ES2275365B1 - DNA MOLECULA THAT CODIFIES A CHLAMYDOMONAS P-HYDROXYPHYLENE PIRUVATE DIOXYGENASE AND ITS APPLICATIONS. - Google Patents
DNA MOLECULA THAT CODIFIES A CHLAMYDOMONAS P-HYDROXYPHYLENE PIRUVATE DIOXYGENASE AND ITS APPLICATIONS. Download PDFInfo
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- ES2275365B1 ES2275365B1 ES200301759A ES200301759A ES2275365B1 ES 2275365 B1 ES2275365 B1 ES 2275365B1 ES 200301759 A ES200301759 A ES 200301759A ES 200301759 A ES200301759 A ES 200301759A ES 2275365 B1 ES2275365 B1 ES 2275365B1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000008557 oxygen metabolism Effects 0.000 description 1
- KXFJZKUFXHWWAJ-UHFFFAOYSA-N p-hydroxybenzoylformic acid Natural products OC(=O)C(=O)C1=CC=C(O)C=C1 KXFJZKUFXHWWAJ-UHFFFAOYSA-N 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 108700022487 rRNA Genes Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000015639 rosmarinus officinalis Nutrition 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 150000003772 α-tocopherols Chemical class 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physiology (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Molécula de ADN que codifica una p-hidroxifenilpiruvato dioxigenasa de chlamydomonas y sus aplicaciones. Dicha molécula comprende (a) una secuencia de ADN identificada por SEQ ID No.1, o (b) una secuencia de ADN análoga a la misma que (i) es sustancialmente homóloga a ella, y/o que (ii) codifica para un polipéptido que es sustancialmente homólogo a la proteína codificada por dicha secuencia de ADN definida en (a). Dicha molécula introducida en una planta productora de tocoferoles por técnicas habituales en biotecnología, permite aumentar el contenido de vitamina E total en dicha planta, así como el contenido en {al} y {ga}-tocoferol, entre otras aplicaciones, como aumentar la resistencia a ciertos herbicidas seleccionados del grupo formado por las tricetonas y los isoxazoles.DNA molecule encoding a chlamydomonas p-hydroxyphenylpyruvate dioxygenase and its applications. Said molecule comprises (a) a DNA sequence identified by SEQ ID No.1, or (b) a DNA sequence analogous to it that (i) is substantially homologous to it, and / or that (ii) encodes for a polypeptide that is substantially homologous to the protein encoded by said DNA sequence defined in (a). Said molecule introduced into a tocopherol producing plant by usual techniques in biotechnology, allows to increase the total vitamin E content in said plant, as well as the content in {al} and {ga} -tocopherol, among other applications, such as increasing resistance to certain herbicides selected from the group consisting of tricetons and isoxazoles.
Description
Molécula de ADN que codifica una p-hidroxifenilpiruvato dioxigenasa de Chlamydomonas y sus aplicaciones.DNA molecule encoding a Chlamydomonas p-hydroxyphenylpyruvate dioxygenase and its applications
La presente invención se encuadra dentro del campo de la biotecnología. Más específicamente la invención se refiere a una molécula de ADN que codifica una p-hidroxifenilpiruvato dioxigenasa de Chlamydomonas y al empleo de la misma tanto para modular la expresión de tocoferoles, como para obtener plantas transgénicas resistentes a ciertos herbicidas. La invención también se refiere al contenido de vitamina E total, \gamma- y \alpha-tocoferol obtenido por represión, expresión y traducción de dicha molécula de ADN.The present invention fits within the biotechnology field. More specifically the invention is refers to a DNA molecule that encodes a Chlamydomonas p-hydroxyphenylpyruvate dioxygenase and the use of it both to modulate the expression of tocopherols, such as to obtain transgenic plants resistant to certain herbicides The invention also relates to the content of total vitamin E, γ- and α-tocopherol obtained by repression, expression and translation of said molecule of DNA
Los radicales libres son compuestos muy reactivos producidos en los organismos vivos en procesos normales como consecuencia del metabolismo del oxígeno. Unos niveles normales de radicales libres en nuestras células tienen cierto papel beneficioso en el organismo; en cambio, unos niveles elevados son perjudiciales para la salud. Los radicales libres pueden atacar a los ácidos grasos poliinsaturados de los fosfolípidos de membrana y dañar así la estructura y funciones de las membranas celulares, también causan daños al ADN y a las proteínas. Los radicales libres conducen a un estrés oxidativo que está implicado en el desarrollo de una serie de enfermedades relacionadas con la edad como la debilitación cognitiva y la enfermedad de Alzheimer, la debilidad del sistema inmune, cataratas, arteriosclerosis, cáncer y artritis (Cross, 1987). Existen numerosos factores ambientales que pueden inducir una elevada producción de radicales libres; entre estos se encuentran las radiaciones, los humos y los pesticidas (Jacobson, 1987; Halliwell, 1996) (los datos completos de éstas y las demás citas bibliográficas se dan al final de la descripción para no hacer demasiado farragosa la presente exposición).Free radicals are very compound reagents produced in living organisms in normal processes as a consequence of oxygen metabolism. Some levels normal free radicals in our cells have true beneficial role in the body; instead, high levels They are harmful to health. Free radicals can attack to polyunsaturated fatty acids of phospholipids of membrane and thus damage the structure and functions of the membranes cellular, also cause damage to DNA and proteins. The Free radicals lead to oxidative stress that is involved in the development of a series of diseases related to age such as cognitive impairment and Alzheimer's disease, weakness of the immune system, cataracts, arteriosclerosis, cancer and arthritis (Cross, 1987). There are numerous factors environmental factors that can induce high radical production free; among these are the radiations, the fumes and the pesticides (Jacobson, 1987; Halliwell, 1996) (full data of these and the other bibliographic citations are given at the end of the description not to make this present too cumbersome exposition).
Los seres vivos presentan varios mecanismos con función antioxidante. Entre las diversas sustancias con capacidad antioxidante se encuentra la vitamina E. Su papel como atrapador de radicales libres es crucial en la prevención de la oxidación de los ácidos grasos insaturados situados en la membrana plasmática y es considerada la primera línea de defensa contra la peroxidación de lípidos (Horwitt, 1986; Packer et al., 1995, Halliwel, 1996).Living things have several mechanisms with antioxidant function. Among the various substances with antioxidant capacity is vitamin E. Its role as a free radical scavenger is crucial in preventing the oxidation of unsaturated fatty acids located in the plasma membrane and is considered the first line of defense against peroxidation of lipids (Horwitt, 1986; Packer et al ., 1995, Halliwel, 1996).
La vitamina E es sintetizada sólo por las plantas; por tanto se encuentra fundamentalmente en productos vegetales (aceites vegetales, semillas ...). Existen 4 tocoferoles con actividad vitamina E (\alpha, \beta, \gamma y \delta-tocoferol). Todas las plantas superiores tienen \alpha-tocoferol (hojas y otras partes verdes), mientras que el \gamma-tocoferol (también el \beta- y el \delta-tocoferol) está presente en concentraciones muy inferiores (Combs, 1992). Las proporciones individuales de los tocoferoles varían ampliamente entre los diferentes aceites de semillas.Vitamin E is synthesized only by plants; therefore it is found mainly in products Vegetables (vegetable oils, seeds ...). There are 4 tocopherols with vitamin E activity (?,?,? and δ-tocopherol). All upper floors have α-tocopherol (leaves and other parts green), while γ-tocopherol (also β- and δ-tocopherol) is present at much lower concentrations (Combs, 1992). The proportions individual tocopherols vary widely between Different seed oils.
La vitamina E se distribuye por todos los tejidos del cuerpo humano a través del plasma y elementos celulares de la sangre, como eritrocitos, leucocitos y plaquetas. Bajo ciertas circunstancias la vitamina E es transportada por la linfa y la sangre como tocoferol libre o unido a \beta-lipoproteína (Bjoerneboe, 1990). La forma predominante en plasma humano es \alpha-tocoferol, que constituye el 90% de la concentración total de vitamina E, y el resto está formado por \gamma-tocoferol y \beta-tocoferol en una proporción de 5:1. De \delta-tocoferol y tocotrienoles se encuentran sólo trazas en plasma humano.Vitamin E is distributed by all tissues of the human body through plasma and cellular elements of blood, such as erythrocytes, leukocytes and platelets. Low certain circumstances vitamin E is transported by lymph and blood as free tocopherol or attached to β-lipoprotein (Bjoerneboe, 1990). The shape predominant in human plasma is α-tocopherol, which constitutes 90% of the total concentration of vitamin E, and the rest is formed by γ-tocopherol and β-tocopherol in a 5: 1 ratio. From δ-tocopherol and tocotrienols are found only traces in human plasma.
