MXPA99009123A - Strawberry fruit promoters for gene expression - Google Patents

Abstract

Promoters isolated from genomic DNA of strawberry plants are disclosed. The promoters are capable of tissue-specific expression in transgenic plants. A plant promoter that is a nucleic acid region located upstream of the 5'end of a plant DNA structural coding sequence that is transcribed at high levels in ripening fruit. This promoter region is capable of conferring high levels of transcription in ripening fruit tissue and in developing seed tissues when used as a promoter for a heterologous coding sequence in a chimeric gene. The promoter and any chimeric gene in which it may be used can be used to obtain transformed plants or plant cells. Chimeric genes including the isolated promoter region, transformed plants containing the isolated promoter regions, transformed plant cells and seeds are also disclosed.

Description

PROMOTERS FOR EXPRESSION OF GENES OF STRAWBERRY FRUIT INTRODUCTION Technical Field The present invention relates to expression of fruit tissue genes for the modification of the fruit phenotype. The invention is exemplified by the use of Fragaria sp. Promoters. which is expressed selectively in tissue of the receptacle. Background One of the goals of plant genetic engineering is to obtain plants that have improved characteristics or traits. Many different types of features or traits in plants are considered advantageous. Those of particular importance with respect to the plants containing fruits, include control of fruit ripening, improvements in the nutritional characteristics of the edible portions thereof, resistance to plant diseases, insect resistance, cold tolerance and stability or Improved storage life of the product for the final consumption obtained from the plant. At least two key components are required to stably treat a desired trait, or control of said trait, in a plant. The first key component comprises identifying and isolating the genes that encode or regulate a particular trait. The second component comprises identifying and isolating the genetic elements essential for the actual expression and / or selective control of the newly isolated genes so that the plant will manifest the desired trait and, ideally, will manifest the trait in a controlled or controllable manner. This second component, which controls or regulates gene expression, typically comprises transcriptional control elements known as promoters. Although a generic class of promoters that drive the expression of heterologous genes has been identified, a wide variety of active promoters in specific white tissues or plant cells are still to be described. The identification of said tissue-specific or white promoters is critical for the introduction of the tissue-specific improvements mentioned above in plants such as fruit plants. Several promoters useful for expressing heterologous genes in selected fruits have already been identified. For example, promoters E4 and E8 (Deikman, et al.), The actinidin promoter of kiwifruit (Lin and others) and the polygalacturonase promoter are known to be specific for fruits. The Patent of E.U.A. No. 4,943,674 (Houck et al., July 24, 1990) discloses a 2AII promoter as being useful in the expression of a heterologous gene in tomato fruit. These promoters, however, have been isolated from fruit tissue comprising mature or maturing ovaries (hereinafter referred to as "traditional fruit"). As such, these traditional fruit promoters could be ineffective in controlling desired traits in such accessory plants having fruits such as strawberry, apple, pear, quince and the like wherein the main portion of the edible fruit comprises tissue of the receptacle (see An Introduction to Plant Biology, 2nd Edition, Braungart &Arnett, eds., CV Mosby Co. 1965). Similarly, to date, genes that are thought to be active in fruit tissues have been isolated from traditional fruit tissues instead of the tissue that contains the receptacle. Promoters involved in the expression of the fruit have been identified in the PCT application WO 97/27295. There is a need for selective promoters for receptacle tissues that provide increased or decreased expression during the development, maturation and complete development of the fruit in the art. Access to said selective promoters for receptacle tissues could allow the genetic treatment of the fruit tissue of commercially important plants such as strawberry, apple and pear. Two cDNAs have previously been identified as selective for receptacle tissues (Reddy and Poovaiah, 1990, Plant Molecular Biology, 14: 127-136 and Wilkinson et al., 1995, Plant Molecular Biology 27: 1097-1108). The promoters for these two cDNAs were cloned and sequenced. The expression of reporter genes in strawberry plants will be used as an analysis of the tissue specificity of the isolated promoters. Of particular interest are promoters that provide selective tissue expression of gene pools. The ability to increase or modify the properties of other promoters is also of interest.

Relevant Literature Reddy and Poovaiah, Plant Mol. Biol., (1990) 14: 127-136, report the cloning of a cDNA from a repressed strawberry auxin mRNA. Wilkinson, and others, Plant Mol. Biol., (1995) 27: 1097-1108 report the identification of mRNA in strawberries that improves expression in fruit ripening, for example RJ39. COMPENDIUM OF THE INVENTION The present invention provides novel promoters called "SAR5 and RJ39", which cause the decrease or increase of selective expression of heterologous DNA tissues in the tissue of plant receptacles. The present invention also provides novel chimeric genes comprising a receptacle-selective promoter operably coupled to a heterologous DNA sequence. The present invention further provides a method for the expression of a heterologous gene, the improvement comprising the use of an accessory fruit plant promoter that causes the decrease or increase in selective expression for seed tissues, weir and tissue from receptacles of plants during the development and maturation and complete development of the fruit, said promoter of accessory fruit plants having a sequence is selected from the group consisting of those sequences shown in SEQ ID NOS. 1 and 2 and sequences substantially homologous thereto.

Additionally, cells of novel transformed plants and transgenic plants comprising the heterologous genes of the present invention or produced by the methods of the present invention are provided. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of the full-length promoter region of the genomic DNA clone of SAR5. Figure 2 shows the nucleotide sequence (SEQ ID NO: 2) of the promoter region of the RJ39 genomic DNA clone. Figure 3 shows a schematic representation of the primary DNA vector pCGN8045, which contains a full-length SAR5 promoter for plant transformation. Figure 4 shows a schematic representation of the DNA vector of pCGN8054, which contains the truncated SAR5 promoter for plant transformation. Figure 5 shows a schematic representation of the DNA vector, pCGN8052 containing the RJ39 promoter for plant transformation. DETAILED DESCRIPTION OF THE PREFERRED MODALITIES In accordance with the present invention, nucleic acid constructs are provided that are active in the tissue of plant receptacles, and in particular, in plants that have accessory fruits. The novel promoter sequences of the present invention provide for the increasing or decreasing expression of heterologous genes during the development and maturation and complete development of fruits. The phrase "heterologous gene" means that the DNA encoding the sequence does not exist in nature in the same gene with the promoter to which it is not attached. The promoter sequences of the present invention will now provide an opportunity to genetically treat agriculturally and commercially important traits in a class of fruits, fruit fabrics and fruit-bearing plants. More specifically, this class of fruits includes those plants comprising accessory fruits and other plants in which the regulation of the receptacle function or the expression genetically treated in the tissue of receptacles is convenient. Constructs can be included in a transcription cassette or an expression cassette in which the downstream region of the regulated transcriptional initiation is a nucleotide sequence of interest that provides for the regulated modification of the plant phenotype, modulating production in a product endogenous, in terms of quantity, relative distribution or similar, or the production of an exogenous expression product to provide a novel function or product. One or more introns may also be present. Depending on the form of the introduction of the nucleic acid construct in a host plant, other DNA sequences may be required so that they are a sufficient T-DNA of an Agrobacterium plasmid to transfer a plant host. Guests of plants of particular interest are fruit plants, such as strawberry.

