substance, shrunken 1, shrunken 2, and globulin 1, US soybean legume (Baumlein et al, 1992), and US seed storage protein of C. camphora (Marraccini et al, 1999, Plant Physiol., Biochem. 37: 273-282). See also WO 00/12733, where the preferred seed promoters from the endl and end2 genes are described. Other promoters specific for Coffea seeds can also be used, including without restriction the deirdirdine gene promoter described in copending, commonly assigned United States patent application No. 60 / 696,890. Examples of other tissue-specific promoters include, without restriction: the gene promoters of the small subunit of ribulose-bisphosphate-carboxylase (RuBisCo) (e.g., the promoter of the coffee subunit as described by Marracini et al., 2003) or gene promoters of the chlorophyll a / b binding protein (CAB) for expression in photosynthetic tissue; and the specific root glutamine synthetase gene promoters, where expression in the roots is desired. The coding region is also operably linked to an appropriate 3 'regulatory sequence. In embodiments where the native 3 'regulatory sequence is not used, the polyadenylation region of nopaline synthetase can be used. Other 3 'regulatory regions, useful include, without restriction, the polyadenylation region of octopine synthase. The selected coding region, under the control of appropriate regulatory elements, is operably linked to a nuclear drug resistance marker, such as resistance to kanamycin. Other selectable, useful marker systems include genes that confer resistance to herbicides or antibiotics (eg, resistance to hygromycin, sulfonylurea, phosphinothricin, or glyphosate) or genes that confer selective growth (eg, phosphomannose isomerase, which make it possible to grow plant cells on mannose). Selectable marker genes include, without limitation, genes encoding Resistance to antibiotics, such as those encoding neomycin phosphotransferase II (NEO), dihydrofolate reductase (DHFR) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glyphosate-resistant EPSPS and / or glyphosate-oxidoreducatase (GOX), Bromoxinyl-Nitrylase (BXN) for bromoxynil resistance, AHAS genes for imidazolinone resistance, sulfonylurea resistance genes, and resistance genes. , 4-dichlorophenoxyacetate (2,4-D). In certain embodiments, the promoters and other expression regulatory sequences, encompassed by the present invention, are operably linked to reporter genes. Reporter genes contemplated for use in the invention include, without restriction, genes encoding green fluorescent protein (GFP), red fluorescent protein (DsRed), fluorescent protein cyano (CFP), yellow fluorescent protein (YFP), orange fluorescent protein Cerian thus (cOFP), alkaline phosphatase (AP), ß-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside-phosphotransferase (neo1, G418t) dihydrofolate-reductase (DHFR), hygromycin-B-phosphotransferase ( HPH), thymidine kinase (TK), lacZ (which codes for a-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT), Beta-Glucuronidase (gus), placental alkaline phosphatase (PLAP), secreted embryonic alkaline phosphatase (SEAP) ), or bacterial or firefly luciferase (LUC). As with many of the standard procedures associated with the practice of the invention, those skilled in the art will realize that additional sequences may serve for the function of a marker or reporter. Additional sequence modifications are known in the art to increase gene expression in a cellular host. These modifications include deletion of sequences encoding superfluous polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be harmful to gene expression. Alternatively, if necessary, the G / C content of the coding sequence can be adjusted to average levels for a given coffee plant host cell, as calculated by reference to known genes, expressed in a coffee plant cell. . Also, when possible, the coding sequence is modified to avoid the predicted secondary structures such as hairpin mRNA. Another alternative to improve gene expression is to use 5 'leader sequences. The translation guide sequences are well known in the art and include the cis (omega ') derivative of the 5' (omega) guide sequences of the tobacco mosaic virus, the 5 'guide sequences of the bromine mosaic virus, mosaic virus of alfalfa, and turnip yellow mosaic virus.

The plants are transformed and then selected for one or several properties, including the presence of the transgenic product, the mRNA encoding the transgene, or an altered phenotype, associated with the expression of the transgene. It should be recognized that the amount of expression, as well as the tissue and temporal specific expression pattern of transgenes in transformed plants, may vary depending on the position of their insertion in the nuclear genome. Such positional effects are well known in the art. For this reason, several nuclear transformants must be regenerated and tested for the expression of the transgene.