La mayor parte de la vitamina E procede de la toma diaria de aceites y margarina. La absorción de la vitamina E depende de la habilidad del cuerpo para absorber grasas; por tanto, alguna enfermedad que afecte a la digestión, absorción o transporte de estas grasas puede conducir a deficiencias en vitamina E. Una deficiencia severa y crónica puede dar lugar a un característico síndrome neurológico de neuropatía progresiva, con ausencia o disminución de reflejos, debilidad de los miembros, alteración en la marcha y pérdida sensorial en brazos y piernas. En los roedores, el déficit de vitamina E produce esterilidad, parálisis y distrofia muscular (Sokol, 1988). El exceso en el consumo de vitamina E no parece producir efectos nocivos.Most of vitamin E comes from the Daily intake of oils and margarine. Vitamin E absorption It depends on the body's ability to absorb fat; so, any disease that affects digestion, absorption or transport of these fats can lead to deficiencies in vitamin E. A severe and chronic deficiency can lead to a characteristic Neurological syndrome of progressive neuropathy, with absence or decreased reflexes, weakness of the limbs, alteration in gait and sensory loss in arms and legs. In rodents, Vitamin E deficiency produces sterility, paralysis and dystrophy muscle (Sokol, 1988). Excess vitamin E intake does not It seems to produce harmful effects.
Estudios recientes que comparan la vitamina E natural con la forma sintética sugieren que la biodisponibilidad de la forma natural es dos veces superior a la de la vitamina E sintética. La vitamina E natural y la sintética muestran las siguientes diferencias (Horwitt, 1986; Cheng et al., 1987; Ingold et al., 1987):Recent studies comparing natural vitamin E with the synthetic form suggest that the bioavailability of the natural form is twice that of synthetic vitamin E. Natural and synthetic vitamin E show the following differences (Horwitt, 1986; Cheng et al ., 1987; Ingold et al ., 1987):
1.- La vitamina E natural es derivada de aceites vegetales, principalmente de aceite de soja, mientras que la vitamina E sintética se produce a partir de derivados del petróleo.1.- Natural vitamin E is derived from oils vegetables, mainly from soybean oil, while the Synthetic vitamin E is produced from derivatives of Petroleum.
2.- La vitamina E natural es un estereoisómero, por el contrario la vitamina E sintética es una mezcla de ocho estereoisómeros.2.- Natural vitamin E is a stereoisomer, on the contrary synthetic vitamin E is a mixture of eight stereoisomers.
3.- La vitamina E natural es más biodisponible que la forma sintética.3.- Natural vitamin E is more bioavailable That synthetic form.
4.- La vitamina E natural queda retenida durante más tiempo en tejidos del cuerpo que la forma sintética.4.- Natural vitamin E is retained for more time in body tissues than the synthetic form.
En la actualidad existe un gran interés hacia el uso de antioxidantes en la industria alimentaria, farmacéutica y de cosmética (Combs, 1992; Halliwell et al., 1992). Esto produce una gran demanda de antioxidantes estables, a lo que hay que añadir la preferencia por parte de los consumidores y de las autoridades sanitarias por los de carácter natural, básicamente vitamina C (ácido ascórbico), vitamina E (tocoferoles y tocotrienoles), y extractos relativamente complejos de varias especies de plantas (Rosmarinus officinalis, Nerium oleander y Myrtus communis).At present there is great interest towards the use of antioxidants in the food, pharmaceutical and cosmetic industry (Combs, 1992; Halliwell et al ., 1992). This produces a high demand for stable antioxidants, to which we must add the preference on the part of consumers and health authorities for those of a natural nature, basically vitamin C (ascorbic acid), vitamin E (tocopherols and tocotrienols), and relatively complex extracts of several plant species ( Rosmarinus officinalis, Nerium oleander and Myrtus communis ).
Los trabajos de Chipault y colaboradores (Chipault et al., 1952, 1955, 1956) fueron los precursores de muchos estudios sobre la capacidad antioxidante de un número de extractos de plantas con aplicaciones potenciales como conservantes en industrias alimentarias, farmacéuticas y de cosmética (Taga et al., 1984; Written et al., 1984; Wu et al., 1984; Economou et al., 1991; Mallet et al., 1994; Daood et al., 1996; Schwants et al., 1996).The works of Chipault et al . (Chipault et al ., 1952, 1955, 1956) were the precursors of many studies on the antioxidant capacity of a number of plant extracts with potential applications as preservatives in food, pharmaceutical and cosmetic industries (Taga et al ., 1984; Written et al ., 1984; Wu et al ., 1984; Economou et al ., 1991; Mallet et al ., 1994; Daood et al ., 1996; Schwants et al ., 1996).
El alga unicelular Chlamydomonas reinhardtii es un organismo modelo para estudios fisiológicos, bioquímicos y genético-moleculares. Su tiempo de generación relativamente corto (6 horas) permite un rápido crecimiento y una fácil disponibilidad de material celular, lo que unido a la existencia de técnicas sencillas y eficientes que permiten la transformación tanto de genes nucleares como cloroplásticos, la convierte en un candidato idóneo para la producción y posterior extracción de tocoferoles.Unicellular algae Chlamydomonas reinhardtii is a model organism for physiological, biochemical and genetic-molecular studies. Its relatively short generation time (6 hours) allows rapid growth and easy availability of cellular material, which together with the existence of simple and efficient techniques that allow the transformation of both nuclear and chloroplast genes, makes it an ideal candidate for the production and subsequent extraction of tocopherols.
La p-hidroxifenilpiruvato
dioxigenasa (EC 1.13.11.27) es la enzima que cataliza la
transformación del p-hidroxifenilpiruvato en ácido
homogentísico, que es el primer compuesto de las rutas de síntesis
de plastoquinonas y tocofe-
roles.P-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) is the enzyme that catalyzes the transformation of p-hydroxyphenylpyruvate into homogentisic acid, which is the first compound of the synthesis pathways of plastoquinones and tocopher-
roles
Una alternativa para obtener algas con mayor capacidad antioxidante sería el desarrollo de estirpes transgénicas que tuvieran unos niveles elevados de ARNm correspondientes a la p-HPPD con el fin de aumentar los niveles totales de tocoferol mediante la sobreexpresión del gen de la p-HPPD, habida cuenta de los resultados mostrados por Shintani y DellaPenna (1998), en los que los niveles totales de vitamina E no se incrementan en transformantes antisentido carentes de actividad \gamma-tocoferol metiltransferasa, y en los que sólo se ve afectada la proporción relativa de los distintos isómeros del tocoferol. Para ello, es necesario identificar, aislar y caracterizar el ADN genómico (ADNg) o complementario (ADNc), que codifica la p-HPPD de Chlamydomonas y/o el ARNm correspondiente a dicha enzima. Igualmente, la obtención de organismos caracterizados por una alta producción de \gamma-tocoferol puede plantearse mediante estrategias antisentido que permitan disminuir la expresión del gen de la \gamma-tocoferol metiltransferasa (\gamma-TMT).An alternative to obtain algae with greater antioxidant capacity would be the development of transgenic lines that had high levels of mRNA corresponding to the p-HPPD in order to increase the total levels of tocopherol by overexpression of the gene of the p-HPPD, taking into account the results shown by Shintani and DellaPenna (1998), in which the total levels of Vitamin E does not increase in antisense transformants lacking of γ-tocopherol methyltransferase activity, and in which only the relative proportion of the different isomers of tocopherol. For this, it is necessary identify, isolate and characterize genomic DNA (gDNA) or Complementary (cDNA), which encodes the p-HPPD of Chlamydomonas and / or the mRNA corresponding to said enzyme. Likewise, obtaining organisms characterized by high γ-tocopherol production may arise through antisense strategies that reduce expression of the γ-tocopherol methyltransferase gene (γ-TMT).
La p-hidroxifenilpiruvato dioxigenasa (HPPD, EC 1.13.11.27, EC 1.14.2.2) es un sitio diana para algunos herbicidas (Schulz et al., 1993; Lee et al., 1997, 1998; Pallet et al., 1998; Viviani et al., 1998). Los inhibidores de esta enzima interrumpen la biosíntesis de carotenoides y tiene como resultado un blanqueamiento de la hoja debido a la pérdida de clorofila. Aunque estos síntomas son similares a los observados en plantas tratadas con inhibidores de la fitoeno desaturasa (Lee et al., 1997), la inhibición de la HPPD tiene un mecanismo de acción diferente. La inhibición de la HPPD afecta indirectamente a la actividad fitoeno desaturasa reduciendo el pool de plastoquinonas disponibles (Pallet et al., 1998). El subsiguiente descenso de los niveles de carotenoides causa el blanqueamiento foliar, ya que el aparato fotosintético deja de ser estabilizado por esos pigmentos. En condiciones de alta intensidad lumínica, no se captura el exceso de energía y se destruyen las moléculas de clorofila.P-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27, EC 1.14.2.2) is a target site for some herbicides (Schulz et al ., 1993; Lee et al ., 1997, 1998; Pallet et al ., 1998; Viviani et al ., 1998). Inhibitors of this enzyme disrupt carotenoid biosynthesis and result in leaf whitening due to the loss of chlorophyll. Although these symptoms are similar to those observed in plants treated with phytoen desaturase inhibitors (Lee et al ., 1997), HPPD inhibition has a different mechanism of action. HPPD inhibition indirectly affects the phytoeno desaturase activity by reducing the pool of available plastoquinones (Pallet et al ., 1998). The subsequent decrease in carotenoid levels causes leaf whitening, since the photosynthetic apparatus is no longer stabilized by these pigments. Under conditions of high light intensity, excess energy is not captured and chlorophyll molecules are destroyed.
