Modulation of coffee aroma precursors in raw (non-fried) coffee grain

FIELD: chemistry; biochemistry.

SUBSTANCE: invention concerns biotechnology and claims polynucleotide encoding polypeptide with cysteine protease effect. Also invention concerns coffee tree cell transformed by the indicated polynucleotide.

EFFECT: possibility to modulate coffee aroma precursor level in raw (non-fried) coffee grain.

14 cl, 22 dwg, 7 tbl, 1 ex

 

The level of technology.

Coffee contains a very complex mixture of aromatic molecules. Up to the present time in the course of intensive studies of the composition of the beverages from instant and ground coffee identified more than 850 compounds, many of which are active aromatic molecules (Flament I. (2002) Coffee Flavor Chemistry, John Wiley and Sons, UK). However, some of the aromatic molecules found in a Cup of coffee ready, present in the raw materials, i.e. in raw beans (green beans) plant species Coffea arabica or Coffea canephora (robustd). In fact, most aromatic coffee is generated during one or more of the multiple processing stages, starting with the collection of ripe red fruits of the coffee tree to the finished product of fried coffee beans or extracts from them, for example, instant coffee.

The various stages of coffee production described A.W. Smith, in Coffee; Volume 1: Chemistry, pp.1-41, dark R.J. and Macrea R. Eds. Elsevier Applied Science, London and New York, 1985; Clarke R.J., in Coffee: Botany, Biochemistry and Production of Beans and Beverage, pp.230-250 and pp.375-393; and M.N. Clifford and K.C. Willson, Eds. Croom Helm Ltd., London. In a nutshell, the process begins with collecting Mature, matured red fruits (Kotenok) of the coffee tree. Can then be removed outer layer, or pericarpel, using either a dry or wet method. The dry method is the most simple and includes 1) the classification and wash the fruit, 2) drying of the fruit after the classification (or natural air drying or mechanically) and (3) peeling the dried fruits in order to remove dry pericardia. The wet method is more complex and usually leads to the production of high quality green beans. The wet method is more commonly associated with fruits of C. arabica. The wet method involves (1) the classification of the fruit of the coffee tree, (2) removal of the fruit pulp (pulp), this stage is carried out immediately after harvesting and usually involves mechanical removal of the "pulp"or pericardia from Mature fruits, (3) "fermentation", i.e. removing mucus, remaining on the beans fruit after removal of the pulp, by incubating them together with the rest of the slime water tanks in periodic mode (the process of "fermentation" can last for up to 80 hours, although 24 hours is sufficient to achieve acceptable fermentation and reducing pH from about 6.8 or 6.9 4.2-4,6 under the action of various enzyme activities and metabolic action of microorganisms whose growth is happening in the fermentation process), (4) drying (this stage includes either air or mechanical drying with heated air fermented coffee beans) and (5) "shelling", this stage involves the mechanical removal of "parchment shell with dried beans (with t is called dried beans with parchment shell); at this stage it is often removed and also the silver seed coat. Obtained after wet or dry processing of raw coffee bean is often subjected to sorting, the majority of processes sorted based on the sorting by size and/or shape of the grain.

The next stage of processing coffee roasting raw coffee beans after hulling or peeling of treated dry or wet method of coffee, respectively. This process is time dependent and induces significant chemical changes in the grain. In the first phase of roasting is the evaporation of the remaining grain water by supplying heat. After evaporation of the main mass of the water begins roasting, when the temperature rises up to 190-200°C. the Degree of roasting, which is normally regulated by changing the color of coffee beans, plays an important role in the formation of the aromatic characteristics of the finished coffee beverage. Therefore, the time and temperature of roasting strictly controlled in order to achieve the desired aromatic coffee profile. After roasting hold the ground coffee in order to facilitate the extraction in the process of manufacturing a coffee beverage or coffee extracts (the latter are used for the production of instant coffee products). Kind of grinding can also affect the final flavor of the drink.

At that time the AC is on the problem of identification of aromatic molecules in coffee has been considerable number of research studies, studying the physical and chemical reactions occurring inside the coffee beans at every stage of their processing, devoted much less research. To a greater extent this applies to roasting, during which a large number of components of the grains exposed to extremely complex series induced by heating of the reaction (Homma S. 2001, in "Coffee: Recent Developments", R.J. dark and O.G. Vitzthum, Eds. Blackwell Science, London; Yeretzian, C., et al. (2002) Eur. Food Res. Technol. 214, 92-104; Flament I. (2002) Coffee Flavor Chemistry, John Wiley and Sons, UK; G.A. Reineccius, "The Maillard Reaction and Coffee Flavor", Conference Proceedings of ASIC, 16thColloque, Kyoto, Japan 1995).

Although the details of most of the reactions that occur at different stages of coffee processing, remain relatively unexplored, it is assumed that the most important aroma generating reaction, responsible for many of the nuances associated with the aroma of coffee is the Maillard reaction (Maillard reaction) in the course of roasting coffee. Intensive Maillard reaction occurs between contained in the coffee bean decay products of sugars/polysaccharides containing amino group of molecules (in particular, proteins, peptides and amino acids) in the roasting process.

It is obvious that the Maillard reaction is an important contribution to the formation of the coffee aroma and aromatic molecules in the process of roasting coffee, to the extent there is a correlation between the level of primary reagents-the private owners of the Maillard reaction in the raw coffee bean and quality of fragrance, emerging after roasting.

As noted above, an important group of substrates in the Maillard reaction are amino acids, peptides and proteins. Using a two-dimensional (2-D) electrophoresis, it was shown that there are differences in the levels and quantities of major spare proteins in raw (unroasted) coffee bean between varieties of Arabica (arabica) and Robusta (robusta), but the relationship between these differences in the content of the spare proteins and quality of aroma could not be established (Rogers et al., 1999, Plant Physiol. Biochem. Vol.37, 261-272). Recently it was found that small differences exist between the spare proteins immature and Mature coffee beans with different quality of flavor (P. Montavon et al., 2003, J. Agric. and Food Chemistry Vol.51, 2328-2334). Because in the process of ripening of the grains, there are many changes in this latest work suggests the possible existence of a link between better quality caused by the ripening of the grains, and differences in migration of the main spare protein coffee beans in 2-D gel.

Recently it was found that there are differences in the profiles of peptides extracted from raw coffee beans varieties of Arabica and Robusta (Ludwig et al., 2000, Eur. Food Res. Technol., Vol.211, 111-116). Although the results of these authors showed that the peptide extracts from Arabica and Robusta differ in the profile before the of estevanico scent the data presented in their work, do not identify which component (s) in the extracts is/are responsible for these differences in the profiles of the precursors of aroma. These scientists have also discovered at least two different protease activity in crude extracts from raw (unroasted) coffee beans, which, however, did not correlate with any specific activity associated with the quality of flavor (Ludwig et al., 2000, Eur. Food Res. Technol., Vol.211, 111-116). And finally, it is also hypothesized that the very high temperatures in the later stages of roasting raw coffee beans cause considerable breakdown of proteins present in the coffee bean (Homma S. 2001, "Coffee: Recent Developments". R.J. dark and O.G. Vitzthum, Eds. Blackwell Science, London; Montavon, P., et al., 2003, "Changes in green coffee protein profiles during roasting = Changes of protein profiles of raw coffee beans during roasting", J. Agric. Food Chem. 51, 2335-2343). However, the General scheme of this protein breakdown are very poorly known, but presumably it depends, inter alia, on the specific state of the main proteins of coffee in the raw materials before roasting. To the authors ' knowledge this application, other significant publications on the possible participation of the peptide profile of coffee in the formation of the aroma of coffee is not available.

In the process of frying the fermented seeds of Theobroma cacao (cocoa beans), as PR is polagaetsa, contained in the seeds of amino acids and peptides are involved in the formation of aroma via the Maillard reaction. In comparison with the seeds of other plants, seeds of T. cacao have been shown to be extremely high specific for aspartic acid by activity (Biehl Century, J. Voigt, Voigt G., Heinrichs, H., Senyuk V and Bytof, G. (1994) "pH-dependent enzymatic formation of oligopeptides and amino acids, the aroma precursors in raw cocoa beans = pH-dependent enzymatic formation of oligopeptides and amino acids, precursors of aroma in raw cocoa beans"; in The Proceedings of the 11thInternational Cocoa Research Conference, 18-24 July 1993, Yamoussoukro, Ivory Coast). To get cocoa beans with a high level of precursors of aroma of cocoa should be natural fermentation stage (when roasting unfermented cocoa beans is formed very weak aroma). During this stage of fermentation of the sugars are fermented fruit pulp, generating a high level of acid, in particular acetic acid (Carr J.G. (1982) Cocoa. In Fermented Foods. Economic Microbiology. Vol.7, pages 275-292, A.H. Rose ed., Academic Press). As a continuation of the fermentation the pH of seeds is reduced, and the cell structure is destroyed. Low pH triggers the mobilization and/or activation specific for aspartic acid protease contained in large quantities in the seeds of cacao, which leads to a massive collapse of the cellular protein (Biehl Century, Passem D. and Sagemann W. (1982) "Effect of Acetic Acid n Subcellular Structures of Cocoa Bean Cotyledons = Effect of acetic acid on cellular structures cotyledons of cacao beans". J. Sci. Food Agric. 33, 1101-1109; Biehl Century, Brunner E., Passem D., Quesnel V.C. and D. Adomako (1985) "Acidification, proteolysis and flavour potential in fermenting cocoa beans = Acidification, proteolysis and aromatic potential in fermented cocoa beans". J. Sci. Food Agric. 36, 583-598). Peptides and amino acids, as shown, are precursors of aroma cocoa (Rohan T., 1964, "The precursors of chocolate aroma: a comparative study of fermented and unfermented cocoa beans = the Precursors of chocolate aroma: a comparative study of fermented and unfermented cocoa beans". J. Food Sci., 29, 456-459; Voigt and J. Biehl C., 1995, "Precursors of the cocoa specific aroma components are derived from the vicilin-class (7S) globulin of the cocoa seeds by proteolytic processing = Predecessors components specific aroma of cocoa are formed from the globulin class vicilin (7S) cocoa beans by proteolysis". Bot. Acta, 108, 283-289). Thus, specific for aspartic acid protease seeds of T. cacao together with contained in the seeds serine carboxypeptidase are assumed to be critical for the formation of precursors of aroma of cocoa fermentation process (Voigt and J. Biehl C., 1995, "Precursors of the cocoa specific aroma components are derived from the vicilin-class (7S) globulin of the cocoa seeds by proteolytic processing. Bot. Acta 108, 283-289; Voigt J., Heinrichs, H., Voigt G. and Biehl C., 1994, "Cocoa-specific aroma precursors are generated by proteolytic digestion of the vicilin-like globulin of cocoa seeds = Predecessors specific aroma of cocoa are formed as a result of proteolysis of the globulin class vicilin (7S) cocoa beans". Food Chemstry, 50, 177-184). Identified gene that encodes a specific aspartic acid protease contained in large quantities in the seeds of the cocoa, and recently published in the application for international patent No. 02/04617 included in full in the reference list to this application, describes a method overexpression of the indicated protein in the seeds of cacao, which can generate an increased level of amino acids and peptides precursors of aroma of cocoa in fermented cocoa beans. However, the content of the published application of international patent No. 02/04617 aimed at cocoa seeds, which are specific long stage acid fermentation, in contrast to coffee beans that are fermented.

Important systemprocesses (WED, cysteine proteinase) vacuoles is KDEL-containing cysteinate. This type protease was characterized in several plants. Recently been discovered three genes encoding cysteinate with a C-terminal KDEL sequences in arabidopsis (Gietl S. and Schmid M., 2001, Sciences), 88, 49-58). One is expressed in senescent ovules, the second - in vessels, and the third in Mature pods. However, more detailed studies of this protein have been conducted in other plants. For example, CF, called sulfhydryl endoproteases (SH-EP), was characterized in zamadol the x seed Vigna mungo (Toyooka, K., Okamoto T. and T. Minamikawa, 2000, J. Cell Biol. 148, 453-463). SH-EP is expressed de novo in germinating cotyledons of V. mungo', it is assumed that it is involved in the collapse of spare proteins accumulated in the storing proteins vacuoles (T. Okamoto and T. Minamikawa J. Plant Physiol. 152, 675-682). The key difference SH-EP polypeptide is that it has a specific COOH-terminal sequence KDEL, which controls the transport of this protein from the endoplasmic reticulum (ER) in storing proteins to the vacuole (Toyooka et al., 2000). Recently put forward the assumption that the SH-EP protein is indeed involved, due to the presence of the KDEL sequence in the formation of specific vesicles, called KV (KDEL Vesicles), not previously described in the literature vesicular transport system (Okamoto T., Shimada T., Hara-Nishimura I, Nishimura M. and T. Minamikawa, 2003, Plant discrimination, 132, 1892-1900).

