ES2339094B1 - PROCEDURE FOR THE PRODUCTION OF TRANSGENIC PLANTS THAT PRESENT HIGH CONTENT IN ANTIOXIDANT COMPOUNDS, HIGH ANTIOXIDANT CAPACITY AND RESISTANCE TO EMPARDECIMIENTO. - Google Patents

Abstract

Procedure for the production of plants transgenic that have a high content of compounds antioxidants, high antioxidant capacity and resistance to blistering The present invention protects the process for the production of transgenic plants, as well as the plants themselves, that are high in antioxidant compounds (such as polyphenols and carotenoids), high antioxidant capacity and resistance to browning, by increasing the activity UDP-glucose: d-fructose-2-glucosyltransferase (UGFT) producer of UDPGlucose in plants. In this invention it remains demonstrated that UGFT is involved in the production of an important pool of UDPGlucose necessary for the production of forms glycosylated polyphenols and carotenoids, highly stable and with high antioxidant capacity

Description

Procedure for the production of plants transgenic that have a high content of compounds antioxidants, high antioxidant capacity and resistance to blistering

Field of the Invention

The present invention relates to a procedure for the production of transgenic plants that have a high content of antioxidant compounds, high capacity antioxidant and resistance to browning, as well as own transgenic plants. Therefore, the present invention can be included in the field of genetic engineering, the plant physiology, food technology and nutraceutical

State of the art

The UDP-glucose: D-fructose-2-glucosyl Transferase (UGFT) plays a fundamental role in both the rate of Nutrient utilization as in sucrose conversion in starch and cell wall polysaccharides (1,2) (Figure 1). UGFT is highly regulated and catalyzes the production of UDPGlucose and fructose from sucrose and UDP (3-6).

UDPGlucose is the glucose donor molecule for transglycosylation reactions involved in the biosynthesis of glycolipids, glycoproteins and cell wall polysaccharides such as cellulose and callose (1,7-9). In addition, tests carried out in vitro have shown that UDPGlucose acts as a glucose donor for glycosylation reactions of antioxidant substances such as polyphenols and carotenoids. These reactions are determinants for the stability, conjugation with other molecules and prevention of the oxidation of the polyphenols that give rise to the browning effect of the vegetables (10). Polyphenols and carotenoids are responsible for the coloring of flowers, fruits and senescent leaves, and also protect the plant against microbial agents and against cell wall degrading enzymes (11). In addition, these compounds have antioxidant and anti-inflammatory activity and are known for their properties against cancer and the accumulation of blood cholesterol (12-15). Organs such as potato tubers are characterized by accumulating large amounts of polyphenols and carotenoids (16-18), and constitute an important source of antioxidant substances in the diets of numerous societies (19).

The glycosylation reactions of polyphenols from UDPGlucose take place in the cytosol (20). Although UGFT is an important source of cytosolic UDPGlucose and that it is important in the production and stability of antioxidant substances in vitro , to date there are no studies describing that the increase in UGFT activity producing UDPGlucosa is involved in the production and accumulation of antioxidant substances in vivo . There is also no experimental evidence or scientific work that suggests the implication of the UGFT activity producing UDPGlucose, in the production of antioxidant substances in the cell. There are also no works in which the intracellular levels of UDPGlucose are altered as a strategy to alter the levels of antioxidant substances in vivo . In this sense, the present invention describes for the first time the production of plants with (a) high content in UDPGlucose, (b) high content in polyphenols and carotenoids, (c) high antioxidant capacity and (d) low browning effect (browning effect) as a consequence of the UGFT activity producing UDPGlucose. With all this it is demonstrated that, as illustrated in Figure 1, UGFT is involved in the production of UDPGlucose necessary for the production of highly stable glycosylated polyphenols and carotenoids with high antioxidant capacity.

Description of the invention Brief Description of the Invention

The present invention relates to a procedure for the production of transgenic plants that have a high content of antioxidant compounds, high capacity antioxidant and resistance to browning, through increase in UGFT activity producing UDPGlucose in plants. In addition the present invention relates to the own ones transgenic plants characterized by said properties.