Los inhibidores de la HPPD han permitido
desarrollar una nueva clase de herbicidas basados en el esqueleto
de la tricetona que aparentemente imita un intermedio de la
reacción. Estos compuestos son inhibidores dependientes del tiempo
de esta enzima. La sulcotriona
(2-[cloro-4-metanosulfonilbenzonil]-ciclohexano)-1,3-diona
y el isoxaflutol
(5-cilopropil-4-(4-trifluorometil-2-metanosulfonilbenzonil)isoxazol)
son productos comerciales pertenecientes a dos de esas clases. La
sulcotriona posee la clásica estructura requerida para un inhibidor
de la HPPD, mientras que el isoxaflutol debe de ser activado a la
forma conjugada dicetonitrilo después de la apertura del anillo
isoxazol (Viviani et al., 1998). Estos son potentes
herbicidas utilizados en el control pre- y
post-emergente de malas hierbas en
maíz.HPPD inhibitors have allowed the development of a new class of herbicides based on the tricetone skeleton that apparently mimics a reaction intermediate. These compounds are time dependent inhibitors of this enzyme. Sulcotrione (2- [chloro-4-methanesulfonylbenzonyl] -cyclohexane) -1,3-dione and isoxaflutol (5-cylopropyl-4- (4-trifluoromethyl-2-methanesulfonylbenzonyl) isoxazole) are commercial products belonging to two of those lessons. Sulcotrione possesses the classic structure required for an HPPD inhibitor, while isoxaflutol must be activated to the conjugated form dikettonitrile after the opening of the isoxazole ring (Viviani et al ., 1998). These are potent herbicides used in pre- and post-emergent weed control in
corn.
La invención proporciona una solución a la necesidad existente de conseguir la clonación de un gen de Chlamydomonas, así como el correspondiente ADNc, que codifica para una HPPD de Chlamydomonas.The invention provides a solution to the existing need to achieve the cloning of a gene from Chlamydomonas, as well as the corresponding cDNA, which codes for an HPPD of Chlamydomonas.
El principal objeto de esta invención lo constituye una molécula de ADN que codifica una HPPD de Chlamydomonas.The main object of this invention is constitutes a DNA molecule that encodes an HPPD of Chlamydomonas
Un objeto adicional de esta invención lo constituye el empleo de dicha molécula de ADN para modular la expresión de la HPPD en Chlamydomonas.A further object of this invention is constitutes the use of said DNA molecule to modulate the HPPD expression in Chlamydomonas.
Otro objeto adicional de esta invención lo constituye una construcción de ADN que comprende la totalidad o una parte de dicha molécula de ADN, así como un vector que contiene dicha molécula o construcción de ADN y una célula transformada con dicho vector.Another additional object of this invention is constitutes a DNA construct that comprises all or one part of said DNA molecule, as well as a vector containing said DNA molecule or construct and a cell transformed with said vector.
Otro objeto adicional de esta invención lo constituye el empleo de dicha molécula de ADN, o de dicha construcción de ADN, en la obtención de algas transgénicas que expresan una actividad enzimática HPPD modulada. Las algas transgénicas resultantes constituyen otro objeto adicional de esta invención.Another additional object of this invention is constitutes the use of said DNA molecule, or of said DNA construction, in obtaining transgenic algae that express a modulated HPPD enzyme activity. The seaweed resulting transgenic constitute another additional object of this invention.
La presente invención proporciona una molécula de ADN que codifica una p-hidroxifenilpiruvato dioxigenasa (HPPD) de Chlamydomonas, en adelante molécula de ADN de la invención, seleccionada entre:The present invention provides a molecule of DNA encoding a p-hydroxyphenylpyruvate Chlamydomonas dioxygenase (HPPD), hereinafter DNA molecule of the invention, selected from:
- a)to)
- una secuencia de ADN que comprende la secuencia de nucleótidos identificada como SEQ ID No 1.a DNA sequence comprising the nucleotide sequence identified as SEQ ID No 1.
- b)b)
- una secuencia de ADN análoga a la secuencia definida en a) quea DNA sequence analogous to the sequence defined in a) that
- i)i)
- es sustancialmente homóloga a la secuencia de ADN definida en a) y/o, queis substantially homologous to the DNA sequence defined in a) and / or, that
- ii)ii)
- codifica un polipéptido que es sustancialmente homólogo a la proteína codificada por la secuencia de ADN definida en a).encodes a polypeptide that is substantially homologous to the protein encoded by the sequence of DNA defined in a).
En el sentido utilizado en esta descripción, el término "análogo/a" pretende incluir a cualquier secuencia de ADN que codifica para una enzima que posee, al menos, actividad HPPD que tiene las propiedades i)-ii) arriba mencionadas. Típicamente la secuencia de ADN análoga:In the sense used in this description, the term "analogous" is intended to include any sequence of DNA that codes for an enzyme that has at least activity HPPD that has the properties i) -ii) above mentioned. Typically the analogous DNA sequence:
- se puede aislar de otra especie que produce una HPPD en base a la secuencia de nucleótidos mostrada en la SEQ ID No 1, o- can be isolated from another species that produces an HPPD based on the nucleotide sequence shown in the SEQ ID No 1, or
- se construye en base a la secuencia de nucleótidos mostrada en la SEQ ID No 1, por ejemplo, mediante la introducción de sustituciones de nucleótidos conservativas, es decir, que dan lugar a la misma secuencia de aminoácidos de la HPPD que la codificada por la secuencia de nucleótidos mostrada en la SEQ ID No 1, pero que corresponde al empleo de codones del organismo hospedador destinado a la producción de la proteína, o bien mediante la introducción de sustituciones de nucleótidos que dan lugar a una secuencia de aminoácidos diferente y, por tanto, posiblemente a una estructura proteica diferente que pudiera dar lugar a una proteína mutante con propiedades diferentes a las de la proteína nativa. Otros ejemplos de posibles modificaciones incluyen la inserción de uno o más nucleótidos en cualquiera de los extremos de la secuencia, la adición de uno o más nucleótidos en cualquiera de los extremos de la secuencia, o la deleción de uno o más nucleótidos en cualquier extremo o en el interior de la secuencia. Por ejemplo, la secuencia de ADN análogo puede ser una sub-secuencia de nucleótidos mostrada en la SEQ ID No 1.- is built based on the sequence of nucleotides shown in SEQ ID No 1, for example, by the Introduction of conservative nucleotide substitutions, is that is, they give rise to the same amino acid sequence of the HPPD than the one encoded by the nucleotide sequence shown in the SEQ ID No 1, but that corresponds to the use of codons of the host organism intended for the production of the protein, or either by introducing nucleotide substitutions that they give rise to a different amino acid sequence and therefore possibly to a different protein structure that could give place to a mutant protein with different properties than those of the native protein Other examples of possible modifications include the insertion of one or more nucleotides at either end of the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides at any end or inside the sequence. For example, the analog DNA sequence may be a nucleotide sub-sequence shown in SEQ ID No 1
En general, la secuencia de ADN análoga es sustancialmente homóloga a la secuencia de nucleótidos identificada como la SEQ ID No 1. En el sentido utilizado en esta descripción, la expresión "sustancialmente homóloga", aplicada a secuencias de nucleótidos, significa que las secuencias de nucleótidos en cuestión tienen un grado de identidad de, al menos un 70%, preferentemente de al menos un 85%, o más preferentemente de al menos un 95%.In general, the analog DNA sequence is substantially homologous to the nucleotide sequence identified as SEQ ID No 1. In the sense used in this description, the "substantially homologous" expression, applied to sequences of nucleotides, means that the nucleotide sequences in issue have a degree of identity of at least 70%, preferably at least 85%, or more preferably at minus 95%.
La molécula de ADN de la invención puede proceder de cualquier estirpe de Chlamydomonas o bien de un organismo hospedador transformado con dicha molécula de ADN.The DNA molecule of the invention can come from any line of Chlamydomonas or from a host organism transformed with said DNA molecule.
Alternativamente, la molécula de ADN de la invención puede ser aislada, mediante técnicas convencionales, a partir de ADN de cualquier otra especie mediante el empleo de sondas o de oligonucleótidos preparados a partir de la información sobre la secuencia de ADN proporcionada en esta descripción.Alternatively, the DNA molecule of the invention can be isolated, by conventional techniques, to from DNA of any other species by employing probes or oligonucleotides prepared from the information about the DNA sequence provided in this description.
En una realización particular, la molécula de ADN de la invención es una molécula de ADNc del ARNm correspondiente al gen de la HPPD de Chlamydomonas, relacionado con la biosíntesis de los tocoferoles y plastoquinonas, que se ha caracterizado molecular y fisiológicamente y cuya secuencia de nucleótidos comprende la secuencia de nucleótidos mostrada en la SEQ ID No 1. El análisis comparativo de la secuencia deducida de la proteína ha puesto de manifiesto que este gen corresponde a un gen que codifica una HPPD. La SEQ ID No 1 corresponde a la secuencia completa del ADNc del ARNm de la HPPD de Chlamydomonas.In a particular embodiment, the molecule of DNA of the invention is a mRNA cDNA molecule corresponding to the Chlamydomonas HPPD gene, related to the biosynthesis of tocopherols and plastoquinones, which has been characterized molecular and physiologically and whose sequence of nucleotides comprise the nucleotide sequence shown in the SEQ ID No 1. The comparative analysis of the sequence deduced from the protein has revealed that this gene corresponds to a gene that encodes an HPPD. SEQ ID No 1 corresponds to the sequence Complete mRNA cDNA from Chlamydomonas HPPD.