Methods The nucleic acids and polypeptides of the present invention can be used in any of a range of methods, whereby protein products can be produced in coffee plants so that the proteins can play a role in improving the taste and / or the aroma of the coffee beverage or coffee products finally produced from the coffee plant grain expressing the protein. In one aspect, the present invention provides methods for altering the profile of oleosin or steroleosin in a plant, preferably coffee, comprising increasing or decreasing an amount or activity of one or more oleosins or steroleosins in the plant. For example, in one embodiment of the invention, a gene encoding oleosin under the control of its own expression control sequences is used to transform a plant in order to increase the production of that oleosin in the plant. Alternatively, an oleosin or steroleosin coding region is operably linked to regions that control heterologous expression, such as constitutive or inducible promoters. The profile of the oily body of a plant can also be altered by decreasing the production of one or several oleosins or steroleosins in the plant, or by selecting variants of natural origin to decrease the expression of oleosin or steroleosin. For example, mutant plants with loss of function (null) can be created or selected from populations of currently available plant mutants. It should also be appreciated by those skilled in the art that populations of mutants can also be selected for mutants overexpressing a particular oleosin, using one or more of the methods described herein. Mutant populations can be produced by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertions, or by directing local lesions induced in genomes (TILLING, see, for example, Henikoff et al, 2004, Plant Physiol. ): 630-636; Gilchrist &Haughn, 2005, Curr Opin. Plant Biol. 8 (2): 211-215). Methods for producing mutant populations are well known in the art. The nucleic acids of the invention can be used to identify oleosin or steroleosin mutants in various plant species. In species such as corn or Arabidopsis, where transposon insertion lines are available, the oligonucleotide primers can be designated to select lines for insertions in the oleosin or steroleosin genes. Through reproduction, a plant line can then be developed to be heterozygous or homozygous for the interrupted gene. A plant can also be engineered to show a phenotype similar to that seen in null mutants, created by mutagenic techniques. A transgenic null mutant can be created by an expression of a mutant form of a protein selected from oleosin or steroleosin, to create a "dominant negative effect". While not limiting the invention to some mechanism, this mutant protein will compete with the wild-type protein to interact with proteins or other cellular factors. Examples of this type "dominant negative effect" are well known for both insect and vertebrate systems (Radke et al, 1997, Genetics 145: 163-171; Kolch et /. , 1991, Nature 349: 426-428). Another type of transgenic null mutant can be created by inhibiting the translation of an mRNA encoding oleosin or steroleosin by "silencing the transcriptional gene". The gene encoding oleosin or steroleosin of the target species for down-regulation, or a fragment thereof, can be used to control the production of the encoded protein. Full-length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides directed to specific regions of the mRNA that are critical for translation can be used. The use of antisense molecules to decrease the expression levels of a predetermined gene is known in the art. Antisense molecules can be provided in themselves by transforming plant cells with a DNA construct which, after transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full length or partial antisense sequences. This silencing effect of the gene can be enhanced by the transgene overproduction of both sense and antisense RNA of the sequence encoding the gene, so that a large amount of dsRNA is produced (e.g., see Waterhouse et al., 1998, PNAS 95: 13959-13964). In this regard, sequences containing dsRNA that correspond to part or all of at least one intron, have been found to be particularly effective. In one embodiment, part or all of the antisense strand of the oleosin or steroleosin coding sequence is expressed by a transgene. In another embodiment, the antisense and hybridization strands of part or all of the oleosin or steroleosin coding sequence are transgenically expressed. In another embodiment, the oleosin and steroleosin genes can be silenced through the use of a range of different post-transcriptional gene silencing techniques (RNA silencing) that are currently available for plant systems. RNA silencing involves the processing of double-stranded RNA (dsRNA) into small fragments of 21-28 nucleotides by an enzyme based on RNAseH ("cutter" or "Cutter-like"). The cleavage products, which are siRNA (small interfering RNA) or miRNA (micro-RNA) are incorporated into protein effector complexes that regulate gene expression in a sequence-specific manner (for reviews of RNA silencing in plants, see Horiguchi , 2004, Differentiation 72: 65-73, Baulcombe, 2004, Nature 431: 356-363, Herr, 2004, Biochem. Soc. Trans. 32: 946-951). The small interfering RNAs can be chemically synthesized or transcribed and amplified in vi tro and then distributed to the cells. The distribution can be through microinjection (Tuschl T et al, 2002), chemical transfection (Agrawal N et al, 2003), electroporation or transfection mediated by cationic liposome (Brummelkamp TR et al, 2002; Elbashir SM et al., 2002 ), or any other means available in the art, which will be appreciated by the person skilled in the art. Alternatively, the siRNA can be expressed intracellularly by inserting DNA templates for siRNA in the cell of interest, for example, by means of a plasmid (Tuschl T et al, 2002), and can be targeted specifically to selected cells. Small interfering RNAs have been successfully introduced into plants (Klahre U et al., 2002). A preferred method of silencing RNA in the present invention is the use of short hairpin RNA (shRNA). A vector containing a DNA sequence encoding a particular desired siRNA sequence is distributed in a target cell by anti-monomer means. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that curl on themselves and form hairpin structures through intramolecular base pairing. These fork structures, once processed by the cell, they are equivalent to RNA molecules and are used by the cell to mediate the silencing of the RNA of the desired protein. Several constructs of particular utility for the silencing of RNA in plant are described by Horiguchi, 2004, supra. Commonly, such a construct comprises a promoter, a sequence of the target gene to be silenced in the "sense" orientation, a spacer, the antisense of the target gene sequence, and a terminator. Another type of synthetic null mutant can also be created by the "cosuppression" technique (Vaucheret et al, 1998, Plant J. 16 (6): 651-659). The plant cells are transformed with a full-length copy or a partial sequence of endogenous gene targeted for repression. In many cases, this results in complete repression of the native gene as well as the transgene. In one embodiment, a coding gene for oleosin or steroleosin from the plant species of interest is isolated and used to transform cells of the same species. Mutant or transgenic plants, produced by any of the methods described above, are also provided in accordance with the present invention. In some modalities, such plants will be useful as a research tool for the additional clarification of the participation of oleosins and steroleosins in the flavor, aroma and other characteristics of coffee beans, associated with oily profiles. Preferably, the plants are fertile, so they are useful for breeding purposes. Thus, mutants or plants that show one or more of the desirable phenotypes mentioned above can be used for plant reproduction, or directly in agriculture or horticultural applications. Plants that contain a specified transgene or mutation can also be crossed with plants that contain a complementary transgene or genotype, in order to produce plants with improved or combined phenotypes. The coffee plants produced according to the methods described above have practical utility for the production of coffee beans with flavored, aroma, improved or other characteristics as described above. Commonly, the beans are roasted and ground to drink. However, other uses for the grains will be apparent to those skilled in the art. For example, the oily bodies can be harvested from the grains (uncooked or lightly roasted), according to known methods, for example, the oily bodies of different purity levels can be purified as described in Guilloteau et al. 2003, Plant Science 164: 597-606, or for example as described in U.S. Pat. 6,146,645 to Deckers et al. and EP 0883997 to Wkabayashi et al. Similar to the isolated oleosin proteins, described above, these oily bodies can be used in the food industry to add flavor and nutrition, for example, to baked goods, yogurt or ice cream (for example, published application US No. 2005/0037111 Berry et al.) And the like, or in the cosmetics industry to produce soaps, skin creams, make-up and the like. The present invention also provides compositions and methods for producing, in a seed-specific or seed-preferred manner, a selected heterologous gene product in a plant. A coding sequence of interest is placed under the control of a coffee oleosin or other seed-specific promoter and other suitable regulatory sequences, to produce a seed-specific chimeric gene. The chimeric gene is introduced into a plant cell by any of the transformation methods described herein or known in the art. These chimeric genes and methods can be used to produce a range of gene products of interest to the plant, including without restriction: (1) detectable gene products such as GFP or GUS, as stated above; (2) gene products that confer an agricultural or horticultural benefit, such as those whose enzymatic activities result in the production of micronutrients (eg provitamin A, also known as beta carotene) or antioxidants (eg, ascorbic acid, fatty acids) omega, lycopene, isoprenes, terpenes); or (3) gene products to control pathogens or pests, such as those described by Mourgues et al., (1998), TibTech 16: 203-210 or others known to be protective for plant seeds or harmful to pathogens. Additionally, due to the expression of oleosin genes, such as the CcOle-1 gene, are also introduced under drought conditions, the oleosin gene promoters may also prove useful in directing gene expression in other tissues, such as mature leaves. , when these are subjected to osmotically severe stress. For example, these promoters can be used to express recombinant proteins, specifically in the leaves of plants (for example tobacco) at the end of maturation, as they suffer from senescence and begin to dry out. The following examples are provided to illustrate the invention in greater detail. The examples are for illustrative purposes, and are not intended to limit the invention.

Example 1 Plant material for RNA extraction Freshly harvested roots, young leaves, stems, flowers and fruits in different stages of development, were harvested from Coffea arabica, L. cv. Ca turra T-2308 developed under greenhouse conditions (25 ° C, 70% RH) and Coffea canephora (robusta) BP-409 developed in the field in Indonesia. The stages of development are defined as follows: small green fruit (SG), large green fruit (LG), yellow fruit (Y) and red fruit (R). The fresh tissues were immediately frozen in liquid nitrogen, then stored at -80 ° C until they were used for RNA extraction.

EXAMPLE 2 Total RNA extraction and generation of cRNA Samples stored at -80 ° C were grown in a powder and the total RNA was extracted from this powder using the method described by Gilloteau et al, 2003. The samples were treated with DNase using the "Qiagen RNase-Free DNase" equipment according to the manufacturer's instructions to eliminate DNA contamination. All RNA samples were analyzed by formaldehyde-agarose gel electrophoresis and visual inspection of the ribosomal RNA bands, after staining with ethyl bromide. Using oligo (dT20) as a primer, the cDNA was prepared from approximately 4 μg of total RNA according to the protocol in the Superscript II reverse transcriptase kit (Invitrogen, Carlsbad, CA). To test the presence of contaminating genomic DNA in the cDNA preparations, a primer pair spanning a known intron of a ubiquitously expressed cDNA, calconisomerase (CHI), was designed. RT-PCR was carried out using 10-fold dilution of cDNA corresponding to 0.1 μg of the original RNA. Conventional PCR reactions contained lx buffer and 5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, and 1 polymerase unit, and 800 nM of each of the gene-specific primers-front-CCCACCTGGAGCCTCTATTCTGTT (SEQ ID NO: 83) and inverse-CCCCGTCGGCCTCAAGTTTC (SEQ ID NO: 84) for 35 cycles. An expected cDNA band of 272 bp was observed after PCR. No second band corresponding to the cDNA + intron was observed in 750 bp, indicating an absence of genomic DNA in the samples (data not shown). The conventional PCR reactions for the genes of interest were carried out using a 100-fold dilution of cDNA corresponding to 0.01 μg of original RNA. PCR was carried out using 800 nM of each of the primers specific for the CcOLE-1 gene (forward-TTCGTTATCTTTAGCCCCATTT; inverse-CATAGGCAAGATTAACAAGGAT353) (SEQ ID NOS: 43, 44, respectively), CcOLE-2 (forward-GTGGCAGCGTTGAGCGT; inverse-GACAATAATGCATGAATACCACAA309) (SEQ ID NOS: 45, 46, respectively), CcOLE-3 (forward-GAGATCAAGGTGGAAGGGAA; inverse-GAAAACCCTCAACAAACAAAGA; 228) (SEQ ID NOS: 47, 48, respectively), CcOLE-4 (forward-CTGACACTGGCTGGAACAATA; inverse-GCACAACATTCCATCAAGTATCT337) (SEQ ID Numbers: 49, 50, respectively), and CcOLE-5 (forward-TGGCATCCTACTTCTCCTCACT; inverse-CTCTCTAGCATAATCCTTCACCTG295) (SEQ ID NOS: 51, 52, respectively). Amplification of the RPL39 gene (forward-TGGCGAAGAAGCAGAGGCAGA; inverse-TTGAGGGGGAGGGTAAAAAG187) (SEQ ID NOS: 53, 54, respectively) was used as a positive control for reverse transcription. The samples were subjected to electrophoresis in a 1.5% randomza gel. The numbers of supra indices in each primer group indicate the size of the amplicon.

