WO2007132040A1 - Transgenic plants with altered starch levels as a result of the variation of the activity of vegetable nudix that hydrolyse adp-glucose - Google Patents
Transgenic plants with altered starch levels as a result of the variation of the activity of vegetable nudix that hydrolyse adp-glucose Download PDFInfo
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- WO2007132040A1 WO2007132040A1 PCT/ES2007/000272 ES2007000272W WO2007132040A1 WO 2007132040 A1 WO2007132040 A1 WO 2007132040A1 ES 2007000272 W ES2007000272 W ES 2007000272W WO 2007132040 A1 WO2007132040 A1 WO 2007132040A1
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- 229920002472 Starch Polymers 0.000 title claims abstract description 43
- 235000019698 starch Nutrition 0.000 title claims abstract description 43
- 239000008107 starch Substances 0.000 title claims abstract description 43
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 39
- 230000000694 effects Effects 0.000 title claims abstract description 15
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- 239000008103 glucose Substances 0.000 title description 3
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- 230000002018 overexpression Effects 0.000 claims description 15
- 230000002255 enzymatic effect Effects 0.000 claims description 14
- 241000219194 Arabidopsis Species 0.000 claims description 13
- 244000061456 Solanum tuberosum Species 0.000 claims description 13
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- 108090000623 proteins and genes Proteins 0.000 claims description 10
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- 241000209094 Oryza Species 0.000 claims description 8
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
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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
- C12N15/8245—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 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
Definitions
- the present invention falls within the field of genetic engineering. Specifically, the invention comprises the use of a plant enzymatic product of the Nudix family, with ADP glucose hydrolytic activity, to obtain plants with altered starch levels.
- glycogen and starch are forms of carbohydrate storage. Specifically, glycogen is the form of carbohydrate storage in animals and bacteria and starch is in plants. In plants, starch accumulates in large quantities in organs such as seeds and tubers, and is a fundamental constituent of the human diet. On the other hand, starch is frequently used in the paper, cosmetic, pharmaceutical and food industries, as well as being used as a fundamental component for the manufacture of biodegradable plastics, low environmental impact paints and bioethanol.
- ADPG ADPglucose
- ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.).
- Pyrophosphatase (EC 3.6.1.21), belonging to the family of enzymes Nudix hydrolases and with hydrolytic activity of ADPG, in the achievement of transgenic plants with altered levels of starch.
- the invention describes the production of transgenic plants that overexpress ASPP, of plant origin, which, as is the case with plants that overexpress ASPP of bacterial origin (Baroja-Fernández, E., Mu ⁇ oz , FJ, Zandueta-Criado, A., Morán-Zorzano, MT, Viale, AM, Etxeberria, E., Alonso-Casaj ⁇ s, N., Pozueta-Romero, J. (2004) "Most of ADPglucose linked to starch biosynthesis occurs outside the chloroplast in source leaves. Proc. Nati. Acad. Sel USA.
- Another objective of the invention is to obtain plants with high starch content after reducing or canceling the endogenous ASPP activity. As with bacteria with low ASPP activity, these plants have high levels of starch.
- FIG. 1 Construction stages of plasmid pET-AtASPP.
- FIG. 10 Construction stages of plasmid pK2GW7,0-StASPP.
- ADPG hydrolytic activity and glycogen (B) content in bacteria transformed with pET-28c (+) and with pET-StASPP.
- ADPG Hydrolytic activity of ADPG (A), starch content (B) and ADPG (C) in Arabidopsis leaves and in different clones of transgenic Arabidopsis plants (8, 5, 3, 7) that overexpress At4gll980 after being transformed making use of strain DSM 18035.
- ADPG (A) 5 contained in starch (B) and ADPG (C) in potato leaves, and in different clones of transgenic potato plants (7,6,2) that express POADP80 after being transformed using of strain DSM 18036.
- AtASPP Arabidopsis
- StASPP potato
- OsASPP rice
- At4gll980 EMBL: AJ748742
- POADP80 EMBL: AMl 80509
- Q9SNS9 Q9SNS9
- At4gll980, POADP80 and Q9SNS9 allowed the creation of specific primers to amplify by RT-PCR complete cDNAs that code for AtASPP, StASPP and OsASPP from total RNA of Arabidopsis, potato and rice leaves, respectively.
- the cDNAs were cloned into the pGemT-easy vector (Promega), giving rise to plasmids pAtASPP ( Figure 3) and pStASPP ( Figure 4).
- pET-AtASPP and pET-StASPP were introduced by electroporation in E. coli BL21 (DE3).
- Over-expression of AtASPP and StASPP took place independently by adding 1 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) in 100 ml of cell culture. After six hours of induced culture, the bacteria were collected and resuspended in 6 ml of "binding buffer" (Novagen, His-bind purification kits), sonicated and centrifuged at 10,000 g for ten minutes. Supernatants containing recombinant ASPPs with a histidine tail were passed through an affinity column of Novagen's "His-bind" protein purification kit. Following the instructions in the kit, recombinant AtASPP and StASPP were eluted with 6 ml of the recommended elution buffer.
- binding buffer Novagen, His-bind purification kits
- the plant ASPP enzyme product was identified by the following functional patterns:
- Adenosine diphosphate-sugar pyrophosphatase (EC 3.6.1.21) that catalyzes the hydrolysis of ADPG in equimolar amounts of GlP and AMP.
- ADPG In addition to ADPG, it recognizes ADPmanosa and ADPribosa, but does not recognize other sugar-nucleotides such as UDPglucose, GDPmanosa, UDPglucoronic, etc. It also does not recognize nucleotides such as 5'-phosphosulfate, ATP, ADP, UTP, GTP or the artificial bis-paranitrophenyl phosphate substrate used for the characterization of phosphodiesterases.
- pET-AtASPP was digested sequentially with the enzymes Xhol, T4 DNA polymerase and Ncol.
- the released fragment (At4gll980) was cloned into the Ncó ⁇ / Smal sites of p35S-NOS (Baroj a-Fernández, E., Mu ⁇ oz, FJ, Zandueta-Criado, A., Moran-Zorzano, MT, Viale, AM, Alonso- Casajus, N., Pozueta- Romero, J. (2004) Most of ADP-glucose linked to starch biosynthesis occurs otuside the chloroplast in source leaves. Proc. Nati.
- p35S-AtASPP-NOS Figure 7
- p35 S-AtASPP-NOS was sequentially digested with the enzymes HindIII and EcoRI and was cloned into the binary plasmid pBIN 20 (Hennegan, K., Danna, KJ. (1998) pBIN20: An improved binary vector for Agrobacterium-mediated transformation. Plant Molecular Biology Repórter 16, 129-131) that had previously been digested sequentially with the enzymes HindIII and EcoRI. The plasmid thus obtained was designated as pBIN35S-AtASPP-NOS ( Figure 8).
- pBIN35 S-AtASPP-NOS and pK2GW7,0-StASPP were introduced into Agrobacterium tumefaciens, giving rise to strains DSM 18035 and DSM 18036, respectively, which were used to transform species such as Arabidopsis, potato, corn and rice.
- DSM 18035 and DSM 18036 were deposited on 10.3.2006 at the "German National Resource Center for Biological Material", located at DMSZ, Mascheroder Weg Ib D-38124 (Braunschweig, Germany). Obtaining transgenic plants deficient in plant ASPPs
- At4gll980 knockout plants from Arabidopsis thaliana were obtained from the "European Arabidopsis Stock Center" Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen,
- Soluble sugars were extracted according to (Heim, U., Weber, H., Baumlein, H., Wobus, U. (1993) "A sucrose-synthase gene of V. Faha L. Expression pattern in developing seeds in relation to starch synthesis and metabolic regulation "Plant 191, 394-401).
- Glucose, sucrose, fructose, glucose-1-phosphate, glucose-6-phosphate and ADPG were determined using an HPLC fitted to a CarboPac PAlO column and a DX500 amperometric detector (Baroja-Fernández, E., Mu ⁇ oz, FJ, Saikusa , T., Rodr ⁇ guez- Lopez, M., Akazawa, T., Pozueta-Romero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissue of plants, Plant CeIl Physiol. 44, 500-509).
- ADPG was determined using an HPLC system adjusted to a Partisil-10-SAX column (Mu ⁇ oz, FJ., Baroja-Fernández, E., Morán-Zorzano, MT, Viale, AM, Etxeberria, E., Alonso- Casaj ⁇ s, N., Pozueta-Romero, J. (2005) Sucrose synthase controls the intracellular levéis of ADPglucose linked to transitory starch biosynthesis in source leaves. Plant CeIl Physiol. 46, 1366-1376).