The technical effects shown in the present invention are extrapolated to any type of plant organ such as: tubers, leaves, fruits and seeds, as well as to any type of plant such as: potato, tobacco, tomato, rice, barley, wheat and corn. The results shown in the present invention were achieved, both constitutively expressing UGFT under the control of the 35S promoter, and expressing UGFT under the control of a specific tuber promoter used (promoter of the patatin gene). It should be said that constitutive expression was particularly preferred. The results shown in the present invention were achieved after over-expressing any isoform and UGFT sequence (the UGFTX variant of the potato UGFT4 isoform being particularly preferred). That is, any plant-expressible promoter that produces UGFT overexpression, as well as any-
Any isoform of UGFT with UDPGlucose producing activity would be comprised in the present invention.

For the purposes of the present invention, they are made include the following terms:

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Transgenic plant: plant whose genome has been modified by genetic engineering in order to achieve different and / or improved biological characteristics compared to those of the wild plant (WT).

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Transformed plant cell : they are plant cells that have a genetic alteration resulting from the introduction and expression of external genetic material in their genome.

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Antioxidant compounds : These are substances capable of retarding or preventing the oxidation of other molecules. They are responsible for capturing free radicals produced in the oxidation reactions of the metabolism. These compounds are widely used in the treatment of cerebrovascular and neurodegenerative diseases, as well as dietary supplements, food preservatives, cosmetics and for the prevention of degradation of rubber and gasoline. In the present invention the antioxidant compounds are selected from polyphenols and carotenoids.

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Polyphenols : they are chemical substances present in plants and characterized by the presence of more than one phenol group (-OH group attached to an aromatic ring) per molecule. The presence of these phenolic rings gives it the ability to act as antioxidants since they can capture oxygen radicals.

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Carotenoids : they are organic pigments that are found naturally in plants and other photosynthetic organisms. Most carotenoids are tetraterpenoids, composed of 40 carbon atoms formed by eight isoprenoid units and contain rings at one or both ends of the molecule. They can function as antioxidants, protecting against self-oxidation.

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Anthocyanins : are organic polyphenolic pigments that provide coloration to many plants and especially many flowers. Its chemical structure consists of a flavil group formed by a benzopyran ring attached to a phenolic ring. They have antioxidant properties preventing the production of free radicals.

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Overexpression of the UGFT enzyme producing UDPGlucose : Overexpression of the UGFT enzyme producing UDPGlucose is understood when the activity of said enzyme is significantly superior to that in wild plants or controls (WT) grown under the same conditions and in the same moment. By way of illustration, Figure 3 shows that the UGFT activity producing UDPGlucose in the tubers of the control potato plants (WT) is 590-710 milli units (mU) / g (fresh weight), while the UGFT activity Producer of UDPGlucose in potato plant tubers constitutively expressing UGFT is 730-1300 mU / g fresh weight. Therefore, the present invention relates to the over-expression of the UGFT enzyme, by way of example, when the production of UDPGlucose in the tubers of potato plants is greater than 710 mU / g (fresh weight) (Figure 3).

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High content in UDPGlucose : as used in the present invention, this expression is directly referred to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. By way of illustration, Figure 4 shows that the average content in UDPGlucose in the tubers of the control potato plants is 95-105 nmol / g (fresh weight), while the tubers of transgenic potato plants expressing UGFT constitutively accumulate 135-160 nmol / g (fresh weight) of UDPGlucose. Therefore, the present invention relates to "high content in UDPGlucose", by way of example, when it is greater than 105 nmol / g (fresh weight) (Figure 4).