Quantitative TaqMan PCR was carried out with the cDNA described above and the protocol recommended by the manufacturer (Applied Biosystems, Perkin-Elmer) was used. All reactions contained TaqMan lx buffer (Perkin-Elmer) and 5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, and 0.625 units of AmpliTaq gold polymerase. PCR was carried out using 800 nM of each of the gene-specific primers, forward and reverse, 200 nM of TaqMan probe, and 1000-fold dilution of cDNA corresponding to 0.001 μg of original RNA. Primers and probes were designed using the PRIMER EXPRESS software (Applied Biosystems: see table 3 below). The cross-specificity of the primers and probes is summarized in the following table. The reaction mixture was incubated for 2 minutes at 50 ° C, then 10 minutes at 95 ° C, followed by 40 amplification cycles of 15 seconds at 95 ° C / 1 minute at 60 ° C. The samples were quantified in the GeneAmp 7500 sequential detection system (Applied Biosystems). The levels of transcription were normalized to the levels of the control gene, rpl39.

Example 3: Isolation of the promoter and construction of the vector The upstream region 5 'of OLE-1 from Coffea canephora was recovered using the Genewalker equipment (BD from Biosciences) and the primers OLE-IA (5'-AAGTTGATGGACCCTTCTGAGGAAGG-3') (SEQ ID No.:55) followed by nested PCR using the OLE-IB primer (5'-AGCTGGTAGTGCTCAGCCATGAAGG-3 ') (SEQ ID NOS .: 56). PCR reactions contained lx buffer and 5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, and 1 unit of LA Taq polymerase (Takara, Combrex Bio, Belgium) with 200 nM of primer OLE-1A and 200 nM of API primer (Genewalker team). The reaction mixture was incubated for 10 minutes at 94 ° C, followed by 7 cycles of 25 seconds amplification at 94 ° C / 4 minutes at 72 ° C and then 32 cycles of 25 seconds amplification at 94 ° C / 4 minutes at 67 ° C. The PCR reaction was diluted 1/200 and used for a second PCR reaction using 200 nM of the nested primer OLE-IB and 200 nM of the nested primer AP2. Nested PCR was incubated for 10 minutes at 94 ° C, followed by 5 cycles of 25 seconds amplification at 94 ° C / 4 minutes at 72 ° C and then 22 cycles of 25 seconds amplification at 94 ° C / 4 minutes at 67 ° C. A 1075 bp genomic fragment was recovered and cloned into the pCR4-TOPO vector (Invitrogen) to produce pCR4-pOLEl and the insert of this plasmid was sequenced.

Example 4 Isolation and identification of oleosin genes from developing coffee bean More than 47,000 EST species of coffee libraries, produced with RNA isolated from young leaves and grain and pericarp tissues from grains harvested at different stages of development. Overlapping ESTs were subsequently "accumulated" into "unigenes" (ie contigs) and the sequences were annotated by making a BLAST investigation of each individual sequence against the non-redundant protein database. The ORF of five of the unigenes were expressed during grain development, scored as glycine-rich proteins / oleosins. EST representing the full length cDNA for each ungen, they were isolated and sequenced. These cDNAs were designated CcOLE-1 to CcOLE-5 (SEQ ID NOS: 2-6) (clones cccs46w9j5, cccs 6w20 22, cccs46w31f3, cccs30wl7hll and cccs30w33 respectively) depending on the number of ESTs obtained. These ESTs were all from libraries obtained from the grain either at 30 and at 46 weeks after fertilization. The deduced amino acid sequences (figure 1) of CcOLE-1 to CcOLE-5 have molecular masses of 15.7, 14.1, 18.6, 15.3 and 17.9 kDa respectively. Each of these proteins contains a hydrophobic region of 81, 73, 80, 72 and 75 amino acids respectively with the KNOT signature portion containing 3 conserved prolines and 1 serine conserved at its center. Figures 9A to 9E show the coffee oleosins, each aligned with the four most homologous sequences in the non-redundant protein database GenBank and Table 1 shows the percent identity for each coffee protein with the base proteins of more closely related data.

Table 1: identity of the oleosin amino acid sequence of Coffea canephora with the homologous GenBank sequences. (NP = not published). The access number of the Coffea oleosins was deposited in the Genbank NCBI.

Oleosin Name of the gene (access number) Publication% identity Coffea camphora (A Y841271) 100 Coffea arabica (AY928084) 99 1 Sesam m indicum (U97700 and JC5703) Chen eí a / 1997 69 Olea enropaea (AAL92479) NP 55 Perilla frutescens ( AAG43516) NP 51 Coffea canephora (A Y841272) 100 Citrus sinensis (T \ 0 \ 2 \) Naot et al 1995 80 2 Prunus dulcis (Q43804) García-Mas et al 1995 79 Corylus avellana (AAO65960) NP 77 Sesamum indicum (AF091840, AAD42942) Tai et al 2002 77 Coffea canephora (A YS41273) 100 Olea europaea (AAL92479) NP 64 3 Sesamum mdicum (AF302807, AAG23840) Tai et al 2002 62 Perilla frutescens (A AG24455) NP 59 Perilla frutescens (AAG09751) NP 58 Coffea canephora (A Y841274) 100 Sesamum mdicum (AF091840, AAD42942) Chen et al 1997 56 4 Citru sinensis (T \ 0 \ 2 \) Naot et al 1995 56 Corylus avellana (AAO65960) NP 54 Prunus dulcis (S51940) García-Mas et al 1995 53 Coffea canephoi a (A Y841275) 100 Arabidopsis thal? Ana-SM2 ( BAB02215) Kim et al 2002 56 Arabidop s thal? Ana-SM \ (AAF69712) Kim et al 2002 53 Theobroma cacao (AF466103) Guilloteau et al 2003 46 Corylus avellana (AAQ67349) NP 39_ The different coffee oleosm sequences were examined in greater detail. The hydrophobicity plots for each coffee oleosm clearly indicate the presence of a large region with a negative value, which is equivalent to the central hydrophobic region (Figure 10). These hydrophobic profiles are similar to the previous published profiles of specific oleoresins of seed (S) from T. cacao (Guilloteau et al, 2003) and Arabidopsis (Kim et al, 2002) and specific oleosins from microspores (SM) and seed of Arabidopsis (Kim et al, 2002). It was previously discovered by Tai et al. (2002) that oleosins expressed during the development of the seed are classified into two classes, which can be called the H and L forms, and are distinguished by the presence or absence of an insertion of 18 amino acids in the C-terminal region. Therefore, the alignment of the C-terminal region around the insertion site of the five coffee oleosins with the equivalent regions of a number of other oleosins found in the Genebank database was made (Figure 4). This alignment led to the classification of OLE-1, OLE-3 and OLE-5 as oleosins H and OLE-2 and OLE-4 as oleosins L. It is observed that the insertion of 18 C-terminal residues of OLE-5 was less homologous to the H insertions of the other oleosins, which include the absence of a highly conserved glycine in position 6 of the insert. Previous work on oily bodies assembled in vi tro showed that any of the H or L oleosins from rice and sesame can stabilize the oily bodies, although the oily bodies reconstituted with the oleosin L alone, were more stable than those reconstituted with the oleosin H or a mixture of oleosins H and L (Tzen et al, 1998; Tai et al, 2002).