- the present invention relates to a process for obtaining transgenic plants, which have an altered expression, either an over-expression or an under-expression, of the plant-derived enzymatic product ASPP (Adenosine Diphosphate Sugar Pyrophosphatase) inside its cells with respect to the expression of said enzyme in the wild plant.
- ASPP Addenosine Diphosphate Sugar Pyrophosphatase
- over-expression of the ASPP plant-based enzyme product is considered when an expression greater than or equal to twice the expression achieved in the wild plant (wt) is achieved.
- infra-expression when an expression less than or equal to 80% of the expression achieved in the wild plant (wt) is achieved.
- the transformation process of the wild plant described above is carried out using Agrobacterium tumefaciens as a transformation vector.
- the transformation vector used, in the process for obtaining transgenic plants is specifically Agrobacterium tumefaciens strains DSM 18035 or DSM 18036.
- a second aspect of the present invention refers to an enzymatic product of plant origin with ASPP activity that contains an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived by degeneration of their genetic code.
- the present invention relates to an enzymatic product of plant origin with ASPP activity containing an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived from degeneration of the genetic code of these, characterized by not hydrolyzing molecules of the group comprised of: GlP 5 G6P, AMP, 3-phosphoglycerate, cAMP, UDPglucose, GDPglucose, adenosine 5'-phosphosulfate, bis-p-nitrophenyl phosphate or long chain nucleic acids .
- the present invention relates to an enzymatic product of plant origin with ASPP activity containing an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived by degeneration of the genetic code of these, characterized by not hydrolyzing molecules of the group comprised of: GlP 5 G6P, AMP, 3-phosphoglycerate, cAMP 5
- UDPglucose, GDPglucose, adenosine 5'-phosphosulfate, bis-p-nitrophenyl phosphate or long chain nucleic acids and for presenting an apparent molecular weight determined by gel filtration between 70-75 kDa and that in denaturing gel migrates as a protein of between 35-40 kDa.
- Another aspect of the present invention relates to the use of the cDNA sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9, or any other sequence derived therefrom by degeneracy of the genetic code, or that can hybridize with some of them, in obtaining transgenic plants that overexpress or under-express the enzymatic product of vegetable origin ASPP.
- Another aspect of the invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP.
- transgenic plant and its progeny comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is of the genus Arabidopsis, preferably Arabidopsis thaliana.
- transgenic plant and its progeny comprising the overexpression or infra-expression of the enzyme product of plant origin, ASPP, where said plant is of the genus Solanum, preferably Solanum tuberosum.
- the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, wherein said plant is of the genus Nicotiana, preferably Nicotiana tabacum.
- the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, wherein said plant is of the genus Lycopersicom, preferably Lycopersicom sculentum.
- the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is of the genus Oryza, preferably Oryza sativa.
- the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is Zea mays.
- transgenic plant and its progeny which comprises the overexpression of the ASPP plant-based enzyme product, as a dietary food or in the manufacture of low starch dietary products.
- transgenic plant and its progeny which comprises the infra-expression of the ASPP plant-derived enzyme product, for the production of starch.
- Example 1 Obtaining complete cDNAs coding for AtASPP, StASPP and OsASPP
- the knowledge of the nucleotide sequence of the gene coding for AtASPP allowed the creation of two specific primers whose sequences are, in the 5'-3 'sense, SEQ ID NO: 1 and SEQ ID NO: 2.
- Using these primers and Arabidopsis leaf RNA was amplified by conventional RT-PCR methods a complete cDNA of At4gll980, which was cloned into pGemT-easy (Promega).
- the nucleotide sequences of the amplified DNA and the deduced amino acid sequence are depicted in SEQ ID NO: 3.
- Vegetable ASPP is a pyrophosphatase that catalyzes the hydrolysis of ADPG producing equimolar amounts of GlP and AMP.
- ADPG In addition to ADPG, it also hydrolyses ADPribosa and ADPmanosa.
- B Does not hydrolyse molecules with phosphomonoester bonds such as GlP, G6P, AMP, 3-phosphoglycerate, and the like. Nor do they hydrolyze cyclic AMP or long chain nucleic acids such as DNA and RNA.
- Example 4 Obtaining transgenic plants that express plant ASPPs
- the external morphology of these plants is not aberrant, after being compared with that of non-transformed plants.
- Table 1 Sugar content (in nanomoles / gram of fresh weight) in unprocessed Arabidopsis leaves and in 4 independent clones of transformed plants with the 35 S-At ASPP-NOS construction.
- Example 5 Plants with high starch content as a result of low plant ASPP activity
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Abstract
The present invention relates to the production of transgenic plants with altered levels of starch as a result of the variation of the activity of vegetable nudix that hydrolyse ADP-glucose. Therefore, a description is given in this invention of how to obtain not only transgenic plants that over-express vegetable ASPPs and therefore accumulate little starch, but also transgenic plants that have a high starch content as a result of the reduction of the vegetable ASPP activity.
Description
PLANTAS TRANGÉNICAS CON NIVELES ALTERADOS DE ALMIDÓN TRANGENIC PLANTS WITH ALMIDON ALTERED LEVELS
COMO RESULTADO DE LA VARIACIÓN DE LA ACTIVIDAD DE NUDIXAS A RESULT OF THE VARIATION OF NUDIX ACTIVITY
VEGETALES QUE HIDROLIZAN ADPGLUCOSAVEGETABLES THAT HYDROLYZE ADPGLUCOSE
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La presente invención se engloba dentro del campo de la ingeniería genética. Concretamente la invención comprende la utilización de un producto enzimático vegetal de la familia Nudix, con actividad hidrolítica de ADP glucosa, para la obtención de plantas con niveles alterados de almidón.The present invention falls within the field of genetic engineering. Specifically, the invention comprises the use of a plant enzymatic product of the Nudix family, with ADP glucose hydrolytic activity, to obtain plants with altered starch levels.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Tanto el glucógeno como el almidón constituyen formas de almacenamiento de carbohidratos. Concretamente, el glucógeno es la forma de almacenamiento de carbohidratos en animales y bacterias y el almidón lo es en las plantas. En plantas el almidón se acumula en grandes cantidades en órganos tales como semillas y tubérculos, y es un constituyente fundamental de la dieta del ser humano. Por otro lado, el almidón es utilizado frecuentemente en las industrias papelera, cosmética, farmacéutica y alimentaria, además de utilizarse como componente fundamental para la fabricación de plásticos biodegradables, pinturas de bajo impacto medioambiental y bioetanol.Both glycogen and starch are forms of carbohydrate storage. Specifically, glycogen is the form of carbohydrate storage in animals and bacteria and starch is in plants. In plants, starch accumulates in large quantities in organs such as seeds and tubers, and is a fundamental constituent of the human diet. On the other hand, starch is frequently used in the paper, cosmetic, pharmaceutical and food industries, as well as being used as a fundamental component for the manufacture of biodegradable plastics, low environmental impact paints and bioethanol.