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High content in polyphenols, anthocyanins and carotenoids : as used in the present invention, this expression is directly referred to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. Thus, the present invention relates to "high content of total polyphenols", by way of illustration, when this is greater than 0.19 mg GAE / g (fresh weight) in the fleshy part of the tubers of transgenic potato plants expressing UGFT constitutively and greater than 0.52 mg GAE / g (fresh weight) in the skin of the tubers of transgenic potato plants that express constitutively UGFT (Figure 5 and 6). Similarly, the present invention relates to "high anthocyanin content", by way of illustration, when it is greater than 4.0 mg / g (fresh weight) in the skin of transgenic potato plant tubers constitutively expressing UGFT (Figure 7 ). On the other hand, the present invention refers to "high carotenoid content" when this is, by way of illustration, of 1.0 µg / g fresh weight in tubers of transgenic potato plants constitutively expressing UGFT (Figure 8).

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High antioxidant capacity : as used in the present invention, this expression is directly referred to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time. Thus, the present invention relates to "high antioxidant capacity", by way of illustration, when it is greater than 6.8 µm Trolox eq / g fresh weight in the fleshy part of the transgenic potato plant tubers constitutively expressing UGFT (Figure 9) and greater than 12.4 µm Trolox eq / g fresh skin weight of transgenic potato plant tubers expressing constitutively UGFT (Figure 10).

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Description of the figures

Figure 1. Proposed conversion mechanisms for sucrose in starch and cell wall polysaccharides in plants.

Figure 2. Construction stages of the plasmid of pBIN35S-UGFT-NOS expression starting of pBIN20 and p35S-UGFT-NOS.

Figure 3. UGFT activity in tubers of wild or control (WT) potato plants, in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of potato plants constitutively expressing UGFT after integrating the 35S-UGFT-NOS construct into its genome after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851. The activity is referred to in milliunits (mU) per gram of fresh weight (y axis). The unit is defined as the amount of UGFT needed to produce a micromol of UDPGlucose per minute.

Figure 4. Content in UDPGlucose (nmol / g fresh weight) in tubers of wild or control potato plants (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of plants constitutively expressing UGFT after integrating the 35S-UGFT-NOS construct into its genome after being transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 5. Content of total polyphenols (in mg equivalent of gallic acid (GAE) / g fresh weight) in the fleshy part of tubers of wild or control potato plants (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in potato plant tubers constitutively expressing UGFT after integrating into its genome the 35S-UGFT-NOS construct after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851.

Figure 6. Content of total polyphenols (in mg GAE / g fresh weight) in skin of tubers of wild or control potato plants (WT) and in the skin of tubers of potato plants constitutively expressing UGFT after integrating into its genome the 35S-UGFT-NOS construct after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851.

Figure 7. Total anthocyanin content (in mg / g fresh weight) in skin of tubers of wild or control potato plants (WT) and in the skin of tubers potato plants constitutively expressing UGFT after integrating the 35S construction into its genome -UGFT-NOS after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851.

Figure 8. Total carotenoid content (in? Cups equivalent of beta-carotene (? EC) / g \ fresh weight) in tubers of wild or control (WT) potato plants and potato plants constitutively expressing UGFT after integrating into its Genome construction 35S-UGFT-NOS (by action of the strain of Agrobacterium tumefaciens CECT: 5851).

Figure 9. Antioxidant capacity (referred to? Equivalent of Trolox / g fresh weight) in the fleshy part of tubers of wild or control potato plants (WT) and in the fleshy part of tubers of potato plants constitutively expressing UGFT after integrate the 35S-UGFT-NOS construct into its genome after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851.

Figure 10. Antioxidant capacity (based on equivalent µm of Trolox / g fresh weight) in the skin of tubers of wild or control potato plants (WT) and in the skin of tubers of potato plants constitutively expressing UGFT after integrating into its genome the 35S-UGFT-NOS construct after being transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.

Figure 11. Polyphenol oxidase activity (in Units / g fresh weight) (A) in the fleshy part and (B) in the skin of tubers of wild or control potato plants (WT) and in the fleshy part and in the skin of potato plant tubers constitutively expressing UGFT after integrating the 35S-UGFT-NOS construct into its genome after being transformed with the Agrobacterium tumefaciens CECT expression vector: 5851.

