[go: up one dir, main page]

WO2010055186A1 - Procédé de production de plantes transgéniques présentant une teneur élevée en composés anti-oxydants, une capacité anti-oxydante élevée et une résistance au brunissement - Google Patents

Procédé de production de plantes transgéniques présentant une teneur élevée en composés anti-oxydants, une capacité anti-oxydante élevée et une résistance au brunissement Download PDF

Info

Publication number
WO2010055186A1
WO2010055186A1 PCT/ES2009/070496 ES2009070496W WO2010055186A1 WO 2010055186 A1 WO2010055186 A1 WO 2010055186A1 ES 2009070496 W ES2009070496 W ES 2009070496W WO 2010055186 A1 WO2010055186 A1 WO 2010055186A1
Authority
WO
WIPO (PCT)
Prior art keywords
ugft
plants
antioxidant
polyphenols
antioxidant compounds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/ES2009/070496
Other languages
English (en)
Spanish (es)
Inventor
Francisco José MUÑOZ PÉREZ
Miren Edurne BAROJA FERNÁNDEZ
Francisco Javier Pozueta Romero
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Iden Biotechnology SL
Original Assignee
Iden Biotechnology SL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Iden Biotechnology SL filed Critical Iden Biotechnology SL
Publication of WO2010055186A1 publication Critical patent/WO2010055186A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1062—Sucrose synthase (2.4.1.13)