Example 5 Tissue specificity and distribution of the development of CcOLE gene expression Table 2 shows that there are 52 ESTs in the unigen representing the most abundant oleosin (CcOLE-1) and only 5 ESTs in the unigen represent the least abundant oleosin ( CcOLE-5). Except for the CcOLE-5 EST found in the leaf library, all the ESTs of oleosin were detected only in the seed libraries and not in the pericarp or leaf libraries.

Table 2. Number and distribution of EST in the unigen containing the full-length Coffea canephora oleosin cDNA To confirm that the coffee oleosins were specific to the grain, the expression of each gene was studied by RT-PCR, using the methods described in example 2. The transcription levels of oleosin in the grain and in the fruit were analyzed in four different stages of development, as well as the leaves, stems, flowers and roots of C. canephora (robusta, BP409) and C. arábi ca (T-2308). The results of the RT-PCR experiment confirm that the five coffee oleosins were expressed mainly in the seed (Figures 5A to 5E). The expression of RPL39, a constitutively expressed ribosomal protein cDNA, was used as a positive control to show the success of the RT-PCR amplification in each RNA sample (Figure 5F). To quantify the transcription levels for each OLE gene at different stages of coffee bean development, as well as in several different coffee tissues, specific transcription assays were developed for each gene based on fluorescent real-time RT-PCR (TaqMan : Applied Biosystems), and the relative transcript levels in each RNA sample were quantified against the expression of a constitutively transcribed gene (RPL39) in the same sample. TaqMan quantitative PCR was carried out with the cDNA using the protocol recommended by the manufacturer (Applied Biosystems, Perkm-Elmer). All reactions contained TaqMan lx buffer (Perkm-Elmer) and 5 mM MgCl2, 200 μM each of dATP, dCTP, dGTP and dTTP, and 0.625 units of AmpliTaq golden polymerase. PCR was carried out using 800 nM of each of the gene-specific primers, forward and reverse, and 200 nM of TaqMan probe, and 1000-fold dilution of cDNA corresponding to 0.001 μg of the original RNA. Primers and probes were designed using the PRIMER EXPRESS software (Applied Biosystems). The primers and gene-specific probes are shown in Table 3. The reaction mixture was incubated for 2 minutes at 50 ° C, then 10 minutes at 95 ° C, followed by 40 cycles of 15 seconds amplification at 95 ° C. / 1 minute at 60 ° C. The samples were quantified in the GeneAmp 7500 sequential detection system (Applied Biosystems). The transcription levels were normalized to the levels of the RPL39 control gene.

Table 3 Gene sequence size SEQ ID No OLE- 1 Front CCGACTCATGAAGGCGTCTT 57 Reverse GTCCTGCAGCGCCACTTT 58 Probe (l) CCAGGAGCAAATGG 60 59 OLE-2 Front GACCGGGCAAGGCAAAA 60 Reverse GCTCAGCCCTGTCCTTCATC 61 Probe (l) CTGCTCTTAAGGCTAGGG 56 62 OLES Front CCGCCACAACAGCTTCAAG 63 Reverse ACACCGCCTTCCCCATATC 64 Probe (l) ACACCATCAGCACCTG 56 65 OLE-4 Front ATTGCTCATGCAGCTAAGGAGAT 66 Reverse TGAGCCTGCTGCCCAAA 67 Probe (l) AGGGACAAAGCTGAAC 59 68 OLES Front GGTTCGGACCGGGTTGAC 69 Reverse TCACCTGACTTGCCGTATTGC 70 Sonda0 'ATGCAAGAAGCCGAATT 56 71 US Front CGTGCTGGCCGCATTAC 72 Reverse GGAGGCTGCTGAGGATAGGA 73 Probe (l) ACTGTTAATAGCCAAAAGA 58 74 STO-1 Front GCACTGGAAGGCCTCTTTTG 75 Reverse GGACTTGCACCAGTGAGAAGTTT 76 Probe (2) AGGGCTCCCCTCCG 61 77 RPL39 Front GAACAGGCCCATCCCTTATTG 78 Reverse CGGCGCTTGGCAATTGTA 79 Probe (2) ATGCGCACTGACAACA 69 80 (1) MGB probes were labeled at 5 'with fluorescent reporter dye 6-carboxyfluorescein (FAM) and at 3' with the inactive dye 6-CARBOXI-N-N-N '-N-tetramethylrhodamine (TAMRA). All sequences are given 5 'to 3'. (2 > RPL39 and CcSTO-1 probes were labeled at 5 'with the VIC fluorescent reporter dye and at the 3' end with the TAMRA inactivator.

The results of the cross-specificity test of the OLE primer / probe groups were determined as described by Tan et al. (2003) and Simkin et al. (2004b) are summarized in table 4. A standard curve was made from the corresponding cDNA. The data represents the equivalent amount of signal reproduced for each primer / probe group with each cDNA. In the paired tests with other Coffea oleosins, each probe provided a minimum of 104-fold discrimination in detection of related transcripts.

Table 4. Specificity of each group of primers and probes by real-time PCR Taiman from CcOLE in the detection of the related sequence. ND = not detected.

Transcription CcOLE-1 CcOLE-2 CcOLE-3 CcOLE-4 CcOLE-5 OLE-1 probe < 2.8 x 10-7 < 7.1 x 10"7 &4. 4. 1 x 10'9 < 1 .8 x 10" 8 OLE-2 < 1.9 x 10"6 1 < 1.2 x 10" 6 < 1.3 x 10-6 < 6.4 x 10"7 OLE-3 < 1 .3 x l O-5 < 1.8 x 10"5 1 ND ND OLE-4 ND ND ND 1 < 5.6 x l 0" 14 OLE-5 ND ND ND < 1 .3 x 10-4 1 Plasmid containing each cDNA was added by reaction in a pairwise test against each primer and probe group. The data represents the equivalent amount of signal produced by 400 pg of each specific gene. Using the TaqMan assays, the levels of OLE transcripts were quantified in the same cDNA samples previously hardened for conventional RT-PCR. The results presented in Figures 5A to 5E (histograms) confirm that each OLE gene shows significant expression only in the grain. However, weak expression of several oleosin genes was also detected in certain different tissues. This is more likely to be due to the existence of oily bodies in other tissues.