A similar assumption was made in respect of KDEL-containing WED protein found in germinating cotyledons of castor bean (Ricinus communis). In this plant, as the authors imply, specified KDEL-containing protease in the framework of programmed cell death (apoptose) endosperm continues to deliver nutrients for the germinating embryo castor bean (Gietl S. and Schmid M., 2001, Sciences), 88, 49-58). These authors suggest that in castor bean KDEL protease formed the ER germinating seeds before the onset of 3 days. When the seed coat is reset for about 3 days, KDEL-containing CF is Packed in specific vesicles, called retinotomy. Later, when the endosperm becomes soft (between 4 and 5 days), KDEL-CP cleaved adherent sequence (KDEL), and this protease migrates to the cytoplasm, where it is involved mainly the collapse of the cellular protein.

Disclosure of inventions

The aim of the present invention is a modification of the pool of protein/peptide/amino acid precursors of flavor in the coffee.

More specifically, the present invention is a modification of the predecessors of the fragrance raw materials (raw coffee bean) so that during the subsequent post-harvest processing and roasting could be achieved by a modified flavor. Not stopping theory, the authors suggested that, if between coffees with a completely different flavor there are differences in the level of peptides and protein degradation, these differences are most likely due to different endogenous by activity in these varieties of coffee beans. These differences can be detected by the difference in the level of mRNA expression with the expression of certain genes protease coffee beans.

Thus, the present invention includes the identification of posledovatel the values of genes encoding-specific protease coffee beans (seeds), and evidence that differences in expression of these genes in the Arabica and the Robusta actually exist.

More specifically, the present invention discloses two main cysteinate coffee (USSR-1 and the USSR-4), four major inhibitor of cysteinate coffee (CcCPI-1, CcCPI-2, CcCPI-3 and CcCPI-4) and two specific for aspartic acid protease coffee (SAR-1 and SSR-2), all of which are expressed in a coffee bean. The authors show how the overexpression of these proteins, especially at the late stage of grain development, or reduced expression of these proteins, especially at the late stage of grain development, can change the amino acid/peptide/protein profile of the Mature grain. Using one or more of the disclosed sequences and gene structures to change the amino acid/peptide/protein profile of ripe coffee beans, the authors have discovered a new way to change the profile of the precursors of aroma in Mature coffee bean.

In the first aspect of the present invention provides an isolated polynucleotide containing the nucleotide sequence encoding the polypeptide having systemprocesses activity, in which the amino acid sequence of the polypeptide and the amino acid sequence, the choice is aema of SEQ ID No 2 or 16, identical, at least 70%, preferably at least 80%, sequence identity is based on the ClustalW alignment; or the complement of the nucleotide sequence whose complement contains the same number of nucleotides and nucleotide sequence, and the complement and the nucleotide sequences are 100% complementary. Preferably the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No 2 or 16 identical, at least 85%, more preferably at least 90%, and optionally at least 95%, sequence identity is based on the ClustalW alignment. Preferably the nucleotide sequence contains the nucleotide sequence of SEQ ID No 1 or 15. Preferably, the polypeptide contains the amino acid sequence of SEQ ID No 2, or 16.

The second aspect is provided an isolated polynucleotide containing the nucleotide sequence encoding the polypeptide having the activity of inhibitor cysteinate, in which the amino acid sequence of the polypeptide and the amino acid sequence selected from SEQ ID nos 4, 10, 12 and 14 are identical, at least 70%, preferably at least 80%, while the identity of p is sledovatelnot based, using the ClustalW alignment; or the complement of the nucleotide sequence, in which the complement contains the same number of nucleotides and nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary. Preferably the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No 4, 10, 12 and 14 are identical, at least 85%, preferably at least 90%, and optionally at least 95%, sequence identity is based on the ClustalW alignment. Preferably the nucleotide sequence has a nucleotide sequence selected from SEQ ID nos 3, 9, 11 or 13, not necessarily from SEQ ID nos 9, 11 or 13, also optional from SEQ ID No 9 or 13, or, which is also optional, SEQ ID No 9. Preferably, the polypeptide contains an amino acid sequence selected from SEQ ID nos 4, 10, 12 and 14, not necessarily from SEQ ID nos 10, 12 and 14, also optional from SEQ ID No 10, or 14, or, which is also optional, SEQ ID No 10.

In the third aspect is provided an isolated polynucleotide containing the nucleotide sequence encoding the polypeptide having specific for aspartic acid endoprotease activity, in which the amino acid sequence of the polypeptide and the amino acid sequence, select the C SEQ ID No 6 or 8, preferably from SEQ ID No 8, identical, at least 75%, preferably at least 80%, sequence identity is based on the ClustalW alignment; or the complement of the nucleotide sequence, in which the complement contains the same number of nucleotides and nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary. Preferably the amino acid sequence of the polypeptide and the amino acid sequence selected from SEQ ID No 6 or 8, preferably from SEQ ID No 8, identical, at least 85%, preferably at least 90% and, optionally, at least 95%, sequence identity is based on the ClustalW alignment. Preferably the nucleotide sequence contains the nucleotide sequence of SEQ ID No 5 or 7, preferably SEQ ID No 7. Preferably, the polypeptide contains the amino acid sequence of SEQ ID No 6 or 8, preferably SEQ ID No 8.

The following aspect is provided a vector containing polynucleotide according to any of the preceding (first to third aspects of the invention.

The following aspect is ensured by the design of the alien recombinant DNA containing polynucleotide according to any of the preceding (with p the pout on third aspects of the invention, operatively associated with a regulatory sequence. It is important that alien design or polynucleotide was alien, or a regulatory sequence was alien, or both of them were alien.

The following aspect is provided a method of transforming cells, providing for the transformation of cells by polynucleotide according to any of the preceding (first to third aspects of the present invention.

The following aspect is provided a cell containing the above-mentioned construction of the alien recombinant DNA, in which the cell is preferably a prokaryotic cell, a eukaryotic cell or a plant cell, preferably a cell of the coffee tree.

The following aspect is provided a transgenic plant containing such a transformed cell.

In the context of the present description, the term "fruit of the coffee tree" (coffee cherry) denotes the drupe (fruit) coffee trees; whole fruit; ectocarpus (peel); pericarpel (fleshy core outer layer of the fruit); the coffee bean. A more detailed explanation of the specified term, see Clarke R.J., Coffee: Botany,Biochemistry and Production of Beans and Beverage, pp.230, M.N. Clifford and K.C. Willson eds, Croom Helm Ltd., London (specified source in full included in the list of references to this application).

The invention can be better understood from nigelec the subsequent detailed description and the accompanying sequence Listing, which is part of this application.

The following table 1 lists described in this invention polypeptides together with the ID of the corresponding sequences (SEQ ID No).

Table 1
SEQ ID No 1 (CcCP1: cysteinate, nucleic acid and corresponding amino acid)
SEQ ID No 2 (CcCP1: cysteinate, amino acids)
SEQ ID No 3 (CcCPI-1: inhibitor cysteinate, nucleic acid and corresponding amino acid)
SEQ ID No 4 (CcCPI-1: inhibitor cysteinate, amino acids)
SEQ ID No 5 (CcAPl: specific for aspartic acid endoprotease 1, nucleic acid and corresponding amino acid)
SEQ ID No 6 (CcAP1: specific for aspartic acid endoprotease 1, amino acids)
SEQ ID No 7 (SAR: specific for aspartic acid protease 2, nucleic acid and corresponding amino acid)
SEQ ID No 8 (SAR: specific for aspartic acid protease 2, amino acids)
SEQ ID No 9 (CcCPI-2 inhibitor sistei the protease, nucleic acid and corresponding amino acid)
SEQ ID No 10 (CcCPI-2 inhibitor cysteinate, amino acids)
SEQ ID No 11 (CcCPI-3: inhibitor cysteinate, nucleic acid and corresponding amino acid)
SEQ ID No 12 (CcCPI-3: inhibitor cysteinate, amino acids)
SEQ ID No 13 (CcCPI-4: inhibitor cysteinate, nucleic acid and corresponding amino acid)
SEQ ID No 14 (CcCPI-4: inhibitor cysteinate, amino acids)
SEQ ID No 15 (USSR-4: cysteinate, nucleic acid and corresponding amino acid)
SEQ ID No 16 (USSR-4: cysteinate, amino acids)

In the list of sequences of single-letter codes indicate nucleotide sequence and three-letter codes denote amino acids, as they are defined in the standards IUPAC-IUBMB and how they are described in Nucleic Acids Research 13: 3021-3030 (1985), which is included in the list of references to this application.

A brief description of the drawings.

Figure 1 shows Northern blot analysis of gene cysteinate in various tissues Coffea arabica, in which tracks in the gel is indicated as follows : the m: R-rhizome; S - stem; L - young leaves; SG, LG, Y -, and Red - seed from a small green fruit, large, green fruit, yellow fruit and red fruit, respectively. Loading of total RNA in each lane of the gel was five micrograms. MW marker indicates a "staircase" of RNA. The panel is autoradiogram after 24 hours of exposure, showing the emergence of the USSR-1 mRNA in the tested tissues, as panel a shows the gel, bromide stained with ethidium before blotting.

Figure 2 shows Northern blot analysis of gene expression of the USSR-1 cysteinate in various tissues Coffea arabica, in which tracks in the gel are indicated as follows: R - root; S, stem; L - young leaves; F - flowers; SG(g), LG(g), Y(g) and Red(g) correspond to RNA isolated from the grain of small green, large green, yellow and red fruits, respectively, and lanes in the gel, denoted by SG(p), LG(p), Y(p) and Red(p)correspond to RNA extracted from tissue pericardia small green, large green, yellow and red fruits, respectively. Loading of total RNA in each lane of the gel was five micrograms. Panel a shows bromide stained with ethidium large subunit ribosomal RNA prior to blotting, which are load control, and the panel is autoradiogram showing the appearance of the USSR-1 mRNA in specific IP is lechenich tissues.

Figure 2A: the alignment of the full sequence of the protein encoded by the USSR-1 cDNA, compared to other full-size systemprocesses from NCBI database. It was performed using the program CLUSTAL package program MegAlign (DNASTAR). Black blocks indicate identical amino acids. Access numbers in the database EMBL indicated in parentheses: Arabidopsis thaliana (AY070063); Vicia sativa (Z99172); Glycine max. GMCP3 (Z32795); Glycine max GmPM33 (AF 167986); Phaseolus vulgaris Moldavain (Z99955); Solanum melongena (AF082181); Nicotiana tabacum (AJ242994); Lycopersicon esculentum (Z14028); Vicia faba (AU).

Figure 3 shows Northern blot analysis of gene inhibitor cysteinate (CcCPI-1) in different tissues of Coffea arabica in which tracks in the gel are indicated as follows: R - root; S, stem; L - young leaves; SG, LG, Y -, and Red - seed from a small green fruit, large, green fruit, yellow fruit and red fruit, respectively. Loading of total RNA in each lane of the gel was five micrograms. MW marker indicates a "staircase" of RNA. The panel is autoradiogram after 24 hours exposure, as panel a shows the gel, bromide stained with ethidium before blotting.

Figure 4 shows Northern blot analysis of gene inhibitor cysteinate (CcCPI-1) at different stages of development of the fruits of Coffea arabica (ARA) and Coffea robusta (ROB). Track while marked as follows: small green square is d (SG), large green fruit (LG), yellow fruit (Y) and red fruit (Red), respectively. Loading of total RNA in each lane of the gel was five micrograms. MW marker indicates a "staircase" of RNA. The panel is autoradiogram after 24 hours of exposure, showing the appearance of the CcCPI-1 mRNA in specific the investigated tissues. Panel a shows the gel, bromide stained with ethidium before blotting.

Figure 5 shows the analysis by polymerase chain reaction with the matrix cDNA obtained from mRNA using reverse transcription reaction (the so-called RT-PCR) gene expression of the USSR-1 in the process of sprouting beans of Coffea arabica. Polymerase chain reaction (PCR) was performed using 10 μl of each reaction mixture to obtain cDNA, diluted in a ratio of 1/100. Modes PCR cycles: 2 min at 94°and then 35 cycles: 94°and 61°C for 1.5 min, 72°for 2.5 minutes final stage of completion chains lasted 7 min at 72°C. Used PCR primers:

A4-43-Upper: 5'-ACCGAGGAGGAGTTTGAGGCTACG-3'

A4-43-lower: 5'-ASSETSASSETS-3'.

mRNA amplified by the method of RT-PCR using specific primers (USSR-1 top/USSR-1 bottom) in different matrices: cDNA of sterilized grain (T0and grains, selected after 2 days (2d), 3 days (3d), 5 days (5d), 1 month (1m) and 2 months (m) germination, respectively. PCR products were separated in 1% (weight/volume) agarose gel and stained bromide by ethidium. RPL39: amplificatory fragment of cDNA encoding protein L39 are effective large subunit of ribosomal 60S.