Definitions

  • the present invention relates to a process for the production of transgenic plants that have a high antioxidant compound content, high antioxidant capacity and resistance to browning, as well as to the transgenic plants themselves. Therefore, the present invention can be encompassed within the field of engineering genetics, plant physiology, food technology and nutraceuticals
  • UDP-glucose D-fructose-2-glucosyl transferase is highly regulated and catalyzes the production of UDPGlucose (UDPG) and fructose from the sucrose and UDP (3-6)
  • UDPGlucosa is the glucose donor molecule for the transglycosylation reactions involved in the biosynthesis of ghcolipids, glycoproteins and cell wall polysaccharides such as cellulose and callose (1, 7-9) ( Figure 14)
  • UDPGlucose acts as a glucose donor for the glycosylation reactions of antioxidant substances such as polyphenols and carotenoids
  • the present invention describes for the first time the production of plants with (a) high content of UDPGlucose, (b) high content of polyphenols and carotenoids, (c) high antioxidant capacity and (d) low browning effect (browning effect) as a consequence of the UGFT activity producing UDPGlucosa.
  • the present invention relates to a process for the production of transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning, by increasing the UGFT activity producing UDPGlucose in plants. Furthermore, the present invention relates to the transgenic plants themselves characterized by said properties.
  • the technical effects shown in the present invention can be extrapolated to any type of organ of the plant 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 by constitutively expressing UGFT under the control of the 35S promoter ( Figure 1 A, B and C), and by expressing UGFT under the control of a tuber-specific promoter (B33 promoter of the patatin gene ) ( Figure 2). It should be noted that the constitutive expression was particularly preferred since with this it was possible to analyze changes in the antioxidant activity in various organs of the plant such as leaves and tubers.
  • Transgenic plant plant whose genome has been modified by genetic engineering with the aim of achieving different biological characteristics and / or improved compared to those of the wild plant (WT).
  • Transformed plant cell they are plant cells that present a genetic alteration resulting from the introduction and expression of external genetic material in their genome.
  • Antioxidant compounds are substances capable of retarding or preventing the oxidation of other molecules. They are responsible for capturing the free radicals produced in the oxidation reactions of the metabolism. These compounds are widely used in the treatment of cerebrovascu- lar and neurodegenerative diseases, as well as dietary supplements, food preservatives, cosmetics and for the prevention of the degradation of rubber and gasoline.
  • the antioxidant compounds are selected among polyphenols and carotenoids.
  • Polyphenols are chemical substances present in plants and characterized by the presence of more than one phenol group (group -OH linked to an aromatic ring) per molecule. The presence of these phenolic rings confers the ability to act as antioxidants since they can pick up oxygen radicals.
  • Carotenoids are organic pigments that are found naturally in plants and other photosynthetic organisms. Most of the 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 auto-oxidation.
  • Anthocyanins polyphenolic organic pigments that provide coloration to many plants and especially to many flowers. Its chemical structure consists of a flavial group formed by a benzopyran ring attached to a phenolic ring. They have antioxidant properties avoiding the production of free radicals.
  • the present invention refers 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) (FIG. 3).
  • this expression is directly related to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time.
  • the present invention refers to "high content of total polyphenols", by way of illustration, when this is higher than 0.19 mg GAE / g (fresh weight) in the fleshy part of the tubers of the transgenic potato plants that express UGFT constitutively and superior to 0.52 mg GAE / g (fresh weight) in the skin of the tubers of the transgenic potato plants that express UGFT constitutively ( Figure 5 and 6).
  • the present invention refers to "high anthocyanin content", by way of illustration, when this is higher than 4.0 mg / g
  • the present invention refers to "high content of carotenoids" when it is higher, for example, of 1.0 ⁇ g / g fresh weight in tubers of transgenic potato plants that express UGFT constitutively ( Figure 8).
  • High antioxidant capacity as used in the present invention, this expression is directly related to a statistically significant value, higher than the values observed in WT plants grown under the same conditions and at the same time.