It was shown that oily body biogenesis can occur outside the embryo in tobacco leaf cells (Wahlroos et al., 2003), European Olea fruit (Donaire et al., 1984) and in mature rice rushes (Wu et al. , 1998). Olesoins were also found associated with ER (Abell et al, 1997, Beaudoin and Napier, 2002). The most significant level of oleosin transcripts detected outside of grain was observed for OLE-5, where the expression was detected very clearly in fully mature flowers. This last observation is consistent with the previously presented alignments that indicate that OLE-5 may belong to the SM group of oleosins (Kim et al, 2002). In fact, this last observation is supported by the results obtained from a sequential comparison between the 16 known sequences of Arabidopsis and the 5 oleosin sequences of coffee (Figure 2). It was observed that OLE transcripts appear to be induced early in Coffea arabica when compared to C. camphora (robusta). Similar results are shown in table 2 above, which indicate that no oleosin genes encoding EST were detected in the robust sample at 18 weeks after fertilization. Taken together, these data indicate that the robusta grains in the small green stage develop less than the grains of arabica with a similar appearance. This difference in development could be closely linked to the slower ripening of robusta beans against arabica beans; Robusta beans develop in a period of 9 to 11 months while the Arabica fruit develops in a period of 6 to 8 months (Costa, 1989). To confirm the previous interpretation, a quantitative Taqman RT-PCR assay was designed to examine the expression of another coffee gene, the US storage protein gene, which is also strongly induced during the late stages of grain development ( Marraccini et al., 1999). The results presented in Figure 6A again indicate that the small green bean sample of robusta also exhibits undetectable expression of US, while the comparable sample of arabica indicates significant expression of this gene. In addition, the additional expression profile of specific grain genes using Taqman assays also showed that the expression profile of the small green robusta grain sample is different from the profile associated with the small green arabian grain (data not shown). The slight differences observed between the results for TaqMan and conventional RT-PCR (figure 5) are probably due to the non-quantitative nature of the latter during 40 cycles.

When the expression pattern for each oleosin gene exclusively in robusta grain was examined, it appeared that CcOLE-1, and to a lesser degree CcOLE-5, showed a different pattern of expression than that of the other three genes; the transcription levels of CcOLE-I and 5 were higher in the large green stage and then progressively decreased to maturity, although the decrease was less pronounced in CcOLE-5. It is noted that CcOLE-1 and CcOLE-5 are oleosins H, and therefore the observed expression pattern was different from another coffee oleosin H (CcOLE-3). The expression patterns found for CcOLE-2, CcOLE-3, and CcOLE 4 in robusta grains indicate that the transcription levels for these genes reached their highest level in the yellow stage, and that the levels before and after that stage they were somewhat inferior. When the levels of transcription of the five oleosins in arabica and robusta beans were compared, the transcription expression patterns were relatively similar, once the difference in development time (ie, the small green Arabica grain) was taken into account. was roughly equivalent to the large grain of robusta). However, after the close examination, some differences in the levels of transcription between the arabica and robusta beans could be observed. Assuming that the level of RPL39 transcripts are similar in both Arabic and robust, the peak transcript levels of OLE-1, OLE-2 and OLE-4 appeared to be approximately twice as high in arabica as in robusta. In contrast, the opposite appeared for the case of OLE-3, where the transcript levels were approximately twice lower in the yellow stage of arabica grain compared to the yellow stage of robusta grain. Note that the US transcript levels were relatively similar between these two species. This latter observation implies that the differences between arabica and robusta in the accumulation of oleosin transcripts are probably not due to differences in the expression of RPL39. Wu et at (1998) showed that the levels of transcription of the two rice oleosins appeared seven days after pollination and vanished in the mature seeds. A similar result was ined by Guilloteau et al, (2003), which showed that oleosin transcripts decreased in mature seeds, reaching a peak at 146 days after fertilization (dpf) and decreased to lower levels by 160 dpf. In the present example, all levels of transcription from OLE-1 to OLE-5 were shown to decrease in the final stages of maturation, although not to the same degree. OLE-1 and OLE-5 showed the greatest decrease during the course of the maturation period. Without pretending to be limited by any explanation of the mechanism, the high level of expression of OLE-1 found in the early stage of endosperm development, could imply that this oleosin has some important role in the initiation / formation of the oily body. In addition, it is notorious that samples of higher (arabic) oil content also have higher levels of OLE-1 expression. While it has been proposed by Ting et al (1996) the oleosin content is not related to the oil content, this may still be the case that the oil content may be related to the O-type oleosin H-type level. 1 expressed in the stage of initiation of the formation of the oily body.

Example 6 Expression of oleosin during germination of; Seed The transcription levels for each OLE gene were quantified in different stages of germination in C. arabica. The results of the RT-PCR experiment showed that the OLE-1 to OLE-4 transcripts were detected in the seeds in the early stages of germination (figure 7). OLE-I, OLE-2 and OLE-3 transcript levels were observed in the peak at three days after inhibition (3DAI). In the case of OLE-2, transcription levels were observed to increase up to levels observed in the final stages of seed maturation (see figure 7). In 5DAI, the OLE-1 and OLE-3 oleosin transcript levels decreased significantly along with OLE-2 and remained low throughout the rest of the germination. The levels of transcription of OLE-2 and OLE-3 were undetectable in 60DAI. OLE-5, previously identified as being probably an SM oleosin, was not detected in germinating grain. In addition, the quantitative RT-PCR also showed a concomitant increase in the transcription of STO-1 during germination, when compared with the expression of oleosins (see Figure 6C and Example 9). Example 7 Number of copies of CcOLE-1, the genome of C. canephora It is known that individual oleosins are usually encoded by any single genes, or genes with low copy number (Tai et al, 2002). In this example, it was confirmed that Coffea canephora OLE-1 is encoded by a simple copy number gene downstream in the coffee genome. Southern blot experiments were performed to estimate the copy number of CcOLE-1. The complete insertion of CcOLE-1 cDNA, which includes the 3 'untranslated region, was labeled with P32 and then hybridized under very stringent conditions to the genomic DNA of the BP-409 variety of robusta, which had been digested with several restriction enzymes as described above. The results obtained after 10 days of exposure (figure 11) showed that the single and double digestions resulted in the detection mainly of a larger band, except for the digestion of Hind III + SspI, where a second band was also detected. It is believed that this second band is due to the presence of a HindIII cut site in 123 bp from the transcription start site (see figure 8). The presence of weaker bands were also detected in the simple Dral and SspI digestions, which were lost from the double digestions. This was probably due to partial digestion of the genomic DNA, or to a very weak cross-hybridization with one or more of the other oleosins. These data strongly indicate that only one, or possibly two, genes in the coffee genome code for CcOLE-1.

Example 8 Identification of specific regulatory elements of seed in the 5 'region of OLE-1 from Coffea canephora The promoter region of OLE-1 was isolated from the genome of C canephora (robusta BP-409). A sequence of approximately 1075 bp upstream of the CcOLE-1 ATG site was recovered by a primer path aided by fully sequenced PCR as described in the previous examples. The promoter sequence obtained was then analyzed for the presence of known regulatory sequences (Figure 8). This analysis indicated the presence of a number of DNA regulatory sequences. For example, a TTTAAAT portion is located 39 bp upstream of the 5 'end of the CcOLE-1 cDNA (indicated by an arrow), and is a likely candidate for the TATA framed sequence. Other regulatory elements that previously showed to be responsible for the spatial and temporal specificity of the gene expression of the storage protein in a variety of plants were also found. The TGTAAAGT sequence (456/463) has been identified as a so-called "endosperm portion" and is involved in the control of endosperm-specific expression of glutamine in the promoters of barley (Thomas, 1993) and wheat (Hammond-Kosack et ah, 1993), legume legume (Shirsat et ah, 1989) and corn zein (Maier et ah, 1987). Other sequences such as the framed CANNTG E (CAAATG 738/743; CATGTG 914/919), which is believed to be involved in the seed-specific expression of French bean phaseolin (Kawagoe and Murai, 1992) and the protein, were also identified. Storage S2 of Douglas-fir (Chatthai et al, 2004). A CATGCAAA element (886/894) is similar to the so-called RY CATGCA (T / a) repeat region (A / g); the core region of the legume case (Dickinson et al, 1988; Shirsat et al, 1989). This portion is essential for the specific expression of the seed of US soy legume genes (Baumlein et al, 1992), β-conglycinin (Chamberlan et al, 1992) and glycinin (Lelievre et al, 1992). The region of the CCATGCA sequence (885/891) is similar to the RY GCATGC repeat element of the 2S albumin promoter, essential for seed-specific expression in transgenic tobacco seeds (Chatthai et al. 2004) and the CATGCC and CATGCC sequence detected in the US specific promoter of C. arabica seed (Marraccini et al, 1999). A AT rich AT ATTATTATT (504/512) similar to the specific seed enhancer identified in the upstream sequence of the soybean β-conglycinin subunit A gene (Alien et al, 1989) was also observed.