El ADPglucosa (ADPG) es el precursor de la biosíntesis del almidón en plantas y del glucógeno bacteriano. Los mecanismos de degradación de este azúcar-nucleótido han sido poco estudiados, aunque se conoce que existen enzimas que hidrolizan ADPG tanto en plantas (Rodríguez-López, M., Baroj a-Fernández, E., Zandueta-Criado, A., Pozueta-ADPglucose (ADPG) is the precursor of starch biosynthesis in plants and bacterial glycogen. The degradation mechanisms of this sugar-nucleotide have been poorly studied, although it is known that there are enzymes that hydrolyse ADPG both in plants (Rodríguez-López, M., Baroj a-Fernández, E., Zandueta-Criado, A., Pozueta -
Romero, J. (2000) "Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevenís starch biosynthesis". Proc. Nati. Acad. ScL, 97, 8705- 8710; Baroja-Fernández, E., Zandueta-Criado, A., Rodríguez-López, M., Akazawa, T.,Romero, J. (2000) "Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis". Proc. Nati Acad. ScL, 97, 8705-8710; Baroja-Fernández, E., Zandueta-Criado, A., Rodríguez-López, M., Akazawa, T.,
Pozueta-Romero, J. (2000) "Distinct isoforms of ADPglucose pyrophosphatase andPozueta-Romero, J. (2000) "Distinct isoforms of ADPglucose pyrophosphatase and
ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer
pseudoplatanus L.). FEBS Lett. 480, 277-282; PCTVESO 1/00021; PCT: ES03/00363) como en bacterias (Moreno-Bruna, B., Baraja-Fernández, E., Muñoz, F.J., Bastarrica- Berasategui, A., Zandueta-Crido, A., Rodríguez-López, M., Lasa, L, Akazawa, T., Pozueta-Romero, J. (2001) "Adenosine diphosphate sugar pyrophosphatase prevenís glycogen biosynthesis in Escherichia coli" Proc. Nati. Acad. Sci. 98, 8128-8132; PCT ES02/00174) sin embargo estas enzimas que hidrolizan ADPG pertenecen a una clasificación diferente al objeto de la presente invención. Además, se sabe que en bacterias existe un enzima hidrolítico de ADPG perteneciente a las "Nudix" hidrolasas (Bessman, MJ., Fricks, D.N. and OΗandley, S.F. (1996) The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, housecleaning enzymes. J. Biol. Chem. 271: 25059-25062) que tiene acceso al pool de ADPG necesario para la biosíntesis del glucógeno. Aunque se conoce la existencia de enzimas hidrolíticos de ADPG vegetales pertenecientes a la familia de las núcleotido pirofosfatasas-fosfodiesterasas (E.C. 2.4.1.21) (Francisco José Muñoz Pérez, Milagros Rodríguez López, Edurne Baraja Fernández, Javier Pozueta Romero, Toshiaki Mitsui, Yohei Nanjo (2003) "Plant Nucleotide sugar pyrophosphatase/phosphodiesterase (NPPase), Method of production, use in the manufacture of testing devices and its application in the production of transgenic plants", PCT/ES03/00363), todavía no se han descrito Nudix vegetales que hidrolizan ADPG. La identificación de Nudix vegetales que controlen el flujo metabólico hacia la biosíntesis del almidón mediante la hidrólisis del ADPG permitirá obtener plantas genéticamente modificadas con niveles alterados de almidón tras la alteración de la expresión de genes propios de la planta.ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.). FEBS Lett. 480, 277-282; PCTVESO 1/00021; PCT: ES03 / 00363) as in bacteria (Moreno-Bruna, B., Baraja-Fernández, E., Muñoz, FJ, Bastarrica- Berasategui, A., Zandueta-Crido, A., Rodríguez-López, M., Lasa , L, Akazawa, T., Pozueta-Romero, J. (2001) "Adenosine diphosphate sugar pyrophosphatase prevenís glycogen biosynthesis in Escherichia coli" Proc. Nati. Acad. Sci. 98, 8128-8132; PCT ES02 / 00174) however these enzymes that hydrolyse ADPG belong to a different classification to the object of the present invention. In addition, it is known that in bacteria there is an ADPG hydrolytic enzyme belonging to the "Nudix" hydrolases (Bessman, MJ., Fricks, DN and OΗandley, SF (1996) The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, housecleaning enzymes J. Biol. Chem. 271: 25059-25062) that has access to the ADPG pool necessary for glycogen biosynthesis. Although the existence of vegetable ADPG hydrolytic enzymes belonging to the pyrophosphatases phosphodiesterase nucleotide family (EC 2.4.1.21) is known (Francisco José Muñoz Pérez, Milagros Rodríguez López, Edurne Baraja Fernández, Javier Pozueta Romero, Toshiaki Mitsui, Yohei Nanjo (2003) "Plant Nucleotide sugar pyrophosphatase / phosphodiesterase (NPPase), Method of production, use in the manufacture of testing devices and its application in the production of transgenic plants", PCT / ES03 / 00363), no plant Nudix have yet been described that hydrolyse ADPG. The identification of plant Nudix that control the metabolic flow towards the biosynthesis of starch through the hydrolysis of ADPG will allow to obtain genetically modified plants with altered levels of starch after altering the expression of the plant's own genes.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
Breve descripción de Ia invenciónBrief description of the invention
En la presente invención se describe por primera vez la aplicación del producto enzimático de origen vegetal denominado ASPP "Adenosine Diphosphate SugarIn the present invention, the application of the enzymatic product of plant origin called ASPP "Adenosine Diphosphate Sugar" is described for the first time
Pyrophosphatase", (EC 3.6.1.21), perteneciente a la familia de enzimas Nudix hidrolasas
y con actividad hidrolítica de ADPG, en la consecución de plantas transgénicas con niveles alterados de almidón.Pyrophosphatase ", (EC 3.6.1.21), belonging to the family of enzymes Nudix hydrolases and with hydrolytic activity of ADPG, in the achievement of transgenic plants with altered levels of starch.
Así, por un lado la invención describe la producción de plantas transgénicas que sobre- expresan ASPP, de origen vegetal, las cuales, al igual que ocurre con las plantas que sobre-expresan ASPP de origen bacteriano (Baroja-Fernández, E., Muñoz, F.J., Zandueta-Criado, A., Morán-Zorzano, M.T., Viale, A.M., Etxeberria, E., Alonso-Casajús, N., Pozueta-Romero, J. (2004) "Most of ADPglucose linked to starch biosynthesis occurs outside the chloroplast in source leaves. Proc. Nati. Acad. Sel USA. 101, 13080-13085; PCT/ES02/00174), acumulan poco almidón. Teniendo, en cuenta la sensibilidad social y el rechazo existente ante las plantas que expresan genes bacterianos o víricos, esta invención supone un avance ya que la alteración de los niveles de almidón son debidos a la variación de la expresión de un gen existente en la planta.Thus, on the one hand the invention describes the production of transgenic plants that overexpress ASPP, of plant origin, which, as is the case with plants that overexpress ASPP of bacterial origin (Baroja-Fernández, E., Muñoz , FJ, Zandueta-Criado, A., Morán-Zorzano, MT, Viale, AM, Etxeberria, E., Alonso-Casajús, N., Pozueta-Romero, J. (2004) "Most of ADPglucose linked to starch biosynthesis occurs outside the chloroplast in source leaves. Proc. Nati. Acad. Sel USA. 101, 13080-13085; PCT / ES02 / 00174), accumulate little starch, taking into account the social sensitivity and rejection of plants that express genes Bacterial or viral, this invention represents a breakthrough since the alteration of starch levels is due to the variation in the expression of a gene existing in the plant.
Además, otro objetivo de la invención es la obtención de plantas con alto contenido en almidón tras reducir o anular la actividad endógena ASPP. Al igual que ocurre con las bacterias con baja actividad ASPP, estas plantas poseen altos niveles de almidón.In addition, another objective of the invention is to obtain plants with high starch content after reducing or canceling the endogenous ASPP activity. As with bacteria with low ASPP activity, these plants have high levels of starch.
Descripción de las figurasDescription of the figures
Figura 1Figure 1
Dendrograma de secuencias aminoacídicas "Nudix" de Arabidopsis thaliana y de E. coli (GenBank accession number AJ298136), H. influenzae (NP_438560), Homo sapiens (NM_016918) y Mus musculns (NM_014142). El dendrograma se obtuvo con el programa MegAlign (DNAStar, Madison, WI, USA). Las áreas sombreadas incluyen secuencias de Arabidopsis incluidas dentro del grupo ASPP.Dendrogram of "Nudix" amino acid sequences of Arabidopsis thaliana and E. coli (GenBank accession number AJ298136), H. influenzae (NP_438560), Homo sapiens (NM_016918) and Mus musculns (NM_014142). The dendrogram was obtained with the MegAlign program (DNAStar, Madison, WI, USA). Shaded areas include Arabidopsis sequences included within the ASPP group.
Figura 2 Comparación de secuencias aminoacídicas entre AtASPP y secuencias de arroz y patataFigure 2 Comparison of amino acid sequences between AtASPP and rice and potato sequences
(GenBank accession number Q9SNS9 and POADP80, respectivamente). El motivo Nudix está sombreado.
Figura 3(GenBank accession number Q9SNS9 and POADP80, respectively). The Nudix motif is shaded. Figure 3
Etapas de construcción del plásmido p At ASPP.Plasmid construction stages p At ASPP.
Figura 4Figure 4
Etapas de construcción del plásmido pStASPP.Construction stages of plasmid pStASPP.
Figura 5 Etapas de construcción del plásmido pET- AtASPP.Figure 5 Construction stages of plasmid pET-AtASPP.
Figura 6Figure 6
Etapas de construcción del plásmido pET-StASPPPET-StASPP Plasmid Construction Stages
Figura 7Figure 7
Etapas de construcción del plásmido p35S-AtASPP-NOS.Construction stages of plasmid p35S-AtASPP-NOS.
Figura 8Figure 8
Etapas de construcción del plásmido pBIN35 S-AtASPP-NOS.Construction stages of plasmid pBIN35 S-AtASPP-NOS.
Figura 9Figure 9
Etapas de construcción del plásmido pDONR/Zeo-StASPP.Construction stages of plasmid pDONR / Zeo-StASPP.
Figura 10 Etapas de construcción del plásmido pK2GW7,0-StASPP.Figure 10 Construction stages of plasmid pK2GW7,0-StASPP.
Figura 11Figure 11
Actividad hidrolítica de ADPG (A) y contenido en glucógeno (B) en bacterias transformadas con pET-28c(+) y con pET-StASPP.