La molécula de ADN de la invención puede obtenerse utilizando métodos convencionales conocidos por los técnicos en la materia, mediante un procedimiento que comprende la extracción del ARNm correspondiente a la transcripción del gen que codifica la HPPD a partir de un organismo productor de dicha enzima, la obtención de una primera cadena de ADNc por transcripción inversa del correspondiente ARNm, la síntesis de una segunda cadena de ADNc, complementaria a la primera, para obtener un ADNc de doble cadena, la unión de unos adaptadores para su inserción en plásmidos o fagos para su propagación en, por ejemplo, un sistema bacteriano y la identificación de los clones que portan el ADNc deseado.The DNA molecule of the invention can Obtained using conventional methods known to technicians in the field, through a procedure that includes the mRNA extraction corresponding to the transcription of the gene that encodes HPPD from an organism producing said enzyme, obtaining a first cDNA chain by transcription inverse of the corresponding mRNA, the synthesis of a second chain cDNA, complementary to the first, to obtain a cDNA of double chain, the union of adapters for insertion in plasmids or phages for propagation in, for example, a system bacterial and the identification of the clones that carry the cDNA wanted.
En una realización particular (véase el Ejemplo 1), se ha caracterizado una EST de Chlamydomonas, a partir de la cual se han diseñado oligonucleótidos específicos (SEQ ID No 3 y SEQ ID No 4) que han permitido obtener un clon casi completo de ADNc correspondiente al ARNm de la HPPD de Chlamydomonas mediante un procedimiento que comprende aplicar la técnica RT-PCR (Retrotranscriptase-PCR) a ARNm procedentes de algas cultivadas en medio autotrófico (Harris, 1989) a 25ºC con iluminación continua de 15-20 W/m^{2} proporcionada por tubos fluorescentes de luz blanca y burbujeadas con aire enriquecido con CO_{2} (5% V/V). Para ello, se extrajeron los ARNm de dichas células, se sometieron a una reacción de transcripción inversa (RT) para obtener los correspondientes ADNc de cadena simple y se realizó PCR utilizando los oligonucleótidos específicos previamente diseñados. Los productos de la amplificación se subclonaron en unos vectores apropiados que se utilizaron para transformar bacterias: seguidamente se extrajo el ADN correspondiente al plásmido recombinante, que se purificó y secuenció. La secuencia obtenida (SEQ ID No 1) se comparó con las secuencias depositadas en la base de datos utilizando el programa BLAST del National Center for Biotechnology Information (NCBI, Estados Unidos). La comparación de las secuencias puso de manifiesto que la secuencia de ADN obtenida presentaba una relativamente elevada homología de secuencia única y exclusivamente con otras secuencias de organismos que codifican una p-HPPD.In a particular embodiment (see Example 1), an EST of Chlamydomonas has been characterized, from the which specific oligonucleotides have been designed (SEQ ID No 3 and SEQ ID No 4) that have allowed to obtain an almost complete clone of CDNA corresponding to Chlamydomonas HPPD mRNA by a procedure that involves applying the technique RT-PCR (Retrotranscriptase-PCR) a MRNA from algae grown in autotrophic medium (Harris, 1989) at 25ºC with continuous illumination of 15-20 W / m2 provided by white light fluorescent tubes and bubbled with air enriched with CO2 (5% V / V). For it, mRNAs were extracted from said cells, subjected to a reverse transcription reaction (RT) to obtain the corresponding single chain cDNA and PCR was performed using the specific oligonucleotides previously designed. The amplification products were subcloned into vectors appropriate that were used to transform bacteria: the DNA corresponding to the plasmid was then extracted recombinant, which was purified and sequenced. The sequence obtained (SEQ ID No 1) was compared with the sequences deposited in the base data using the National Center for BLAST program Biotechnology Information (NCBI, United States). The comparison of the sequences showed that the DNA sequence obtained had a relatively high single sequence homology and exclusively with other sequences of organisms that encode a p-HPPD.
La invención proporciona, además, una construcción de ADN, en adelante, construcción de ADN de la invención, que comprende la totalidad de la molécula de ADN de la invención o un fragmento de, al menos, 8 nucleótidos consecutivos de la molécula de ADN de la invención y una región iniciadora de la transcripción funcional en plantas. En dicha construcción, cualquiera de los extremos (3' ó 5') de la totalidad o del fragmento de la molécula de ADN de la invención puede estar unido al extremo 3' de dicha región iniciadora de la transcripción. La construcción de ADN de la invención también puede contener, operativamente enlazada, una secuencia de terminación de la transcripción. En una realización particular, dicha región iniciadora y terminadora de la transcripción sería funcional en células de Chlamydomonas.The invention also provides a DNA construction, henceforth, DNA construction of the invention, which comprises the entire DNA molecule of the invention or a fragment of at least 8 consecutive nucleotides of the DNA molecule of the invention and an initiating region of the functional transcription in plants. In such construction, any of the ends (3 'or 5') of the whole or of the fragment of the DNA molecule of the invention can be attached at the 3 'end of said transcription initiation region. The DNA construct of the invention may also contain, operably linked, a sequence of termination of the transcription. In a particular embodiment, said region initiator and terminator of transcription would be functional in Chlamydomonas cells.
La molécula de ADN de la invención, o la construcción de ADN de la invención, puede ser insertada en un vector apropiado; por tanto, la invención también se refiere a un vector, tal como un vector de expresión, que comprende dicha molécula de ADN, o una construcción que la contiene. La elección del vector dependerá de la célula hospedadora en la que se va a introducir posteriormente. A modo de ejemplo, el vector donde se introduce dicha secuencia de ADN puede ser un plásmido o un vector que, cuando se introduce en una célula hospedadora, se integra en el genoma de dicha célula y se replica junto con el cromosoma (o cromosomas) en el (o en los que) se ha integrado.The DNA molecule of the invention, or the DNA construct of the invention, can be inserted into a appropriate vector; therefore, the invention also relates to a vector, such as an expression vector, comprising said DNA molecule, or a construct that contains it. The chose of the vector will depend on the host cell in which it is going to enter later. As an example, the vector where enter said DNA sequence can be a plasmid or a vector which, when introduced into a host cell, is integrated into the genome of said cell and replicates along with the chromosome (or chromosomes) in which (or in which) it has been integrated.
En el vector proporcionado por esta invención,
la molécula de ADN de la invención estará conectada operativamente
a un promotor y a una secuencia terminadora. El promotor puede ser
cualquier secuencia de ADN que muestre actividad transcripcional en
la célula hospedadora elegida y puede derivar bien de genes que
codifican para proteínas homólogas o heterólogas de la célula
hospedadora. Los procedimientos utilizados para ligar la secuencia
de ADN de la invención al promotor y a la secuencia terminadora,
respectivamente, y para insertar dicha construcción en un vector
son bien conocidos por los técnicos en la materia y han sido
descritos, por ejemplo, por Sambrook et al.
(1989).In the vector provided by this invention, the DNA molecule of the invention will be operatively connected to a promoter and terminator sequence. The promoter can be any DNA sequence that shows transcriptional activity in the chosen host cell and can be derived well from genes encoding homologous or heterologous proteins of the host cell. The methods used to link the DNA sequence of the invention to the promoter and the terminator sequence, respectively, and to insert said construct into a vector are well known to those skilled in the art and have been described, for example, by Sambrook et al. .
(1989).
La invención también proporciona una célula que comprende una secuencia de ADN de la invención, o una construcción de ADN que contiene a dicha secuencia o dicho vector mencionado más arriba. Las células hospedadoras que se pueden transformar con la secuencia de ADN de la invención pueden ser células procarióticas o, preferentemente, eucarióticas, tales como células de tejidos vegetales. La transformación de células de tejidos vegetales también puede realizarse por métodos convencionales. Para una revisión de la transferencia génica a plantas, incluyendo vectores, métodos de transferencia de ADN, etc., véase, por ejemplo, el libro titulado "Gene Transfer to Plants", de I. Potrykus y G. Spangenberg. Ed. Springer Lab. Manual (1995).The invention also provides a cell that comprises a DNA sequence of the invention, or a construct of DNA containing said sequence or said vector mentioned more above. Host cells that can be transformed with the DNA sequence of the invention can be prokaryotic cells or, preferably, eukaryotic, such as tissue cells vegetables. The transformation of plant tissue cells also It can be done by conventional methods. For a review of the gene transfer to plants, including vectors, methods of DNA transfer, etc., see, for example, the book entitled "Gene Transfer to Plants", by I. Potrykus and G. Spangenberg. Ed. Springer Lab. Manual (1995).