Figure 12. Bold effect on tubers of wild potato plants or controls (WT) and in potato plant tubers constitutively expressing UGFT after mechanical cutting and exposure to air for 28 hours.

Detailed description of the invention Obtaining transgenic potato plants that over-express UGFT

In the present invention UGFT has been expressed constitutively by the action of the constitutive promoter 35S of the tobacco mosaic virus. For this, the 35S-UGFT-NOS construction was introduced into the potato genome ( Solanum tuberosum ), the design of which is detailed below.

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Known nucleotide sequence encoding for the UGFT4 isoform of wild potato UGFT (22) were created two specific primers corresponding to the 5 'and 3' ends of the gene, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Making use of these primers was amplified by conventional methods of PCR a 2418 pair DNA fragment of bases, designated with UGFTX, from a sheet cDNA library Potato Such a PCR fragment was introduced into plasmid pSK Bluescript (Stratagene) giving rise to the pUGFT construction, which It was amplified in the host bacteria XL1 Blue. Sequence UGFTX nucleotide is SEQ ID NO: 3 which is slightly different than UGFT4 (access number in GenBank U24087).

The sequential introduction in pUGFT of the promoter 35S and the NOS terminator in the 5 'and 3' regions of UGFTX, resulted in to plasmid production p35S-UGFT-NOS whose map of restriction is represented in Figure 2A.

In order to transfer this construction to the plant genome via Agrobacterium tumefaciens , it must first be cloned into a binary plasmid. To do this, p35S-UGFT-NOS was digested sequentially with the enzymes NotI, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 (Figure 2B) (23) that had previously been digested sequentially with the EcoRI, T4 DNA polymerase enzymes and HindIII. The plasmid thus obtained was designated as pBIN35S-UGFT-NOS (Figure 2C), which was introduced by electroporation into the strain of A. tumefaciens suitable for transforming plants according to conventional protocols.

Thus, for example, for the production of transgenic potato plants pBIN35S-UGFT-NOS, electroporation was introduced in A. tumefaciens with the deposit number CECT 5851, which acted as a transfer vector of the 35S-UGFT-NOS construct to the nuclear genome of potato when used by conventional methods of genetic transformation of potato (24).

Cultivation, processing and estimation of the characteristics of transgenic potato plants with altered levels of UGFT

Control potato plants (WT) were not used transformed and lines 4, 5, 6 and 12 that express UGFT constitutively as a result of integration into its genome of 35S-UGFT-NOS construction. Transgenic potato plants were also used with reduced UGFT activity (21). The plants were grown between May and September 2006 and 2007 on a plot of the term 25 of Sartaguda (Navarra, Spain). The plants were randomized in plots of 50 square meters, using 30 plants per line. The separation between rails was 90 cm. The separation between floor and floor of the same lane was 35 cm. Be they collected the tubers, those of a weight were selected approximately 100 grams and cylinders were obtained that crossed the whole tuber making use of a cylindrical steel perforator (2 cm in diameter). Such cylinders were frozen in nitrogen liquid and pulverized using a mortar.

For the measurement of UGFT activity, the powder resulting after homogenization of the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 ° C at 100 mM  HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder). After dialysate extracts, UGFT activity was measured as described in the literature (25). Briefly, the vegetable homogenate was incubated for 5 minutes at 37 ° C in 50 mM HEPES (pH 7.0), 1 mM MgCl 2, 15 mM KCl, 50 mM sucrose and 2 mM UDP. After stopping the reaction at 100 ° C the amount of UDPGlucose produced was determined using a Waters Associate's HPLC system set to a Partisil-10-SAX column.

The UDPGlucose existing in tuber powder of potato obtained after homogenization with liquid nitrogen is extracted and measured as described in the literature (25). For thus, 0.5 g of the frozen powder was resuspended in 0.4 mL of 1.4 M HClO 4, left at 4 ° C for 1 hour and centrifuged at 10,000 x g for 30 minutes. The supernatant was neutralized with K 2 CO 3 and centrifuged at 10,000 x g for 30 minutes. He UDPGlucose existing in the supernatant was measured using a HPLC Waters Associate's system set to a column Partisil-10-SAX.