Figura 12ADPG (A) hydrolytic activity and glycogen (B) content in bacteria transformed with pET-28c (+) and with pET-StASPP. Figure 12
Actividad hidrolítica de ADPG (A), contenido en almidón (B) y ADPG (C) en hojas de Arabidopsis y en diferentes clones de plantas transgénicas de Arabidopsis (8, 5, 3, 7) que sobre-expresan At4gll980 tras haber sido transformadas haciendo uso de la cepa DSM 18035.Hydrolytic activity of ADPG (A), starch content (B) and ADPG (C) in Arabidopsis leaves and in different clones of transgenic Arabidopsis plants (8, 5, 3, 7) that overexpress At4gll980 after being transformed making use of strain DSM 18035.
Figura 13Figure 13
Actividad hidrolítica de ADPG (A)5 contenido en almidón (B) y ADPG (C) en hojas de patata, y en diferentes clones de plantas transgénicas de patata (7,6,2) que sobre expresan POADP80 tras haber sido transformadas haciendo uso de la cepa DSM 18036.Hydrolytic activity of ADPG (A) 5 contained in starch (B) and ADPG (C) in potato leaves, and in different clones of transgenic potato plants (7,6,2) that express POADP80 after being transformed using of strain DSM 18036.
Descripción detallada de Ia invenciónDetailed description of the invention
Obtención de cDNAs que codifican para ASPPs vegetalesObtaining cDNAs that code for plant ASPPs
Las ASPPs de Arabidopsis (AtASPP), patata (StASPP) y arroz (OsASPP) están codificadas por At4gll980 (EMBL: AJ748742), POADP80 (EMBL: AMl 80509) y Q9SNS9, respectivamente. Estas secuencias tienen función desconocida pero fueron identificadas tras observar que presentan cierta homología con ASPPs de origen bacteriano y animal (Figura 1 y Figura 2). El conocimiento de las secuencias nucleotídicas de At4gll980, POADP80 y Q9SNS9 permitió la creación de cebadores específicos para amplificar por RT-PCR cDNAs completos que codifican para AtASPP, StASPP y OsASPP a partir de RNA total de hojas de Arabidopsis, patata y arroz, respectivamente. En el caso concreto de Arabidopsis y patata, los cDNAs se clonaron en el vector pGemT-easy (Promega), dando lugar a los plásmidos pAtASPP (Figura 3) y pStASPP (Figura 4). Los fragmentos amplificados se clonaron en el vector pET28c(+) (Novagen), dando lugar a los plásmidos pET-AtASPP (Figura 5) y pET-StASPP (Figura 6), respectivamente.
Obtención de ASPPs vegetales recombinantesThe ASPPs of Arabidopsis (AtASPP), potato (StASPP) and rice (OsASPP) are encoded by At4gll980 (EMBL: AJ748742), POADP80 (EMBL: AMl 80509) and Q9SNS9, respectively. These sequences have an unknown function but were identified after observing that they have a certain homology with ASPPs of bacterial and animal origin (Figure 1 and Figure 2). The knowledge of the nucleotide sequences of At4gll980, POADP80 and Q9SNS9 allowed the creation of specific primers to amplify by RT-PCR complete cDNAs that code for AtASPP, StASPP and OsASPP from total RNA of Arabidopsis, potato and rice leaves, respectively. In the specific case of Arabidopsis and potato, the cDNAs were cloned into the pGemT-easy vector (Promega), giving rise to plasmids pAtASPP (Figure 3) and pStASPP (Figure 4). The amplified fragments were cloned into the vector pET28c (+) (Novagen), giving rise to plasmids pET-AtASPP (Figure 5) and pET-StASPP (Figure 6), respectively. Obtaining recombinant plant ASPPs
pET-AtASPP y pET-StASPP fueron introducidos por electroporación en E. coli BL21(DE3). La sobre-expresión de AtASPP y StASPP tuvo lugar de manera independiente mediante la adición de 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) en 100 mi de cultivo celular. Tras seis horas de cultivo inducido se recogieron las bacterias y se resuspendieron en 6 mi de "binding buffer" (Novagen, His-bind purification kits), se sonicaron y se centrifugaron a 10.000 g durante diez minutos. Los sobrenadantes que contienen las ASPPs recombinantes con una cola de histidinas se hicieron pasar a través por una columna de afinidad del kit de purificación de proteínas "His-bind" de Novagen. Siguiendo las instrucciones del kit se eluyeron AtASPP y StASPP recombinantes con 6 mi del tampón de elución recomendado.pET-AtASPP and pET-StASPP were introduced by electroporation in E. coli BL21 (DE3). Over-expression of AtASPP and StASPP took place independently by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) in 100 ml of cell culture. After six hours of induced culture, the bacteria were collected and resuspended in 6 ml of "binding buffer" (Novagen, His-bind purification kits), sonicated and centrifuged at 10,000 g for ten minutes. Supernatants containing recombinant ASPPs with a histidine tail were passed through an affinity column of Novagen's "His-bind" protein purification kit. Following the instructions in the kit, recombinant AtASPP and StASPP were eluted with 6 ml of the recommended elution buffer.
Identificación del producto con actividad enzimática ASPP vegetalProduct identification with plant ASPP enzymatic activity
El producto enzimático ASPP vegetal, se identificó mediante los siguientes patrones funcionales:The plant ASPP enzyme product was identified by the following functional patterns:
B Es una "adenosine diphosphate-sugar pirofosfatase" (EC 3.6.1.21) que cataliza la hidrólisis del ADPG en cantidades equimolares de GlP y AMP.B It is an "adenosine diphosphate-sugar pyrophosphatase" (EC 3.6.1.21) that catalyzes the hydrolysis of ADPG in equimolar amounts of GlP and AMP.
B Además del ADPG, reconoce ADPmanosa y ADPribosa, pero no reconoce otros azúcares-nucleótidos tales como UDPglucosa, GDPmanosa, UDPglucorónico, etc. Tampoco reconoce nucleótidos tales como 5 '-phosphosulfate, ATP, ADP, UTP, GTP ni el sustrato artificial bis-paranitrofenil-fosfato utilizado para la caracterización de fosfodiesterasas.B In addition to ADPG, it recognizes ADPmanosa and ADPribosa, but does not recognize other sugar-nucleotides such as UDPglucose, GDPmanosa, UDPglucoronic, etc. It also does not recognize nucleotides such as 5'-phosphosulfate, ATP, ADP, UTP, GTP or the artificial bis-paranitrophenyl phosphate substrate used for the characterization of phosphodiesterases.
M No hidroliza moléculas con enlaces fosfomonoéster tales como la GlP, G6P, AMP, 3- fosfoglicerato, y otras similares. Tampoco hidroliza AMP cíclico, ni ácidos nucleicos de larga cadena, tales como ADN o ARN.
Obtención de plantas transgénicas que sobre-expresan ASPPs vegetalesM Does not hydrolyse molecules with phosphomonoester bonds such as GlP, G6P, AMP, 3-phosphoglycerate, and the like. It also does not hydrolyze cyclic AMP, or long-chain nucleic acids, such as DNA or RNA. Obtaining transgenic plants that overexpress plant ASPPs
Para la sobre-expresión de AtASPP, pET-AtASPP fue digerido secuencialmente con los enzimas Xhol, T4 DNA polimerasa y Ncol. El fragmento liberado (At4gll980) fue clonado en los sitios Ncóí/Smal de p35S-NOS (Baroj a-Fernández, E., Muñoz, F.J., Zandueta-Criado, A., Moran-Zorzano, M.T., Viale, A.M., Alonso-Casajus, N., Pozueta- Romero, J. (2004) Most of ADP-glucose linked to starch biosíntesis occurs otuside the chloroplast in source leaves. Proc. Nati. Acad. Sci. USA, 101, 13080-13085) dando lugar a p35S-AtASPP-NOS (Figura 7), el cual posee el promotor constitutivo 35S, At4gll980 y el terminador NOS. p35 S-AtASPP-NOS fue digerido secuencialmente con los enzimas HindIII y EcoRI y se clonó dentro del plásmido binario pBIN 20 (Hennegan, K., Danna, KJ. (1998) pBIN20: An improved binary vector for Agrobacterium-mediated transformation. Plant Molecular Biology Repórter 16, 129-131) que previamente había sido digerido secuencialmente con los enzimas HindIII y EcoRI. El plásmido así obtenido se designó con el nombre de pBIN35S-AtASPP-NOS (Figura 8).For the overexpression of AtASPP, pET-AtASPP was digested sequentially with the enzymes Xhol, T4 DNA polymerase and Ncol. The released fragment (At4gll980) was cloned into the Ncóí / Smal sites of p35S-NOS (Baroj a-Fernández, E., Muñoz, FJ, Zandueta-Criado, A., Moran-Zorzano, MT, Viale, AM, Alonso- Casajus, N., Pozueta- Romero, J. (2004) Most of ADP-glucose linked to starch biosynthesis occurs otuside the chloroplast in source leaves. Proc. Nati. Acad. Sci. USA, 101, 13080-13085) giving rise to p35S-AtASPP-NOS (Figure 7), which has the 35S constitutive promoter, At4gll980 and the NOS terminator. p35 S-AtASPP-NOS was sequentially digested with the enzymes HindIII and EcoRI and was cloned into the binary plasmid pBIN 20 (Hennegan, K., Danna, KJ. (1998) pBIN20: An improved binary vector for Agrobacterium-mediated transformation. Plant Molecular Biology Repórter 16, 129-131) that had previously been digested sequentially with the enzymes HindIII and EcoRI. The plasmid thus obtained was designated as pBIN35S-AtASPP-NOS (Figure 8).