La invención también proporciona una proteína con actividad p-hidroxifenilpiruvato dioxigenasa, en adelante HPPD de la invención, que tiene una secuencia de aminoácidos seleccionada entre:The invention also provides a protein. with p-hydroxyphenylpyruvate dioxygenase activity, hereinafter HPPD of the invention, which has a sequence of amino acids selected from:
- a)to)
- una secuencia de aminoácidos que comprende la secuencia de aminoácidos mostrada en la SEQ ID No 2;a amino acid sequence comprising the amino acid sequence shown in SEQ ID No 2;
- b)b)
- la secuencia de aminoácidos deducidos a partir de la secuencia de nucleótidos mostrada en la SEQ ID No 1; ythe amino acid sequence deduced from the sequence of nucleotides shown in SEQ ID No 1; Y
- c)C)
- una secuencia de aminoácidos sustancialmente homóloga y funcionalmente definidas en a) o en b).a substantially homologous and functionally amino acid sequence defined in a) or b).
En el sentido utilizado en esta descripción, la expresión "sustancialmente homóloga" significa que las secuencias de aminoácidos en cuestión tienen un grado de identidad de, al menos un 70%, preferentemente de al menos un 85%, y, más preferentemente de al menos un 95%.In the sense used in this description, the "substantially homologous" expression means that amino acid sequences in question have a degree of identity of at least 70%, preferably at least 85%, and, more preferably at least 95%.
Asimismo, en el sentido utilizado en esta descripción, la expresión "funcionalmente equivalente" significa que la proteína en cuestión tiene una actividad p-hidroxifenilpiruvato dioxigenasa.Also, in the sense used in this description, the expression "functionally equivalent" means that the protein in question has an activity p-hydroxyphenylpyruvate dioxygenase.
En una realización particular, la HPPD de la invención es una HPPD de Chlamydomonas (Chlamydomonas reinhardtii) que tiene una secuencia de aminoácidos que comprende la secuencia de aminoácidos mostrada en la SEQ ID No. 2. La SEQ ID No 2 corresponde a la secuencia de aminoácidos de un fragmento de una HPPD de Chlamydomonas y ha sido deducida a partir de la secuencia de nucleótidos del fragmento parcial del ADNc del ARNm correspondiente al gen de la HPPD de Chlamydomonas mostrada en la SEQ ID No. 1. La comparación entre la SEQ ID No. 1 y las secuencias presentes en las bases de datos puso de manifiesto una identidad de secuencia a nivel de aminoácidos significativa con secuencias correspondientes a HPPD de plantas superiores. El análisis por ordenador del ORF del gen HPPD mostró una proteína deducida de 432 aminoácidos.In a particular embodiment, the HPPD of the invention is a Chlamydomonas HPPD ( Chlamydomonas reinhardtii ) having an amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 2. SEQ ID No. 2 corresponds to the sequence of amino acids of a fragment of a Chlamydomonas HPPD and has been deduced from the nucleotide sequence of the partial mRNA fragment of the mRNA corresponding to the Chlamydomonas HPPD gene shown in SEQ ID No. 1. The comparison between SEQ ID No. 1 and the sequences present in the databases revealed a significant amino acid level sequence identity with sequences corresponding to HPPD from higher plants. Computer analysis of the ORP of the HPPD gene showed a protein deduced from 432 amino acids.
La HPPD de la invención puede obtenerse mediante un método que implica cultivar una célula hospedadora adecuada que contiene la molécula de ADN de la invención, o una construcción de ADN de la invención, bajo condiciones que permiten la producción de la proteína y su recuperación del medio de cultivo.The HPPD of the invention can be obtained by a method that involves cultivating a suitable host cell containing the DNA molecule of the invention, or a construct of DNA of the invention, under conditions that allow production of the protein and its recovery from the culture medium.
La HPPD de la invención puede obtenerse, alternativamente, a partir de un organismo productor de la misma mediante un procedimiento que comprende el cultivo del organismo productor, en las condiciones apropiadas para la expresión de dicha enzima, y, posteriormente, recuperar dicha enzima.The HPPD of the invention can be obtained, alternatively, from a body producing it through a procedure that includes organism culture producer, under the appropriate conditions for the expression of said enzyme, and subsequently recover said enzyme.
La HPPD de la invención tiene importancia en la industria alimentaria, en particular, en la industria del procesado y de la conservación de alimentos, tales como atún, sardinas, etc. El empleo de la HPPD de la invención en la industria alimentaria podría dar lugar a alimentos con unos niveles superiores de antioxidantes naturales otorgando así un valor añadido a dichos productos alimenticios puesto que se podría aumentar el tiempo de conservación de los mismos. La molécula de ADN de la invención puede ser utilizada en procesos de mejora de la conservación de diferentes alimentos, modulando la expresión por sobreexpresión de la HPPD, alterando con ello por un lado la mayor capacidad vitamínica de los alimentos y por otro la resistencia al deterioro de dichos alimentos. Por otro lado, la HPPD de la invención también podría utilizarse en la industria agronómica. Su uso podría dar lugar a plantas que presentasen tolerancia a ciertos herbicidas como los de la familia de los isoxazoles o de las tricetonas. En una realización particular, la molécula de ADN de la invención se utiliza en la obtención de plantas transgénicas que poseen unos niveles de ARNm correspondientes muy elevados o muy reducidos o inexistentes. Para la obtención de estas plantas transgénicas se puede proceder con las técnicas convencionales de ARNm antisentido y/o sobreexpresión (silenciamiento en sentido), u otras.The HPPD of the invention is important in the food industry in particular in the processing industry and food preservation, such as tuna, sardines, etc. The use of the HPPD of the invention in the food industry could lead to food with higher levels of natural antioxidants thus giving added value to these food products since it could increase the time of conservation of them. The DNA molecule of the invention It can be used in processes to improve the conservation of different foods, modulating the expression by overexpression of HPPD, thereby altering the greater capacity on the one hand vitamin of food and on the other the resistance to deterioration of these foods. On the other hand, the HPPD of the invention also Could be used in the agronomic industry. Its use could give place to plants that had tolerance to certain herbicides like those of the isoxazoles or tricetonas family. In a particular embodiment, the DNA molecule of the invention is used in obtaining transgenic plants that have some corresponding very high or very low mRNA levels or nonexistent To obtain these transgenic plants, you can proceed with conventional antisense mRNA techniques and / or overexpression (sense silencing), or others.
Por tanto, la invención también se refiere a una célula transgénica de una planta que comprende una construcción de ADN de la invención que tiene un promotor, funcional en dicha planta, operativamente enlazado a una sub-secuencia de ADN de, al menos, 8 nucleótidos, derivada de una molécula de ADN de la invención, estando unida dicha sub-secuencia de ADN al promotor en una orientación opuesta a la de su expresión.Therefore, the invention also relates to a transgenic cell of a plant comprising a construction of DNA of the invention having a promoter, functional in said plant, operatively linked to a sub-sequence DNA of at least 8 nucleotides, derived from a DNA molecule of the invention, said sub-sequence being joined of DNA to the promoter in an orientation opposite to that of its expression.
Una planta transgénica que comprende, al menos, una de dichas células transgénicas, constituye un objeto adicional de esta invención. En una realización particular, las plantas transgénicas son plantas que producen diferentes niveles tanto de \gamma-tocoferol como de \alpha-tocoferol. En otra realización particular las plantas transgénicas son plantas con distinto grado de tolerancia a ciertos herbicidas.A transgenic plant comprising at least one of said transgenic cells constitutes an additional object of this invention. In a particular embodiment, the plants GMOs are plants that produce different levels of both γ-tocopherol as of α-tocopherol. In another particular embodiment transgenic plants are plants with different degrees of tolerance to certain herbicides.
La utilización de la molécula de ADN de la invención, en particular, de una molécula de ADNc que codifica una HPPD de Chlamydomonas, de longitud completa o parcial, mediante cualquier tipo de técnica, puede generar células transgénicas que tengan alterados los niveles de tocoferoles o de vitamina E total, lo que redunda en un valor económico añadido a las mismas.The use of the DNA molecule of the invention, in particular, of a cDNA molecule encoding a Chlamydomonas HPPD, full or partial length, by Any type of technique can generate transgenic cells that have altered levels of tocopherols or total vitamin E, which results in an economic value added to them.
Por lo tanto, otro objeto de la presente invención lo constituye una preparación enzimática resultante de la ruptura de la célula u organismo hospedador que contiene la molécula o construcción de ADN de la invención, así como al resultante de someter a esta preparación a diversos pasos de purificación o enriquecimiento por los métodos conocidos por los técnicos en la materia. Una preparación resultante de mezclar la preparación anterior con otros componentes también es objeto de la invención.Therefore, another object of the present invention constitutes an enzyme preparation resulting from the rupture of the host cell or organism that contains the DNA molecule or construct of the invention, as well as at resulting from subjecting this preparation to various steps of purification or enrichment by the methods known to the technicians in the field. A preparation resulting from mixing the Previous preparation with other components is also subject to the invention.
Otro objeto adicional de la presente invención lo constituye un método basado en la sobreexpresión del gen que codifica la HPPD mediante los métodos conocidos por los técnicos en la materia, que permita el aumento en el contenido de vitamina E, o de cualquiera de los isómeros en particular.Another additional object of the present invention it is a method based on the overexpression of the gene that encode the HPPD by the methods known to the technicians in matter, which allows the increase in vitamin E content, or of any of the isomers in particular.
Por último, otro objeto de la invención comprende un método basado en cualquiera de las técnicas conocidas que permita la sobreexpresión del gen que codifica la HPPD y así conferir resistencia a los herbicidas que actúan inhibiéndola, lo que permite además su utilización como gen marcador en procesos de transformación.Finally, another object of the invention comprises a method based on any of the known techniques that allows overexpression of the gene encoding the HPPD and thus confer resistance to herbicides that act by inhibiting it, which which also allows its use as a marker gene in processes of transformation.