In one embodiment, DNA sequences are provided for the promoters which are active in strawberry plants. Strawberry plants are an important commercial fruit crop in many temperate regions of the world and are especially suitable for improvement by genetic engineering techniques such as clonal propagation, against conventional cultivation and selection. High heterozygosity and polyploidy associated with commercial lines of strawberry plants hinder the improvement of such plants by traditional growing methods. In contrast, clonal propagation of strawberry plants provides stable transformation of a single dominant gene for a desired trait into a commercially important genotype without sexual recombination. The novel promoters of the present invention now provide an opportunity to genetically treat such plants that have receptacle fruits such as strawberries such as commercially and agriculturally convenient traits including the delayed maturity of the fruit, increased sugar content, modified color and resistance to fungi. as described more specifically below. In another embodiment of the present invention, the selective promoter sequences for receptacle tissues are isolated from a genomic bank created from Fragaria vesca DNA. Specifically, a probe hybridizes to genomic DNA fragments of Fragaria vesca under medium to high restriction hybridization conditions (Meniatis et al., 1982). The identified genomic fragments are isolated and purified. Confirmation of selective activity for receptacle tissues can then be achieved by transforming plants, or plant tissues, with chimeric genes containing said substantially homologous sequences according to the examples described below. In one important embodiment of the present invention, two different novel promoters are provided, each individually capable of directing the transcription to a high level of a second DNA sequence expressly coupled thereto in the tissue of receptacles of plants having accessory fruits. These promoters are designated SAR5 and RJ39 (also referred to as RJ39C). The nucleotide sequences of these promoters are provided in SEQ ID NOS. 1 and 2, respectively. It is understood by those skilled in the art that the DNA sequences shown in any of SEQ ID NOS. 1 and 2 include any active promoter in development, maturity and complete tissue development of fruit receptacles having a DNA sequence substantially homologous to any of the promoter sequences. The fruit of the strawberry develops from the tissue of receptacles at the base of the flowers. From the base of the flower, the tissue of receptacles develops in the tissue of receptacles of the ripe fruit through the stages of: small green, large green, green-white, changing red and full red. Selective promoters of novel fruits have been isolated that exhibit diminishing or increasing expression and selective for tissues during the development of strawberry fruit. An mRNA, RJ39, has been identified as having increased expression during fruit maturity, selectivity in the tissue of receptacles in strawberry fruit tissue (Wilkinson et al., Plant Mol. Biol. (1995) 27: 1097 -1108). The expression of RJ39 mRNA was observed at low levels of fruit development at the green-white stage. The expression of RJ39 was increased during fruit maturity through the full red stage of development. The SAR5 mRNA was expressed during the development and maturation of the fruit. SAR5 was identified by maturing by auxin and exhibited decreased expression during ripening of the strawberry fruit (Reddy and Poovaiah, Plant Mol. Biol. (1990) 14: 127-136). The expression was initiated during the development of the flowering bulb during the development of the flower and early fruit development, then it decreases linearly during the ripening of the fruit and was expressed at very low or absent levels in the fruits of changing red and full red. These mRNAs were abundantly expressed in the tissue of maturation receptacles of accessory fruit plants (RJ39) or early in the development and maturation of fruits (SAR5) and showed little or no detectable expression in the tissues of the leaves. The low number of hybridization fragments and the lack of sequence variability indicated a low gene copy number. The cDNA clones of RJ39 and SAR5 were used to isolate genomic clones that contained a genomic copy of the cDNA and nucleic acid sequences corresponding to the transcriptional initiation region. The expression controlled by these promoters can be confirmed by the fusion to the gene of β-glucuronidase (GUS) and then the expression of the GUS enzyme during several stages of the development, maturation and development of the fruit, in the transgenic fruit. The promoters of the present invention can be used to increase the sugar content in the fruit. In particular, the action of the plant glucose-6-phosphatase gene can be inhibited by controlling the transcription of a counter-sense sequence corresponding to one or both of the glucose-6-phosphatase subunits. Other genes that could be usefully fused to a promoter of the present invention include sucrose phosphatase synthase (SPS), which is thought to control the overall regimen of sucrose biosynthesis in plant cells. Expression of an SPS gene, driven by SAR5 or RJ39 may result in a developing fruit with superior carbohydrate composition. The use of the promoters of the present invention with other genes such as ADP Glucose pyrophosphorylase, glgC16, which encodes an enzyme that synthesizes starch, may also be of interest. Expression of glgC16 driven by SAR5 or RJ39 may result in a developing fruit with superior carbohydrate composition.