Para la sobre-expresión de StASPP, se clonó en pDONR/Zeo (Invitrogen) un cDNA completo que codifica para StASPP, dando lugar al plásmido pDONR/Zeo-StASPP (Figura 9). A partir de pDONR/Zeo-StASPP y ρK2GW7,0 (Karimi, M., Inze, D., Depicker, A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7: 193-195) se obtuvo el plásmido pK2GW7,0-StASPP (Figura 10), el cual posee el promotor constitutivo 35S, un cDNA completo de POADP80 y el terminador 35S.For over-expression of StASPP, a complete cDNA encoding StASPP was cloned into pDONR / Zeo (Invitrogen), giving rise to plasmid pDONR / Zeo-StASPP (Figure 9). From pDONR / Zeo-StASPP and ρK2GW7.0 (Karimi, M., Inze, D., Depicker, A (2002) GATEWAY ™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7: 193-195) obtained plasmid pK2GW7,0-StASPP (Figure 10), which possesses the 35S constitutive promoter, a complete cDNA of POADP80 and the 35S terminator.
pBIN35 S-AtASPP-NOS y pK2GW7,0-StASPP fueron introducidos en Agrobacterium tumefaciens, dando lugar a las cepas DSM 18035 y DSM 18036, respectivamente, las cuales fueron utilizadas para transformar especies tales como Arabidopsis, patata, maíz y arroz. DSM 18035 y DSM 18036 fueron depositadas el 10.3.2006 en el "Germán National Resource Centre for Biological Material", sita en el DMSZ, Mascheroder Weg Ib D-38124 (Braunschweig, Alemania).
Obtención de plantas transgénicas deficientes en ASPPs vegetalespBIN35 S-AtASPP-NOS and pK2GW7,0-StASPP were introduced into Agrobacterium tumefaciens, giving rise to strains DSM 18035 and DSM 18036, respectively, which were used to transform species such as Arabidopsis, potato, corn and rice. DSM 18035 and DSM 18036 were deposited on 10.3.2006 at the "German National Resource Center for Biological Material", located at DMSZ, Mascheroder Weg Ib D-38124 (Braunschweig, Germany). Obtaining transgenic plants deficient in plant ASPPs
Plantas knockouts de At4gll980 de Arabidopsis thaliana se obtuvieron del "European Arabidopsis Stock Centre" Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen,At4gll980 knockout plants from Arabidopsis thaliana were obtained from the "European Arabidopsis Stock Center" Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen,
H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Check, R., Gadrinab, C,H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Check, R., Gadrinab, C,
Heller, C, Jeske, A., Koesema, E., Meyers, CC, Parker, H., Prednis, L., Ansari, Y.,Heller, C, Jeske, A., Koesema, E., Meyers, CC, Parker, H., Prednis, L., Ansari, Y.,
Choy, N., Denn, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C,Choy, N., Denn, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C,
Ndubaku, R., Schmidt, L, Weigel, D., Cárter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, CC, Ecker, J.R. (2003) Genome-Wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653-657).Ndubaku, R., Schmidt, L, Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, CC, Ecker, J.R. (2003) Genome-Wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653-657).
Determinación del contenido en azúcares solubles y almidónDetermination of soluble sugar and starch content
Los azúcares solubles se extrajeron según (Heim, U., Weber, H., Baumlein, H., Wobus, U. (1993) "A sucrose-synthase gene of V. Faha L. Expression pattern in developing seeds in relation to starch synthesis and metabolic regulation" Planta 191, 394-401). Glucosa, sacarosa, fructosa, glucosa- 1 -fosfato, glucosa-6-fosfato y ADPG fueron determinados haciendo uso de un HPLC ajustado a una columna CarboPac PAlO y un detector amperométrico DX500 (Baroja-Fernández, E., Muñoz, F. J., Saikusa, T., Rodríguez- Lopez, M., Akazawa, T., Pozueta-Romero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosíntesis in heterotrophic tisúes of plants, Plant CeIl Physiol. 44, 500-509). Además, el ADPG fue determinado haciendo uso de un sistema de HPLC ajustado a una columna Partisil-10-SAX (Muñoz, FJ., Baroja-Fernández, E., Morán-Zorzano, M.T., Viale, A.M., Etxeberria, E., Alonso- Casajús, N., Pozueta-Romero, J. (2005) Sucrose synthase controls the intracellular levéis of ADPglucose linked to transitory starch biosíntesis in source leaves. Plant CeIl Physiol. 46, 1366-1376). Para confirmar las medidas de ADPG, éste fue eluido de las columnas Partisil-10-SAX y CarboPac PAlO, enzimáticamente hidrolizado con ASPP de E. coli purificada y se midió su conversión en AMP y glucosa- 1 -fosfato. El almidón se midió haciendo uso de kits comerciales que se basan fundamentalmente en la acción de la amiloglucosidasa sobre las unidades de glucosa del almidón y posterior detección
espectrofotométrica del NADH producido tras una reacción enzimática acoplada (Rodríguez-López, M., Baroj a-Fernández, E., Zandueta-Criado, A., Pozueta-Romero, J. (2000) "Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis" Proc. Nati. Acad. Sci. USA 97, 8705-8710).Soluble sugars were extracted according to (Heim, U., Weber, H., Baumlein, H., Wobus, U. (1993) "A sucrose-synthase gene of V. Faha L. Expression pattern in developing seeds in relation to starch synthesis and metabolic regulation "Plant 191, 394-401). Glucose, sucrose, fructose, glucose-1-phosphate, glucose-6-phosphate and ADPG were determined using an HPLC fitted to a CarboPac PAlO column and a DX500 amperometric detector (Baroja-Fernández, E., Muñoz, FJ, Saikusa , T., Rodríguez- Lopez, M., Akazawa, T., Pozueta-Romero, J. (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissue of plants, Plant CeIl Physiol. 44, 500-509). In addition, the ADPG was determined using an HPLC system adjusted to a Partisil-10-SAX column (Muñoz, FJ., Baroja-Fernández, E., Morán-Zorzano, MT, Viale, AM, Etxeberria, E., Alonso- Casajús, N., Pozueta-Romero, J. (2005) Sucrose synthase controls the intracellular levéis of ADPglucose linked to transitory starch biosynthesis in source leaves. Plant CeIl Physiol. 46, 1366-1376). To confirm the ADPG measurements, it was eluted from the Partisil-10-SAX and CarboPac PAlO columns, enzymatically hydrolyzed with purified E. coli ASPP and its conversion to AMP and glucose-1-phosphate was measured. Starch was measured using commercial kits that are primarily based on the action of amyloglucosidase on starch glucose units and subsequent detection NADH spectrophotometric produced after a coupled enzymatic reaction (Rodríguez-López, M., Baroj a-Fernández, E., Zandueta-Criado, A., Pozueta-Romero, J. (2000) "Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis "Proc. Nati. Acad. Sci. USA 97, 8705-8710).
Así, en un primer aspecto la presente invención se refiere a un procedimiento para la obtención de plantas transgénicas, las cuales presentan una expresión alterada, bien sea una sobre-expresión o una infra-expresión, del producto enzimático de origen vegetal ASPP (Adenosine Diphosphate Sugar Pyrophosphatase) en el interior de sus células con respecto a la expresión de dicho enzima en la planta silvestre. El procedimiento comprende la transformación de la planta silvestre con un vector que comprenda alguna de las siguientes secuencias de ADN:Thus, in a first aspect the present invention relates to a process for obtaining transgenic plants, which have an altered expression, either an over-expression or an under-expression, of the plant-derived enzymatic product ASPP (Adenosine Diphosphate Sugar Pyrophosphatase) inside its cells with respect to the expression of said enzyme in the wild plant. The procedure comprises the transformation of the wild plant with a vector comprising any of the following DNA sequences:
a) Una secuencia que codifique para los polipéptidos mostrados en SEQ ID NO: 3,a) A sequence encoding the polypeptides shown in SEQ ID NO: 3,
SEQ ID NO: 6 ó SEQ ID NO: 9. b) Secuencias nucletídicas SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9.; c) Secuencias que hibriden con las definidas en "a" o "b" y que codifiquen para un producto enzimático con actividad ASPP; d) Secuencias que difieran de las definidas en "a", "b" o "c" debido a la degeneración del código genético.SEQ ID NO: 6 or SEQ ID NO: 9. b) Nucletide sequences SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 .; c) Sequences that hybridize with those defined in "a" or "b" and that code for an enzymatic product with ASPP activity; d) Sequences that differ from those defined in "a", "b" or "c" due to the degeneracy of the genetic code.