Figure 6 shows Western blot analysis of protein expression of the USSR-1 (A). Total protein was extracted from grains (g) and pericardia (p)selected from the developing fruits of a coffee tree on the stages of small green (SG), large green (LG), yellow (Y) and red (Red) fruits. Panel: Division 50 μg of total protein in 12% SDS-PAGE gel and staining Kumasi blue". Panel a: Detection of protein was performed using anti-SCR polyclonal antibodies (rabbit), as described in methods. The approximate size of the colored bars in the panel indicated by arrows on the left. Large arrow in each panel indicates the presence of a primary spare protein that participates in cross-binding assays with one of the antibodies.

Figure 6A shows the optimal alignment of the full-size protein encoded CcCPI-1 cDNA, in relation to other homologous full-size systemprocesses from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database EMBL and the percentage of identity is given in parentheses. Malus x domestica (AAO; identity 42,3%), Common sunflower (JE0308; the identity of 41.5%), Arabidopsis thaliana (AAM64985; identichnosti 30%) and Rumex obtusifolius (CAD21441; the identity of 29.3%).

Figure 7 shows RT-PCR analysis of gene expression CcCPI-1 in various tissues Coffea arabica CCCA2 (a) and Coffea robusta FRT-32 (B). PCR was performed using 10 μl of each mixture obtained cDNA diluted in a ratio of 1/1000. Modes of cycles: 2 min at 94°and then 40 cycles: 94°C, 1 min - 60°C, 1.5 min - 72°C, 1 min final stage of completion chains lasted 7 min at 72°C. Used PCR primers:

CcCPI-1 (top): 5'-AGGAAAGTGGGAGCAAGGGAGAAGA-3'

CcCPI-1 (bottom): 5'-TAGTATGAACCCAAGGCCGAACCAC-3'.

The lanes in the gel are indicated as follows: M - marker; +R - diluted plasmid containing the gene CcCPI-1; R - root; S, stem; L - young leaves; F - flowers. SGG test, LGg, Yg and Rg - grain selected from the small green, large green, yellow and red fruits, respectively. SGp, LGp, Yp and Rp - fabric pericardia isolated from a small green, large green, yellow and red fruits, respectively.

Figure 8 shows the optimal alignment of the full-size protein encoded CcCPI-2 cDNA, in relation to other homologous full-size systemprocesses from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database EMBL and the percentage of identity is given in parentheses. Rumex obtusifolius (CAD21441; identity 66,7%), Dianthus caryophyllus (AAK30004; identity 71,7%), Manihot esculenta (AAF72202; identity 65,2%).

Figure 9 is it shows RT-PCR analysis of gene expression CcCPI-2 in various tissues Coffea arabica CCCA2 (a) and Coffea robusta FRT-32 (B). PCR was performed using 10 μl of each mixture to obtain cDNA, diluted in a ratio of 1/1000. Modes of cycles: 2 min at 94°and 40 cycles: 94°C, 1 min at 57°s, 1.5 minutes - 72°C, 1 min final stage of completion chains lasted 7 min at 72°C. Used PCR primers:

CcCPI-2 (top): 5'-GTGAAGCCATGGTTGAACTT-3'

CcCPI-2 (bottom): 5'-GTAATGATACTCAAGCCAGA-3'.

The lanes in the gel are indicated as follows: M - marker; +R - diluted plasmid containing the gene CcCPI-2; R - root; S, stem; L - young leaves; F - flowers. SGG test, LGg, Yg and Rg - grain selected from the small green, large green, yellow and red fruits, respectively. SGp, LGp, Yp and Rp - fabric pericardia isolated from a small green, large green, yellow and red fruits, respectively.

Figure 10 shows the optimal alignment of the full-size protein encoded CcCPI-3 cDNA, in relation to other homologous full-size systemprocesses from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database EMBL and the percentage of identity is given in parentheses. Citrus x paradisi (AAG38521; identity 42,4%), Actinidia deliciosa (AAR92223; identity 44,4%) and Arabidopsis thaliana (AAM64661; the identity of 44%).

Figure 11 shows the optimal alignment of the full-size protein encoded CcCPI-4 cDNA, in relation to other homologous to the full-size C is teenpreteen from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database EMBL and the percentage of identity is given in parentheses. Citrus x paradisi (AAG38521; identity 23,6%) and Arabidopsis thaliana (AAM64661; the identity of 20%).

Figure 12 shows RT-PCR analysis of gene expression CcCPI-4 in various tissues Coffea arabica CCCA2 (a) and Coffea robusta FRT-32 (B). PCR was performed using 10 μl of each mixture to obtain cDNA, diluted in a ratio of 1/100. Modes cycles were as follows: 2 min at 94°and 40 cycles: 94°C, 1 min - 60°C, 1.5 min - 72°C, 1 min final stage of completion chains lasted 7 minutes at 72°C. Used PCR primers:

CcCPI-4 (top): 5'-CTACGGTCGCAGCCAAATC-3'

CcCPI-4 (bottom): 5'-ACAACTGCACCTTCAATGTAC-3'.

The lanes in the gel are indicated as follows: M - marker; +R - diluted plasmid containing the gene CcCPI-4; R - root; S, stem; L - young leaves; F - flowers. SGG test, LGg, Yg and Rg - grain selected from the small green, large green, yellow and red fruits, respectively. SGp, LGp, Yp and Rp - fabric pericardia isolated from a small green, large green, yellow and red fruits, respectively.

Figure 13 shows Northern blot analysis of genes specific for aspartic acid protease 2 (SAR) in various tissues of Coffea arabica in which tracks in the gel are indicated as follows: R - root; S, stem; L - young leaves; F-flowers; SG (G) and (R), LG (G) is (R), Y (G) and (R) and Red (G) and (R) respectively grains and pericarpel small green, large green, yellow and red fruits; SG (G), LG (G), Y (G) and R (G) - pericarpel small green, large green, yellow and red fruit of the coffee tree. In each lane of the gel was loaded with five micrograms of total RNA. Panel a shows the control load bromide stained with ethidium large ribosomal RNA prior to blotting, and the panel is autoradiogram showing the appearance of Star mRNA in specific the investigated tissues.

Figure 14 shows the cDNA sequence and predicted it amino acid sequence of the USSR-4. Lowercase letters: 5' and 3' untranslated region of the gene. Uppercase: open reading frame. Bold: amino acid sequence; * - termination broadcast codon (stop codon).

Figure 15 shows the alignment of the full sequence of the protein encoded by the USSR-4 cDNA, compared to other full-size systemprocesses from NCBI database. It was performed using the program CLUSTAL W from the MegAlign software package (the package Lasergene, DNASTAR). Black blocks indicate identical amino acids. Access numbers are given in parentheses: Dacus carrota (JC7787); Ricinus communis (AF050756); Vicia sativa (Z34895); Phaseolus vulgaris (X56753); Helianthus annuus (AB109188); Glycine max Cysl (AB092555); Glycine max Cys2 (AB092557); Canavaia ensiformis (P49046); Oryza sativa (AB004648); Vigna mungo (PI 2412); Pisum sativum (AJ004985).

Figure 16 shows the alignment of the full cDNA sequence of the USSR-4 (KDDL) and partial cDNA sequence of the USSR-4 (KDEL) using the ClustalW program in the MegAlign software package.

Figure 17 shows the alignment of the full open reading frame of the USSR-4 (KDDL) and incomplete open reading frame of the USSR-4 (KDEL) using the ClustalW program in the MegAlign software package.

Figure 18 shows the chromatogram for the determination of DNA sequences amplified by PCR of genomic DNA encoding KDEL/KDDL region gene of the USSR-4. "Rob" means the grade Robusta coffee, "Arab" - grade Arabica.

Figure 19 shows Northern blot analysis of gene expression of cysteinate USSR-4 in various tissues Coffea arabica. The lanes in the gel are indicated as follows: R - root; S, stem; L - young leaves; F - flowers; SG(g), LG(g), Y(g) and Red(g) grain selected from the small green, large green, yellow and red fruits, respectively. SG(p), LG(p), Y(p) and Red(p) - fabric pericardia isolated from a small green, large green, yellow and red fruit of the coffee tree, respectively. In each lane of the gel was loaded with five micrograms of total RNA. Panel a shows the control load of colored bromism by ethidium large subunit ribosomal RNA prior to blotting, and the panel is In is autoradiogram, showing the appearance of the USSR-3 mRNA in specific the investigated tissues.

Figure 20 shows RT-PCR analysis of the expression of the USSR-4 whole-grain during germination. The samples were taken after 0 days, i.e. immediately after the sterilization processing, 2D - 2 days after processing, 3D - 3 days after treatment, 5D - 5 days after treatment, 1M - one month after treatment, 2M - two months after treatment, "-" control without DNA; +R - diluted DNA plasmids of the USSR-4; M - molecular weight markers.

Figure 21 shows the optimal alignment of the full-size protein encoded by SAR-1 cDNA, in relation to other homologous full-length sequences specific for aspartic acid proteases from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database are indicated in parentheses. Arabidopsis thaliana (AY099617) and Arabidopsis thaliana (BAB09366).

Figure 22 shows the optimal alignment of the full-size protein encoded by the cDNA SAR-2, relative to other homologous full-length sequences specific for aspartic acid proteases from NCBI database. Black blocks indicate identical amino acids. Access numbers in the database are indicated in parentheses: Glycine max (BAB64296), Ipomoea batatas (AAC), Lycopersicon esculentum (S71591) and Nepenthes alata (BAB20972).

The implementation of the invention.

P is hinata terminology.

In the context of describing the term "polynucleotide" refers to a nucleotide sequence, for example, a fragment of the nucleic acid. Polynucleotide may be a polymer of RNA or DNA, i.e. one or donativum which doesn't contain synthetic, non-natural or modified bases of the nucleotides. Polynucleotide in the form of a polymer of DNA may contain one or more segments of cDNA, genomic DNA, synthetic DNA, or a mixture thereof.

Similar nucleic acid fragments are characterized in the present invention the percentage identity of the coded amino acid sequences disclosed here with amino acid sequences defined by algorithms that are traditionally used by qualified specialists in this field. Suitable for this purpose, the nucleic acid fragments (or selected polynucleotide according to the aspects from the first to the third present invention) encode polypeptides that are identical, at least 70%, preferably at least 80%amino acid sequence disclosed in the present description. Preferred nucleic acid fragments encode amino acid sequences that are identical, at least 85% amino acid sequence disclosed in the present description. More preference is sustained fashion the nucleic acid fragments encode amino acid sequences, are identical, at least 90% amino acid sequence disclosed in the present description. Even more preferred are nucleic acid fragments that encode amino acid sequences that are at least 95% identical to the amino acid sequences disclosed in the present description. Multiple sequence alignment should be performed using the ClustalW alignment (Thompson et al., 1994, Nucleic Acids Research, Vol.22, p.4673-4680; Higgins &Sharp, 1989, Cabios. 5: 151-153).

In the context of describing the term "similar nucleic acid fragments" refers to polynucleotide sequences that change in one or more nucleotide base lead to the replacement of one or more amino acids, but these changes either do not affect the function of the polypeptide encoded by the nucleotide sequence, or do not affect the ability of the fragment of the nucleic acid to serve as an intermediary for silencing ("zamykanie") gene expression using, for example, antisense technology or technology of co-expression. The term "similar nucleic acid fragments" refers to a modified polynucleotide sequences in which one or more nucleotide base is delegated (delegated) or embedded (embed the SJ) provided that modifications to either not affect the function of the polypeptide encoded by the nucleotide sequence, or do not affect the ability of the fragment of the nucleic acid to serve as an intermediary for silencing gene expression. It can be seen that the scope of the present invention beyond the scope of polynucleotide and polypeptide sequences disclosed in the description.

Similar nucleic acid fragments may be selected by screening nucleic acid fragments in the form of subfragments or modified nucleic acid fragments for their ability to affect the level of the polypeptide encoded by the non-modified nucleic acid fragments in the plant or plant cell.

The term "operatively linked" defines the linking of two or more nucleic acid fragments into a single fragment of the nucleic acid so that the function of one of them affect the function of another.