Example 9 Isolation and characterization of a coffee steroleosin cDNA A simple member of the steroleosin family designated CcSTO-1 (cccs46wllol5, AY841276), CcSTO-1 herein, was detected in the grain at 30 weeks and at 46 weeks after flowering (table 5).

Table 5. Distribution number of ESTs in the unigen containing the full-length steroleosin cDNA Steroleosins have previously been identified in association of oily seed bodies (Lin et al, 2002, Lin and Tzen, 2002). Steroleosins are stearyl dehydrogenases, which bind NADP +, which manifest dehydrogenase activity on both estradiol and corticosterone in vi tro (Lin et al, 2002). Without intending to limit itself to any explanation of mechanism, steroleosins may be involved in the regulation of signal transduction, a specialized biological function related to the oily bodies of seeds, which could be linked to the mobilization of oily bodies during the germination of the seed (Lin et al, 2002). Lin et al (2002) and Lin and Tzen, (2004) identified two different steroleosins associated with oily bodies in Sesame indi cum, designated steroleosin A and steroleosin B. Lin et al, (2002) also identified 8 members of the steroleosin family in Arabidopsi s thaliana in the non-redundant protein database in NCBI. However, Joliver et al (2004) detected only one steroleosin (steroleosin 1).; BAB09145) associated with oily Arabidopsis bodies in vivo. Figure 2 shows an optimized alignment of the CcSTO-1 protein sequence with the two most homologous GenBank protein sequences. The full-length amino acid sequence of CcSTO-l has 79% and 66% homology with steroleosin B associated with the oily body of S. indi cum (AF498264; Lin and Tzen, 2004) and steroleosin 7 of A. thaliana (CAB39626; see Lin et al, 2002) respectively. The active site S- (12X) -Y- (3X) -K conserved is indicated. In addition, a KNOT proline portion within the N-terminal domain having two conserved prolines also indicates and is believed to function as an anchor in a manner similar to that previously reported for the KNOT portion of oleosin (Lin et al, 2002). A Taqman-specific quantitative RT-PCR assay of STO-1 transcription levels gene in both arabica and robusta showed that this transcript is expressed mainly in low levels in the grain, although they were also observed in other tissues approximately 16 times lower levels of expression (figure 6). When steroleosin transcript levels in Arabica and Robusta grains are compared, the levels of STO-1 transcript showed to be relatively similar, between these two species, once development synchronization was taken into account. The appearance of STO-1 transcription at a later stage in robusta is similar to the results observed for all the genes tested here. Both in robusta and arabica beans, STO-1, the type levels of transcription in the large green stage, and then decrease in the later stages of development. A similar result was obtained by Lin and Tzen (2004) who showed that the transcription of steroleosin A associated with the oily body of sesame seed accumulated during the development of the seed.

Example 10 Functional analysis of the coffee oleosin CcDH2 promoter in Arabidopsis thaliana using a GUS-promoter fusion A functional analysis of the CcDH2 coffee oleosin promoter in Arabidopsis tha liana was conducted. The promoter was linked to a reporter gene, specifically a sequence encoding beta-glucuronidase (GUS).

Materials and methods The oleosin CcOlel promoter sequence was amplified using Pful polymerase under the conditions described by the supplier (Stratagene) and the following primers: TG-702 ttgaagcttACGACAGGTTTCCCGACTG (SEQ ID NO: 81) and TG-703 gcagatctaccatggGCGGTGGACGGTAGCTTAT (SEQ ID NO. : 82). The PCR fragment obtained in this way was then cut with HindIII and BglII and cloned into the HindIII / BglII sites of the plant transformation vector pCAMBIA1301. This places the approximately 1 kb fragment containing the oleosin promoter sequence, which contains the nearly complete 5 'untranslated region (only minus 3 bp) found in the oleosin cDNA (approximately 70 bp) in ATG for the GUS ( first exon of GUS). The correct position of the promoter was verified by sequencing. In the new oleosin promoter that contains the vector, it is named pCAMBIA1301UCD3.1 Transformation of the plant. The transformation vector pCAMBIA1301UCD3.1 after transformed into the strain EHA105 of Agroba cteri um tumefa ciens using standard procedures. The hygromycin resistance gene, activated by a 2 x 35S promoter, was the selectable marker for plant in pCambial301. The transformation mediated by Agrobacteri um tumefaciens of Arabidopsis (with the plasmid pCAMBIAlS01UCD3.1) was carried out by the floral bath method (Clough and Bent, 1998).

Transformed plants were identified by plating seeds on 0.8% agar containing 1 mM potassium nitrate and 50 μg per ml of hygromycin. Transformed plantings were identified 7 days after plating, as plants with an extended primary root. Seeds were transferred to 0.5X M &S salts containing 0.8% agar. The plants were transferred to the soil, when the second pair of leaves was developed and left to mature and prepare the seed (TI). In some cases, the Ti seeds germinated, and then they were allowed to develop into seeds (T2). Staining with GUS. The almacigos and silicas examined for GUS staining came from either TI or T2 seeds, and were in different stages of development. The GUS staining solution was prepared by dissolving 5 mg of X-Gluc in 50 μl of dimethyl formamide, and then adding this to lOml of 50 mM NaP04 pH 7.0. With a fine forceps, the almacigos were transferred from the germination plates to a 1.5 ml microfuge tube containing 1.0 ml of GUS stain. The tubes were transferred to a desiccator and placed under vacuum for 10 minutes and incubated at 37 ° C (in the dark) for 24 or 48 hours. The staining was removed and replaced with the decolorization solution (70% EtOH). The clarification was accelerated by placing the tubes at 37 ° C.

Depending on the amount of pigment in the fabric, several changes of 70% EtOH were required. Dyed almacigos and other tissues were observed in a dissecting microscope and the images were digitally recorded. In the case of silicas, the silicas were removed from the plants and opened with a scalpel to allow the penetration of the dye. The previous GUS dye was modified to include 0.5% Triton XIOO. After staining, the silicas were decolorized by incubation in EtOH: acetic acid (2: 1) and then incubated in light Hover medium (100 g, chlorine hydrate in 60 ml of water). The silicas with younger seeds were preincubated in the Ethanol: acetic acid solution for 4 hours, and the silicas with older seeds for 8 hours. The silicas were rinsed in Hoover's light medium for 24 hours to several days.

Results GUS expression was observed in Arabidopsis thaliana transformed with pCaml301UCD3.1 in seedlings at different stages of development. The expression was observed in cotyledons, hypocotyledons of very young almacigos and in the first true leaves of older almacigos. No significant expression was detected in the roots. No GUS activity was detected in the mature leaves. GUS expression was also detected in the silica wall, but GUS staining in the silica wall was not as intense as in the young germinating seed tissues. It was not possible to fully clarify the silica in Hoyler's medium, so that the residual green pigmentation remained on the silica wall, giving the tinted silica a bluish-green color. The activity of GUS was restricted to silica and did not extend to the floral stem. These data confirm that the CcOLE-1 coffee oleosin promoter activates the expression of the linked coding sequence in seeds, in silicas, as well as in the first cotyledons and the first true leaves of the germinating seeds. Importantly, this result demonstrates that the CcOIe-1 promoter sequence described herein contains all the functional elements required to activate specific gene expression of seed in plants. The data also indicate that the CcOIe-1 promoter can be used to activate the expression of genes in immature tissues such as the first two cotyledons derived from the germinating seed embryo. In addition, the data indicate that the CcDH2 promoter is activated in other tissues such as silicas. It is observed that the activation level in the silicas and the grain seems to be relatively lower than in the cotyledons of the seed in germination, although at least part of this difference could be due to the differences in the ability to stain GUS in these types of very different fabric. Finally, given the relatively large evolutionary distance between Arabidopsis and Coffea, the demonstration in the present that the CcDH2 promoter of coffee functions in Arabidopsis implies that this promoter must be activated in a relatively broad variety of plants.