En este aspecto, se considera sobre-expresión del producto enzimático de origen vegetal ASPP cuando se consigue una expresión mayor o igual al doble de la expresión conseguida en la planta silvestre (wt). Por otro lado se considera infra-expresión cuando se consigue una expresión menor o igual al 80% de la expresión conseguida en la planta silvestre (wt).In this regard, over-expression of the ASPP plant-based enzyme product is considered when an expression greater than or equal to twice the expression achieved in the wild plant (wt) is achieved. On the other hand it is considered infra-expression when an expression less than or equal to 80% of the expression achieved in the wild plant (wt) is achieved.
En una realización preferida de la presente invención el procedimiento de transformación de la planta silvestre arriba reseñado se lleva a cabo utilizando como vector de transformación Agrobacterium tumefaciens.
En una realización aún más preferida de la presente invención el vector de transformación utilizado, en el procedimiento para la obtención de plantas transgénicas, es Agrobacterium tumefaciens concretamente las cepas DSM 18035 o DSM 18036.In a preferred embodiment of the present invention, the transformation process of the wild plant described above is carried out using Agrobacterium tumefaciens as a transformation vector. In an even more preferred embodiment of the present invention the transformation vector used, in the process for obtaining transgenic plants, is specifically Agrobacterium tumefaciens strains DSM 18035 or DSM 18036.
En un segundo aspecto de la presente invención se refiere a un producto enzimático de origen vegetal con actividad ASPP que contiene una secuencia aminoacídica seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9 u otra secuencia derivada por degeneración del código genético de éstas.In a second aspect of the present invention it refers to an enzymatic product of plant origin with ASPP activity that contains an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived by degeneration of their genetic code.
En una realización preferida la presente invención se refiere a un producto enzimático de origen vegetal con actividad ASPP que contiene una secuencia aminoacídica seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9 u otra secuencia derivada por degeneración del código genético de éstas, caracterizado por no hidrolizar moléculas del grupo comprendido por: GlP5 G6P, AMP, 3-fosfoglicerato, AMPc, UDPglucosa, GDPglucosa, adenosina 5'-phosphosulfato, bis-p-nitrofenil-fosfato o ácidos nucleicos de larga cadena.In a preferred embodiment the present invention relates to an enzymatic product of plant origin with ASPP activity containing an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived from degeneration of the genetic code of these, characterized by not hydrolyzing molecules of the group comprised of: GlP 5 G6P, AMP, 3-phosphoglycerate, cAMP, UDPglucose, GDPglucose, adenosine 5'-phosphosulfate, bis-p-nitrophenyl phosphate or long chain nucleic acids .
En otra realización preferida la presente invención se refiere a un producto enzimático de origen vegetal con actividad ASPP que contiene una secuencia aminoacídica seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9 u otra secuencia derivada por degeneración del código genético de éstas, caracterizado por no hidrolizar moléculas del grupo comprendido por: GlP5 G6P, AMP, 3-fosfoglicerato, AMPc5 In another preferred embodiment the present invention relates to an enzymatic product of plant origin with ASPP activity containing an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived by degeneration of the genetic code of these, characterized by not hydrolyzing molecules of the group comprised of: GlP 5 G6P, AMP, 3-phosphoglycerate, cAMP 5
UDPglucosa, GDPglucosa, adenosina 5'-phosphosulfato, bis-p-nitrofenil-fosfato o ácidos nucleicos de larga cadena y por presentar un peso molecular aparente determinado por filtración en gel entre 70-75 kDa y que en gel desnaturalizante migra como una proteína de entre 35-40 kDa.UDPglucose, GDPglucose, adenosine 5'-phosphosulfate, bis-p-nitrophenyl phosphate or long chain nucleic acids and for presenting an apparent molecular weight determined by gel filtration between 70-75 kDa and that in denaturing gel migrates as a protein of between 35-40 kDa.
Otro aspecto de la presente invención se refiere al uso de la secuencia de cDNA seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9, o cualquier otra secuencia derivada de éstas por degeneración del código genético, o que pueda hibridar
con alguna de ellas, en la obtención de plantas transgénicas que sobre-expresen o infra- expresen el producto enzimático de origen vegetal ASPP.Another aspect of the present invention relates to the use of the cDNA sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9, or any other sequence derived therefrom by degeneracy of the genetic code, or that can hybridize with some of them, in obtaining transgenic plants that overexpress or under-express the enzymatic product of vegetable origin ASPP.
Otro aspecto de la invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP.Another aspect of the invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP.
En una realización preferida de la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es del género Arabidopsis, preferentemente Arabidopsis thaliana.In a preferred embodiment of the present invention it refers to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is of the genus Arabidopsis, preferably Arabidopsis thaliana.
En otra realización preferida de la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es del género Solanum, preferentemente Solanum tuberosum.In another preferred embodiment of the present invention it refers to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the enzyme product of plant origin, ASPP, where said plant is of the genus Solanum, preferably Solanum tuberosum.
En otra realización preferida la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es del género Nicotíana, preferentemente Nicotiana tabacum.In another preferred embodiment, the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, wherein said plant is of the genus Nicotiana, preferably Nicotiana tabacum.
En otra realización preferida la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es del género Lycopersicom, preferentemente Lycopersicom sculentum.In another preferred embodiment the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, wherein said plant is of the genus Lycopersicom, preferably Lycopersicom sculentum.
En otra realización preferida la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es del género Oryza, preferentemente Oryza sativa.
En otra realización preferida la presente invención se refiere a una planta transgénica y su progenie, que comprende la sobre-expresión o la infra-expresión del producto enzimático de origen vegetal, ASPP, donde dicha planta es Zea mays.In another preferred embodiment the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is of the genus Oryza, preferably Oryza sativa. In another preferred embodiment the present invention relates to a transgenic plant and its progeny, comprising the overexpression or infra-expression of the plant-derived enzyme product, ASPP, where said plant is Zea mays.
En otro aspecto de la presente invención se refiere al uso de una planta transgénica y su progenie, que comprende la sobre-expresión del producto enzimático de origen vegetal ASPP, como alimento dietético o en la fabricación de productos dietéticos con bajo contenido en almidón.In another aspect of the present invention it refers to the use of a transgenic plant and its progeny, which comprises the overexpression of the ASPP plant-based enzyme product, as a dietary food or in the manufacture of low starch dietary products.
A este respecto se considera bajo contenido en almidón cuando la planta transgénica presenta un contenido en almidón sustancialmente menor al contenido que presenta la planta silvestre (wt). En el caso concreto de Arabidopsis (figura 12) se considera bajo contenido en almidón cuando la planta transgénica presenta un contenido en almidón menor al 50% del contenido en almidón que presenta la planta silvestre (wt). En el caso de la patata (figura 13) se considera bajo contenido en almidón cuando la planta transgénica presenta un contenido en almidón menor al 70% del contenido en almidón que presenta la planta silvestre (wt). Estos valores tienen carácter meramente indicativo.In this regard, it is considered low starch content when the transgenic plant has a starch content substantially less than the content of the wild plant (wt). In the specific case of Arabidopsis (figure 12), it is considered low starch content when the transgenic plant has a starch content less than 50% of the starch content of the wild plant (wt). In the case of potatoes (Figure 13), it is considered low starch content when the transgenic plant has a starch content of less than 70% of the starch content of the wild plant (wt). These values are merely indicative.
En un último aspecto de la presente invención se refiere al uso de una planta transgénica y su progenie, que comprende la infra-expresión del producto enzimático de origen vegetal ASPP, para la producción de almidón.In a final aspect of the present invention it refers to the use of a transgenic plant and its progeny, which comprises the infra-expression of the ASPP plant-derived enzyme product, for the production of starch.
A continuación se procede a la exposición de los ejemplos. La exposición detallada de los modos de realización, ejemplos y de las figuras que siguen se proporcionan a modo de ilustración y no pretenden ser limitantes de la presente invención.The examples are presented below. The detailed exposition of the embodiments, examples and figures that follow are provided by way of illustration and are not intended to be limiting of the present invention.