  • the present invention refers to "high antioxidant capacity", by way of illustration, when this is higher than 6.8 ⁇ mol Trolox eq / g fresh weight in the fleshy part of the tubers of transgenic potato plants that express UGFT constitutively ( Figure 9) and superior to 12.4 ⁇ mol Trolox eq / g fresh weight in the skin of the tubers of transgenic potato plants that express UGFT constitutively ( Figure 10).
  • Figure 1 Construction steps of the expression plasmid pBIN35S-UGFT-NOS.
  • FIG. 3 UGFT activity in tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of potato plants that express UGFT constitutively after integrating the 35S-UGFT-NOS construct into its genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 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 micromole of UDPGlucose per minute.
  • Figure 4 Content in UDPGlucose (nmol / g fresh weight) in tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • FIG. 5 Content of total polyphenols (in mg equivalents of gallic acid (GAE) / g fresh weight) in the fleshy part of tubers of wild potato plants or controls (WT), in tubers of potato plants transformed with an antisense vector of UGFT (anti-UGFT) (21) and in tubers of potato plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • GENE gallic acid
  • Figure 6 Content of total polyphenols (in mg GAE / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers of potato plants that constitutively express UGFT after integrating into their genome Ia 35S-UGFT-NOS construction after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • Figure 7 Content of total anthocyanins (in mg / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers potato plants that constitutively express UGFT after integrating the 35S construction into their genome -UGFT- NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • Figure 8 Content of total carotenoids (in ⁇ g equivalents of beta-carotene ( ⁇ CE) / g fresh weight) in tubers of wild potato plants or controls (WT) and potato plants that constitutively express UGFT after integrating the construction into their genome 35S-UGFT-NOS (by action of the Agrobacterium tumefaciens strain CECT: 5851).
  • Figure 9 Content of total carotenoids (in ⁇ g equivalents of beta-carotene ( ⁇ CE) / g fresh weight) in tubers of wild potato plants or controls (WT) and potato plants that constitutively express UGFT after integrating the construction into their genome 35S-UGFT-NOS (by action of the Agrobacterium tumefaciens strain CECT: 5851).
  • Antioxidant capacity (referred to ⁇ mol equivalent of Trolox / g fresh weight) in the fleshy part of tubers of wild potato plants or controls (WT) and in the fleshy part of tubers of potato plants constitutively expressing UGFT after integrating in its genome Ia construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • FIG. 10 Antioxidant capacity (referred to ⁇ mol equivalent of Trolox / g fresh weight) in the skin of tubers of wild potato plants or controls (WT) and in the skin of tubers of potato plants constitutively expressing UGFT after integrating into their genome Ia construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • Antioxidant capacity (referred to ⁇ mol equivalent of Trolox / g fresh weight) in leaves of wild potato plants or controls (WT) and potato plants constitutively expressing UGFT after integrating into their genome the construction 35S-UGFT-NOS after having been transformed with the expression vector Agrobacterium tumefaciens CECT.5851.
  • FIG. 12 Polyphenol oxidase activity (in Units / g fresh weight) (A) in the fleshy part and (B) in the skin of tubers of wild potato plants or controls (WT) and in the fleshy part and in the skin of tubers of potato plants that constitutively express UGFT after integrating the 35S-UGFT-NOS construct into their genome after having been transformed with the expression vector Agrobacterium tumefaciens CECT: 5851.
  • Figure 13 Effect of browning on tubers of wild potato plants or controls (WT) and on tubers of potato plants constitutively expressing UGFT after mechanical cutting and exposure to air for 28 hours.
  • Figure 14 Proposed mechanisms for the conversion of sucrose into cell wall polysaccharides, glycoproteins and glycosylated polyphenols in plants.
  • UGFT has been expressed constitutively by the action of the 35S constitutive promoter of tobacco mosaic virus.
  • the 35S-UGFT-NOS construction was introduced into the genome of the potato (Solanum tuberosum) whose design is detailed below.
  • two specific primers corresponding to the 5 ' and 3 ' ends of the gene were created, whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • UGFTX a 2418 base pair DNA fragment, designated with UGFTX, was amplified by conventional PCR methods from a potato leaf cDNA library.
  • p35S-UGFT-NOS was digested sequentially with the enzymes Notl, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 ( Figure 1B) (23) that had previously been digested sequentially with the enzymes EcoRI, T4 DNA polymerase and Hindlll.
  • the plasmid thus obtained was designated pBIN35S-UGFT-NOS ( Figure 1C), which was introduced by electroporation in the A. tumefaciens strain suitable for transforming plants according to conventional protocols.
  • pBIN35S-UGFT-NOS was introduced by electroporation into A. tumefaciens with deposit number CECT 5851, which acted as transfer vector of the 35S-UGFT-NOS construct to the genome nuclear potato when used by conventional methods of genetic transformation of potatoes (24).