Another possible use is with the invertase gene. The expression of invertase in a spill cell such as in a fruit is a method to increase a cell's ability to act as a stronger spill by decomposing sucrose to metabolites that can be used in carbon routes, v.gr ., starch biosynthesis. The sucrose is then immobilized in the tissue of the landfill. The expression of invertase in the appropriate tissue and cellular compartments when the fruit is a strong dump, that is, in a green fruit, is highly convenient. The use of the promoters of the present invention with a gene for sucrose synthase could be convenient for the reasons given for SPS. Other genes can be used in constructs with SAR5 and RJ39 promoter sequences to achieve resistance to pathogens. For example, genes encoding the production of phytoalexins (eg, hydroxystilbenes), the expression of disease resistance genes, such as R genes, defense induction genes, such as avr genes and genes for insect resistance . The promoters of the present invention can also be used in constructions containing two genes of complementary functions. For example, plants can be transformed with a construct that contains two genes that can be complementary to each other to increase sugars in a fruit. A gene may be required in early fruit development, and the second gene product may act on the last gene product early in the ripening of the fruit. For example, the SAR5 promoter can be used to boost glgC16 early in fruit development to increase the amount of starch in developing fruit and the RJ39 promoter can be used to boost late SPS in fruit development to further increase the soluble solids in the tissue of fruit receptacles. Alternatively, the promoters can be used together to drive the same gene. Using the SAR5 and RJ39 promoters to boost the same gene, a sustained level of gene expression can be achieved during the development of the fruit. For example, the invertase can be driven by SAR5 and RJ39 to provide a constant landfill resistance, increased during the development and maturation of the fruit. Plants containing two or more promoter gene fusions can be produced by various methods for one skilled in the art. A single construct containing two promoter gene functions can be used for transformation or alternatively, two constructs of promoter genes can be used (co-transformed). In addition, transgenic plants containing one of the promoter gene fusions can be used as explants material for re-transformed with the second promoter gene fusion Transgenic plants containing two promoter gene fusions can also be obtained by crossing transgenic plants containing a fusion of promoter genes integrated into their genome using the promoter sequences provided herein. The person skilled in the art is now able to isolate, or chemically or enzymatically synthesize, by conventional methodologies, promoters that have sequences essentially identical to those sequences described herein and promoters substantially homologous thereto, eg, the isolation of said s Promoter sequences can be achieved using conventional techniques to synthesize a hybridization probe comprising all or a portion of a promoter sequence displayed in any of SEQ ID NOS. 1 and 2. The hybridization probe preferably is about 20 to 600 nucleotides in length. A double-stranded DNA molecule containing one or more promoters of the present invention can be inserted into the genome of a plant by any suitable method. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those described, eg, by Herrera-Estrella, L., and others, Klee, H.J., and others, and EPO publication 120,516 (Schilperoort et al.). In addition to the transformation vectors of plants derived from Ti plasmids or root inducers (Ri) from Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase uptake of free DNA, delivery of free DNA via microprojectile bombardment and transformation using virus or pollen. A sequence of nucleotides of interest is inserted downstream from and below the regulation of the transcriptional initiation region. The nucleotide sequence of interest provides for plant phenotype modification for example by altering the production of an endogenous product, in terms of amount, relative distribution or the like, or by coding a structurally or functionally novel gene product. The nucleotide sequence can have any open reading frame that encodes a peptide of interest, for example, an enzyme, or a sequence complementary to a genomic sequence, wherein the genomic sequence can be an open reading frame, an intron, a sequence of reading without coding, or any other sequence in which the complementary sequence inhibits transcription, the processing of messenger RNA, e.g., division or translation. The nucleotide sequence of interest may be of natural or synthetic origin, or combinations thereof. Depending on the nature of nucleotide sequences of interest, it may be convenient to synthesize the sequence with preferred codons of plants. The preferred codons of plants can be determined from the highest frequency codons in the proteins expressed in the longest amount in the particular plant species of interest. The termination region is one that is functional in a host cell of plants. In addition to containing at least one termination sequence, the termination region may include a poly A signal. In view of the relative exchangeability of the termination regions, the selection of a termination region to be used in the termination construction is primarily It is based on convenience. The region of termination in the transcriptional initiation region or the termination region and the nucleotide sequence of interest may originate from the same sources or from different sources. Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the termination regions of the octopine synthase gene and the nopaline synthase gene. Additional DNA sequences can be included in the transcription cassette, for example, adapters or linkers by joining the DNA fragments in the appropriate orientation and as appropriate, in the appropriate reading frame. Other DNA sequences can be nested to transfer the transcription constructs in organisms used to transform plant cells, e.g., A. tumefaciens. In this regard, the use of T-DNA of the Ti or Ri plasmids as a flanking region in a transcription construct is described in EPO application No. 116,718 and PCT application Nos.