Example 11 Induction of the expression of the CcOle-1 coffee gene by osmotic stress To explore the role of coffee oleosin CcOle-1 in the response to osmotic stress, the CcOle-1 expression was examined in plants subjected to water deficit (drought).

Materials and methods: Dehydration experiments were carried out using clonally propagated small clump-grown Cephaea catimor trees grown in a greenhouse. The trees were approximately three years old and developed on the ground. Several weeks before the experiments, the trees were grown together in the greenhouse at a temperature of about 25 ° C, with a relative humidity of about 70%, and were watered daily using automatic irrigation. At the beginning of the experiment, three trees acted as controls and were watered daily. The other three trees were not watered and thus suffered progressive dehydration. Sampling two young leaves (5-8 cm in size, taken from the emerging growth on top of the plant), was carried out each week for each tree. The samples were frozen directly in liquid nitrogen. RNA extraction and cDNA synthesis. The extraction of tissue samples subjected to the various stress treatments and controls was carried out using the RNEASY® Plant mini-kit from Qiagen GmbH (Hilden, Germany). The frozen tissue samples were initially ground in a mortar and pestle using liquid nitrogen in order to obtain a powder. The RNA in this frozen powder was then extracted according to the protocol of the RNEASY® Plant mini-equipment. In summary, a maximum of 100 mg of frozen powder was mixed with the cell lysis buffer and β-mercaptoethanol. For tissues that showed significant necrosis, 2 μM PMSF was also added. To eliminate the low levels of contaminating genomic DNA, a treatment using DNase without DNase contained in the Plant RNEASY® mini-equipment (as described by the supplier) was used, ie a 15-minute treatment at room temperature in the column. At the end the RNA was eluted from the column in 50 μl of water without RNase. The amount of RNA was determined by spectrophotometric measurement at 260 nm and the quality of the RNA was estimated by calculating the absorbance ratio at 260 nm / 280 nm. The quality of the RNA was also verified by electrophoresis in 1% agarose gels. The reverse transcription reactions were carried out for these RNA samples as follows; approximately 1 μg of total RNA and 12.4 μM of oligo-dT [2.3 μl of oligo-dT 70 μM (Proligo)] with water without RNase up to a final volume of 13 μl. This mixture was incubated at 65 ° C for 5 minutes. Then, 7 μl of a 5X buffer mixture (transcribed RT reaction buffer), 20 U of RNAse inhibitor, 1 mM of the four dNTPs (250 μm each and 10 U of TRANSCRJPTOR® reverse transcriptase (Roche, Nutley) were added. , NJ) This mixture was incubated at 55 ° C for 40 minutes Finally, 0.5 μl of RNaseH (Invitrogen, Carlsbad, CA) was added to the 20 μl of mixture and the reaction was subsequently incubated for 30 minutes at 37 ° C. The generated cDNAs were purified using the SNAPMR gel purification equipment from Invitrogen (Carlsbad, CA) according to the protocol provided by the provider: Primers and MGB probe design The primers and MGB probe groups were designed using the PRIMER software EXPRESS ™ (Applied Biosystems, Foster City, Calif.) Hybridization temperatures of the primers were approximately 60 ° C, whereas the MGB probe was approximately 70 ° C. The size of the amplicons was approximately 80 bp. The primers were synthesized by PROLIGO and the MGB probe were synthesized according to the supplier's instructions (Applied Biosystems, Foster City, CA). The sequences of the primers and probes for Cc01e-1 and CcRpl39 have been presented previously in Table 3. Real-time quantitative RT-PCR. The cDNA used for these experiments was prepared as described above. The TaqMan PCR was performed as described in several previous sections. The absence of some significant level of residual genomic DNA in the cDNA preparations was verified by measuring the level of quantitative PCR amplification signal for a genomic specific primer / probe group for the GOS gene versus the signal for a GOS cDNA probe .

Results: Figure 2 shows the induction of the expression of the Cc01e-1 gene in the leaves of small trees developed in greenhouse when irrigation was stopped (drought conditions). After three weeks, it was discovered that the expression of Cc01e-1 was induced slightly by the water tension in a plant versus the average expression of 01e-l between three well-hydrated control plants. Small induction was observed in the other two plants treated in week 3. By week 4, expression of Ole-1 was induced in two of the three treated plants. At week 6, the three treated plants showed an elevation in the expression of 01e-1. The increased levels of Ole-1 expression discovered for all three plants subjected to water stress varied between one RQ > 0.18 and < 0.4. Although these values were several times lower than those observed for Ole-1 in developing grain, nevertheless they were several times higher than those observed for the control leaves without undergoing stress. This last observation indicates that oleosins such as CcOle-1 can contribute to the endogenous protection of leaf tissues under osmotic stress.

References Aalen RB, Opsahl-Ferstad HG, Linnestad C, Olsen OA. (1994). Transcripts encoding an oleosin and a dormancy-related protein are present in both the aleurone layer and the embryo of developing barley (Hordeum vulgare L.) seeds.

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The present invention is not limited to the embodiments described and exemplified above, but has the ability to be varied and modified within the scope of the appended claims.

Claims (75)

1. Isolated nucleic acid molecule of coffee (Coffea spp.), Which has a coding sequence coding for an oleosin.

2. Nucleic acid molecule according to claim 1, wherein the coding sequence codes for an oleosin having a molecular weight between about 14 kDa and about 19 kDa.

3. Nucleic acid molecule according to claim 2, wherein the oleosin has an amino acid sequence comprising one or more fragments selected from the group consisting of: a) residues 1 to approximately 27, approximately 28 to approximately 109, or approximately 110 to end C of SEQ ID NO: 8 or SEQ ID NO: 9; b) residues 1 to approximately 15, approximately 16 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 10; c) residues 1 to approximately 30, approximately 31 to approximately 114, or approximately 115 to the C-terminus of SEQ ID NO: 11; d) residues 1 to approximately 18, approximately 19 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 12; and e) residues 1 to about 40, about 41 to about 115, or about 116 to the C terminus of SEQ ID NO: 134.

Nucleic acid molecule according to claim 3, wherein the oleosin has an amino acid sequence greater than 80% identical to one of SEQ ID NOS: 8-13.

5. Nucleic acid molecule according to claim 4, wherein the oleosin has an amino acid sequence according to any of SEQ ID NOS: 8-13.

6. Nucleic acid molecule according to claim 4, wherein the coding sequence is more than 70% identical to one of the coding sequences set forth in SEQ ID NOS: 1-6.

7. Nucleic acid molecule according to claim 6, wherein the coding sequence comprises any of SEQ ID NOS: 1-6.

8. Nucleic acid molecule according to claim 1, which is a gene having an open reading structure comprising the coding sequence.

9. MRNA molecule produced by transcription of the gene according to claim 8.

10. CDNA molecule produced by reverse transcription of the mRNA molecule according to claim 9.

11. Oligonucleotide between 8 and 100 bases in length, which is complementary to a segment of the nucleic acid molecule according to claim 1.

12. Vector comprising the nucleic acid molecule according to claim 1.

13. Vector according to claim 12, which is an expression vector selected from the group of vectors consisting of plasmid, cosmid, baculovirus, bacmid, bacterial, yeast and viral vectors.