EJEMPLOS DE REALIZACIÓN DE LA INVENCIÓNEXAMPLES OF EMBODIMENT OF THE INVENTION
Ejemplo 1: Obtención de cDNAs completos que codifican para AtASPP, StASPP y OsASPP
El conocimiento de la secuencia nucleotídica del gen que codifica para AtASPP permitió la creación de dos cebadores específicos cuyas secuencias son, en sentido 5' - 3', SEQ ID NO: 1 y SEQ ID NO: 2. Haciendo uso de estos cebadores y de RNA de hojas de arabidopsis se amplificó por métodos convencionales de RT-PCR un cDNA completo de At4gll980, que se clonó en pGemT-easy (Promega). Las secuencias nucleotídica del DNA amplificado y la secuencia aminoacídica deducida se representan en SEQ ID NO: 3.Example 1: Obtaining complete cDNAs coding for AtASPP, StASPP and OsASPP The knowledge of the nucleotide sequence of the gene coding for AtASPP allowed the creation of two specific primers whose sequences are, in the 5'-3 'sense, SEQ ID NO: 1 and SEQ ID NO: 2. Using these primers and Arabidopsis leaf RNA was amplified by conventional RT-PCR methods a complete cDNA of At4gll980, which was cloned into pGemT-easy (Promega). The nucleotide sequences of the amplified DNA and the deduced amino acid sequence are depicted in SEQ ID NO: 3.
El conocimiento de la secuencia nucleotídica del gen que codifica para StASPP permitió la creación de dos cebadores específicos cuyas secuencias son, en sentido 5' - 3', SEQ ID NO: 4 y SEQ ID NO: 5. Haciendo uso de estos cebadores y de RNA de hojas de patata se amplificó por métodos convencionales de RT-PCR un cDNA completo de POADP80, que se introdujo en pGemT-easy. La secuencia nucleotídica del DNA amplificado y la secuencia aminoacídica deducida se representan en SEQ ID NO: 6.The knowledge of the nucleotide sequence of the gene coding for StASPP allowed the creation of two specific primers whose sequences are, in the 5'-3 'sense, SEQ ID NO: 4 and SEQ ID NO: 5. Using these primers and Potato leaf RNA was amplified by conventional RT-PCR methods a complete cDNA of POADP80, which was introduced into pGemT-easy. The nucleotide sequence of the amplified DNA and the deduced amino acid sequence are depicted in SEQ ID NO: 6.
El conocimiento de la secuencia nucleotídica del gen que codifica para OsASPP, permitió la creación de dos cebadores específicos cuyas secuencias son, en sentido 5' - 3', SEQ ID NO: 7 y SEQ ID NO: 8. Haciendo uso de estos cebadores y de RNA de hojas de arroz se amplificó por métodos convencionales de RT-PCR un cDNA completo de Q9SNS9, que se introdujo en pGemT-easy. La secuencia nucleotídica del DNA y la secuencia aminoacídica deducida se representan en SEQ ID NO: 9.The knowledge of the nucleotide sequence of the gene coding for OsASPP, allowed the creation of two specific primers whose sequences are, in the 5'-3 'sense, SEQ ID NO: 7 and SEQ ID NO: 8. Using these primers and Rice leaf RNA was amplified by conventional RT-PCR methods a complete cDNA of Q9SNS9, which was introduced into pGemT-easy. The nucleotide sequence of the DNA and the deduced amino acid sequence are represented in SEQ ID NO: 9.
Ejemplo 2: Obtención de ASPPs vegetales recombinantesExample 2: Obtaining Recombinant Plant ASPPs
Tras seis horas de cultivo en presencia de 1 mM IPTG, BL21(DE3) transformadas con pET-AtASPP o pET-StASPP se recogieron, se resuspendieron en el tampón "His-bind" (Novagen) de purificación de proteínas, y se lisaron mediante sonicación. Los sobrenadantes obtenidos tras la centrifugación de los Usados se pasaron por una columna de afinidad del kit de purificación de proteinas "His-bind" de Novagen. Las bacterias transformadas con pET-AtASPP o pET-StASPP poseen una actividad hidrolítica de ADPG elevada y un bajo contenido en glucógeno (Figura 11).
Ejemplo 3: Identificación del producto con actividad enzimática obtenidoAfter six hours of culture in the presence of 1 mM IPTG, BL21 (DE3) transformed with pET-AtASPP or pET-StASPP were collected, resuspended in the "His-bind" (Novagen) buffer for protein purification, and lysed by sonication The supernatants obtained after centrifugation of the Used ones were passed through an affinity column of the Novagen "His-bind" protein purification kit. Bacteria transformed with pET-AtASPP or pET-StASPP have a high ADPG hydrolytic activity and a low glycogen content (Figure 11). Example 3: Identification of the product with enzymatic activity obtained
El producto así obtenido cumple las siguientes características:The product thus obtained meets the following characteristics:
• La ASPP vegetal es una pirofosfatasa que cataliza la hidrólisis del ADPG produciendo cantidades equimolares de GlP y AMP.• Vegetable ASPP is a pyrophosphatase that catalyzes the hydrolysis of ADPG producing equimolar amounts of GlP and AMP.
B Además del ADPG, también hidroliza ADPribosa y ADPmanosa.B In addition to ADPG, it also hydrolyses ADPribosa and ADPmanosa.
B No hidroliza moléculas con enlaces fosfomonoéster tales como la GlP, G6P, AMP, 3- fosfoglicerato, y otras simila-res. Tampoco hidrolizan AMP cíclico ni ácidos nucleicos de larga cadena tales como ADN y ARN.B Does not hydrolyse molecules with phosphomonoester bonds such as GlP, G6P, AMP, 3-phosphoglycerate, and the like. Nor do they hydrolyze cyclic AMP or long chain nucleic acids such as DNA and RNA.
B Requiere magnesio para su funcionamiento óptimo.B Requires magnesium for optimal functioning.
• No hidroliza bis-para-nitrofenil-fosfato.• Does not hydrolyse bis-para-nitrophenyl phosphate.
M Peso molecular aparente de la proteína purificada en geles desnaturalizantes, en torno a 35-40 kDa.M Apparent molecular weight of the purified protein in denaturing gels, around 35-40 kDa.
B Peso molecular aparente medido por filtración en gel, en torno a 70-75 kDa, indicando que posiblemente se trate de un homodímero.B Apparent molecular weight measured by gel filtration, around 70-75 kDa, indicating that it is possibly a homodimer.
Ejemplo 4: Obtención de plantas transgénicas que sobre expresan ASPPs vegetalesExample 4: Obtaining transgenic plants that express plant ASPPs
Utilizando las cepas de Agrobacterium tumefaciens DSM 18035 y DSM 18036 se obtuvieron plantas de arabidosis (Arabidosis thalianá) tabaco (Nicotiana tabacum), patata (Solanum tuberosum), tomate (Lycopersicon sculentutri), arroz (Oryza sativa) y maíz {Zea mays) que sobrexpresan AtASPP (At4gll980) y StASPP (POADP80), respectivamente. Estas plantas se caracterizan por poseer una actividad ASPP en todos
los órganos analizados (raíz, hoja, frutos, tubérculos y tallo) 6-20 veces superior a la ASPP endógena existente en las plantas no transformadas (Figuras 12A y 13A). Además, presentaron las siguientes características fenotípicas:Using the strains of Agrobacterium tumefaciens DSM 18035 and DSM 18036, plants of arabidosis (Arabidosis thalianá) tobacco (Nicotiana tabacum), potato (Solanum tuberosum), tomato (Lycopersicon sculentutri), rice (Oryza sativa) and corn {Zea mays) were obtained. overexpress AtASPP (At4gll980) and StASPP (POADP80), respectively. These plants are characterized by having an ASPP activity in all the organs analyzed (root, leaf, fruits, tubers and stem) 6-20 times higher than the endogenous ASPP existing in non-transformed plants (Figures 12A and 13A). In addition, they presented the following phenotypic characteristics:
1. Bajo contenido en almidón (Figuras 12B y 13B).1. Low starch content (Figures 12B and 13B).
2. Bajo contenido en ADPG (Figuras 12C y 13C).2. Low content in ADPG (Figures 12C and 13C).
3. Niveles normales de azúcares solubles totales (Tabla 1).3. Normal levels of total soluble sugars (Table 1).
La morfología externa de estas plantas no es aberrante, tras ser comparada con la de las plantas no transformadas.
The external morphology of these plants is not aberrant, after being compared with that of non-transformed plants.