WO84 / 02913, 02919 and 02920. See also Herrera-Estrella, Nature (1983) 303: 209-213; Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80: 4803-4807; Horsch et al., Science (1984) 223: 496-498; and DeBlock et al., EMBO J. (1984) 3: 1681-1689. The expression constructs of the present invention, which contain the regulated 5 'unregulated regions of two selective strawberry receptacle genes, are transformed into plant cells to evaluate their ability to function with a structural gene different from the open reading frame that it is natively associated with the 5 'untranslated region and to evaluate its expression characteristics. A variety of techniques are available for the introduction of DNA into a host of plant cells. These techniques include transformation using Ti plasmid DNA and A. tumefaciens or A. rhizogenes as the transformation agent, protoplast fusion, injection, electroporation and the like. The transcription construct is usually bound to a marker that allows the selection of transformed cells in the treated population, for example, resistance to antibiotics such as kanamycin, G418, bleomycin, chloramphenicol and others. Any variety of plants can be employed as a host cell according to this invention. Of particular interest are agricultural fruit crops, such as strawberries and tomatoes, although the use of the transcriptional initiation regions of RJ39 and SAR5 in other plants, including other plants that have fruits are also considered. Examples of plants in which the promoters of the present invention may find use include, but are not limited to, any plant weed tissue that includes strawberry seeds, raspberry, tomato, potato tuber, tobacco, soybean, cotton capsule and cotton seed Host cells of transformed plants are used to regenerate plants. See, e.g., McCormick et al., Plant Cell Reports (1986) 5: 81-84. These plants grow and polinate with the same transformed strain or with different strains and the resulting hybrid that have the desired phenotypic characteristics identified. Two or more generations can be developed to ensure that the desired phenotypic characteristic is stably maintained and inherited and then the seeds are harvested to ensure that the desired phenotype or other property is achieved. The embodiments described above and the following examples are provided to better provide for the practice of the present invention. It should be that these embodiments and examples are provided for illustrative purposes only and not by way of limitation of the scope of the invention. The following experimental protocol describes the identification and isolation of the promoter of a gene differentially expressed in the tissue of plant receptacles. One skilled in the art will recognize that substitutions and alterations can be made in the components, conditions, and procedures presented herein without departing from the scope or intent of the protocol. The recombinant DNA techniques employed are familiar to those skilled in the art of manipulating and cloning DNA fragments and are used in accordance with the teachings of Sambrook et al. EXAMPLES Example 1 Isolation of Promoters A. Genomic DNA Isolation from Strawberries The genomic DNA of strawberries was isolated from Fragaria vesca (2n = 14) and Fragaria x ananassa (Redcoat variety) as described herein. The tissue of fresh or frozen strawberry leaves was ground in a cooled mortar and two volumes (v / p fresh tissue) of pH extract buffer from DNA extract (500 mM sorbitol, 100 mM Tris, 5 mM) were added. of EDTA, 2% ß-mercaptoethanol, pH 7.5) and ground again in a short time. The homogenate was transferred to a centrifuge tube and 2.5 volumes of pH-regulating solution of the Nucleus Lysis were added (200 mM Tris, 50 mM EDTA, 2 M NaCl, and 2% C (hexadecyltrimethylammonium bromide)., pH 7.5) with 0.5 volumes of 5% Sarcosyl solution. The homogenates were mixed in a short time by inversion, then incubated at 65 ° C for 15 minutes. The DNA solutions were mixed again at room temperature for 5 minutes by inversion. The samples were extracted with chloroform once with an equal volume of chloroform / isoamyl alcohol (24 1). The samples were centrifuged at 12,000 rpm for 15 minutes and the upper aqueous phase was transferred to a new tube. The genomic DNA was precipitated by adding a volume of isopropanol, incubated for 30 minutes on ice and centrifuged for 10 minutes at 12,000 xg. The DNA pellets were washed with 70% ethanol and air dried. Two additional steps were used to remove contaminating polysaccharides. The dried pellets were resuspended in TE, and volume Vt of 5M NaCl was added. The samples were then incubated on ice for 30 minutes and centrifuged for 10 minutes at 12,000 xg. The supernatant was transferred to a new tube and the DNA was precipitated with 2.5 volumes of ethanol. The dried pellets were resuspended in TE. Potassium acetate (final concentration 2M) was added and the samples were again incubated on ice for 30 minutes and centrifuged for 10 minutes at 12,000 xg. The DNA was precipitated in the supernatant, dried and resuspended in TE B Promoter Isolation of SAR5 A genomic clone of the SAR5 gene was obtained by PCR amplification of genomic DNA from strawberries using primers designed according to the sequence of SAR5 (Reddy and Poovaiah, 1990, Plant Molecular Biology, 14 127-136). Frontal PCR SAR5-5C1 (5'-TCGAATTCAGAGCAAAGATGGTTCTGC-3 ', SEQ ID NO 3) contains the SAR5 coding sequence of the 5' end of the cDNA, including the ATG start codon (underlined above) and the restriction cloning sites SAR5-3N2 (5'-ACCTCGAGGGATCCTCATCACTTGTCG-3 ', SEQ ID NO 4) is the reverse primer that contains complementary sequences in bases 369 to 386 in the 3' untranslated region (numbering according to Reddy and Poovaiah) and the sites of restriction cloning. Genomic DNA is prepared from tissue of strawberry leaves (Redcoat variety) using the method described in 1A above. The PCR amplification was carried out according to the manufacturer's recommendations (Perkin-Elmer) at 40 cycles of 94 ° C, 1 minute, 49 ° C, 1 minute, and 72 ° C 1 minutes. The PCR product was purified from an agarose gel plate using a Prep-A-Gene (BioRad) kit, digested with EcoRI and BamHl and cloned into the EcoRI and BamHl sites of pBluescript SK- (Stratagene) creating the clone pCGN8023. Sequence analysis obtained using an automatic ABI sequencer showed that a complete genomic DNA copy of SAR5 was obtained and contained two introns. In order to reduce some of the complexities involved in the cloning of an octoploid species, such as Fragaria x ananassa (variety Redcoat, 8n = 56), the DNA of a diploid species, Fragaria vesca was used to clone the SAR5 promoter. Strawberry genomic DNA was prepared from Fragaria vesca as described in Example 1A above. The DNA was digested with the restriction enzyme BglII and the digestion products were prepared on a 0.7% agarose gel. The digested fractionated DNA fragments were transferred to a positively charged nylon membrane (Nitran) by capillary spot analysis overnight in 10X SSC buffer (Southern blot). A radioactive probe from the first 450 bp of the genomic clone SAR5 (up to the BglII site) was prepared using a Prime-lt kit (Stratagene) and the filter was incubated overnight in a hybridization solution at 60 degrees centigrade. The filter was subsequently washed to remove the binding of non-specific probes, with the final wash being 0.25X SSC, 0.1% SDS at 60 degrees centigrade. The results after exposure of X-ray film indicated that SAR5 is a single copy gene in F. vesca. The genomic DNA of F. vesca was digested with BglII in combination with EcoRI, PstI, Xhol or Spel, followed by analysis of Southern blots and hybridization as described above. From the size of the hybridization bands in each line, a restriction map of the SAR5 promoter region (extending down to the