Los siguientes ejemplos sirven para ilustrar la presente invención y no deben ser considerados como limitadores del alcance de la misma.The following examples serve to illustrate the present invention and should not be considered as limiting the scope of it.
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Se ha clonado el gen hppd1, que codifica una p-HPPD de Chlamydomonas, relacionado con la biosíntesis de la vitamina E y plastoquinonas y se ha caracterizado molecular y fisiológicamente. El análisis comparativo de la secuencia deducida de la proteína ha puesto de manifiesto que este gen corresponde a un gen que codifica una p-HPPD.The hppd1 gene, which encodes a Chlamydomonas p-HPPD, related to biosynthesis of vitamin E and plastoquinones and has been characterized molecular and physiologically The comparative analysis of the deduced sequence of the protein has revealed that this gene corresponds to a gene that encodes a p-HPPD.
Para obtener el gen hppd1 se siguió una estrategia que comprendía el empleo de la técnica RT-PCR, amplificando por PCR los fragmentos de ADNc obtenidos a partir de ARNm de células de Chlamydomonas cultivadas en medio autotrófico (Harris, 1989) a 25ºC con iluminación continua de 15-20 W/m^{2} proporcionada por tubos fluorescentes de luz blanca y burbujeadas con aire enriquecido con CO_{2} (5% v/v). Los productos amplificados se subclonaron en unos vectores y con dichos vectores se transformaron células de Escherichia coli, seleccionándose las células transformantes que contenían el vector con el inserto de ADNc correspondiente a la p-HPPD de Chlamydomonas, que se aisló, purificó y secuenció. La secuencia obtenida se comparó con otras secuencias de ADN que codifican p-HPPD de otros organismos.To obtain the hppd1 gene, a strategy was followed that included the use of the RT-PCR technique, amplifying by PCR the cDNA fragments obtained from mRNA of Chlamydomonas cells grown in autotrophic medium (Harris, 1989) at 25 ° C with continuous illumination 15-20 W / m2 provided by white light fluorescent tubes and bubbled with air enriched with CO2 (5% v / v). The amplified products were subcloned into vectors and with said vectors Escherichia coli cells were transformed, the transforming cells containing the vector being selected with the cDNA insert corresponding to the Chlamydomonas p-HPPD, which was isolated, purified and sequenced. The sequence obtained was compared with other DNA sequences encoding p-HPPD from other organisms.
Seguidamente se explica con detalle este proceso:This is explained in detail below. process:
Para la obtención del ARNm correspondiente a la transcripción del gen que codifica la p-HPPD de Chlamydomonas se extrajo el ARN total procedente de células de Chlamydomonas cultivadas en medio autotrófico (Harris, 1989) a 25ºC con iluminación continua de 15-20 W/m^{2} proporcionada por tubos fluorescentes de luz blanca y burbujeadas con aire enriquecido con CO_{2} (5% v/v). Para la extracción del ARN total se siguió una modificación del método descrito por Newman et al. (1990).To obtain the mRNA corresponding to the transcription of the gene encoding the Chlamydomonas p-HPPD, the total RNA from Chlamydomonas cells grown in autotrophic medium (Harris, 1989) was extracted at 25 ° C with continuous illumination of 15-20 W / m 2 provided by white light fluorescent tubes and bubbled with air enriched with CO2 (5% v / v). A modification of the method described by Newman et al . Was followed for total RNA extraction. (1990).
Una vez extraído el ARN total se sometió a una reacción de transcripción inversa (RT-PCR). Para ello se utilizó el kit comercial PowerScript Reverse Transcriptase (Clontech, CA, Palo Alto). Las condiciones de la RT-PCR incluyen: síntesis de las subpoblaciones ancladas de ADNc de simple cadena, amplificación de las mismas por PCR utilizando iniciadores arbitrarios; separación y comparación de las poblaciones resultantes de ADNc de doble cadena mediante electroforesis en geles de agarosa; recuperación, desde el gel, de los fragmentos de ADNc amplificados; las condiciones fueron las que se indican en el manual de uso del citado kit "PowerScript Reverse Trancriptase"Once the total RNA was extracted, it underwent a reverse transcription reaction (RT-PCR). For this was used the commercial kit PowerScript Reverse Transcriptase (Clontech, CA, Palo Alto). The conditions of the RT-PCR include: subpopulation synthesis single-stranded cDNA anchored, amplifying them by PCR using arbitrary primers; separation and comparison of the resulting populations of double stranded cDNA by agarose gel electrophoresis; recovery, from the gel, of the amplified cDNA fragments; the conditions were what are indicated in the user manual of the aforementioned "PowerScript Reverse Trancriptase "
A partir de la secuencia de una EST de Chlamydomonas, se sintetizaron dos oligonucleótidos específicos, SEQ ID No. 3 y SEQ ID No. 4, que se utilizaron para la reconstrucción mediante PCR del ADNc casi completo correspondiente al gen de la p-HPPD de Chlamydomonas.From the sequence of an EST of Chlamydomonas, two specific oligonucleotides were synthesized, SEQ ID No. 3 and SEQ ID No. 4, which were used for the PCR reconstruction of the corresponding almost complete cDNA to the Chlamydomonas p-HPPD gene.
Una vez amplificados por PCR los fragmentos de ADNc, éstos se purificaron de la mezcla de PCR mediante el kit "High Pure PCR Product Purification Kit" de la casa comercial ROCHE DIAGNOSTICS GMBH, siguiendo la metodología descrita en su manual de instrucciones.Once the PCR fragments were amplified CDNA, these were purified from the PCR mixture by the kit "High Pure PCR Product Purification Kit" of the commercial house ROCHE DIAGNOSTICS GMBH, following the methodology described in its manual.
Posteriormente, los productos de la amplificación, que se correspondían con los ARNm que contenían la secuencia de nucleótidos que codifica la p-HPPD de Chlamydomonas, se subclonaron en un vector pBluescript II KS^{+} de la casa comercial STRATAGENE siguiendo las instrucciones del fabricante.Subsequently, the products of the amplification, which corresponded to mRNAs containing the nucleotide sequence encoding the p-HPPD of Chlamydomonas, were subcloned into a pBluescript II KS + vector STRATAGENE commercial house following the instructions of the maker.
Los fragmentos de ADNc contenidos en el plásmido recombinante se secuenciaron en un secuenciador automático ABI 310 utilizando el kit Taq DyeDeoxy Terminator Cycle Sequencing de APPLIED BIOSYSTEMS (California, Estados Unidos), y así se obtuvo la secuencia de nucleótidos mostrada en la SEQ ID No. 1 (que corresponde a la secuencia de ADN que codifica para una HPPD de Chlamydomonas).The cDNA fragments contained in the plasmid recombinant were sequenced in an ABI 310 automatic sequencer using the Taq DyeDeoxy Terminator Cycle Sequencing kit from APPLIED BIOSYSTEMS (California, United States), and thus the nucleotide sequence shown in SEQ ID No. 1 (which corresponds to the DNA sequence that codes for an HPPD of Chlamydomonas).
A continuación, se transformaron células de E. coli con dicho vector, y se comprobó que las células transformantes contenían el vector con el inserto de ADNc correspondiente a la p-HPPD de Chlamydomonas mediante técnicas convencionales descritas en Sambrook et al., (1989). Seguidamente se extrajo el ADN correspondiente al plásmido recombinante, se purificó y secuenció utilizando un secuenciador automático de ADN mediante técnicas convencionales descritas en Sambrook et al. (1989). La secuencia obtenida (SEQ ID No 1) se comparó con las secuencias depositadas en las bases de datos utilizando el programa BLAST del National Center for Biotechnology Information (NCBI, Estados Unidos). La comparación de las secuencias puso de manifiesto que la secuencia de ADN obtenida que codifica para la HPPD de Chlamydomonas presentaba una relativamente alta homología de secuencia única y exclusivamente con otras secuencias de diferentes organismos que codifican una HPPD.Then, E. coli cells were transformed with said vector, and it was found that the transforming cells contained the vector with the cDNA insert corresponding to the Chlamydomonas p-HPPD by conventional techniques described in Sambrook et al ., (1989) . The DNA corresponding to the recombinant plasmid was then extracted, purified and sequenced using an automatic DNA sequencer by conventional techniques described in Sambrook et al . (1989). The sequence obtained (SEQ ID No 1) was compared with the sequences deposited in the databases using the BLAST program of the National Center for Biotechnology Information (NCBI, United States). The comparison of the sequences showed that the DNA sequence obtained that encodes the Chlamydomonas HPPD had a relatively high sequence homology only and exclusively with other sequences of different organisms encoding an HPPD.