Total polyphenols existing in the powder of tuber obtained after homogenization in liquid nitrogen extracted and measured essentially using the method Modified Folin-Ciocalteu described in the literature (26).

Total carotenoids in the powder of tuber obtained after homogenization in liquid nitrogen extracted and measured essentially using the method described in (27), measuring the absorbance of the organic extract at 450 nm using a calibration curve of β-carotene.

Total anthocyanins existing in the powder tuber obtained after homogenization in liquid nitrogen is extracted and measured using the method pH differential (28).

The antioxidant capacity of tuber powder obtained after homogenization in liquid nitrogen was measured by Use of the TEAC (Trolox equivalent antioxidant capacity) method according to It is described in the literature (29) using Trolox as a standard and 2,2'-acino-bis- (3-ethylbenzothiazolin-6-sulfonate (ABTS)

The polyphenol oxidase activity of the powder tuber obtained after homogenization in liquid nitrogen is measured according to the method described in (30).

Thus the first object of the present invention is refers to a procedure for obtaining transgenic plants high in antioxidant compounds, high capacity antioxidant and resistance to browning, in relation to wild plant phenotype (WT), which comprises the transformation of the wild plant (WT) with a vector of expression that over-expresses the UGFT enzyme.

In a preferred embodiment the process of the invention is characterized in that the antioxidant compounds are select between polyphenols and carotenoids.

In another preferred embodiment, the process of the invention is characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS.

The second object of the present invention relates to the use of the Agrobacterium tumefaciens CECT 5851 expression vector comprising plasmid pBIN35S-UGFT-NOS for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning. in relation to the phenotype of the wild plant.

In a preferred embodiment, the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS, is used to obtain transgenic plants with the aforementioned characteristics, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids

The third object of the present invention relates to the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising plasmid pBIN35S-UGFT-NOS for obtaining transformed plant cells with high content of antioxidant compounds and high antioxidant capacity in relation thereto unprocessed cells. In a preferred embodiment, the use of the vector described above for the transformation of plant cells is characterized in that the antioxidant compounds are selected from polyphenols and carotenoids.

The fourth object of the present invention is refers to transgenic plants transformed with a vector of expression that over-expresses the UGFT enzyme, characterized by presenting high content in compounds antioxidants, high antioxidant capacity and resistance to blaring over wild plants not transformed.

In a preferred embodiment, the plants of the invention, are characterized in that the compounds Antioxidants are selected from polyphenols and carotenoids.

In another preferred embodiment, the transgenic plants of the present invention are characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS.

In another preferred embodiment, the plants of the present invention, are characterized in that select from any of the following species: potato, Rice, tomato, corn and barley.

The fifth object of the present invention is refers to the use of the transgenic plants mentioned previously, to obtain high concentrations of UDPGlucose and high levels of antioxidant compounds, specifically selected from carotenoids and polyphenols.

The sixth object of the present invention is refers to plant cells transformed with a vector of expression that over-expresses the UGFT enzyme, characterized by presenting high content in compounds antioxidants and high antioxidant capacity compared to cells unprocessed vegetables

In a preferred embodiment, the cells transformed vegetables of the invention are characterized in that the antioxidant compounds are selected from polyphenols and carotenoids

In another preferred embodiment, the transformed plant cells of the present invention are characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS.

In another preferred embodiment, the cells Vegetables of the present invention are characterized 1 because they select from any of the following species: potato, Rice, tomato, corn and barley.

The seventh object of the present invention is refers to the use of the transformed plant cells mentioned previously, to obtain high concentrations of UDPGlucose and high levels of antioxidant compounds, specifically selected from carotenoids and polyphenols.

Deposit of microorganisms according to the Budapest Treaty

The microorganisms used herein invention were deposited in the Spanish Crop Collection Type (CECT), located in the University Research Building of Valencia, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with deposit number CECT 5851.