site of internal BglII) was generated. The restriction map indicated that a promoter fragment of approximately 6kb (PstI), 3.6kg (Spel), 2kb (Xhol) or 0.5kb (EcoRI) could be obtained by cutting with the appropriate enzyme in combination with BglII. Genomic DNA from F. vesca was cut with Xhol and BglII, fractionated on a 0.7% agarose gel and the gel region around 2.5 kb (promoter region of 2kb + 450pb coding) was excised with a razor blade . The DNA of the gel piece was purified using a Prep-A-Gene kit and ligated to pBluescript SK DNA digested with XhoI and BamHI. The binding mixture was transformed into competent E. coli cells that were plated in selection medium. The ampicillin-resistant colonies were patched in a grid format to new plates and then used in nitrocellulose filters for in situ hybridization (Sanmbrook et al., Molecular Cloning). A probe corresponding to the first 450 bp of the SAR5 genomic clone was prepared using ECL Marking and Detection equipment and used to screen the filters according to the manufacturer's instructions (Amersham). After the X-ray film, a positive colony was identified. This colony was inoculated in liquid medium to be grown overnight and the plasmid DNA was prepared using a Qiaprep Spin Miniprep (Qiagen). The plasmid DNA was analyzed by restriction enzyme analysis and then sequenced using an ABI automatic sequencer. The clone was verified to contain the 5 'flanking sequence (the SAR5 promoter) and a portion of the SAR5 coding region (up to the internal BglII site). The clone was designated pGSARd-16. The restriction site BamHl was introduced upstream of the initiation codon SAR5 (ATG) by PCR amplification of the previous promoter clone with the primers pSAR5-BH1 (5'-TAGGATCCGTCTTTGCTCTGAACTC-3 ', SEQ ID NO: 5) and pSAR5-R4 (5'-ACCTTGTACCTAAGAAAGCC-3 \ SEQ ID NO: 6). The PCR product was purified from an agarose gel plate using a Prep-A-Gene kit, digested with Xbal and BamHl and cloned into the Xbal and BamHl sites of pBluescript SK, yielding the plasmid pSAR5RCP / pSK (also referred to as the truncated promoter or TSAR5). To facilitate other cloning experiments, the TSAR5 promoter is subcloned as an Xbal fragment for EcoRI at the Xbal sites for EcoRI, creating the plasmid pCGN8046. The full-length SAR5 promoter, designated as FSR or PCGN8047 (SEQ ID NO: 1), was recreated by cloning the region upstream of the original promoter as a Xbal to XbaI fragment at the SalI to Xbal sites of pCGN8046. Plasmids containing the full-length promoter reconstructed with a BamHI site introduced just upstream of the initiation codon SAR5 were identified by restriction enzyme analysis. B. Isolation of Promoter RJ39 Originally the RJ39 clone of cDNA induced for strawberry ripening isolated by Wilkinson was isolated. Lanahan, Conner and Klee (Plant Molecular Biology 27: 1097-1108, 1995) using a polymerase chain reaction differential display technique to compare mRNA populations of white berries against completely red berries. Northern blot analysis revealed that the RJ39 mRNA is strongly enhanced for fruit or fruit-specific, with expression levels growing as the fruit develops from the small green to fully red stage. Little or no expression was detected on the leaf, petiole or root tissues. The cDNA clone that was isolated did not have the full length of 5 ~ 1 bp against almost 850 bp of transcription in Northern blot analysis) and the translation product of this cDNA did not show significant homology for any known proteins. A genomic clone containing the RJ39 coding region and the 5 'flanking sequences (promoter) was obtained in a manner similar to that described for the SAR5 promoter. Strawberry genomic DNA was prepared from Fragaria vesca (2n = 14) as described in Example 1A. An aliquot of DNA was digested with the Xbal restriction enzyme and then the DNA fragments were separated on a 0.7% agarose gel. The digested fractionated DNA fragments were transferred to a positively charged nylon membrane (Nitran) by capillary spot analysis overnight in 10X SSC buffer (Southern blotting). A radioactive probe was prepared from the first 400 bp of the RJ39 cDNA clone (up to the Xbal site) using a Prime-It (Stratagene) kit and the filtrate was incubated overnight in a hybridization solution at 60 degrees centigrade. The filter was subsequently washed to remove (non-specific probe binding), with the final wash being 0.25X SSC, 0.1% SDS at 60 degrees Celsius. The results after exposure of X-ray film indicated that RJ39 is a single copy gene in F. vesca. The aliquots of F. vesca genomic DNA were digested with Xbal in combination with EcoRI, PstI, Xhol, Spel, BglII. Hindül, EcoRV Sphl or Salí followed by analysis of Southern blots and hybridization as described above. From the site of the hybridization bands in each line, it was determined that the only restriction site within the Xbal site of 5 'flanking was EcoRI (band of approximately 1.1 kb). The other restriction enzymes tested gave the band the same size (approximately 1.9kb) as an F. vesca genomic DNA cut with Xbal alone. In order to obtain a functional promoter fragment, a genomic clone of approximately 1.8-2kb should be obtained, which contains the entire RJ39 coding region and almost 900pb of the promoter sequence. The genomic DNA of F. vesca was cut with Xbal, fractionated on a 0.7% agarose gel, and the gel region between 1.6-2kb was excised with a shaving blade. The DNA of the gel piece was purified using a Prep-A-Gene kit and ligated to the SK DNA of pBluescript treated with alkaline phosphatase, digested with Spel. The binding mixture was transformed into competent E. coli cells which were then plated on selection media containing IPTG and X-GAL. While. the ampicillin-resistant colonies were fished in a grid format to new plates and then used in nitrocellulose filters for in situ hybridization (Sambrook et al., Molecular Cloning). A probe corresponding to the first 400 bp of the RJ39 cDNA clone was prepared using a Prime-lt kit (Stratagene) and used to screen the filters in a manner similar to that used for Southern blot analysis. The hybridization temperature was 65 degrees centigrade and the final wash was 0.15X SSC, 0.1% SDS at 65 degrees Celsius. After exposure of the X-ray film, several positive colonies were identified. These colonies were inoculated in liquid medium for overnight development and the plasmid DNA was prepared using a Qiaprep Spin Miniprep (Qiagen) kit. Plasmid DNA was analyzed by restriction enzyme analysis and two clones were selected. The sequence analysis of the two clones showed that both clones contained 5 'flanking sequence (the RJ39 promoter) and the complete RJ39 coding region (downstream of the Xbal site in the 3' flanking sequence). One clone, designated pRJ39C # N-91, was selected for additional cloning. The BglII restriction site was introduced upstream of the RJ39 initiation codon (ATG) by PCR amplification (with Pfu polymerase) from pRJ39 # N-91 with the primers RJ39C-3N1 (5'-AACTGCAGATCTAGTGTGGCAGTAGGTCTG-3 ', SEQ ID NO: 7) and the promoter of the T7 promoter (5'-TAATACGACTCACTATAGGG-3 \ SEQ ID NO: 