  • UGFT has been expressed specifically in the potato tuber by the action of the B33 promoter that governs the expression of a gene coding for the patatin (24).
  • the B33-UGFT construction was introduced into the potato genome (Solanum tuberosum), the design of which is detailed below.
  • primers SEQ ID NO: 4 and SEQ ID NO: 5 a DNA fragment of 2418 base pairs was amplified by PCR from the p35S-UGFT-NOS plasmid.
  • Such a PCR fragment was introduced into the pDONR / Zeo plasmid (Invitrogen) by a Gateway BP recombination reaction giving rise to the pDONR-UGFT construct, which was amplified in the host bacterium TOP 10.
  • the PCR was amplified by PCR.
  • B33 promoter of the patatin using the specific primers corresponding to the 5 ' and 3 ' ends of the promoter whose sequences are SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
  • the PCR fragment obtained contains 1551 base pairs and was introduced into the plasmid pDONR / P4-P1 R (Invitrogen) by a recombination reaction Gateway BP giving rise to the construction pDONR-B33, which was amplified in the host bacterium TOP 10.
  • the B33 promoter and UGFT were transferred to the expression plasmid pKAN R4-R2 to produce plasmid pKAN-B33-UGFT (also designated pKAN-B33-SuSy).
  • the plasmid thus obtained was introduced by electroporation in the A. tumefaciens strain suitable for transforming plants according to conventional protocols.
  • B33-UGFT was introduced by electroporation into A. tumefaciens with the deposit number DSM 22756, which acted as transfer vector of the B33-UGFT construction to the potato nuclear genome at be used by conventional methods of genetic transformation of potato (24).
  • Controlled potato plants (WT) not transformed and lines 4, 5, 6 and 12 constitutively expressing UGFT were used as a result of the integration in their genome of the construction 35S-UGFT-NOS.
  • Transgenic potato plants with reduced UGFT activity were also used (21). The plants were cultivated between May and September
  • the powder resulting after the homogenization of the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 0 C in 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder).
  • the UGFT activity was measured as described in the literature (25). Briefly, the plant homogenate was incubated for 5 minutes at 37 0 C in 50 mM HEPES (pH 7.0), 1 mM MgCl 2, 15 mM KCI, 50 mM sucrose and 2 mM UDP. After stopping the reaction at 100 0 C the amount of UDPglucose produced was determined using an HPLC system Waters Associates' s adjusted to a Partisil SAX-10-column.
  • the UDPGlucose in the potato tuber powder obtained after homogenization with liquid nitrogen was extracted and measured as described in the literature (25). For this, 0.5 g of the frozen powder was resuspended in 0.4 ml_ of 1.4 M HCIO 4 , left at 4 0 C for 1 hour and centrifuged at 10,000 xg for 30 minutes. The supernatant was neutralized with K 2 CO 3 and centrifuged at 10,000 xg for 30 minutes. The UDPGlucose existing in the supernatant was measured using a Waters Associate ' s HPLC system fitted to a Partisil-10-SAX column. The total polyphenols existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the modified Folin-Ciocalteu method described in the literature (26).
  • the total carotenoids existing in the tuber powder obtained after homogenization in liquid nitrogen were 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 ⁇ -carotene.
  • the total anthocyanins existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured using the pH-differential method (28).
  • the antioxidant capacity of the tuber powder obtained after homogenization in liquid nitrogen was measured using the TEAC (Trolox equivalent antioxidant capacity) method as described in the literature (29) using Trolox as standard and 2,2 ' -acinobis- ( 3-ethylbenzothiazolin-6-sulfonate (ABTS).
  • the polyphenol oxidase activity of the tuber powder obtained after homogenization in liquid nitrogen was measured according to the method described in (30).
  • the first object of the present invention refers to a process for the production of transgenic plants with a high content of antioxidant compounds, high antioxidant capacity and resistance to browning, in relation to the phenotype of the wild plant (WT), which comprises the transformation of the wild plant (WT) with an expression vector that over-expresses the UGFT enzyme.
  • the method is characterized in that the antioxidant compounds are preferably selected from among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • the method of the invention is characterized in that the expression vectors used for the transformation of the plants are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefaciens DSM 22756, which comprise the plasmids pBIN35S-UGFT-NOS and pKAN-B33 -UGFT respectively.