The term "regulatory sequence" refers to nucleotide sequences localized before, inside or after the coding sequence, and which influence the transcription, processing or stability of the RNA or broadcast coding sequences associated with them. Regulatory sequences may include promoters, leader sequences of the translation is AI, introns, transcription terminators and sequence recognition of polyadenylation. If the regulatory sequence in the form of a promoter operatively linked to the coding sequence, such a regulatory sequence capable of influencing the expression of the coding sequence. Coding sequences can be operatively linked to regulatory sequences in sense or antisense orientation.

The term "expression" refers to transcription (and stable accumulation) sense RNA (mRNA) or antisense RNA isolated from nucleic acid fragments of the present invention. Expression may also refer to translation of mRNA into a polypeptide. "Overexpression" refers to such production of the gene product in a transgenic cell that exceeds levels of production in normal or normal, cells. The term "altered levels" refers to the production of gene product (s) in the transgenic cell in amounts or proportions that differ from his (their) number or proportion in normal, or normal, cells.

The term "transformation" refers to the transfer of the fragment of the nucleic acid into the genome of the host cell, resulting in genetically stable inheritance. Cell owners who, containing the transformed nucleic acid fragments are referred to in the context of describing as "transgenic cells".

Standard recombinant DNA and molecular cloning used in the present invention, are well known in the art and are described in detail in the book of Sambrook et al. "Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989, which is included in the list of references to this application.

Examples

The following examples illustrate the invention and do not limit the scope of the invention. In the examples, the number in the parts and percentages indicated in mascotech and % wt., the degrees in Celsius, if there is no reference to the other dimension.

In the following examples, the following abbreviations are used:

PCR: polymerase chain reaction (PCR, polymerase chain reaction);

RACE: rapid amplification of cDNA ends (rapid amplification cDNA ends);

RT-PCR: RT-PCR prior to obtaining cDNA from the matrix RNA due to the reaction of reverse transcription (reverse transcriptase PCR).

From the above discussion and the following examples of qualified professionals in this field can evaluate the significant differences between the present invention and, without departing from the scope of the invention, can make various changes and modifications to adapt it, if necessary, to different areas of the applications and conditions.

Obtaining cDNA libraries and screening cDNA

Obtaining specific RNA from coffee beans

Coffee fruits varieties of Robusta Q 121 were collected after 30 WAF (weeks after flowering) in ICCRI, Indonesia. Then, from the collected fruits were removed pericarpel, and the remaining material perisperm/endosperm was frozen and crushed into powder in liquid nitrogen. RNA was extracted from frozen powdered material using the method described previously for the extraction of RNA from cacao beans (Guilloteau M. et al., 2003, "Oil bodies in Theobroma cacao seeds: cloning and characterization of cDNA encoding the 15.8 and 16.9 kDa oleosins". Plant Science, Vol.164, 597-606). Poly And+RNA was prepared from about 250 μg total RNA using ready-made set of reagents "PolyA Purist™" AMBION kit (manufacturer Ambion, Inc.) in accordance with the instruction to the specified collection.

The first series of cDNA clones from the coffee bean.

Approximately 50-100 ng of poly specified And+RNA was used for synthesis of the first chain cDNA with the use of the finished product reverse transcriptase "Superscript™ II RNase H-(GIBCOBRL™) and a set of reagents for the synthesis of cDNA SMART™ PCR cDNA synthesis (Clontech). Was preparing a reaction mixture containing 2 μl of 30 WAF poly And RNA, 1 μl of CDS oligo ("SMART™ PCR cDNA kit, Clontech), 1 μl of Smart II oligo ("SMART™ PCR cDNA kit, Clontech) and 8 ál of deionized H20. The resulting mixture was warmed up with 72°C for 5 minutes, and then placed the do on the ice. Then added the following: 1 μl of 10 mm blend desoxyribonuclease (dNTP), 4 μl of SuperScriptII™ buffer for 1 chain and 2 μl of dithiothreitol (DTT). This mixture is kept at 42°C for 2 minutes, after which was added 1 μl of the finished product reverse transcriptase "SuperScriptII™ RNaseH" reverse transcriptase" (200 units/μl, GIBCO BRL™) and incubated the mixture at thermostat with air circulation at 42°C for 50 minutes.

After the reaction reverse transcription PCR was performed. 98 ál of Master Mix, as described in "SMART™ PCR cDNA kit (Clontech)containing polymerase Advantage™2 (ready set "Advantage™ 2 PCR kit, ClonTech), was placed on ice, and then added 3 μl of the above reaction mixture for synthesis of the 1st chain cDNA. These 100 ál of the mixture for PCR was placed in the apparatus MJ Research PTC-150" (HB), which was supported by the following PCR conditions: 95°C for 1 min, then 16 cycles of: 95°, 15 seconds - 65°C, 30 seconds - 68°C, 6 minutes. Amplified DNA was purified using a set of "Strataprep™ PCR Purification Kit" (Stratagene) in accordance with the instructions of the supplier. DNA was suirable 50 μl deionized water, and then DNA "expiation" (polished, in this case, a process of "blunting" of DNA ends) using Pfu-polymerase 1 reagents contained in the ready set "PCR-Script™ Amp cloning kit (Stratagene) in the following mixture: 50 μl DNA, 5 μl 10 mm dNTP mixture, and 6.5 μl of 10 × Pfu-1 "polished" is the buffer, 5 μl of cloned Pfu-1 DNA polymerase (with 0.5 units/μl). This reaction mixture was incubated at 72°C for 30 minutes in Poland machine with heated lid (Perkin Elmer). Using the methodology described in the instructions to finish the set of reagents "pPCR-Script™ Amp kit" (Stratagene), blunt PCR products ligated with hydrolyzed Srf-1 vector pPCR-Script™ Amp SK(+) in the presence of Srf-1 enzyme, and the reaction products of ligation were transformed into XL-10 Gold™ Kan" ultracompetent E. coli cells. Selection of clones transformed with plasmids containing inserts was performed using LB-Amp Petri dishes, a IPTG and Xgal distributed across the surface, as indicated in the instructions to finish the set of reagents "pPCR-Script™ Amp kit. Selected colonies are white and marked as clones Davl-1, etc.

A second series of clones cdnis coffee beans from selected based on the size of the cDNA.

Kernels Express a small number of proteins, such as spare proteins grains (White et al., 2000, Plant Discrimination, Vol. 124, 1582-1594). If cDNA derived from such tissue, very high level of spare proteins and other specific proteins grains leads to a high level of "redundancy" encoding them cDNA, i.e. the population of the obtained cDNA contains a high relative amount of the same cDNA. To reduce the redundancy of cDNA derived from mRNA coffee beans, and sat in the active characterization of extended and weakly expressed cDNA used strategy for cloning the second cDNA. The products of the above reactions with reverse transcriptase was used as template for subsequent PCR using a set of reagents "Advantage™ 2 PCR" (ClonTech): 3 μl of the reaction mixture for reverse transcriptase, 5 µl of 10 x Advantage™ 2 PCR buffer, 1 ál dNTP mix (10 mm each desoxycholate), 2 μl of PCR primer (ready set "SMART™ PCR cDNA kit, ClonTech), and 39 μl of deionized water and 1 μl of 50 × Advantage™ 2 polymerase mix. The obtained PCR mixture was placed in the apparatus "MJ ResearchPTC-150" (HB), which supported the following modes PCR: 95°C for 1 minute, and then 16 cycles of: 95°, 15 seconds - 65°C, 30 seconds - G8°C, 6 minutes. At the end of the PCR was added 1 μl of 10% SDS in the gel-loading buffer, the sample was heated to 37°C for 10 minutes. Then the sample was divided into parts for download in a 0.7% agarose gel without ethidium bromide: 10% loaded into a small hole next to the hole marker DNA, and the remaining 90% was loaded into the neighboring, larger preparative well. After electrophoresis the area gel with markers of size plus 10% of the reaction sample was stained with bromism by ethidium. This painted area of the gel was then used as a marker for cutting thin layers of gel containing PCR amplified cDNA of various sizes, from the rest unpainted (preparative) part of the gel. Received six thin layers of gel, with the shown range of sizes of PCR fragments: A1A (0.8 to 1 kb) A1B (1-1,5 kb), A2 (1,5-2,25 kb), A3 (2,25-of 3.25 kb), A4 (3.25 to 4 kb) and A5 (4-6,5 kb).

DNA from each layer was suirable from agarose using a set of "QIAEX II kit from the company Qiagen according to the supplier's instructions (samples 3A, 4A and 5A were heated for 10 minutes at 50°and samples 1A, 1B and 2 And was heated for 10 minutes at room temperature). Purified Dunaeva cDNA re-amplified using PCR with TAQ enzyme mix, which produces fragments with 3' T overhang, as follows: 30 ál isolated from gel double-cDNA, 5 μl 10 × TAQ buffer (supplied with polymerase mix TAQ PLUS precision polymerase mix, Stratagene), 1 μl of 40 mm dNTP mix (no 10 mm each), 2 μl of PCR primer (ready set "SMART™ PCR cDNA", Clontech)and 0.5 μl of polymerase mixture TAQ PLUS precision polymerase mix (Stratagene) and 11.5 μl of deionized water. Modes PCR: 95°C for 1 minute, then 7 cycles: 95°, 15 seconds - 65°, 1 minute - 72°C, 8 minutes, and then 1 cycle: 95°, 15 seconds - 65°, 1 minute - 72°C, 10 minutes.

The obtained amplified DNA is then ligated into the vector pCR™-TOPO™ and cloned into TOP10 E. coli cells using a set of "TORO™ TA kit (Invitrogen) according to the instructions of the supplier. The clones were named in order of their selection and positioning in dimensional gel (e.g., A2-1, A2-2 and so on).

Screening and preliminary identification of cDNA from the coffee is th grain.

The first series of white colonies obtained in the library of the Dav-1 were subjected to screening in order to determine first the size of each insert by PCR amplification of the insert using primers T3 and T7, adjacent to the cloning site, and verification of the amplified PCR fragments by gel-electrophoresis.

Each colony white resuspendable in 200 µl of sterile water for 10-30 μl of the resulting suspension were added 5 μl of 10X Taq polymerase buffer (Stratagene), I ál 10 mm dNTP mix, and 2.5 μl of 20 μm primer T3, and 2.5 μl of 20 μm primer T7, 1 μl of dimethyl sulfoxide (DMSO)and 0.5 μl of Taq polymerase (Stratagene) and the site, located between a final volume of 50 µl. Used the following PCR program: 94°C for 1 min, then 30 cycles of: 94°C, 1 min at 55°C, 1.5 min - 72°, and 3.5 min, and a final cycle of 7 min at 72°C. To reduce the level of "redundancy", PCR inserts of the same size were subjected to hydrolysis by restriction enzyme Pai III. PCR fragments from the same sample fragments Nai III restriction has not been further studied. Plasmid clones with PCR fragments >500 BP and have unique designs fragments Nai III enzyme was purified by using a set of "Qiawall 8 ultra plasmid kit (Qiagen) for dideoxy-sequencing from the 5'end using appropriate sequencing primers T7 or T3 encoded in flanking insertion sequences of the vector. As insert napryamuya was cloned, the first step was to determine the 5'end of each clone by hydrolysis of purified plasmid DNA by restriction enzyme Seal (CDS primer contains SMART Sea 1 website that allows you to define the orientation of the insert in the vector). Data generated DNA sequences were subsequently used to search for homologous proteins in devoid of repeats database GENEBANK order to obtain a preliminary annotation of each cDNA clone using the program BLASTX™.

Banks cDNA of the grains are characterized by a high level of repetitions. That is, a small number of mRNA grains has an extraordinarily high level of expression, for example, those that encode spare protein grain, and therefore cDNA in great abundance, see banks cDNA from grains (White et al., 2000, Plant discrimination, Vol.124, 1582-1594). Therefore, as soon as in the first series sequencing cDNA from coffee beans have been identified major abundant (recurring) cDNA, it took the introduction of the pre-screening of inserts containing colonies of white, before determining the size of the inserts. Four DNA sequences expressibility very much and for each of these abundant sequences were designed with the following specific primers:

1) 2S protein, contig 8A 5'-AGCAACTGCAGCAAGGTGGAG-3' and contig 8V 5'-CGATTTGGCACTGCTGTGGTTC-3' (55°C for PCR, fragment 14 BP),

2) 2S protein, contig 15A 5'-GCCCGTGCTCCTGAACCA-3' and contig 15V 5'-GTATGGTTGCGGTGGCTGAA-3' (55°C for PCR, a fragment of 256 BP),

3) oleosin 15,5, contig, SOA 5'-ACCCCGCTTTTCGTTAT-3' and contig 30V 5'-TCTGGCTACATCTTGAGTTCT-3' (55°C for PCR, a fragment of 261 BP),

4) protein 11S, contig 37A 5'-GTTTCCAGACCGCCATCAG-3' and contig 37V 5'-ATINSTALESANAS-3' (59°for PCR, a fragment of 261 BP).