14. Vector according to claim 12, wherein the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter.

15. Vector according to claim 12, wherein the coding sequence of the nucleic acid molecule is operably linked to an inducible promoter.

16. Vector according to claim 12, wherein the coding sequence of the nucleic acid molecule is operably linked to a tissue-specific promoter.

17. Vector according to claim 16, wherein the tissue specific promoter is a seed-specific promoter.

18. Vector according to claim 17, wherein the seed-specific promoter is a specific promoter of coffee seed.

19. Vector according to claim 18, wherein the coffee seed specific promoter is a promoter of the oleosin gene.

20. Vector according to claim 19, wherein the oleosin gene promoter comprises SEQ ID NO: 15.

21. Host cell transformed with the vector according to claim 12.

22. Host cell according to claim 21, selected from the group consisting of plant cells, bacterial cells, fungal cells, insect cells and mammalian cells.

23. Host cell according to claim 21, which is a plant cell selected from the group of plants consisting of coffee, tobacco, Arabidopsis, corn, wheat, rice, soybeans, rye, barley, oats, sorghum, alfalfa, clover, canola, safflower , sunflower, peanut, cacao, tomatillo, potato, pepper, eggplant, beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, zinnia, and pastures.

24. Fertile plant produced from the plant cell according to claim 23.

25. Method for modulating the flavor or aroma of coffee beans, which comprises modulating the production of one or several oleosins within the coffee seeds.

26. The method according to claim 25, which comprises increasing the production of one or several oleosins.

27. The method according to claim 26, which comprises increasing the expression of one or several endogenous oleosin genes within the coffee seeds.

28. The method according to claim 26, comprising introducing a transgene encoding oleosin in the plant.

29. The method according to claim 25, which comprises reducing the production of one or more oleosins.

30. The method according to claim 29, comprising introducing a nucleic acid molecule in coffee, which inhibits the expression of one or more genes encoding oleosin.

31. Isolated promoter of a coffee plant gene that codes for an oleosin.

32. Promoter according to claim 31, wherein the gene codes for an oleosin, has a molecular weight of between about 14 kDa and about 19 kDa.

33. Promoter according to claim 32, wherein the gene encodes an oleosin, has an amino acid sequence comprising one or more fragments selected from the group consisting of: a) residues 1 to approximately 27, approximately 28 to approximately 109, or approximately 110 to the C-terminus of SEQ ID NO: 8 or SEQ ID NO: 9; b) residues 1 to approximately 15, approximately 16 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 10; c) residues 1 to approximately 30, approximately 31 to approximately 114, or approximately 115 to the C-terminus of SEQ ID NO: 11; d) residues 1 to approximately 18, approximately 19 to approximately 89, or approximately 90 to the C-terminus of SEQ ID NO: 12; and e) residues 1 to about 40, about 41 to about 115, or about 116 to the C terminus of SEQ ID NO: 13.

34. Promoter according to claim 33, wherein the gene coding for an oleosin has an amino acid sequence greater than 80% identical to one of SEQ ID NOS: 8-13.

35. Promoter according to claim 34, which has an amino acid sequence according to any of SEQ ID NOS: 8-13.

36. Promoter according to claim 35, wherein the gene comprising an open reading structure more than 70% identical to one of the coding sequences set forth in SEQ ID NOS: 1-6.

37. Promoter according to claim 36, wherein the gene comprising an open reading structure has any of SEQ ID NOS: 1-6.

38. Promoter according to claim 31, comprising one or more regulatory sequences selected from the group consisting of TTAAAT, TGTAAAGT, CAAATG, CATGTG, CATGCAAA, CCATGCA and ATATTTATT.

39. Promoter according to claim 38, comprising SEQ ID NO: 15.

40. Chimeric gene comprising the promoter according to claim 31, operably linked to one or more coding sequences.

41. Vector for transforming a cell, comprising the chimeric gene according to claim 40.

42. Cell transformed with the vector according to claim 41.

43. Transformed cell according to claim 42, which is a plant cell.

44. Transformed plant cell according to claim 43, which is a cell of Coffea spp.

45. Fertile transgenic plant, produced by regeneration of the transformed plant cell according to claim 43.

46. A fertile transgenic plant according to claim 45, which Coffea spp.

47. Nucleic acid molecule isolated from coffee. { Coffea spp. ), which has a coding sequence that codes for an steroleosin.

48. Nucleic acid molecule according to claim 47, wherein the steroleosin has an amino acid sequence comprising one or more fragments selected from the group consisting of residues 1 to about 50, about 50 to about 80, about 81 to about 102, about 103 to about 307, and about 308 to the carboxy terminus of SEQ ID NO: 14.

49. Nucleic acid molecule according to claim 47, wherein the steroleosin has an amino acid sequence greater than 80% identical to SEQ ID NO: 14.

50. Nucleic acid molecule according to claim 49, wherein the steroleosin has SEQ ID NO: 14.

51. Nucleic acid molecule according to claim 50, wherein the coding sequence is more than 70% identical to the coding sequence set forth in SEQ ID NO: 7.

52. Nucleic acid molecule according to claim 51, wherein the coding sequence comprises SEQ ID NO: 7.

53. Nucleic acid molecule according to claim 47, which is a gene having an open reading structure comprising the coding sequence.

54. MRNA molecule produced by transcription of the gene according to claim 53.

55. CDNA molecule produced by reverse transcription of the mRNA molecule according to claim 54.

56. Oligonucleotide between 8 and 100 bases in length, which is complementary to a segment of the nucleic acid molecule according to claim 47.

57. Vector comprising the nucleic acid molecule according to claim 47.

58. Vector according to claim 57, which is an expression vector selected from a group of vectors such as plasmid, cosmid, baculovirus, beta, bacterial or yeast vectors or viral vectors.

59. Vector according to claim 57, wherein the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter.

60. Vector according to claim 57, wherein the coding sequence of the nucleic acid molecule is operably linked to an inducible promoter.

61. Vector according to claim 57, wherein the coding sequence of the nucleic acid molecule is operably linked to a tissue-specific promoter.

62. Vector according to claim 61, wherein the tissue-specific promoter is a seed-specific promoter.

63. Vector according to claim 62, wherein the seed specific promoter is a coffee seed specific promoter.

64. Vector according to claim 63, wherein the brown seed-specific promoter in an oleosin gene promoter.

65. Vector according to claim 64, wherein the oleosin gene promoter comprises SEQ ID NO: 15.

66. Host cell transformed with the vector according to claim 57.

67. Host cell according to claim 66, selected from the group consisting of plant cells, bacterial cells, fungal cells, insect cells and mammalian cells.

68. Host cell according to claim 67, which is a plant cell selected from the group of plants consisting of coffee, tobacco, Arabidopsis, corn, wheat, rice, soybeans, rye, barley, oats, sorghum, alfalfa, clover, canola, safflower , sunflower, peanut, cacao, tomatillo, potato, pepper, eggplant, beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, zinnia, and pastures.

69. Fertile plant produced from the plant cell according to claim 68.

70. Method for modulating the flavor or aroma of coffee beans, which comprises modulating the production of one or several oleosins within the coffee seeds.

71. Method according to claim 70, which comprises increasing the production of one or several steroleosins.

72. The method according to claim 71, which comprises increasing the expression of one or several endogenous steroleosin genes within the coffee seeds.

73. The method according to claim 72, comprising introducing a transgene coding for steroleosin in the plant.

74. Method according to claim 70, which comprises decreasing the production of one or more steroleosins.

75. The method according to claim 74, comprising the introduction of a nucleic acid molecule into coffee that inhibits the expression of one or more genes coding for steroleosin.