  • Another object of the present invention relates to the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for obtaining transgenic plants that have a high content of antioxidant compounds, high antioxidant capacity and resistance to browning in relationship to the phenotype of the wild plant.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • a further object of the present invention relates to the use of the expression vector Agrobacterium tumefaciens CECT 5851 comprising the plasmid pBIN35S-UGFT-NOS for the production of transformed plant cells which have a high content of antioxidant compounds and a high antioxidant capacity in relation to the untransformed cells
  • the antioxidant compounds are preferably selected from polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • a further object of the present invention refers to the expression vector Agrobacterium tumefaciens DSM 22756, characterized in that it comprises the plasmid pKAN-B33-UGFT that over-expresses the UGFT enzyme, as well as its use for the production of transformed plant cells that have high content in antioxidant compounds and high antioxidant capacity in relation to untransformed cells.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • the expression vector Agrobacterium tumefaciens DSM 22756 is used 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.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • Another object of the present invention relates to the transgenic plant transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having a high content of antioxidant compounds, high antioxidant capacity and resistance to browning with respect to the non-transformed wild plant.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • the transgenic plant is characterized in that the expression vectors used are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefacien DSM 22756, comprising the plasmids pBIN35S-UGFT-NOS and pKAN-B33-UGFT, respectively .
  • the transgenic plant is characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.
  • Another object of the present invention relates to plant cells transformed with an expression vector that over-expresses the UGFT enzyme, characterized by having a high content of antioxidant compounds and high antioxidant capacity compared to untransformed cells.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • the plant cell is characterized in that the expression vectors used are preferably selected from Agrobacterium tumefaciens CECT 5851 and Agrobacterium tumefacien DSM 22756, which comprise the plasmids pBIN35S-UGFT-NOS and pKAN-B33-UGFT, respectively.
  • the plant cell is characterized in that it is selected from any of the following species: potato, rice, tomato, corn and barley.
  • Another object of the present invention refers to the use of the transgenic plants, mentioned above for obtaining high concentrations of UDPGlucosa and high levels of antioxidant compounds.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT.
  • the last object of the present invention refers to the use of the transformed plant cells, mentioned above, for obtaining high concentrations of UDPGlucose and high levels of antioxidant compounds.
  • the antioxidant compounds are preferably selected among polyphenols and carotenoids, without excluding the possibility that other antioxidant substances have been affected by the over-expression of the UGFT. DEPOSIT OF MICROORGANISMS ACCORDING TO THE BUDAPEST TREATY
  • the microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT), located in the Research Building of the University of Valencia, Burjassot Campus, Burjassot 46100 (Valencia, Spain) with deposit number CECT 5851 and in the "German National Resource Center for Biological Material", located in the DMSZ, Mascheroder Weg 1 b D-38124 (Braunschweig, Germany) with deposit number DSM 22756.
  • CECT Spanish Type Culture Collection
  • Example 1 Process for obtaining transgenic potato plants with a high content of antioxidants, high antioxidant capacity and resistance to browning as a consequence of the increase of the UGFT activity producing UDPGlucosa
  • UGFT has been expressed constitutively by the action of the 35S constitutive promoter of tobacco mosaic virus.
  • the 35S-UGFT-NOS construction was introduced into the genome of the potato (Solanum tuberosum) whose design is detailed below.
  • UGFTX The nucleotide sequence of UGFTX is SEQ ID NO: 3 which is different from UGFTX (access number in GenBank U24087).
  • the introduction of UGFT in pDONR / Zeo and the subsequent recombination with pDONR-B33 gave rise to the production of the plasmid pKAN-B33-UGFT, whose restriction map and way of obtaining it is represented in Figure 2.
  • p35S-UGFT-NOS was digested sequentially with the enzymes Notl, T4 DNA polymerase and HindIII and was cloned into the binary plasmid pBIN20 ( Figure 1B) (23) that had previously been digested sequentially with the enzymes EcoRI, T4 DNA polymerase and Hindlll.
  • the plasmid thus obtained was designated pBIN35S-UGFT-NOS ( Figure 1C), which was introduced by electroporation in the A.
  • tumefaciens strain suitable for transforming plants according to conventional protocols for example, for the production of transgenic potato plants that overexpress constitutively UGFT, pBIN35S-UGFT-NOS was introduced by electroporation into A. tumefaciens with deposit number CECT 5851, which acted as transfer vector of the construction 35S-UGFT-NOS to the nuclear potato genome when used by conventional methods of genetic transformation of potatoes (24).