PCR for this preliminary screening was performed in the following conditions: 10-30 ál of white colonies in sterile H3About 5 µl of 10 x Taq buffer (Stratagene), 1 μl 10 mm dNTP, and 2.5 µl of each primer (20 μm), 1 μl DMSO, and 0.5 μl of Taq polymerase (Stratagene 10 units/μl), and sterile site, located between was added to the final total reaction mixture of 50 µl. The PCR program consisted of 1 min at 94°C, then 30 cycles of: 94°C, 1 min to 1.5 min at a specific temperature for each pair of primers - 72°C for 2.5 min in the final cycle of 7 min at 72°C.

Sequencing of inserts full size cDNA and sequence analysis

The cDNA clones whose partial sequence showed initial homology with proteases and protease inhibitors, were completely sequenced on both circuits using the strategy of "walking" standard dideoxy primers. The sequence presented as SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13 and 15. To confirm the preliminary annotation was performed search for sequences homologous obtained Polner smirnym sequences, using BLASTX and database GenBank, excluding repetitions of proteins.

The identities of the sequences in pairs of sequences were calculated using the program ClustalW™ in the MegAlign module™ software package Lasergene™ (DNASTAR Inc.). Were selected the following default settings: (1. The PARAMETERS of the MULTIPLE ALIGNMENT Gap penalty (limitation period) 15,00; Gap length penalty (length limitation period) 6,66; Delay divergent Seqs (divergent delayed posledovatelnostei) (%) 30; DNA transition weight (variation of mass DNA) of 0.5, Protein Weight Matrix (realized during many years the matrix protein) - gonnet on Series; DNA Weight Matrix (weighted matrix DNA) - SWISS. 2 - PARAMETERS of the PAIRWISE ALIGNMENT - Slow/Accurate (Slow/Accurate) (Gap penalty - 15,00;

A Gap length penalty of 6.66; Protein Weight Matrix - gonnet on Series; DNA Weight Matrix - IUB). Used sequence represented or a full-length nucleotide sequence of each cDNA or ORF (open reading frame, open-reading frames) of each cDNA.

Table 2

The percent identity between the sequences of nucleic acids and amino acids of genes USSR-1, CcCPI-1, CcAP-1 and SSR-2 and related genes found in GenBank data Bank, excluding repeat proteins as well as proteins from WO 02/04617.
Consistently hundred cDNAThe nucleotide identity (%) The identity of proteins (%) (ORF)
CcAPl vrs TcAPl2,913,3
CcAPlvrsTcAP22,49,8
CcAP2vrsTcAPl55,061,5
CcAP2 vrs TcAP255,161,3
CcCP-1 vs proposed cysteinate Arabidopsis thaliana (AY070063)51,864,3
CcCP-1 vs containerboards Glycine max (Z32795)49,161,3
CcCP-1 vs predecessor of cysteinate Vicia sativa (Z99172)49,060,9
CcAP2 vs predecessor specific for aspartic acid protease Lycopersicon esculentum (L46681)65,971,1
CcAP2 vs mRNA proposed specific for aspartic acid protease Ipomoea batatas (AF259982)71,769,6
CcAP2vsNaAP4MPHK for specific aspartic acid protease 4 Nepenthes alata (AB045894)58,466,5
CcCPI-1 vs cystatin Malus x domestica (AY176584)38,845,5

5'RACE PCR.

It is established that the cDNA insert of clone A5-812 contains introns. So to confirm coding this protein sequence was necessary to allocate a new cDNA containing the full coding sequence. It was the Delano using a set of reagents for amplification of cDNA SMART™ RACE cDNA amplification Kit (Clontech). The first chain cDNA used for 5' RACE, obtained as described above to create cDNA libraries. Was designed specific primer to gene Gar (5'-CATATAATATTAAAAGCACCACCCATAA-3') - this sequence is localized at a distance of 92 BP from the poly (A) tail clone A5-812. Subsequently, this specific primer was used with a mixture of universal primers (UPM) from a set CLONTECH in PCR under the following conditions: 2,5 μl of the product of the first chain cDNA, 5 ál 10X Advantage 2 PCR buffer (CLONTECH), 1 μl dNTP mixture (10 mm), 1 ál 50X Advantage 2 polymerase mix (CLONTECH), 5 μl of a mixture of universal primers A (10X) (CLONTECH), 1 μl Gar (10 μm) with the addition of sterile water to a final volume of 50 µl. PCR was performed as follows: 20 cycles of 94°C, 30 sec 68°C, 30 sec - 72°C, 3 min, with subsequent final reaction completion circuit for 5 min at 72°C. was obtained fragment length of approximately 1700 BP, to retrieve which of the gel used, the ready set CONCERT™ Rapid Gel Extraction kit (GibcoBRL). The selected fragment cloned in the vector pCR 4-TOPO and transformed into Escherichia coli using the ready set for cloning "Toro-TA cloning kit (Invitrogen). The obtained plasmid was purified using a set of reagents for the extraction of plasmids ("QIAfilter Plasmid Midi Kit, Qiagen, France) and the insert of this plasmid sequenced on two chains.

Establish what about, in DNA clone A5-442 (AP1) is completely absent 5' region of the cDNA. To highlight this area spent 5'RACE using the ready set for amplification of cDNA SMART™ RACE cDNA amplification Kit (Clontech). Was designed specific to the sequence of the primer rAPl (5'-TGGAGTCACAAGATGTCTCGACGAACTG-3'), located at a distance of 396 BP from the poly (A) tail. This specific primer used then with a mixture of universal primers (UPM) from a set of CLONTECH PCR kit under the following conditions: a 2.5 μl of the first chain cDNA, 5 ál 10X Advantage 2 PCR buffer (CLONTECH), 1 μl dNTP mixture (10 mm), 1 ál 50X Advantage 2 polymerase mix (CLONTECH), 5 μl of a mixture of universal primers A (10X) (CLONTECH), 1 μl of rAPl followed by the addition of sterile water to a final volume of 50 µl. Mode cycles of PCR: 20 cycles of 30 sec at 94°C, 30 sec at 68°C and 3 min at 72°followed by reaction of the final completion of the circuit for 5 min at 72°C. the resulting fragment with a length of about 2000 BP, extracted from the gel using a set of reagents for rapid extraction from the gel, CONCERT™ Rapid Gel Extraction kit (GibcoBRL). The selected fragment cloned in the vector pCR 4-TOPO and transformed into Escherichia coli using the ready set for cloning "Toro-TA cloning kit (Invitrogen). The obtained plasmid was then purified using a kit for the extraction of plasmids (QIAfilter Plasmid Midi Kit, Qiagen, France) and the insert is the plasmids sequenced on two chains.

Obtain RNA for large EST libraries.

RNA was isolated from dissected grain and tissues pericardia at different stages of development and from young leaves using the previously described method. To obtain RNA for the purpose of creating different EST libraries (libraries that contain expressed sequences) used the following varieties and fabric coffee: (1) young leaves, one grade (FRT-32); (2) pericarpium (with 8 different stages of development) from 5 varieties (FRT-32, FRT-31, FRT-400, FRT-4001 and Q121); (3) whole fruit of the coffee tree after 22 weeks after fertilization (WAF, weeks after fertilisation) from one grade (FRT-31); (4) the coffee bean after 18+22 WAF from five varieties (FRT-32, FRT-31, FRT-400, FRT-4001 and Q121); (5) the coffee bean after 30 WAF from 5 varieties (FRT-32, FRT-31, FRT-400, FRT-4001 and Q121); (6) the coffee bean after 42 WAF from five varieties (FRT-32, FRT-31, FRT-400, FRT-4001 and Q121) and (7) the coffee bean later 46 WAF from 2 varieties (FRT-32 and Q121).

Obtaining cDNA clones and sequence analysis of DNA.

The cDNA clones for different EST libraries was prepared as follows: poly And+mRNA was isolated using a kit for isolation of mRNA "PolyTrack™ mRNA Isolation System" (TV System, Promega) according to the manufacturer's instructions for small-scale selection. Purified poly (A+the mRNA was used to obtain cDNA, which was then unidirectionally cloned in the vector-based phage lambda, as described in the instructions to g is a Hilbert set to create the library "ZAP-cDNA™ library construction kit (cat.# 200450 Stratagene). The Protocol mass excision (cutting the cloned DNA from the vector) included the excision PBlueScript phagemid (plasmid vector built into phage) from the vector Uni-Zap XR (phage vector) and getting white colonies after sowing in 150 mm plates with LB-ampicillinum agar, to which were added 80 μl of X-gal (20 mg/ml) and 16 μl of IPTG (0.5 M). Single colonies were selected randomly to obtain plasmid DNA, which is then used for sequencing the 5'ends of the cDNA inserts.

The obtained DNA sequences are EST sequences (Expressed Seguence Tags=markers expressed sequences) for each clone. Then all the data for EST sequences from 7 libraries grouped "in-silico", thus creating a unique group of sequences, called the set of sequences of unigenes" (unigene unique genes). Thus, each sequence of unigene theoretically corresponds to a separate genetic product. However, it should be noted that due to the fact that many unigene represent only a partial cDNA sequence, it is likely that some genes may be represented by two or more unigene. Preliminary annotation set unigenes was performed using the program automatically BLAST search in which the search PEFC is the sequences of each unigene is in the database of GenBank, excluding repeat proteins. The specified BLAST approach to finding allowed us to obtain five successful BLAST "hits" (hits, "issue" with the lowest e-values), which belonged to "unigene annotations.

Northern blot analysis.

Freshly harvested roots, young leaves, stem, flowers and fruits at different stages of development [small green fruits (SG), large green fruits (LG), yellow fruit (Y) and red fruit (R)] were selected from varieties of Coffea arabica CCCA2 grown in the greenhouse (25°C, relative humidity, 0V, 70%) in Tours, France, and Coffea canephora FRT32 grown either in Ecuador or in ICCRI, Indonesia. Fresh tissue was immediately frozen in liquid nitrogen and were isolated total RNA from each tissue using the extraction method described above. 5 µg of total RNA was applied by 1.2% (mass/vol) denaturing RNA gel containing formaldehyde. Samples of total RNA from each plant tissue was heated at 65°C for 15 min in the presence of 7 ál of the loading buffer for samples RNA "RNA Sample Loading Buffer" (without ethidium bromide, Sigma), and then immediately placed on ice for 2 minutes before subsequent application of RNA on a 1.2% gel. The gel was summed voltage of 60 volts for 5 hours. Then the gel was twice soaked in 10xSSC within 20 minutes of the RNA from the gel was transferred during the night due to capillary diffusion on "Positive TM Membrane" (Qbiogene) in 10xSSC; RNA was fixed to membrane by heating for 30 min at 80° C. Probes were prepared using a set of "Rediprime™ II random prime labelling system kit (Amersham) in the presence (P32) dCTP. Hybridization was carried out at 65°C for 24 h in a solution for hybridization (5X SSC, 40 μg/ml denatured sperm DNA, salmon, 5% [weight/volume] SDS, and 5x denhardt's solution). The membrane was washed twice for 30 min at 65°using 2X SSC, 0,1% SDS (weight/volume) and IX SSC, 0,1% SDS (weight/volume).

Northern-blot analysis, presented in figure 1, shows that the gene of the USSR-1 cysteinate coffee is expressed in the fruit of C. coffee arabica at all studied stages, with yellow fruits, the expression level was slightly higher than at other stages. Not detected expression of a specified gene in the roots, stem or leaves of C. arabica. Figure 2 shows another Northern blot experiment to control the expression of the USSR-1 in C. arabica using a new medication RNA. In this experiment, the fruit of the four stages were cut to highlight tissues of pericardia and tissues grains, corresponding to each stage of development of the fruit. Then from the obtained tissues were extracted total RNA. The results obtained showed the same temporal pattern of expression of the USSR-1 during development of the fruit, but this new experiment has additionally shown a high level of primary expression of the USSR-1 only in the tissues of the seeds of the fruit. In pericarp fruits coffee der the VA significant gene expression USSR-1 was not observed. This last result emphasizes the role of the specified genetic product in an exclusive change in the protein, peptide and amino acid profile of the coffee beans under normal growing conditions.