8). The PCR product was purified from an agarose gel plate using a Prep-A-Gene kit, digested with BamHl and ligated to pUC119 digested with Smal and BamHl creating the plasmid pCGN8051. Example 2 Preparation of Plant Expression Constructs The expression construct of pCGN8014 was used as a cloning vector for plant transformation. The vector is a derivative of the vector pMON18354 which is described in the patent application of E.U.A. provisional 60/036 131 and the regular request that claims priority, in the application number presented on January 20, 1998, the description of which is incorporated herein by reference. PMON18354 was modified by transposing the fragment containing the nopaline synthase promoter (nos 5 '), the neomycin phosphotransferase kanamycin resistance gene (nptll) and the nos (nos 3') termination sequences between the right border and the SRE49 promoter to the 3 'position between the reporter gene ß-glucuronidase (GUS), elements us 3 'and the sequence on the right bank. The modification gave the vector pCGN8014. Another binary vector for transformation of plants pCGN5928, was constructed using the kanamycin phosphotransferase kanamycin resistance gene (nptll) driven by the nopaline synthase transcriptional initiation region (nos 5 ') and the transcription termination sequences (nos 3') ) (Fraley et al., Proc. Natl. Acad. Sci. (1983) 80: 4803-4807 and Depicker et al., J. Molec. Appl. Genet. (1982) 1: 562-573). Both nos 5 'and nos 3' were amplified by PCR from the C58 strains of Agrobacterium tumafaciens and ligated with the nptl1 gene of pCGN783 (Houck, et al., Frontiers Appl Microbiol (1988) 4) as a fragment of EcoR I for form pCGN5908. The 5'-nptII-nos3 'fragment was then cloned into PCGN1541, containing 3232. right bank (0.5Kb), lacZ, left bank (0.58Kb), as a Xho I fragment between the sequences of the right bank lacZ and the left bank to create the intermediate pCGN5910. The origins of ColEÍ and pRi of replication as well as the gene of resistance to Gentamicin were acquired from a deleted Not I derivative of pCGN1532 (MacBride and Summerfeit, Plant Molecular Biology, (1990), 14: 269-276) as a fragment of BamH I to create pCGN5924. Finally, a linker containing unique restriction sites was synthesized and cloned into the PCGN5924 sites of Asp 718 / Hind III (within the lacZ sequence) to create the binary vector pCGN5928. The plant transformation vectors were constructed to test the tissue resistance and specificity of the TSAR5 and FSR promoters in transgenic plants using the GUS report gene as a marker. The vector pCGN8045 was created by cloning the FSR promoter of pCGN8047 as a fragment of HindIII to BamH1 at the HindIII to BglII sites of pCGN8014. The order of the genetic elements in T-DNA are: RB-pFSR-GUS-nos3'-nos5'-nptII-nos3'-LB. the pCGN8054 vector was cloned into the TSAR5 promoter part of pCGN8046 as a fragment from Xbal to BamHl at the Xbal to BglII sites of pCGN8014. The order of the genetic elements in the T-DNA are: RB-pTSAR5-GUS-nos3'-nos5'-nptII-nos3'-LB. These vectors were transformed into an appropriate Agrobacterium strain and can be used to generate transgenic strawberry plants for the evaluation of the promoters. A binary vector was constructed to test the tissue resistance and specificity of the RJ39 promoter in transgenic plants using the beta-glucuronidase (GUS) reporter gene as a marker. The pCGN8052 vector was created by cloning the RJ39 promoter of pCGN8051 as a fragment from Xbal to BglII at the Xbal to BglII sites of pCGN8014. The order of the genetic elements of the AND-T are: RB-pRJ39C-GUS-nos3'-nos5'-nptII-nos3'-LB. (The RJ39 promoter is also referred to as RJ39C). This vector was transformed into an appropriate Agrobacterium strain and used to generate transgenic strawberry plants for the evaluation of the promoter. Two additional DNA fusion constructs were prepared, one containing the full length SAR5 promoter controlling glgC16 and one containing the truncated SAR5 promoter controlling glgC16. The constructions were prepared in the following manner. The plant transformation vector pCGN8040 contains glgC16 under the control of the truncated promoter SAR5. The truncated promoter SAR5 (TSAR5) was cloned as a fragment of PstI-BamHI at the PstI and BglII sites of pCGN8222 creating the plasmid pCGN8039. The TSAR5 promoter replaces the TFM7 promoter in pCGN8222. pCGN8222 was created by cloning the glgC16 coding sequence of pMON18345 (described in U.S. Patent 5,608,150) as a BglII-SacI fragment in pMON18337 (described in the provisional US patent application 60/036 131 and the regular application claiming priority application number of the same filed on January 20, 1998). The fragment TSAR5-glgC16-nos3 'was cloned from pCGN8039 as a fragment of PstI-Notl in the binary vector pCGN5928, Ssel-Notl sites, to create the plant expression vector pCGN8040. The plant transformation vector pCGN8042 also contains glgC16, but is under the control of the full-length SAR5 promoter. The full-length SAR5 promoter (FSR) was cloned from pCGN8047 as a PstI-BamHI fragment in pCGN8222, PstI-BglII sites, to form the plasmid pCGN8041. The fSAR5-glgC16-nos 3 'fragment was cloned as a PstI-NotI fragment at the Ssel-Notl restriction sites DE pCGN5928 to give the plant expression vector pCGN8042. The plant transformation vectors were transformed into strain LBA4404 of Agrobacterium tumefaciens by the method of Holsters et al., Molecular and General Genetics (1979) 163: 181-187. Example 3 Transformation of Plants Transgenic strawberry plants can be obtained using the methods of Nehra, N. S. and others. (Plant Cell Rep. (1990) 9: 10-13 and Plant Cell Rep. (1990), 9.293-298), Matthews, H.V. and others, (In Vitro Cell, Dev. Biol. (1995) 31.36-43 and WO 95/35388, Dec. 28, 1995) or as described in the patent application of E.U.A. , filed on January 19, 1998. The plant transformation vector pCGN8045 was transformed into strawberry plants using the following method. Strawberry Micropropagation Transformation Protocol A Plant Material In vitro BHN FL90031-30 or BHN 92664-501 strains (adapted with CA) were grown in vitro from strawberry cultures in pre-sterilized Magenta GA7 boxes (Magénta Có., Chicago, IL ) containing micropropagation medium (Table 3). Each unit of tissue contains two to three apical meristems in two units that are placed in each bottle. The cultures were then incubated at approximately 22 ° C, with a cold white light with photoperiod from 16/8 to approximately 34-40 mEinsteins m-2 sec-1. Approximately every four weeks, each tissue unit was subdivided from two to four sets and placed in fresh medium of the same composition. The ideal mother material for the explant is available in two or three weeks after the last subculture. Table 1: Selection medium A Component Concentration Sales MS / vitamins MS (Sigma M0404) 4.4 g / L Glucose 20 g / L Wash Agar 8 g / L Thiadiazurone 2.3 mg / L Indoleaketic acid 1.75 mg / L Timetine 500 mg / L Cefotaxime 100 mg / L Canamycin 50 mg / L pH adjusted to 5.7 Table 2: Elongation Medium A Component Concentration MS Salts / vitamins MS (Sigma