De acuerdo con lo anterior, se ha conseguido clonar mediante RT-PCR el ADNc que codifica la p-hidroxifenilpiruvato dioxigenasa. Esto permitió la reconstrucción de un ADNc de 1838 pb que codifica una p-hidroxifenilpiruvato dioxigenasa (HPPD). La fase abierta de lectura desde el codón de inicio al codón de terminación codifica un péptido de 432 aminoácidos con una masa molecular calculada de 47206 Da, un punto isoeléctrico de 5,50 y una carga neta de -10,331 a pH 7,0.In accordance with the above, it has been achieved clone by RT-PCR the cDNA encoding the p-hydroxyphenylpyruvate dioxygenase. This allowed the reconstruction of an 1838 bp cDNA encoding a p-hydroxyphenylpyruvate dioxygenase (HPPD). The phase open reading from start codon to termination codon encodes a 432 amino acid peptide with a molecular mass calculated from 47206 Da, an isoelectric point of 5.50 and a load net of -10,331 at pH 7.0.
La clonación y caracterización del gen de la p-hidroxifenilpiruvato dioxigenasa constituye un paso previo para la obtención de organismos transgénicos con los niveles de tocoferoles alterados.Cloning and characterization of the gene of the p-hydroxyphenylpyruvate dioxygenase constitutes a previous step to obtain transgenic organisms with altered tocopherol levels.
Por otro lado, el gen de la HPPD gen puede ser utilizado como un gen marcador en procesos de transformación de plantas, ya que confiere resistencia a ciertos herbicidas, o ser utilizado para crear plantas resistentes a dichos herbicidas.On the other hand, the HPPD gene may be used as a marker gene in transformation processes of plants, as it confers resistance to certain herbicides, or be used to create plants resistant to these herbicides.
Seguidamente, se proporciona una relación detallada de las referencias bibliográficas que se han ido citando a lo largo de la exposición anterior:Next, a relationship is provided detailed references that have been cited throughout the previous exhibition:
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- Chipault, J.R., Mizuno, G.R., Hawkins, J.M., Lundsberg, W.O. (1952). Antioxidant properties of natural spices. Food. Res. 17, 46-55.- Chipault , JR, Mizuno , GR, Hawkins , JM, Lundsberg , WO ( 1952 ). Antioxidant properties of natural spices. Food. Res . 17, 46-55.
- Chipault, J.R., Mizuno, G.R., Hawkins, J.M.,1 Lundsberg, W.O. (1955). Antioxidant properties of spices in oil-in-water emulsions. Food Res. 20, 443-448.- Chipault , JR, Mizuno , GR, Hawkins , JM, 1 Lundsberg , WO ( 1955 ). Antioxidant properties of spices in oil-in-water emulsions. Food Res . 20, 443-448.
- Chipault, J.R., Mizuno, G.R., Hawkins, J.M., Lundsberg, W.O. (1956). The antioxidant properties of spices in foods. Food Technol. 10, 209-211.- Chipault , JR, Mizuno , GR, Hawkins , JM, Lundsberg , WO ( 1956 ). The antioxidant properties of spices in foods. Food Technol 10, 209-211.
- Combs, G.F. (1992). The vitamins. Fundamental aspects in nutrition and health. Academic Press, San Diego.- Combs , GF ( 1992 ). The vitamins Fundamental aspects in nutrition and health. Academic Press, San Diego.
- Cross, C.E. (1987). Oxygen radicals and human disease. Ann. Intern. Med. 107, 526-545.- Cross , CE ( 1987 ). Oxygen radicals and human disease. Ann. Intern. Med . 107, 526-545.
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- Economou, K.D., Oreopoulou, V., Thomolupoulos, C.D. (1991). Antioxidant activity of some plant extracts of the family Labiateaea. J. Am. Oil. Chem. Soc. 68, 109-113.- Economou , KD, Oreopoulou , V., Thomolupoulos , CD ( 1991 ). Antioxidant activity of some plant extracts of the family Labiateaea. J. Am. Oil. Chem. Soc . 68, 109-113.
- González, M.J. (1990). Serum concentrations and cellular uptake of vitamin E. Med. Hypotheses 32, 107-110.- González , MJ ( 1990 ). Serum concentrations and cellular uptake of vitamin E. Med. Hypotheses 32, 107-110.
- Halliwell, B. (1996). Antioxidants in human health and disease. Ann. Rev. Nutr. 16, 33-50.- Halliwell , B. ( 1996 ). Antioxidants in human health and disease. Ann. Rev. Nutr . 16, 33-50.
- Halliwell, B., Gutteridge, J.M.C., Cross, C.E. (1992). Free radicals, antioxidants and human disease: Where are we now?. J. Lab. Clin. Med. 6, 598-620.- Halliwell , B., Gutteridge , JMC, Cross , CE ( 1992 ). Free radicals, antioxidants and human disease: Where are we now ?. J. Lab. Clin. Med . 6, 598-620.
- Harris, E. (1989). The Chlamydomonas sourcebook. (Harris E., ed) Academic Press, New York.- Harris , E. ( 1989 ). The Chlamydomonas sourcebook . (Harris E., ed) Academic Press, New York.
- Horwitt, M.K. (1986). The promotion of vitamin E.J. Nutr. 116, 1371-1377.- Horwitt , MK ( 1986 ). The promotion of vitamin EJ Nutr . 116, 1371-1377.
- Horwitt, M.K. (1986). Interpretations of requirements for thiamin, riboflavin, niacin-tryptophan, and vitamin E plus comments on balance studies and vitamin B6. Am. J. Clin. Nutr. 44, 973-985.- Horwitt , MK ( 1986 ). Interpretations of requirements for thiamin, riboflavin, niacin-tryptophan, and vitamin E plus comments on balance studies and vitamin B6. Am. J. Clin. Nutr . 44, 973-985.
- Ingold, K.U., Burton, G.W., Foster, D.O., Hughes, L., Lindsay, D.A., Webb, A. (1987). Biokinetics of and discrimination between dietary RRR- and SRR-alpha- tocopherols in the male. Rat Lipids 22, 163-172.- Ingold , KU, Burton , GW, Foster , DO, Hughes , L., Lindsay , DA, Webb , A. ( 1987 ). Biokinetics of and discrimination between dietary RRR- and SRR-alpha- tocopherols in the male. Rat Lipids 22, 163-172.
- Jacobson, H.N. (1987). Dietary standards and future developments. Free Rad. Bici. Med. 3, 209-213.- Jacobson , HN ( 1987 ). Dietary standards and future developments. Free Rad Bike. Med . 3, 209-213.
- Lee, D.L., Prisbylla, M.P., Cromartie, T.H., Dagarin, D.P., Howard, S.W., Provan, W.M., Ellis, M.K., Fraser, T., Mutter, L.C. (1997) The discovery and structural requirements of inhibitors of p-hydroxyphenylpyruvate dioxygenase. Weed Sci 45, 601-609.- Lee , DL, Prisbylla , MP, Cromartie , TH, Dagarin , DP, Howard , SW, Provan , WM, Ellis , MK, Fraser , T., Mutter , LC ( 1997 ) The discovery and structural requirements of inhibitors of p- hydroxyphenylpyruvate dioxygenase. Weed Sci 45, 601-609.
- Lee, D.L., Knudsen, C.G., Michaely, W.J., Chin, H.L., Nguyen, N.H., Carter, C.G., Cromartie, T.H., Lake, B.H., Shribbs, J.M., Fraser, T. (1998). The structure-activity relationships of the triketona class of HPPD herbicides. Pestic. Sci. 54, 377-384.- Lee , DL, Knudsen , CG, Michaely , WJ, Chin , HL, Nguyen , NH, Carter , CG, Cromartie , TH, Lake , BH, Shribbs , JM, Fraser , T. ( 1998 ). The structure-activity relationships of the triketona class of HPPD herbicides. Pestic Sci . 54, 377-384.
- Mallet, J.F., Cerrati, C., Ucciani, E., Gamisans, J., Gruber, M. (1994). Antioxidant activity of plant leaves in relation to their alpha-tocopherol content. Food Chem. 49, 61-65.- Mallet , JF, Cerrati , C., Ucciani , E., Gamisans , J., Gruber , M. ( 1994 ). Antioxidant activity of plant leaves in relation to their alpha-tocopherol content. Food Chem 49, 61-65.
- Newman, S.M., Boynton, J.E., Gillham, N.W., Randolph-Anderson, B.L., Johnson, A.M., Harris, E.H. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events. Genetics 1990 126: 875-888.- Newman , SM, Boynton , JE, Gillham , NW, Randolph-Anderson , BL, Johnson , AM, Harris , EH Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events. Genetics 1990 126: 875-888.
- Parker, L., Witt, E.H., Tritschler, H.J. (1995). Alpha-lipoic acid as a biological antioxidant. Free Rad. Biol. Med. 19, 227-250.- Parker , L., Witt , EH, Tritschler , HJ ( 1995 ). Alpha-lipoic acid as a biological antioxidant. Free Rad Biol. Med . 19, 227-250.
- Pallett, K.E., Little, J.P., Sheekey, M., Veerasekaran, P. (1998). The mode of action of isoxaflutole I. Physiological effects, metabolism, and selectivity. Pestic. Biochem. And Physiol. 62, 113-124.- Pallett , KE, Little , JP, Sheekey , M., Veerasekaran , P. ( 1998 ). The mode of action of isoxaflutole I. Physiological effects, metabolism, and selectivity. Pestic Biochem And Physiol . 62, 113-124.
- Sambrook, J., Fritsch, E.F., Maniatis, R. (1998) Molecular cloning; A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.- Sambrook , J., Fritsch , EF, Maniatis , R. ( 1998 ) Molecular cloning; A laboratory manual. Cold Spring Harbor Laboratory Press , Cold Spring Harbor.