The authors created EST library from the leaves of the coffee tree, and from the tissues of the grains and pericardia obtained at different stages of development of the fruit of the coffee tree. Detection EST USSR-1 in different libraries (shown below - see table. 3) also shows that this gene is strongly expressed in the grain, but is not expressed to a significant extent in pericarp or in sheets. The pattern of expression of the USSR-1 in the process of seed formation is similar to the expression pattern observed for homologous with her sequence of Vicia sativa (gene CPR4: Fischer, J. et al., 2000. Plant Molecular Biology, 43, 83-101). These authors showed that CPR4 not detected by Northern blot testing nor in sheet nor in the rhizome or stem, which once again confirms the fact that the USSR-1 is specific for coffee beans. It is hardly possible to expect that changes in the expression of the USSR-1, especially in grain, as suggested here, due to the use of, for example, specific to the grain of the promoter in the antisense constructs of the USSR-1 or sverkhekspressiya design of the USSR-1 can prevent the metabolism in other tissues.

Table 3
The name of the geneThe number of EST
Grain 18 weeks.Whole fruits 22 weeks.Grain 30 weeks.Grain 42 weeks.Grain 46 weeks.PericarpelSheet
USSR-100401500

Optimal alignment of sequences in the case of the USSR-1 (figa) shows that this cDNA encodes cysteinate.

Northern-blot analysis, presented in figure 3, shows that the gene inhibitor cysteinate CcCPI-1 coffee is expressed in the fruit of C. arabica in all studied stages. However, in contrast to the expression observed in cysteinate USSR-1, a gene CcCPI-1 shows a higher expression level at two early stages of development of the fruit of coffee (at the stages of small green and large green fruit), and the two later stages of fruit formation specified gene is expressed at a lower level. This expression pattern is consistent with the existing hypothesis that protein inhibitor cysteinate (CcCPI-1) controls the level of activity of cysteinate, which is specifically expressed in the grain, while the USSR-1 - fruits KOF is logo tree. It can be expected that regulatory protein, such as protein inhibitor cysteinate will be expressed earlier than its protein target, when it is necessary to control the level of activity of this protein target continuously, from the time of expression of the protein target. Not found the expression of this gene in the roots, stem or leaves of C. arabica. It is noteworthy that the similarity of the samples in the expression of the USSR-1 and CcCPI-1 is consistent with the existing hypothesis that these proteins might functionally interact.

The results of Northern blotting (figure 3) showed that the CcCPI-1 is expressed at all stages in the fruit of the coffee tree. However, this experiment did not allow to determine if this expression in whole fruits or only in pericarp or grain. Expression in leaves was also unable to control. However, the expression CcCPI-1 in different EST libraries (shown below in the table. 4) shows that this gene is expressed mainly in the grain, in pericarp or sheets of expression of the indicated gene was not detected. This result also suggests that the CcCPI-1 controls the level of activity of cysteinate, which is specifically expressed in grain, for example, USSR-1.

Table 4
The name of the gene The number of EST
Grain 18 weeks.Whole fruits 22 weeks.Grain 30 weeks.Grain 42 weeks.Grain 46 weeks.PericarpelSheet
CcCPI-1001000

Northern-blot analysis, presented in figure 4, shows that the gene inhibitor cysteinate CcCPI-1 coffee is expressed differently in the fruits of C. canephora (robusta) in comparison with the fruits of C. arabica. First, the data of figure 4 show that the gene CcCPI-1 is expressed in C. arabica earlier. Secondly, and more importantly, gene CcCPI-1 is expressed in the fruit of C. canephora at a much higher level. This difference in expression is likely to affect the level of activity of cysteinate found in the fruits of C. arabica, compared to the fruits of C. canephora. Because this class of proteins is closely associated with the resistance of plants to pests, it is likely that the high level of gene expression CcCPI-1 in C. canephora contributes to increased resistance to disease, often seen with grades of "Robusta" compared with grades of "Arabica".

RT-PCR analysis of the expression of the USSR-1 in the process of germination of grain.

To determine the expression of the USSR-1 in the process of germination to anago grain fruit of the coffee tree was collected at the stage of full maturation, was rinsed with water and removed from them pericarpel (each fruit usually contains two seeds). The obtained grains were dried for one week in the open air at room temperature. Before germination with each grain had to manually delete parchment shell and silver seed shell (epidermi), then the grains were sterilized, dropping them in a 1% (weight/volume) solution of sodium hypochlorite for 1 hour, then washed twice with sterile distilled water. For germination 150 sterile seeds were placed individually in tubes containing salt Heller (Heller, 1953) and 7 g/l agar, and incubated at 25°With daily 8-hour day length.

After 2 day, 3 day, 5 day, 1 month and 2 months of germination were selected by three parties of ten grains each, immediately frozen in liquid nitrogen and stored at - 80°C until RNA extraction. From beans, germinated for 1 and 2 months at the time of sampling carved primary (embryonic) roots and frozen separately from the beans. We selected thirty-sterile grains at T=0 and frozen for later use as T(0) of the control.

4 µg treated DNA Asai total RNA extracted from each sample was used for cDNA synthesis with the use of review of the oligonucleotides according to the instructions to finish the set with about atoi transcriptase "Superscript II Reverse Transcriptase" (Invitrogen, Carlsbad, Ca). A fragment of the gene ribosomal protein coffee L39 are effective amplified in each sample cDNA as a control stage cDNA synthesis. PCR was performed using 50 μl reaction mixtures containing 10 μl diluted in a ratio of 1/100 solutions cDNA, 1 μm of each primer, 5 µl of 10X PCR ThermoPol buffer [10 mm (NH4)2SO4, 2 mm MgSO4, 20 mm Tris-HCl with pH 8.8 at 25°C, 10 mm KS1 and 0.1% Triton X-100] and 2.5 units of Taq polymerase (New England Biolabs, Beverly, MA). Modes PCR: 2 min at 94°C, followed by 35 cycles of: 94°C, 1 min - 60°C, 1.5 min - 72°C, 2.5 minutes at the Ultimate stage of completion of the chain - for 7 min at 72°C. the Following primers were used for amplification by PCR:

USSR-1 upper 5'-ACCGAGGAGGAGTTTGAGGCTACG-3' and

USSR-1 lower 5'-ACGCTTCCCCCATGAGTTCTTGA-3',

providing products yield cDNA 726 BP

Primers to protein Rpl39 were:

A5-1750-top 5'-TGGCGAAGAAGCAGAGGCAGA-3'

A5-1750-lower 5'-TTGAGGGGGAGGGTAAAAAG-3'.

RT-PCR was used to determine the expression of the USSR-1 at different stages of germination. The results show that the transcripts of the USSR-1 are found in whole grains almost all studied stages (figure 5). It has been previously shown (Fischer, J. et aL, 2000, Plant Molecular Biology, 43, 83-101), proposed that RNA homolog of the USSR-1 - CPR4 of V, sativa - also known as germ main stem and cotyledons of seeds of V. sativa in processarray.

Western blot analysis

Fabric leaves and fruits for analysis were obtained from Coffea arabica CCCA2, to analysis the tissue was kept frozen at -80°C. Fabric grain and pericardia the fruit of coffee trees at different stages of their development were cut individually insignificant, as far as possible, the release of pericardia. Then these different tissues were quickly crushed into fine powder, which can only be obtained by using liquid nitrogen in a pre-frozen laboratory mortar with pestle. Protein extracts from the indicated tissues were prepared by a modified version of the method of extraction described by Tanaka et al., 1986 (Plant discrimination, 81, 802-806). Used the following buffers:

buffer Tanaka:

sucrose - 0.7 M

Tris-HCl, pH 8, 0.5 M -

P-mercaptoethanol - 2% (about./about.)

NaCl - 0.1 M

Before using the above buffer were added:

EDTU - up to 5 mm

PMSF 2 mm

Buffer for loading in the gel:

Glycerin - 15% (about./about.)

β-mercaptoethanol - 2% (about./about.)

SDS - 3% (about./about.)

Tris-HCl, pH 6.8 - 62.5 mm.

Several hundred milligrams of frozen powdered tissue was added to 650 ál buffer Tanaka. Proteins were extracted by addition of one volume of Tris-saturated phenol, pH 8 (i.e. phenol, saturated with a solution of 10 mm Tris-HCl, pH 8). Each about ASEC was intensively stirred for 20 min, and then were centrifuged for 20 min at room temperature at 13000g. After centrifugation, the proteins present in the phenol phase. Samples of 20 µl were retained for analysis (see below), and the remaining amount of proteins in phenol phase was besieged by precipitation overnight at -20 ° C followed by the addition of five volumes of methanol containing 0.1 M ammonium acetate. After that, the samples were centrifuged for 20 min at room temperature at 13000g, and the resulting precipitates were washed twice in 500 μl of methanol containing 0.1 M ammonium acetate. Then precipitation resuspendable in 30 μl of buffer for loading into the gel to quantify the proteins.

Protein in 20 μl samples of phenolic phase was also besieged by precipitation, as described above, and the resulting precipitate resuspendable in the sample buffer from the ready set for the analysis of proteins BioRad dc Protein assay Kit. Quantification of total protein in the sample was performed using a set of BioRad dc Protein assay Kit according to the instructions of the supplier. After this General samples brought up to 5 µg/µl by addition of a buffer for loading into the gel.

Samples containing approximately 50 μg of protein each, were separated by electrophoresis in SDS-polyacrylamide gel (12% Tris-glycine Novex® Invitrogen™). The proteins transferred electro-blotting onto PVDF membrane by standard methods. Aspecific the ski binding sites on the membrane were blocked by incubation of the membrane in 10% solution of skimmed milk powder in TBS buffer (BioRad™ ) for one hour at room temperature or over night at 4°C. Batirovna proteins were tested for two hours at room temperature or over night at 4°With with a polyclonal antibody (1/5000 dilution in 10% solution of skimmed milk powder in TBS), developed for the proposed homologue - CPR4 of Vicia sativa, which were kindly provided by A.Schlereth and K.Muntz of the Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK, Institute of genetics and the study of cultivated plants, Germany; A.Schlereth, C.Becker, C.Horstmann, J.Tiedmarm and K.Muntz 2000, Journal of Experimental Botany, 51: 1423-1433). Then the membrane is washed three times for 20 minutes in TBS +0.1% of Tween 20 buffer, and then incubated for one hour with secondary antibody labeled with horseradish peroxidase (Ig rabbit against antigens goats, Immunopure®, Pierce). Then the membrane is washed twice for 20 minutes in TBS +0.1% of Tween 20 buffer and once for 20 minutes in TBS. The presence of the enzyme associated with the second antibody, visualized by chemiluminescent detection using the amplifying system ECL+® system (Amersham Life Science) according to the supplier's instructions.

The results indicate that the polypeptide of approximately 41 kDa, which corresponds exactly to the expected molecular mass polypeptide-predecessor of the USSR-1 (43735 Yes), it is found in all studied stages of ripening coffee what about the grain, but not in the tissues of pericardia (6). The expression pattern of this protein is similar to the expression pattern of mRNA USSR-1 (figure 2). Another polypeptide of approximately 22 kDa is also detected at the stages of yellow and red fruit of the coffee tree, but in smaller quantities than the polypeptide of 41 kDa. The size of this second polypeptide coincides with the expected size of the Mature form of the USSR-1 (25239 Yes). The expected Mature size of the USSR-1 after processing was determined by protein alignment between the full-length sequence of the ORF of the USSR-1 and the sequence of the predicted Mature forms CPR4 (Vicia sativa - # access # Z99172, identity with the USSR-1 = 60,9%). The N-terminal site of processing of the polypeptide CRP4 when generating the Mature form was predicted based on sequence comparison with other polypeptides, type CPR papain (J. Fischer, S. Becker, S. Hillmer, S. Horstmann, C. Neubohn, A. Schlereth, Senyuk V, A. Shutov and K. Muntz, 2000, Plant Molecular Biology, 43, 83-101). Interestingly, in contrast to the results presented here, according to which in the process of seed formation are detected and the precursor and Mature forms of the USSR-1, in developing seeds and during germination of seeds of V. sativa was found only Mature form of the polypeptide CPR4 (Fischer et al., 2000).

RT-PCR analysis of gene expression in grade Robusta FRT-32.

Isolated tissues FRT-32 and total RNA was extracted and these tissues previously described method. cDNA was obtained from the treated DNA Asai total RNA, as described above for RT-PCR experiments with cDNA Arabica. Then there was specific PCR conditions described above for RT-PCR experiments with cDNA Arabica. Specific conditions of amplification used primers the oligonucleotides are given in the caption to the figure for each experiment.

CcCPI-1.