Examples of embodiment of the invention

Examples are described below in which the procedure for obtaining the transgenic potato plants with high substance content antioxidants, high antioxidant capacity and resistance to blistering, as a result of increased activity UGFT producer of UDPGlucose, as well as the values obtained for each of the above characteristics in plants transgenic compared to those obtained in non-potato plants transformed.

The examples set out below. they are intended to illustrate the invention without limiting the scope Of the same.

Example 1 Procedure for obtaining transgenic potato plants with high antioxidant content, high capacity antioxidant and resistance to browning as a result of Increase in UGFT activity producing UDPGlucose

In the present invention UGFT has been expressed constitutively by the action of the constitutive promoter 35S of the tobacco mosaic virus. For this, the 35S-UGFT-NOS construction was introduced into the potato genome ( Solanum tuberosum ), the design of which is detailed below.

Known nucleotide sequence encoding for the UGFT4 isoform of wild potato UGFT (22) were created two specific primers corresponding to the 5 'and 3' ends of the gene, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Making use of these primers was amplified by conventional methods of PCR a 2418 pair DNA fragment of bases, designated with UGFTX, from a sheet cDNA library Potato Such a PCR fragment was introduced into plasmid pSK Bluescript (Stratagene) giving rise to the pUGFT construction, the which was amplified in the host bacteria XL1 Blue. The UGFTX nucleotide sequence is SEQ ID NO: 3 which is different from UGFTX (GenBank access number U24087).

The sequential introduction into pUGFT of the 35S promoter and the NOS terminator in the 5 'and 3' regions of UGFTX, resulted in the production of plasmid p35S-UGFT-NOS whose restriction map is represented in Figure
2A.

In order to transfer this construction to the plant genome via Agrobacterium tumefaciens , it must first be cloned into a binary plasmid. To do this, p35S-UGFT-NOS was digested sequentially with the enzymes NotI, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 (Figure 2B) (23) that had previously been digested sequentially with the EcoRI, T4 DNA polymerase enzymes and HindIII. The plasmid thus obtained was designated as pBIN35S-UGFT-NOS (Figure 2C), which was introduced by electroporation into the strain of A. tumefaciens suitable for transforming plants according to conventional protocols.

Thus, for example, for the production of transgenic potato plants pBIN35S-UGFT-NOS, electroporation was introduced in A. tumefaciens with the deposit number CECT 5851, which acted as a transfer vector of the 35S-UGFT-NOS construct to the nuclear genome of potato when used by conventional methods of genetic transformation of potato (24).

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Example 2 Cultivation and processing of transgenic potato plants high in antioxidant substances, high capacity antioxidant and resistance to browning as a result of Increase in UGFT activity producing UDPGlucose

Control potato plants (WT) were not used transformed and lines 4, 5, 6 and 12 that express UGFT constitutively as a result of integration into its genome of 35S-UGFT-NOS construction. Transgenic potato plants were also used with reduced UGFT activity (21). The plants were grown between May and September 2006 and 2007 on a plot of the term 25 of Sartaguda (Navarra, Spain). The plants were distributed to random on plots of 50 square meters, using 30 plants per line The separation between rails was 90 cm. The separation between floor and floor of the same lane was 35 cm. Be they collected the tubers, those of a weight were selected approximately 100 grams and cylinders were obtained that crossed the whole tuber making use of a cylindrical steel perforator (2 cm in diameter). Such cylinders were frozen in nitrogen liquid and pulverized using a mortar.

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Example 3 Measurement of UGFT activity and concentration of UDPGlucose in tubers of transgenic potato plants with high activity UGFT producer of UDPGlucose compared to non-potato plants transformed

For the measurement of UGFT activity, the powder resulting after homogenization of the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 ° C at 100 mM  HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder). After dialysate extracts, UGFT activity was measured as described in the literature (25). Briefly, the homogenate Vegetable was incubated for 5 minutes at 37 ° C in 50 mM HEPES (pH 7.0), 1 mM MgCl2, 15 mM KCl, 50 mM sucrose and 2 mM UDP. After stop the reaction at 100 ° C the amount of UDPGlucose was determined produced using a Waters Associate's HPLC system adjusted to a column Partisil-10-SAX.