Tabla 1: Contenido en azúcares (en nanomoles/ gramo de peso fresco) en hojas de Arabidopsis no transformadas y en 4 clones independientes de plantas transformadas con la construcción 35 S- At ASPP-NOS .Table 1: Sugar content (in nanomoles / gram of fresh weight) in unprocessed Arabidopsis leaves and in 4 independent clones of transformed plants with the 35 S-At ASPP-NOS construction.
Ejemplo 5: Plantas con alto contenido en almidón como resultado de la baja actividad ASPP vegetalExample 5: Plants with high starch content as a result of low plant ASPP activity
Plantas knockouts de At4gll980 de Arabidopsis thaliana poseen una actividad ASPP inferior a la observada en plantas control. Además, estas plantas presentaron las siguientes caracerísticas:Knockouts of At4gll980 from Arabidopsis thaliana have an ASPP activity lower than that observed in control plants. In addition, these plants presented the following features:
1. Alto contenido en almidón.1. High starch content.
2. Niveles normales de azúcares solubles totales.
La morfología extema de las plantas deficitarias en AtASPP no es aberrante, tras ser comparada con la de las plantas control.
2. Normal levels of total soluble sugars. The external morphology of the deficit plants in AtASPP is not aberrant, after being compared with that of the control plants.
Claims
1. Procedimiento para la obtención de plantas transgénicas, las cuales presentan una sobre-expresión o una infra-expresión del producto enzimático de origen vegetal ASPP (Adenosine Diphosphate Sugar Pyrophosphatase) en el interior de sus células, que comprende la transformación de la planta silvestre con un vector que comprenda alguna de las siguientes secuencias de ADN:1. Procedure for obtaining transgenic plants, which have an overexpression or an under-expression of the enzymatic product of vegetable origin ASPP (Adenosine Diphosphate Sugar Pyrophosphatase) inside its cells, which includes the transformation of the wild plant with a vector comprising any of the following DNA sequences:
a) Una secuencia de cADN que codifique para los polipéptidos mostrados en SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9. b) Secuencias nucletídicas de cADN representadas por SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9. c) Secuencias de ADN que hibriden con las definidas en "a" o "b" y que codifiquen para un producto enzimático con actividad ASPP; d) Secuencias de ADN que difieran de las definidas en "a", "b" o "c" debido a la degeneración del código genético.a) A cDNA sequence encoding the polypeptides shown in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9. b) Nucleitic cDNA sequences represented by SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9. c) DNA sequences that hybridize with those defined in "a" or "b" and that code for an enzymatic product with ASPP activity; d) DNA sequences that differ from those defined in "a", "b" or "c" due to the degeneracy of the genetic code.
2. Procedimiento según la reivindicación 1, donde el vector de transformación utilizado es Agrobacterinm tumefaciens.2. Method according to claim 1, wherein the transformation vector used is Agrobacterinm tumefaciens.
3. Procedimiento según la reivindicación 2, donde el vector comprende las cepas DSM 18035 o DSM 18036.3. Method according to claim 2, wherein the vector comprises strains DSM 18035 or DSM 18036.
4. Producto enzimático de origen vegetal con actividad ASPP que contiene una secuencia arninoacídica seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó SEQ ID NO: 9 u otra secuencia derivada por degeneración del código genético de éstas.4. Enzymatic product of plant origin with ASPP activity that contains an arninoacidic sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 9 or another sequence derived by degeneration of their genetic code.
5. Producto enzimático origen vegetal según la reivindicación 4, caracterizado por no hidrolizar moléculas del grupo comprendido por: GlP5 G6P, AMP, 3- fosfoglicerato, AMPc, UDPglucosa, GDPglucosa, adenosina 5 '-phosphosulfato, bis-p-nitrofenil-fosfato o ácidos nucleicos de larga cadena.5. Plant-based enzyme product according to claim 4, characterized in that it does not hydrolyse molecules of the group comprising: GlP 5 G6P, AMP, 3- phosphoglycerate, cAMP, UDPglucose, GDPglucose, 5'-phosphosulfate adenosine, bis-p-nitrophenyl phosphate or long chain nucleic acids.
6. Producto enzimático origen vegetal según la reivindicación 4 ó 5, caracterizado por presentar un peso molecular aparente determinado por filtración en gel entre 70-75 kDa y que en gel desnaturalizante migra como una proteína de entre 35-40 kDa.6. Plant-based enzyme product according to claim 4 or 5, characterized in that it has an apparent molecular weight determined by gel filtration between 70-75 kDa and which in denaturing gel migrates as a protein between 35-40 kDa.
7. Uso de la secuencia de cDNA seleccionada entre SEQ ID NO: 3, SEQ ID NO: 6 ó7. Use of the cDNA sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 or
SEQ ID NO: 9, o cualquier otra secuencia derivada de éstas por degeneración del código genético, o que pueda hibridar con alguna de las anteriores, en la obtención de plantas transgénicas que sobre-expresen o infra-expresen el producto enzimático de origen vegetal ASPP.SEQ ID NO: 9, or any other sequence derived from them by degeneration of the genetic code, or that can hybridize with any of the above, in obtaining transgenic plants that overexpress or under-express the plant-derived enzyme product ASPP .
8. Planta transgénica y su progenie obtenibles según el procedimiento de las reivindicaciones 1-3, que comprende la sobre-expresión del producto enzimático de origen vegetal, ASPP.8. A transgenic plant and its progeny obtainable according to the procedure of claims 1-3, comprising the over-expression of the enzymatic product of plant origin, ASPP.
9. Planta transgénica y su progenie obtenibles según el procedimiento de las reivindicaciones 1-3, que comprende la mira-expresión del producto enzimático de origen vegetal, ASPP.9. Transgenic plant and its progeny obtainable according to the procedure of claims 1-3, which comprises the look-expression of the enzyme product of plant origin, ASPP.
10. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es del género Arabidopsis, preferentemente Arabidopsis thaliana.10. Transgenic plant and its progeny, according to any of claims 8-9, wherein said plant is of the genus Arabidopsis, preferably Arabidopsis thaliana.
11. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es del género Solanum, preferentemente Solanum tuberosum.11. Transgenic plant and its progeny according to any of claims 8-9, wherein said plant is of the genus Solanum, preferably Solanum tuberosum.
12. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es del género Nicotiana, preferentemente Nicotiana tabacum. 12. Transgenic plant and its progeny, according to any of claims 8-9, wherein said plant is of the genus Nicotiana, preferably Nicotiana tabacum.
13. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es del género Lycopersicom, preferentemente Lycopersicom sculentum.13. Transgenic plant and its progeny according to any of claims 8-9, wherein said plant is of the genus Lycopersicom, preferably Lycopersicom sculentum.
14. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es del género Oryza, preferentemente Oryza sativa.14. Transgenic plant and its progeny, according to any of claims 8-9, wherein said plant is of the genus Oryza, preferably Oryza sativa.
15. Planta transgénica y su progenie, según cualquiera de las reivindicaciones 8-9, donde dicha planta es Zea mays.15. Transgenic plant and its progeny, according to any of claims 8-9, wherein said plant is Zea mays.
16. Uso de una planta transgénica y su progenie, según cualquiera de las reivindicaciones 8 y 10-15, como alimento dietético, o en la fabricación de productos dietéticos, con bajo contenido en almidón.16. Use of a transgenic plant and its progeny, according to any of claims 8 and 10-15, as a dietary food, or in the manufacture of dietary products, with low starch content.
17. Uso de una planta transgénica, según cualquiera de las reivindicaciones 9-15, para la producción de almidón. 17. Use of a transgenic plant, according to any of claims 9-15, for the production of starch.
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Non-Patent Citations (6)
Title |
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ALONSO J.M. ET AL.: "Genome-wide insertional mutagenesis of Arabidopsis thaliana", SCIENCE, vol. 301, no. 5633, January 2003 (2003-01-01), pages 653 - 657, XP002300984, DOI: doi:10.1126/science.1086391 * |
BAROJA-FERNANDEZ E. ET AL.: "Most of ADP-glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 101, no. 35, August 2004 (2004-08-01), pages 13080 - 13085 * |
DATABASE UNIPROT [online] OGAWA T. ET AL.: "Comprehensive analysis of cytosolic Nudix hydrolase in Arabisopsis thaliana", Database accession no. (Q9SZ63) * |
DATABASE UNIPROT [online] SASAKI T. ET AL.: "Oryza sativa niponbarne (GA3) genomic DNA, chromosome 6, PAC clone: P0538C01", Database accession no. (Q9SNS9) * |
MUNOZ F.-J. ET AL.: "Cloning, expression and characterization of a Nudix hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to starch biosynthesis in Arabidopsis thaliana", PLANT & CELL PHYSIOLOGY, vol. 47, no. 7, July 2006 (2006-07-01), pages 926 - 934 * |
THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 26, July 2005 (2005-07-01), pages 25277 - 25283 * |
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