  • pKAN-B33-UGFT was introduced by electroporation into A. tumefaciens with the deposit number DSM 22756, which acted as vector of transfer of the B33-UGFT construct to the nuclear genome of the potato.
  • Example 2 Cultivation and processing of transgenic potato plants with high content of antioxidants, high antioxidant capacity and resistance to browning as a consequence of the increase of the UGFT activity producing UDPGlucosa
  • Controlled potato plants (WT) not transformed and lines 4, 5, 6 and 12 constitutively expressing UGFT were used as a result of the integration in their genome of the construction 35S-UGFT-NOS.
  • Transgenic potato plants were also used reduced UGFT activity (21).
  • the plants were cultivated between May and September of 2006 and of 2007 in a plot of the term 25 of Sartaguda (Navarra, Spain).
  • the plants were randomly distributed in plots of 50 square meters, making use of 30 plants per line.
  • the separation between lanes was 90 cm.
  • the separation between plant and plant of the same lane was 35 cm.
  • Leaves and tubers were collected. From the collected tubers, those that had an approximate weight of 100 grams were selected and cylinders were obtained that crossed the entire tubercle using a cylindrical steel perforator (2 cm in diameter). Such cylinders were frozen in liquid nitrogen and pulverized using a mortar.
  • Example 3 Measurement of the UGFT activity and the concentration of UDPGlucose in tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to potato plants not transformed.
  • the powder resulting after the homogenization of the leaf and the fleshy part and / or the skin of the tuber with liquid nitrogen was resuspended at 4 0 C in 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol (5 mL / gram powder).
  • the UGFT activity was measured as described in the literature (25). Briefly, the plant homogenate was incubated for 5 minutes at 37 0 C in 50 mM HEPES (pH 7.0), 1 mM MgCl 2, 15 mM KCI, 50 mM sucrose and 2 mM UDP.
  • the UGFT activity in the tubers of any of the plants that express UGFT constitutively is 1.5-2 times higher than that existing in the same organ of a control plant (WT) grown under the same conditions and at the same time.
  • the tubers of the anti-UGFT plants showed an activity 3-4 times lower than that observed in tubers of the WT plants grown under the same conditions and at the same time.
  • the tubers of plants that constitutively express UGFT accumulate levels of UDPGlucosa (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 under the same conditions and at the same time.
  • potato tubers of UGFT antisense plants accumulate levels of UDPGlucose (15-30 nmol / g fresh weight) significantly lower than those observed in tubers of WT plants grown under the same conditions and at the same time.
  • Example 4 Measurement of the concentration of total polyphenols, total carotenoids and anthocyanins in the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.
  • the total polyphenols existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured essentially using the modified Folin-Ciocalteu method described in the literature (26).
  • the fleshy part of the tubers of plants that express UGFT constitutively accumulate levels of polyphenols (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 the same conditions and at the same time ( Figure 5).
  • the fleshy part of potato tubers of UGFT antisense plants accumulate polyphenol levels (0.14-0.15 nmol / g fresh weight) significantly lower than those observed in tubers of WT plants grown under the same conditions and in the same conditions. moment.
  • the content of total polyphenols present in the fleshy part of the tubers of potato plants that express UGFT constitutively is 25-35% higher than in the tubers of WT plants, and 45% higher than in the tubers of the UGFT antisense plants.
  • the skin of the tubers of plants that express UGFT constitutively accumulates levels of polyphenols (0.58-0.72 mg GAE / g fresh weight) significantly higher than 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.
  • the total carotenoids existing in the tuber powder obtained after homogenization in liquid nitrogen were 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 ⁇ -carotene.
  • the total anthocyanins existing in the tuber powder obtained after homogenization in liquid nitrogen were extracted and measured using the pH-differential method (28).
  • the skin of the tubers of plants that express UGFT constitutively accumulates levels of anthocyanins (4.1-5.5 mg / g fresh weight) higher than those observed in the skin of tubers of WT plants (3.8-4.0 mg / g fresh weight) ( Figure 7) grown under the same conditions and at the same time.
  • tubers of plants that express UGFT constitutively accumulate levels of carotenoids (1.1-1.6 ⁇ g ⁇ CE / g fresh weight) significantly higher than those observed in tubers of WT plants (0.8-1.0 ⁇ g ⁇ CE / g fresh weight) ( Figure 8) grown in the same conditions and at the same time.