As illustrated in Figure 3, the UGFT activity in the tubers of any of the plants that express UGFT constitutively is 1.5-2 times superior to that existing in the same organ of a control plant (WT) grown under the same conditions and at the same time. By the opposite, the tubers of plants anti-UGFT showed an activity 3-4 times lower than that observed in tubers of WT plants grown under the same conditions and in the same moment.

The tubers of plants that express constitutively UGFT accumulate levels of UDPGlucose (135-160 nmol / g fresh weight) significantly higher than those observed in tubers of WT plants (95-105 nmol / g fresh weight) (Figure 4) grown in the same conditions and at the same time. On the contrary, the UGFT antisense plant potato tubers accumulate levels of UDPGlucose (15-30 nmol / g fresh weight) significantly lower than those observed in tubers of WT plants grown in the same conditions and in the same moment.

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Example 4 Measurement of the concentration of total polyphenols, carotenoids total and anthocyanins in the transgenic plant tubers of potato with high UGFT activity producing UDPGlucose with respect to unprocessed wild plants

Total polyphenols existing in the powder of tuber obtained after homogenization in liquid nitrogen extracted and measured essentially using the method Modified Folin-Ciocalteu described in the literature (26).

The fleshy part of plant tubers that express UGFT constitutively accumulate polyphenol levels (0.2-0.25 mg GAE / g fresh weight) significantly higher than those observed in tubers of WT plants (0.17-0.19 GAE / g fresh weight), grown in same conditions and at the same time (Figure 5). For him opposite, the fleshy part of the plant potato tubers UGFT antisense accumulate polyphenol levels (0.14-0.15 nmol / g fresh weight) significantly lower than those observed in tubers of cultivated WT plants in the same conditions and at the same time.

The skin of plant tubers that express UGFT constitutively accumulates polyphenol levels (0.58-0.72 mg GAE / g fresh weight) significantly superior to those observed in the skin of tubers of WT plants (0.48-0.52 mg GAE / g fresh weight) (Figure 6) grown under the same conditions and at the same time.

Total carotenoids in the powder of tuber obtained after homogenization in liquid nitrogen extracted and measured essentially using the method described in (27), measuring the absorbance of the organic extract at 450 nm using a calibration curve of β-carotene.

The tubers of plants that express UGFT constitutively accumulate carotenoid levels (1.1-1.6 \ mug \ betaCE / g fresh weight) significantly higher than those observed in tubers of WT plants (0.8-1.0 \ mug \ betaCE / g fresh weight) (Figure 8) grown under the same conditions and in the same moment.

Total anthocyanins existing in the powder tuber obtained after homogenization in liquid nitrogen is extracted and measured using the method pH differential (28).

The skin of plant tubers that express UGFT constitutively accumulates levels of anthocyanins (4.1-5.5 mg / g fresh weight) significantly superior to those observed in the skin of tubers of WT plants (3.8-4.0 mg / g fresh weight) (Figure 7) grown in the same conditions and at the same time.

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Example 5 Measurement of antioxidant capacity in the fleshy part and in the tuber skin of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to plants wild unprocessed

The antioxidant capacity of tuber powder obtained after homogenization in liquid nitrogen was measured by Use of the TEAC (Trolox equivalent antioxidant capacity) method according to It is described in the literature (29) using Trolox as a standard and 2,2'-acino-bis- (3-ethylbenzothiazolin-6-sulfonate (ABTS)

The fleshy part of plant tubers that express UGFT constitutively possess capacity values antioxidant (7.6-9.0? equivalent of Trolox / g fresh weight) significantly higher than those observed in the fleshy part of tubers of WT plants (6.5-6.8 µmol equivalent of Trolox / g weight fresh) grown under the same conditions and at the same time (Figure 9).