(a) the optimal alignment CcCPI-1 (figa), indicating that this cDNA encodes an inhibitor of cysteinate.

b) Data of RT-PCR for expressii CcCPI-1 (Fig.7) in the Arabica and the Robusta. PCR was performed as described previously; the modes of reactions and used PCR primers is indicated in the legend to the figure. These data confirm and extend the previously presented data on expression in Arabica, which show that CcCPI-1 is expressed only in the grain, and not in pericarp. Weak expression of this gene is also detected in the flowers (a result that could not be determined Northern blot analysis). The method of RT-PCR is defined as the expression Robusta (Fig.7). A sample of this expression is the same as in Arabica, except that the flowers, or at the stage of small green beans no expression was not detected. No expression in the Robusta coffee is at the stage of small green beans were also reported for other genes, i.e., in this regard, gene CcCPI-1 is not unique.

Table 5

The frequency of occurrence of genes inhibitor cysteinate CPI-2, CPI-3 and in different EST libraries.
The name of the geneThe number of EST
Grain 18 weeks.Whole fruits 22 weeks.Grain 30 weeks.Grain 42 weeks.Grain 46 weeks.PericarpelSheet
CcCPI-202120110
CcCPI-30010200
CcCPI-4001about006

CcCPI-2

(a) the optimal alignment CcCPI-2 (Fig), indicating that this cDNA encodes an inhibitor of cysteinate.

b) Data of RT-PCR for expressii CcCPI-2 (Fig.9) in the Arabica and the Robusta. PCR was performed as described above; used PCR primers is indicated in the legend to the figure. These data show that CcCPI-2 is expressed in all tissues, so the protein product of this gene likely plays an important role in the control of one or more cysteinate present in these tissues. The number of ESTs in each library, a CR is entered in the table. 5 (see above), suggests that the CPI-2 can be expressed in the grain (seed) after 30 weeks after fertilization to a greater extent than in the leaves, pericarp or grain after 46 weeks after fertilization.

CcCPI-3

(a) the optimal alignment CcCPI-3 (figure 10), indicating that this cDNA encodes an inhibitor of cysteinate.

b) Data of RT-PCR for the expression of this inhibitor cysteinate at the present time. However, expression of this gene is "in silico", judging by the number of ESTs in each library (table. 5, see above), indicates that the CcCPI-3 is expressed in a coffee grain is present in the libraries of grains Grain 30w" and "Grain 46w", i.e. after 30 and 46 weeks). No ESTs specified gene in pericarp, leaves or whole fruit of the coffee tree suggests that this gene is probably specific to coffee bean genome.

CcCPI-4

(a) the Optimal alignment CcCPI-4 (11), indicating that this cDNA encodes an inhibitor of cysteinate.

b) Data of RT-PCR for expression CcCPI-4 (Fig) in Arabica and Robusta coffee. PCR was performed as described above, used PCR primers is indicated in the legend to the figure. The obtained data indicate that this gene is significantly expressed in the Arabica leaves, flowers and grain at the stage of red fruit. Since a thorough analysis of the original gel (panel A: ar is Bica) shows in the case of small green fruits and pericardia large green beans in protein bands appear fuzzy spots, it can be assumed that this gene may also weakly expressed in the Arabica beans and pericarp at all studied stages of fruit formation. The data obtained for the species, indicate that this gene is significantly expressed in leaves, flowers, small green and large green beans. Only one EST for CcCPI-4 was discovered in the libraries of grains or pericardia (PL. 5, see above), which indicates that the expression of this gene in grain and/or pericarp is relatively low or confined to a small defined areas in these two tissues.

In each case, gene inhibitor cysteinate (CPI), it is assumed that the overexpression or suppression of expression of these genes in the formation of the grains (i.e. under the control of a very strong, specific to grain, promoter such as the promoter 11 S in coffee), will change the protein, peptide and amino acid profiles in Mature grains (as well as the level of precursors of aroma).

Sprouting grains and RT-PCR analysis

Sterilized, dried coffee bean C. arabica CCCA2 (with remote parchment and silver seed shells) were placed one by one into tubes containing 10 ml of solid feeder is th environment Heller HI 5 and 7 g/l agar, and incubated daily at 25°C for 8-hour day length. After 2 day, 3 day, 5 day, 1 month and 2 months of germination were collected on three grain and removed from them the primary (embryonic root, if it existed, and then grain and roots were immediately frozen in liquid nitrogen and stored at -80°before extraction of RNA. Served as control in the same way, dried and sterilized, but not progressie grain. Extraction of RNA from samples of grain were performed as described previously. The treated DNA Asai total RNA extracted from each sample was used for cDNA synthesis using oligo-(C1T)2O as a primer in accordance with manufacturer's instructions to finish the set reverse transcriptase "Superscript II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA). Then there was a panorama using an aliquot of each of the reaction mixtures with cDNA. (50 µl reaction mixtures contained 10 ál of diluted at the ratio of 1/10 solutions cDNA, 1 μm of each primer, 5 µl of 10X PCR ThermoPol buffer, 200 μm dNTP, and 2 units of Taq polymerase (New England Biolabs, Beverly, MA). Modes cycles were as follows: 2 min at 94°C, then 40 cycles: 94°C, 1 min - 54°C, 1.5 min - 72°C for 2.5 minutes at the Ultimate stage of completion of the chain lasted 7 min at 72°C. PCR primers were as follows:

CP-4KDDL61: 5'-GAAGAACTCATGGGGAACAGGAT-3',

CP-4KDDL345: 5'-TTATTCAAACCATCACAGGAGGAG-3'

Genomma the PCR and DNA sequencing of the purified PCR fragments.

Genomic DNA of five different coffees (FRT-07, FRT-19, FRT-32, CCCA2 and GPFA57) used in PCR as described previously in experiments on the study of expressii method RT-PCR in the germinating grain. Were obtained PCR products of the expected size, and these fragments were purified from gel. Then amplified by PCR DNA was subjected to a second round of amplification PCR and obtained in this reaction sequence the DNA sequenced using the same primers that were used for amplification.

Isolation and characterization of the gene cysteinate USSR-4

Using the collection of ESTs obtained from RNA extracted from coffee beans at various stages of its development, tissue pericardia at different stages of development coffee beans and leaves, the authors of this proposal have been able to isolate the full-size cDNA encoding cysteinate coffee, which has a C-terminal KDDL sequence. This cDNA authors called the USSR-4 (KDDL) (Fig). Alignment of the protein encoded by this cDNA, with other vysokomolochnye systemprocesses plants shown in Fig. The data from this alignment and the corresponding search using the BLAST program clearly show that the protein encoded by the sequence of the USSR-4 (KDDL) coffee is a member of the family of plants KDEL containing cysteinate (Fig). Precision Eden who was mentioned USSR-4 (KDDL) and the most homologous sequences from the database are given in tables 6A and 6B.

Table 6A

The identity of the amino acid sequence of the USSR-4 (KDDL) cysteinate Coffea canephora amino acid sequences most homologous sequences from GenBank
Cysteinate Coffea canephoraThe name of the gene (access number)percent identity of protein
Dacus carrota (JC7787)73
Vignamungo(P12412)69
USSR-4 (KDDL)Glycibe max Cysl(AB092555)70
Glycibe max Cys2(AB092557)68
Vicia sativa (Z34895)64

49
Table 6B

The identity of nucleic acid sequences (cDNA) of the USSR-4 (KDDL) cysteinate Coffea canephora sequences of nucleic acids of most homologous sequences from GenBank
Cysteinate Coffea canephoraThe name of the gene (access number)% identity DNA
Dacus carrota (JC7787)55
Vigna mungo (PI 2412)61
USSR-4 (KDDL)Glycibe max Cysl(AB092555)
Glycibe max Cys2(AB092557)62
Vicia sativa (Z34895)60

Hence it is clear that the obtained sequence of the USSR-4 KDDL coffee has one important difference from almost all other sequences shown in Fig, namely it does not contain the expected sequence, which determines the delay (retention) of the protein in the endoplasmic reticulum (ER) - C-terminal KDEL sequence, and contains only a variant of this sequence - KDDL. Based on the study of the ability of the variants of the C-terminal KDEL sequence to determine the delay of the protein in the ER of plant cells Denecke et al. (Denecke J., De Rycke R, Botterman J., 1992, EMBO J. 11, 2345-2355) previously showed that C-terminal variants, such as SDEL, KDDL, KDEI and KDEV, can cause complete loss of function of the delay of the protein of the endoplasmic-reticulum. Thus, the presence of sequence KDDL in coffee plant homologue KDEL cysteinate was a complete surprise. On table. 7 shows that unigen containing cDNA of the USSR-4 (KDDL), has 21 ESTs. Therefore, the authors of this application have studied subsequently, the sequence of other ESTs in this unigene and found that seven of these ESTs contain positive sequence data KDDL plot. Of these seven cDNA sequences contained six KDDL after outermost, and one - KDEL sequence. The proposal was selected clone cDNA with a C-terminal sequence KDEL and received full sequence of this partial cDNA clone. The obtained DNA sequences and protein shown in Fig and 17, respectively.

Table 7

The number of EST unigene. contains full-size cDNA of the USSR-4 (KDDL)
Cysteine-protease NameUnigenThe number of EST
Grain 18wWhole fruit 22 wGrain 30 wGrain 42wGrain 46wPericarpelSheet
USSR-412510300801300

cDNA, the coding sequence of the USSR-4 (KDEL), shown in Fig, represents only part of the cDNA, i.e. only the fragment length 817 P.N. against 1336 BP full-size cDNA of the USSR-4 (KDDL) clone. Partial cDNA of the USSR-4 (KDEL) contains 8 single nucleotide substitutions compared with the equivalent sequence found in the cDNA clone of the USSR-4 (KDDL), although only two of these nucleotide substitutions leading to amino acid change effects the successive open reading frame (Fig). In 3' untranslated region there are 3 distinct nucleotide substitutions. In addition, there is also an insertion of 12 nucleotides in the 3' untranslated region of the USSR-4 (KDEL) cDNA sequence, which is probably within the microsatellite region. These data indicate the presence of two different and very important molecular markers for the two alleles of the USSR-4 coffee, one of which is a SNP associated with functionally important KDEL plot, and the second is a microsatellite marker associated with the 3' untranslated region of the specified gene. The last point is very important because microsatellite sequences are usually considered as genetic markers with high variability, and therefore, it is likely that using this containing the microsatellite region, you can find other alleles of the specified gene.

To study the distribution of the two alleles of the gene of the USSR-4, identified above in different varieties of Arabica and Robusta, a small stretch of genomic sequence covering the USSR-4 gene amplified by PCR from five different genotypes. From each sample of genomic DNA were obtained PCR fragments of the expected size (207 base pairs); the resulting PCR products were purified from the gel and re-amplified in order to receive the Oia sufficient DNA for direct DNA sequencing of PCR products. The results obtained during the sequencing reactions presented on Fig. Chromatogram sequencing five sequences show that the two studied varieties of Arabica clearly contain a KDEL sequence, and the three studied varieties of Robusta - KDDL sequence. This result suggests that KDDL allele, likely, limited varieties of Robusta and can't be found in varieties of Arabica. Although the study for three varieties of Robusta was found KDEL sequence, the opening of a KDEL sequence at Comell EST library indicates that this allele may exist, at least in some clones of some varieties of Robusta.

Gene expression of the USSR-4 were studied using Northern blot and RT-PCR analyses. On Fig shows the result obtained in the experiment with Northern blot analysis using RNA extracted from coffee beans and pericardia at different stages of development, as well as RNA isolated from roots, young leaves, stems and flowers of different varieties of Arabica. The data obtained using the probe of the USSR-4 (KDDL), with approximately 98% homology with known DNA sequence of the USSR-4 (KDEL) allele, showed that the USSR-4 is expressed only in the grain. In pericarp or rhizome, stem, flowers and leaves its expression was not observed. Due to the very high ur is una identity between the two alleles suggested that that probe the USSR-4 (KDDL) will hybridisierung with transcripts of these two alleles. A similar experiment using RT-PCR analysis also revealed the same gene expression profile of the USSR-4.

The expression of the USSR-4 also studied with whole-grains during germination using RT-PCR analysis. In this experiment, we used primers that are common for CESR-4 KDEL, and for the USSR-4 KDDL alleles. The results of this experiment are shown in Fig. Transcripts of the USSR-4 were detected in all studied stages of germination, although the level of transcripts is likely to be somewhat decreased in 3-day-old samples, and then again started to rise as the progression of germination (the highest level observed in 1 - and 2-month-old samples).