  • Example 5 Measurement of the antioxidant capacity in leaves and tubers (fleshy part and skin) of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.
  • the antioxidant capacity of the leaf and tuber powder (fleshy part and skin) obtained after homogenization in liquid nitrogen was measured using the TEAC (Trolox equivalent antioxidant capacity) method as described in the literature (29) using Trolox as standard and 2 , 2 '-acino-bis- (3-ethyl-6-sulfonate (ABTS).
  • Polyphenols and carotenoids are the major determinants of the antioxidant capacity of cells.
  • the increase in the content of polyphenols and carotenoids induced by the ectopic expression of enzymes involved in the biosynthesis of these compounds leads to the increase of the antioxidant activity in tubers of potato plants.
  • the constitutive expression of UGFT in potato plants by the procedure described in the present invention led to an increase of between 20-30% of the total antioxidant activity in the tubers of the same.
  • This result is due to the higher content of polyphenols and carotenoids presented by said plants and not to the increase of other antioxidant compounds, such as ascorbic acid, since the concentration of said acid in the tubers of plants that constitutively express UGFT remains unchanged. in relation to the concentration present in the WT plants.
  • Example 6 Measurement of the polyphenol oxidase activity in the fleshy part and in the skin of the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed wild plants.
  • the polyphenol oxidase activity of the tuber powder obtained after homogenization in liquid nitrogen was measured according to the method described in (30).
  • the polyphenol oxidase activity of the fleshy part of potato tubers that express UGFT in a constitutive manner is normal when compared with the one existing in tubers of WT plants grown under the same conditions and at the same time ( Figure 12A).
  • the polyphenol oxidase activity of the skin of potato tubers that express UGFT constitutively has a tendency to be lower than that observed in the tubers of WT plants grown under the same conditions and at the same time (Figure 12B).
  • Example 7 Measurement of the resistance to browning in the tubers of transgenic potato plants with high UGFT activity producing UDPGlucose with respect to non-transformed WT plants.
  • tubers of plants that overexpress UGFT are more resistant to the browning effect than the tubers of WT plants grown under the same conditions and at the same time, after suffering mechanical damage and exposure to air (Figure 13). This fact is due, at least in part, to the high content of total polyphenols and carotenoids present in the plants that express UGFT and not to the decrease in the activity of polyphenol oxidase, since the maximum polyphenol oxidase activity in tubers of plants that express constitutively UGFT was normal compared to the tubers of the WT plants.
  • Plant Physiol. Biochem. 40, 907-911 7. Kleczkowski, LA (1994) Glucose activation and metabolism through UDP-glucose pyrophosphorylase in plants. Phytochemistry 37, 1507-1515 25 8. Buckeridge, MS, Vergara, CE. , Carpita, N. C (1999). The mechanism of synthesis of a mixed-linkage (1 - 3), (1 - 4) ⁇ -D-glucan in maize. Evidence for multiple sites of glucosyl transfer in the synthase complex. Plant Physiol. 120, 1105-1116 9. Neckelmann, G. and Orellana, A.
  • CM Ghislain, M., Bertin, P., Oufir, M., Herrera, MR, Hoffmann, L., Hausman, J- F., Larondelle, Y., Evers, D. (2007) Andean potato cultivars (Solanum tuberosum L) as a source of antioxidant and mineral micronutrients. J. Agrie. Food Chem. 55, 366-378 20. Vogt, T. and Jones, P. (2000) Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 5, 380-386 21. Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Physiology (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nutrition Science (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne un procédé permettant de produire des plantes transgéniques, et les plantes ainsi obtenues lesquelles présentent une teneur élevée en composés anti-oxydants (tels que les polyphénols et les caroténoïdes), une capacité anti-oxydante élevée et une résistance au brunissement, grâce à l'augmentation de l'activité de UDP-glucose:d-fructose-2- glucosyltransférase (UGFT) productrice de UDP-Glucose dans les plantes. Dans la présente invention, il est démontré que UGFT est impliquée dans la production d'une masse importante de UDP-Glucose nécessaire à la production de formes glycosylées des polyphénols et des caroténoïdes, hautement instables et présentant une capacité antioxydante élevée.
PCT/ES2009/070496 2008-11-13 2009-11-11 Procédé de production de plantes transgéniques présentant une teneur élevée en composés anti-oxydants, une capacité anti-oxydante élevée et une résistance au brunissement Ceased WO2010055186A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200803245A ES2339094B1 (es) 2008-11-13 2008-11-13 Procedimiento para la produccion de plantas transgenicas que presentan alto contenido en compuestos antioxidantes, alta capacidad antioxidante y resistencia al empardecimiento.
ESP200803245 2008-11-13