M0404) 4.4 g / L Glucose 20 g / L Agar Wash 8 g / L Timetine 500 mg / L Cefotaxime 100 mg / L Indoleaketic acid 0.45 mg / L Galacturonic acid 2.5 mg / L Canamycin 50 mg / L pH adjusted to 5.7 Table 3: Micropropagation medium Component MS Sales Concentration (Sigma 0153) 2.2 g / L MS vitamins (Sigma M3900) 1 mL / L MgSO4.7 H20 0.2797 g / L CaCl2.7 H20 0.2739 g / L KH2P04 0.5950 g / L H3B03 18.6 mg / L NaMo04.2 H20 0.7 mg / L Iron stock solution 5 mL / L Mio-inositiol 100 mg / L Ascorbic acid 100 mg / L N6-benzylaminopurine 1 mg / L Indolebutyric acid 0.37 mg / L Sucrose 30 g / L Agar Wash 8 g / L pH adjusted to 5.8 Preparation of Agrobacterium Four days before co-culture, the LBA4404 strain of Agrobacterium tumefaciens containing plant expression vectors was changed from a frozen AB glycerol mother plate (AB medium supplemented with 15 g Difco Bacto Agar, Table 4) containing 150 g / L of streptomycin, 100 mg / L of gentamicin and 100 mg / L of kanamycin. Twenty-four hours before co-culture, the single colonies were placed in 5 mL of MG / L medium (Table 5). The cultures were grown overnight at 30 ° C, shaking at 200 rpm. Table 4: Medium AB Component Quantity 20X Solution AB Mother [120 g / L K2HP04, 46 g / L NaH2P04.H20, 40 g / L NH4CI, 6 g / L KCl] 50 mL MgSO4 1M 1 mL CaCl2 0.1 M 1 mL 20 % Glucose (w / v) 25 mL FeS04.7 H20 (0.25 mg / mL) 10 mL Table 5: MG / L Medium Component Concentration Mannitol 5 g / L L-glutamic acid 1 g / L KH2P04 0.25 g / L NaCl 0.10 g / L MgSO4.7 H20 0.10 g / L Biotin 1 mg / L Tryptone 5 g / L Yeast Extract 2.5 g / L pH adjusted to 7.0 Inoculation of explants. Explant steps and pre-culture The small folded leaves about 2-4 mm in length that have a vitreous, vibrant green appearance were excised at the petiole. They were placed in a petri dish containing approximately 1-1.5 mL of sterile water and a sterilized WHATMAN filter paper. The basal portion of the leaves was removed with a single cut so that 3 leaflets of each leaf were produced. Leaflets (explants) were placed in preculture plates (Table 6). The preculture plates were prepared using a solid medium and 1 mL of TXD liquid medium was pipetted which was supplemented with 200 mM acetosyringone and 100 mM galacturonic acid in the solid plate. Two sterilized WHATMAN filter papers were placed on the plate. Approximately 50 explants were placed in each preculture plate. The plates were placed under low light conditions for approximately three days by placing them in a box covered with aluminum foil. Table 6: Preculture medium / co-culture with top layer Component Concentration Sales MS / vitamins MS (Sigma M0404) 4.4 g / L Glucose 30 g / L Wash Agar 8 g / L Tiadazurone 2.2 mg / L Indoleaketic Acid 1.75 mg / L Acetosyringone 39.28 mg / L Galacturonic acid (100 mM) 4 mL pH adjusted to 5.7 The top layer is 1 mL / TXD liquid medium plate containing 200 mM acetosyringone, 100 mM galacturonic acid, and 2 sterile 8.5 WHATMAN filter papers.

Inoculation and co-culture steps The Agrobacterium suspension was diluted to 5x108 bacteria / mL with MG / L medium just immediately before use. The explants were removed from the pre-culture plate and allowed to settle in 5 mL of bacterial suspension for 5 minutes. The explants were then removed from the bacterial suspension and analyzed dry on sterile paper towels and placed back on the pre-culture plate. The explants spread uniformly to the downstream adaxial side so that there is good contact with the filter paper and does not overlap. These plates were then co-cultivated under low light conditions for an additional 3 days. Selection and regeneration of tissue. The explants moved to retard the medium (Table 7) for 3 days, towards the adaxial descending side. The explants were stored under low light conditions during the delay period. Table 7: Delay Medium Component Concentration MS Salts / vitamins MS (Sigma M0404) 4.4 g / L Glucose 20 g / L Agar Washing 8 g / L Tiadazurone 2.3 mg / L Indolaacetic acid 1.75 mg / L Timentin 500 mg / L Cefotaxime 100 mg / L pH adjusted to 5.7 Table 8: Root formation medium A Component Concentration Sales MS (Sigma 0153) 2.2 g / L MS vitamins (Sigma M3900) 1 mL / L MgSO4.7H2O 0.2797 g / L CaCl2.2 H2O 0.2739 g / L KH2PO4 0.5950 g / L H3BO3 18.6 mg / L NaMoO4.2H20 0.7 mg / L Iron Mother Solution 5 mL / L Mio-inositiol 100 mg / L Ascorbic Acid 100 mg / L Indolebutyric Acid 0.37 mg / L Timetine 500 mg / L Cefotaxime 100 mg / L Glucose 20 mg / L Wash Agar 8 g / L pH adjusted to 5.7 After a delay of three days, the explants (approximately 50 per plate) were transferred to the adaxial downstream side on selection medium A (Table 1) and cultured for approximately 3 weeks in the light (20-40 mEínsteins m-2 sec-1). After about 3 weeks, the explants were placed in selection medium B (Table 9). The subcultures were carried out poop 3 weeks. For 6 weeks, the transformed explants will produce green shoots and green calluses. Only the explants that contain this bud material and green calluses could be moved. If the explants associated with the buds and green calluses are still green and healthy, then you should move the entire explant with the regeneration material. For 9 to 12 weeks, actively growing green shoot units can be selected from the explants and placed on their own in selection medium B (Table 9). Each unit actively in division represents an independent event. Once the unit has tripled in size, the individual shoots can be placed in the middle of elongation (Table 6). This step can take three to six weeks. Buds are formed in the root formation medium (Table 8). This step requires approximately two to three weeks. Table 9: Selection Medium B Component Concentration MS Salts / vitamins MS (Sigma M0404) 4.4 g / L Glucose 30 g / L Washer Agar 8 g / L Tiadazurone 3.4 mg / L Indoleaketic Acid 0.45 mg / L Timet? A 500 mg / L Cefotaxime 100 mg / L Canamycin 50 mg / L pH adjusted to 5.7 The shoots are sown in 6-pack containers of Sunshina # 1 mixture (80% peat). The containers were placed in a humidity tent in trays with dome covers for 3 days. Subsequently, the dome covers are tilted in half to allow air flow for 3 more days. The dome lid was removed after 6 days and the plants remained under moisture activity for an additional 10 to 15 days. The plants were moistened until they were removed from the humidity tent. The plates were then removed and placed on a shelf for 7 days and transplanted into 15.24 cm containers of 25% each: peat, sand, pumice stone and red wood straw. Temperatures on greenhouse day vary from about 20-24.5 ° C and night temperatures are about 10-14.5 ° C. There is no artificial light and the intensity of light decreases from the end of May to the end of September using a shade fabric. Example 4 Determination of Gene Expression Plants transformed with promoter-GUS fusions can be screened for gene expression using methods well known in the art. Expression expression applied to auxin exogenously can also be examined using methods well known in the art. The expression of the GUS enzyme could be expected to be similar to the expression of mRNA of SAR5 and RJ39.