Ling et al. (Ling, J.-Q., Kojima T., Shiraiwa, M. and H. Takahara, 2003, Biochim. Biophys. Acta, 1627,129-139) was able to extract from soybean cotyledons two cDNA encoding the KDEL-containing cysteinate. These two cDNA was 93,5% similarity at the DNA level and expressibility in roots, flowers and seeds during their development. In emerging or Mature seeds, their expression was not observed in the results of Northern blotting, although expression was detected in Mature beans. cDNA encoding KDEL-containing cysteinate, was also isolated from carrots (SakutaC., Oda, A., Konishi M., Yamakawa, S., H. Kamada and S. Satoh, 2001, Biosci. Biotechnol. Biochem., 65, 2243-2248). TRANS is ripti this gene were detected in Mature dried seeds and whole germinating seeds at 2 and 3 days after impregnation with water. Expression of this gene in other tissues of carrot or during seed development were observed. Another KDEL-containing proteins and the corresponding cDNA were isolated from V. sativa (Fischer J., Becker, S., Hillmer, S., Horstmann C., Neubohn Century, Schlereth, A., Senyuk V, Shutov A. and K. Muntz, 2000, Plant Molecular Biol., 43, 83-101). Using Northern blot transcripts of this gene were detected in cotyledons during germination, but not in the main axis of the embryo of the germinating seed. In maturing seeds, Mature seeds or leaves and roots transcripts were not detected.

Results presented here show that cysteinate (CF) KDEL-type of coffee reveals some new and unexpected features. First, the authors discovered that the coffee bean Robusta expresses CF gene KDEL-type, which has a single mutation in the sequence, the coding region KDEL, which leads to a change from KDEL to KDDL. Based on the data Denecke et al. (1992), one might expect that this particular change in the sequence associated with the delay Beck ER, alter the cellular localization and/or control protein USSR-4 (KDDL) Robusta. The authors suggested that the presence of a transcribed copy of the gene of the USSR-4 (KDDL) can produce a significant change in the peptide/amino acid profile in a coffee bean varieties with the USSR-4 (KDEL) sequence. The authors of the bid, medium, small is also revealed, that cysteinate KDEL type of coffee, showing the expected expression during germination of grain, unexpectedly is expressed on all studied stages of grain development. As noted above, to date in the published literature there is no clear data showing significant expression cysteinate KDEL type in other plants during seed development, although its transcripts were detected in Mature seeds of carrot (Sakuta et al., 2001).

Newly discovered properties cysteinate KDEL type of coffee, the above will probably have an important influence on peptide and amino acid profiles in Mature beans of Arabica and Robusta and, thereby, change the pool critical predecessors coffee aroma. Taking into account that the transcripts of cysteinate KDEL type present in Mature coffee bean, it is also possible that the KDEL protein type can be activated during wet processing of coffee and, thereby, change in the future, the peptide/amino acid profile of the coffee bean after wet processing. The experiments described have led to the creation of molecular markers (SNP and microsatellite marker)that can be used in classical breeding breeding coffee with specific alleles of the gene of cysteinate KDEL-type (which will be you Iwate related changes in protein/peptide/amino acid profiles). For example, you can selekcionirovat grade Robusta coffee, which will have only KDEL allele of the USSR-4. Next, using techniques of genetic modification can directly alter the activity of cysteinate KDEL-type coffee, or other plants by specific overexpression of cysteinate KDEL or KDDL type of seeds. Alternative: you can reduce the level of cysteinate KDEL type with the use of antisense, sense or PHKi technologies. In both cases, the protein/peptide/amino acid pool in the newly hatched transformed plants will change, leading to new profiles of protein/peptide/amino acid pools of precursors of aroma.

Northern-blot analysis, presented at Fig, shows that the gene Ssar-2 specific for aspartic acid protease coffee is expressed as in the grain, and pericarp fruit of the coffee tree varieties of C. arabica in all studied stages of fruit development. Gene Ssar-2 also shows relatively high expression in the rhizome. More prolonged exposure of the film at autoradiography expression of SAR-2 can also be detected in the tissues of stems, leaves and flowers of C. arabica.

SAR-1 and SSR-2

On Fig and 22 shows that each of SSR-1 and SSR-2 encodes specific for aspartic acid protease.

Overexpression and incomplete expression of posledovatel the values of the genes of proteases CcCP-1, USSR-4, SAR-1 and SSR-2 and inhibitors of proteases CcCPI-1,2,3 and 4 in coffee beans.

It is assumed that the basic profile of spare protein and amino acid/peptide profile may change in Mature coffee grains as a result of change (either increase or decrease) the expression of one or more of these genes.

Interest methods overexpression of a gene are well known in the prior art. These methods involve the creation of a chimeric gene in three main components:

1) a promoter sequence on the 5' end of the gene, preferably specific promoter seeds that have started to use in recent times, such as, for example, specific promoter coffee beans described Marraccini et al.(Marraccini et al., 1999, Molecular cloning of the complete US storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants=Molecular cloning of the gene of total protein reserve US Coffea arabica and analysis of the promoter in transgenic tobacco plants. Plant Physiol. Biochem., Vol.37, 273-282, and WO 99/026688);

2) full coding sequence expressed gene;

3) at the 3' regulatory region, for example, the 3' region of the gene nepalensis from T-DNA of the Ti-plasmid of Agrobacterium tumefaciens.

Then chimeric gene can be cloned into a transformation vector Agrobacterium tumefaciens, and this vector can be transformed Agrobacterium tumefaciens strain for use when t is informatsii coffee, which detailed the transformation described by Leroy et al., 2000 (Leroy et al., 2000, Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf minor. = Genetically modified coffee plants expressing the gene of Bacillus thuringiensis cry1Ac resistance to the weak sheet. Plant Cell Reposts, 19, 382-389). Then plants with stable inserts transformation can be skanirovat to select seeds of such plants, which sverkhekspressiya specific genes used in the experiment in transformation, especially in Mature seeds, using methods such as methods of detecting overexpression of a gene or overexpression activity of the proteins compared to seeds pseudotranslation plants.

For example, visokokvalifizirovannii specialist in this field can create a recombinant construct comprising (1) the longest promoter sequence of a gene 11S coffee, described Marraccini et al. (1999), (2) full-size cDNA sequence of the gene of the USSR-1 or the USSR-4 (KDDL) without poly (A) tail and (3) the known sequence of the termination of transcription, such as the well-studied terminator nopaline. It is also possible that elevated levels of overexpression of recombinant constructions may be the result of replacement of the 5' non-coding region of the USSR-4 or other cDNA sequences on the 5' coding region is here gene 11S coffee or 5' non-coding region other strong specific promoters seeds or coffee, any other relevant varieties. Then the sequence of the recombinant gene can be incorporated into the appropriate section of the vector Agrobacterium T-DNA, described by Leroy et al. Designed T-DNA vector can be entered in the appropriate Agrobacterium strain, for example, the strain described by Leroy et al., and containing the T-DNA of the Agrobacterium strain can be used for transformation of coffee according to the method detailed described by Leroy et al.

In the prior art it is well known that the expression of a known gene sequences may be reduced or completely blocked by antisense suppression, and gene expression using nucleic acid fragments that are only part of the full coding region of the gene, and the nucleic acids that do not have 100% sequence identity with cupressinum genome. In this case, the sequence is selected for a particular experiment antisense suppression or compressie, will replace the full-size gene in the above diagram the construction of a chimeric gene. The obtained chimeric constructs antisense suppression, or compressie can be cloned into the transformation vector Agrobacterium tumefaciens and to transform Agrobacterium tumefaciens strain for its use for transformation of coffee, as described above. Then the plant is stable transformation inserts can be skanirovat for selection of plants with reduced expression of specific gene sequences, used in the seeds of transformed plants. Reduced expression can be detected by methods such as Northern blotting, RT-PCR with partial and/or full quantitative assessment.

Another method of reducing or suppressing gene expression in plants involves the use of small fragments disclosed here sequences of the gene for the production of RNA silencing the application interfering RNA (PHKi, see, for example, G.J. Hannon, 2002, Nature, Vol.418, 244-251;

Tang et al., 2003, Genes Dev, Vol.17, 49-63). Under this approach, small areas of one or more disclosed here sequences can be cloned into the transformation vector Agrobacterium tumefaciens, as described above, which contains specific seed promoter and an appropriate 3' regulatory region. This new embedded sequence for PHKi shall be designed so that the produced RNA formed RNA structure in vivo, leading to the formation of a small double strand RNA in transformed cells, while the sequence of these Nebojsa double strand RNA will trigger the collapse of homologous them mRNA in these transformed cells.

Squeaks naturally occurring mutations in the genes of the USSR-1. USSR-4, CcAP-1,SAR-2, CcCPI-1, CcCPI-2, CcCPI-3, CcCPI-4 and the creation of new mutations in these genes.

Disclosed sequence here is eljnosti can be used for screening of natural populations allelic variants of these genes. This can be done by using sequences of the USSR-1, USSR-4, SAR-1, SAR-2, CcCPI-1, CcCPI-2, CcCPI-3, CcCPI-4 as probes in the search for naturally arising RFLP (length polymorphism fragments) in the genomic DNA of different coffees. More high-performance discovery of allelic variants is the screening of mutations associated with TILLING method (Till, B.J., et al., 2003, Large scale discovery of induced point mutations with highthroughput TILLING. = Large-scale discovery of induced point mutations with high-TILLING method. Genome Research, Vol.13, 524-530). In this case, the selected and klonirovana one some specific sequence of a gene, for example, disclosed here USSR-1, USSR-4, SAR-1, SAR-2, CcCPI-1, CcCPI-2, CcCPI-3, CcCPI-4 sequences, when screening for mutations associated with TILLING method, can be used to identify variants of the sequences between the cloned sequence and the corresponding sequence of cDNA or genomic sequence in different varieties. Using a pair of PCR primers, the coding segments of DNA from 700-1100 base pairs known cloned gene can be scanned to identify naturally arisen changes in its sequences in different varieties. In the ideal case, one or more vari is new sequences can be correlated with specific phenotypic change, identifying, thus, a genetic marker of this phenotypic variation.

In addition, using the disclosed here, the sequence of the USSR-1, USSR-4, SAR-1, SAR-2, CcCPI-1, CcCPI-2, CcCPI-3 ISSR-4, you can use the full TILLING method for creating and discovering new mutants on these genes and to obtain, thus, the plants containing these specific mutations. For example, using the TILLING method, you can bring the coffee trees, which will have specific mutations, such as missense mutations (leading to the substitution of nonconforming amino acids in the protein chain) in the coding sequence, which inactivates interest gene target.

1. Isolated polynucleotide containing the nucleotide sequence encoding the polypeptide having systemprocesses activity, where the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No.2 identical, at least 70%, preferably at least 80%, sequence identity is based on the ClustalW alignment; or the complement of the nucleotide sequence, where the complement contains the same number of nucleotides and nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.

3. Polynucleotide according to claim 1, where the nucleotide sequence contains the nucleotide sequence of SEQ ID No.1.

4. Polynucleotide according to claim 1, where the polypeptide contains the amino acid sequence of SEQ ID No.2.

5. Isolated polynucleotide containing the nucleotide sequence encoding the polypeptide having systemprocesses activity, where the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No.16 identical, at least 80%, sequence identity is based on the ClustalW alignment; or the complement of the nucleotide sequence, where the complement contains the same number of nucleotides and nucleotide sequence, and the complement and the nucleotide sequence are 100% complementary.

6. Polynucleotide according to claim 5, where the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID No.16 identical, at least 85%, preferably at least 90%, neoba the consequently, at least 95%, sequence identity is based on the ClustalW alignment.

7. Polynucleotide according to claim 5, where the nucleotide sequence contains the nucleotide sequence of SEQ ID No.15.

8. Polynucleotide according to claim 5, where the polypeptide contains the amino acid sequence of SEQ ID No.16.

9. Expressing the vector containing polynucleotide according to any one of claims 1 to 8, encoding a polypeptide having systemprocesses activity.

10. Recombinant design DNA containing polynucleotide according to any one of claims 1 to 8, encoding a polypeptide having systemprocesses activity, which is operatively linked to a regulatory sequence.

11. The way of transformation of the cells of the coffee tree, including the transformation of cells by polynucleotide according to any one of claims 1 to 8, encoding a polypeptide having systemprocesses activity.

12. Transformed cell of the coffee tree containing recombinant design DNA of claim 10, encoding a polypeptide having systemprocesses activity.

13. Transgenic coffee tree, providing the modulation level of the precursors of aroma of coffee in the raw unroasted coffee beans containing cell according to item 12.

14. The modulation method of the predecessors of the aroma of coffee in the raw unroasted coffee beans, including the introduction in coffee tree recombinant constructs DNA of claim 10.



 

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