- Schulz, A., Ort, O., Beyer, P., Kleinig, H. (1993). SC-0051, a 2-benzoyl-cyclohexane-1,3-dione beaching herbicide is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase. FEBS Lett. 318, 162-166.- Schulz , A., Ort , O., Beyer , P., Kleinig , H. ( 1993 ). SC-0051, a 2-benzoyl-cyclohexane-1,3-dione beaching herbicide is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase. FEBS Lett . 318, 162-166.
- Schwants, P., Kimball, B.A., Idso, S.B., Hendrix, D.L., Polle, A. (1996) Antioxidants in sun and shade leaves of sour orange trees (Citrus aurantium) after long-term acclimation to elevated CO_{2}. J. Exp. Bot. 47, 1941-1950.- Schwants , P., Kimball , BA, Idso , SB, Hendrix , DL, Polle , A. ( 1996 ) Antioxidants in sun and shade leaves of sour orange trees (Citrus aurantium) after long-term acclimation to elevated CO_ {2} . J. Exp. Bot . 47, 1941-1950.
- Shintani, D., DellaPenna, D. (1998). Elevating the vitamin E content of plants through metabolic enginering. Science. 11, 2098-2100.- Shintani , D., DellaPenna , D. ( 1998 ). Elevating the vitamin E content of plants through metabolic enginering. Science 11, 2098-2100.
- Sokol, R.J. (1988). Vitamin E deficiency and neurologic disease. Ann. Rev. Nutr. 8, 351-373.- Sokol , RJ ( 1988 ). Vitamin E deficiency and neurologic disease. Ann. Rev. Nutr . 8, 351-373.
- Taga, M.S., Miller, E.E., Pratt, D.E. (1984). Chia seeds as a source of natural lipid antioxidants. J. Am. Oil Chem. Soc. 61, 928-933.- Taga , MS, Miller , EE, Pratt , DE ( 1984 ). Chia seeds as a source of natural lipid antioxidants. J. Am. Oil Chem. Soc . 61, 928-933.
- Viviani, E., Little, J.P., Pallett, K.E. (1998). The mode of action of isoxaflutole II. Characterization of the inhibition of carrot 4-hydroxyphenylpyruvate dioxigenass by the diketonitrile derivate of isoxaflutole. Pestic. Biochem. And Physiol. 62, 125-134.- Viviani , E., Little , JP, Pallett , KE ( 1998 ). The mode of action of isoxaflutole II. Characterization of the inhibition of carrot 4-hydroxyphenylpyruvate dioxigenass by the diketonitrile derivate of isoxaflutole. Pestic Biochem And Physiol . 62, 125-134.
- Written, C., Miller, E.E., Pratt, D.E. /(1984). Cotton-seed flavonoids as lipid antioxidants. J. Am. Oil Chem. Soc. 61, 1075-1078.- Written , C., Miller , EE, Pratt , DE / ( 1984 ). Cotton-seed flavonoids as lipid antioxidants. J. Am. Oil Chem. Soc . 61, 1075-1078.
- Wu, J.W., Lee, M.H., Ho,. T., Chang, S.S. (1984). Elucidation of the chemical structure of natural antioxidants isolated from rosemary. J. Am. Oil Chem. Soc. 59, 339-345.- Wu , JW, Lee , MH, Ho ,. T., Chang , SS ( 1984 ). Elucidation of the chemical structure of natural antioxidants isolated from rosemary. J. Am. Oil Chem. Soc . 59, 339-345.
<110> Universidad de Córdoba<110> University of Córdoba
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<130> Patente de UCO (chlamydomonas)<130> UCO patent (chlamydomonas)
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Claims (28)
- a)to)
- una secuencia de ADN que comprende la secuencia de nucleótidos identificada como SEQ ID No. 1;a DNA sequence comprising the nucleotide sequence identified as SEQ ID No. 1;
- b)b)
- una secuencia de ADN análoga a la secuencia definida en a) quea DNA sequence analogous to the sequence defined in a) that
- i)i)
- es sustancialmente homóloga a la secuencia de ADN definida en a) y/o, queis substantially homologous to the DNA sequence defined in a) and / or, that
- ii)ii)
- codifica para un polipéptido que es sustancialmente homólogo a la proteína codificada por la secuencia de ADN definida en a).encodes for a polypeptide that is substantially homologous to the protein encoded by the sequence of DNA defined in a).
- a)to)
- una secuencia de aminoácidos que comprende la secuencia de aminoácidos mostrada en la SEQ ID No. 2,a amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 2,
- b)b)
- la secuencia de aminoácidos deducida a partir de la secuencia de nucleótidos mostrada en la SEQ ID No. 1, ythe amino acid sequence deduced from the sequence of nucleotides shown in SEQ ID No. 1, and
- c)C)
- una secuencia de aminoácidos sustancialmente homóloga y funcionalmente equivalente a las secuencias de aminoácidos definidas en a) o en b).a substantially homologous and functionally amino acid sequence equivalent to the amino acid sequences defined in a) or in b).
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| US10793872B2 (en) | 2012-09-14 | 2020-10-06 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
| US10876130B2 (en) | 2014-03-11 | 2020-12-29 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| UY33141A (en) | 2009-12-23 | 2011-07-29 | Bayer Cropscience Ag | TOLERANT PLANTS TO INHIBITING HERBICIDES OF HPPD |
| AR079882A1 (en) | 2009-12-23 | 2012-02-29 | Bayer Cropscience Ag | TOLERANT PLANTS TO INHIBITING HERBICIDES OF HPPD |
| ES2658990T3 (en) | 2009-12-23 | 2018-03-13 | Bayer Intellectual Property Gmbh | HPPD-inhibiting herbicide-tolerant plants |
| UY33140A (en) | 2009-12-23 | 2011-07-29 | Bayer Cropscience Ag | TOLERANT PLANTS TO INHIBITING HERBICIDES OF HPPD |
| AR079883A1 (en) | 2009-12-23 | 2012-02-29 | Bayer Cropscience Ag | TOLERANT PLANTS TO INHIBITING HERBICIDES OF HPPD |
| US20120311743A1 (en) | 2010-02-02 | 2012-12-06 | Bayer Cropscience Ag | Soybean transformation using hppd inhibitors as selection agents |
| EP2669371A1 (en) | 2010-11-10 | 2013-12-04 | Bayer CropScience AG | HPPD variants and methods of use |
| CN103981149A (en) | 2011-08-22 | 2014-08-13 | 拜尔作物科学公司 | Methods and means to modify a plant genome |
| MX2018003044A (en) | 2015-09-11 | 2018-04-11 | Bayer Cropscience Ag | Hppd variants and methods of use. |
| US11708565B2 (en) | 2017-03-07 | 2023-07-25 | BASF Agricultural Solutions Seesi US LLC | HPPD variants and methods of use |
| BR112020008096A2 (en) | 2017-10-24 | 2020-11-03 | Basf Se | method for checking tolerance to a GM herbicide and soy plant |
| BR112020008092A2 (en) | 2017-10-24 | 2020-09-15 | BASF Agricultural Solutions Seed US LLC | method for checking tolerance to a GM herbicide and soy plant |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2734842B1 (en) * | 1995-06-02 | 1998-02-27 | Rhone Poulenc Agrochimie | DNA SEQUENCE OF A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE AND OBTAINING PLANTS CONTAINING A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE, TOLERANT TO CERTAIN HERBICIDES |
| US6087563A (en) * | 1996-01-29 | 2000-07-11 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Cloned arabidopsis p-hydroxyphenyl pyruvic acid dioxygenase DNA |
| JP2000513228A (en) * | 1996-06-27 | 2000-10-10 | イー・アイ・デユポン・ドウ・ヌムール・アンド・カンパニー | Plant gene for p-hydroxyphenylpyruvate dioxygenase |
| HUP0000053A3 (en) * | 1996-07-25 | 2003-02-28 | American Cyanamid Co | Hppd gene and inhibitors |
| WO2000032757A2 (en) * | 1998-12-03 | 2000-06-08 | E.I. Du Pont De Nemours And Company | Plant vitamin e biosynthetic enzymes |
| BR0116018A (en) * | 2000-12-07 | 2005-12-13 | Syngenta Ltd | Phenyl pyruvate dioxigenase (hppd) resistant hydroxy enzyme specific for tricetone inhibitor, herbicide resistant plants, methods for selecting a polynucleotide encoding a tricetone resistant enzyme, for selecting polynucleotides comprising a region encoding a hppd enzyme resistant to tricetone and syncharpic acid, and to provide a plant that is tolerant to hppd inhibitor herbicides, morphologically normal fertile whole plant, use of selectable polynucleotide, method for selectively controlling weeds at a site including crop plants and herbs. weeds, polynucleotide and plant cells |
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2003
- 2003-07-25 ES ES200301759A patent/ES2275365B1/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10793872B2 (en) | 2012-09-14 | 2020-10-06 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
| US10876130B2 (en) | 2014-03-11 | 2020-12-29 | BASF Agricultural Solutions Seed US LLC | HPPD variants and methods of use |
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| ES2275365A1 (en) | 2007-06-01 |
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