One method involves the GUS strain of plant tissue and the tinsion pattern examination. The tissue of plants and fruits of plants transformed with promoter-GUS constructions can be harvested at different stages of development and stained for β-glucuronidase activity. The fruit tissue can be selected and stained by infiltrating the tissue with GUS tinsion pH buffer (50 mM Potassium Phosphate (pH 7) 1 mg / ml X-Gluc (5-bromo-4-Chloro-3-indole). β-D-glucuronide) and 0.1% of Trition X-100). The tissue is left to stain in the pH regulating solution of tinsión overnight at 37 ° C. The tissue is then flushed using 70% ethanol washes for 1 hour, then 100% ethanol. The tissue can be examined visually for tinsion. In order to quantify expression, assays can also be used to determine GUS enzyme activity (Jefferson, R.A., Plant Mol. Biol. Repórter (1987) 5: 387-405). The total protein was extracted from the tissue of plants using GUS extraction pH buffer (50 mM Sodium Phosphate, pH 7.0, 10 mM β-mercaptoethanol, 10 mM EDTA, 0.1% Lauryl Sodium Sarcosine and 0.1% Triton X-100). The samples were centrifuged and the supernatant containing the protein was transferred to a new tube. Analyzes were carried out in pH buffer of analysis (1 mM MUG (4-methyl umbelliferil ß-D-glucurinide in pH regulation of GUS extraction). Fluorescence was measured using a fluorometer and relative expression can determine.

In addition, expression of genes at the transcription level can be examined using Northern hybridizations. Total or po! I (A) + RNA can be isolated from fruit tissues, as well as other tissues at different stages of development. The RNA is then separated on a denaturing agarose gel and transferred to the nylon membrane (Sambrook et al., 1989). Hybridizations can then be carried out using a labeled probe and the hybridized membrane is then exposed to radiographic film. The expression of mRNA transcription can be determined by observing the hybridization pattern of the membrane. The above examples provide the SAR5 and RJ39 promoters that can be used to provide selective tissue expression of growing or shrinking fruit receptacles of heterologous genes during the development and maturation and complete fruit development. Said expression pattern is particularly convenient for the expression of genes for traits such as increased sugar content and disease resistance. All publications and patent applications mentioned in this specification indicate the level of skills of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same degree as if each publication or patent application is individual and specifically and individually indicated to be incorporated by reference.

Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (1)

1. CLAIMS 1 A DNA construct comprising a transcription factor operably linked to a heterologous DNA coding sequence of interest, wherein the trans-focal factor selectively directs tissue expression in receptacles and where the expression of the sequence of receptors increases during the maturation of the fruit 2 The construction according to claim 1, wherein the transcppcionai factor is the promoter sequence of RJ39 3 The construct according to claim 2, wherein the transcription factor is at least 90% identical to SEQ ID NO 2 4 The construction according to claim 2, wherein the transcription factor is capable of hybridizing under strict conditions with the DNA sequence indicated in SEQ ID NO 2. The construction according to claim 2, wherein the transcription factor is encoded by the DNA sequence in SEQ ID NO 2 6 A DNA construct that com the transcourse factor is operably linked to a coding sequence of DNA heterogeneous of interest, where the transcription factor is selectively directed to expression in receptacle tissues and where the expression of the sequence of interest decreases during development and maturation 7. The construct according to claim 6, wherein the transcription factor is the promoter sequence of SAR5. 8. The construction according to Claim 6, wherein the transcription factor is at least 90% identical to SEQ. ID NO: 1. 9. The construct according to claim 6, wherein the transcription factor is capable of hybridizing under stringent conditions to the DNA sequence indicated in SEQ ID NO: 1. 10. The construct according to claim 6, wherein the transcription factor is encoded by the DNA sequence in SEQ ID NO: 1. 11. The construct according to claim 6, wherein the transcription factor is repressed in response to endogenous or exogenous auxins. The construction according to claim 1 or claim 6, comprising the 5'-3 'transcription factor transcription direction, the DNA sequence of interest and further comprising a functional transcriptional termination region in plants. 13. The construct according to claim 12, wherein the DNA sequence of interest is an open reading frame that encodes an amino acid sequence. 14. The construct according to claim 12, wherein the DNA sequence of interest is complementary to endogenous mRNA to a plant cell. 15. The construct according to claim 1 or claim 6, wherein the DNA sequence of interest is selected from the group consisting of sucrose phosphate synthase ADP glucose pyrophosphorylase, invertase, glucose-6-phosphatase or sucrose synthase. 16. The construct according to claim 1 or claim 6, wherein the DNA sequence of interest is a sequence encoding a gene for resistance against a plant pathogen. 17. A method for modifying the route phenotype in a transgenic plant, the method comprising the steps of developing a transgenic plant for producing fruit tissue, wherein the tissue cells of fruits comprise in its genome one or more DNA constructs of according to claim 1 and claim 6. 18. A method according to claim 17, comprising a construction according to claim 1 and also comprising a construction according to claim 6. 19. A method according to the claim 18, wherein the DNA sequence of construction interest according to claim 1 comprising sucrose phosphate synthase and wherein the DNA sequence of interest of the construct according to claim 6 comprising glucose ADP of pyrophosphorylase. 20. A transgenic plant produced by the method of claim 17. 21. A transgenic plant according to claim 20, wherein the plant is a strawberry plant.