The skin of plant tubers that express UGFT constitutively possess capacity values antioxidant (14.2-16.2? equivalent of Trolox / g fresh weight) significantly higher than observed on the skin of tubers of WT plants (12.0-12.4 µmol equivalent of Trolox / g weight fresh) grown under the same conditions and at the same time (Figure 10).

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Example 6 Measurement of polyphenol oxidase activity in the fleshy part and on the skin of tubers of transgenic potato plants with high UGFT activity producing UDPGlucose compared to plants wild unprocessed

The polyphenol oxidase activity of the powder tuber obtained after homogenization in liquid nitrogen is measured according to the method described in (30). Polyphenol activity oxidase of the fleshy part of the potato tubers that express UGFT constitutively is normal when compared to the existing one in tubers of WT plants grown under the same conditions and at the same time (Figure 11A). The polyphenol oxidase activity of the skin of potato tubers that express UGFT so constitutive has a tendency to be lower than that observed in tubers of WT plants grown under the same conditions and at the same time (Figure 11B).

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Example 7 Measurement of resistance to browning in tubers transgenic potato plants with high UGFT activity producing UDPGlucose compared to non-transformed WT plants

The tubers of plants that constitutively over-express UGFT are more resistant to the browning effect than the tubers of WT plants grown in them conditions and at the same time, after suffering mechanical damage and exposure to air (Figure 12).

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3. Nakai , T., Konishi , T., Zhang , X.-Q., Chollet , R., Tonouchi , N., Tsuchida , T., Yoshinaga , F., Mori , H., Sakai , F., Hayashi , T. ( 1998 ) An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mtagenesis of Ser 11. Plant Cell Physiol . 39, 1337-1341

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5. Asano , T., Kunieda , N., Omura , Y., Ibe , H., Kawasaki , T., Takano , M., Sato , M., Furuhashi , H., Mujin , T., Takaiwa , F ., Wu , C., Tada , Y., Satozawa , T., Sakamoto , M., Shimada , H. ( 2002 ) Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 14, 619-628

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Claims (19)

1. Procedure for obtaining plants transgenic with high content of antioxidant compounds, high antioxidant capacity and resistance to browning, in relation to to the phenotype of the wild plant, which includes the transformation of the wild plant with an expression vector that over-expresses the UGFT enzyme. 2. Method according to claim 1, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 3. Method according to any one of claims 1 or 2, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising plasmid pBIN35S-UGFT-NOS. 4. Use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS to obtain transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relation to the phenotype of the wild plant . 5. Use according to claim 4, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 6. Use of the Agrobacterium tumefaciens CECT 5851 expression vector comprising plasmid pBIN35S-UGFT-NOS to obtain transformed plant cells that have high content of antioxidant compounds and high antioxidant capacity in relation to unprocessed cells. 7. Use according to claim 6, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 8. Transgenic plant, transformed with an expression vector that over-expresses the UGFT enzyme, characterized by its high content of antioxidant compounds, high antioxidant capacity and resistance to browning with respect to the non-transformed wild plant. 9. Transgenic plant according to claim 8, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 10. A transgenic plant according to any of claims 8 or 9, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS. 11. Transgenic plant according to any of claims 8 to 10, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley. 12. Plant cell transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having high content of antioxidant compounds and high antioxidant capacity with respect to unprocessed cells. 13. Transformed plant cell according to claim 12, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 14. Transformed plant cell according to any of claims 12 or 13, characterized in that the expression vector is Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS. 15. Transformed plant cell according to any of claims 12 to 14, characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley. 16. Use of transgenic plants, of any of claims 8 to 11, for obtaining high concentrations of UDPGlucose and high levels of compounds antioxidants 17. Use according to claim 16 characterized in that the antioxidant compounds are selected from polyphenols and carotenoids. 18. Use of transformed plant cells, of any of claims 12 to 15, for obtaining high concentrations of UDPGlucose and high levels of compounds antioxidants 19. Use according to claim 18, characterized in that the antioxidant compounds are selected from polyphenols and carotenoids.