Publications (1)

Publication Number Publication Date
WO2010055186A1 true WO2010055186A1 (fr) 2010-05-20

Family

ID=42126179

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2009/070496 Ceased WO2010055186A1 (fr) 2008-11-13 2009-11-11 Procédé de production de plantes transgéniques présentant une teneur élevée en composés anti-oxydants, une capacité anti-oxydante élevée et une résistance au brunissement

Country Status (2)

Country Link
ES (1) ES2339094B1 (fr)
WO (1) WO2010055186A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006862A1 (fr) * 1996-08-09 1998-02-19 Calgene Llc Procedes de fabrication de composes carotenoides et d'huiles speciales a partir de graines de plantes
WO2005075649A1 (fr) * 2004-02-05 2005-08-18 Universidad Publica De Navarra Procede de production de saccharose synthase recombinante, son utilisation dans la fabrication de kits de determination de saccharose, production de adpglucose et obtention de plantes transgeniques dont les feuilles et les organes de reserve accumulent une forte teneur en adpglucose et amidon
WO2007045063A2 (fr) * 2005-10-21 2007-04-26 Alellyx S. A. Polynucleotides, structures d'adn et procedes pour la modification de la teneur en cellulose de plantes
WO2008021574A2 (fr) * 2006-08-18 2008-02-21 Ceres, Inc. Modulation de niveaux de caroténoïdes des plantes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006862A1 (fr) * 1996-08-09 1998-02-19 Calgene Llc Procedes de fabrication de composes carotenoides et d'huiles speciales a partir de graines de plantes
WO2005075649A1 (fr) * 2004-02-05 2005-08-18 Universidad Publica De Navarra Procede de production de saccharose synthase recombinante, son utilisation dans la fabrication de kits de determination de saccharose, production de adpglucose et obtention de plantes transgeniques dont les feuilles et les organes de reserve accumulent une forte teneur en adpglucose et amidon
WO2007045063A2 (fr) * 2005-10-21 2007-04-26 Alellyx S. A. Polynucleotides, structures d'adn et procedes pour la modification de la teneur en cellulose de plantes
WO2008021574A2 (fr) * 2006-08-18 2008-02-21 Ceres, Inc. Modulation de niveaux de caroténoïdes des plantes

Also Published As

Publication number Publication date
ES2339094B1 (es) 2011-03-18
ES2339094A1 (es) 2010-05-14

Similar Documents

Publication Publication Date Title
Liu et al. Regulation, evolution, and functionality of flavonoids in cereal crops
Sun et al. Biochemical and molecular characterization of a flavonoid 3-O-glycosyltransferase responsible for anthocyanins and flavonols biosynthesis in Freesia hybrida
Meng et al. Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress
Luo et al. A STAY‐GREEN protein S l SGR 1 regulates lycopene and β‐carotene accumulation by interacting directly with S l PSY 1 during ripening processes in tomato
Mahajan et al. Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco
CA2537065C (fr) Procede de production d'une rose avec des petales de couleur modifiee
Kim et al. Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato
Zhang et al. Anthocyanin accumulation and molecular analysis of correlated genes in purple kohlrabi (Brassica oleracea var. gongylodes L.)
Ebisawa et al. Supplementary ultraviolet radiation B together with blue light at night increased quercetin content and flavonol synthase gene expression in leaf lettuce (Lactuca sativa L.)
Feng et al. Flavonoids are involved in salt tolerance through ROS scavenging in the halophyte Atriplex canescens
Lorenc-Kukuła et al. Engineering flax with the GT family 1 Solanum sogarandinum glycosyltransferase SsGT1 confers increased resistance to Fusarium infection
Kim et al. Down-regulation of sweetpotato lycopene β-cyclase gene enhances tolerance to abiotic stress in transgenic calli
Mierziak et al. Crossbreeding of transgenic flax plants overproducing flavonoids and glucosyltransferase results in progeny with improved antifungal and antioxidative properties
Lorenc-Kukuła et al. Engineering flax with increased flavonoid content and thus Fusarium resistance
Li et al. Purple canola: Arabidopsis PAP1 increases antioxidants and phenolics in Brassica napus leaves
Pandey et al. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato
Hutabarat et al. Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight
Xi et al. Seed‐specific overexpression of antioxidant genes in Arabidopsis enhances oxidative stress tolerance during germination and early seedling growth
ES2301234T3 (es) Procedimiento y composiciones para modular el contenido de flavonoides.
Lim et al. Co-expression of onion chalcone isomerase in Del/Ros1-expressing tomato enhances anthocyanin and flavonol production
Sun et al. VvMYBA6 in the promotion of anthocyanin biosynthesis and salt tolerance in transgenic Arabidopsis
Massa et al. Modifying anthocyanins biosynthesis in tomato hairy roots: a test bed for plant resistance to ionizing radiation and antioxidant properties in space
Woo et al. Expression of tobacco tocopherol cyclase in rice regulates antioxidative defense and drought tolerance
ES2385533T3 (es) Secuencias genéticas que tienen actividad de metiltransferasa y usos de las mismas
Wang et al. Correlation of tyrosinase activity and betacyanin biosynthesis induced by dark in C3 halophyte Suaeda salsa seedlings

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09825794

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09825794

Country of ref document: EP

Kind code of ref document: A1