EP1268831A2 - Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures - Google Patents

Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures

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Publication number
EP1268831A2
EP1268831A2 EP01940107A EP01940107A EP1268831A2 EP 1268831 A2 EP1268831 A2 EP 1268831A2 EP 01940107 A EP01940107 A EP 01940107A EP 01940107 A EP01940107 A EP 01940107A EP 1268831 A2 EP1268831 A2 EP 1268831A2
Authority
EP
European Patent Office
Prior art keywords
seed
plants
gene
plant
dna sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01940107A
Other languages
German (de)
English (en)
Inventor
Hans Weber
Isolde Saalbach
Martin Giersberg
Petra Hoffmeister
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Original Assignee
Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut fuer Pflanzengenetik und Kulturpflanzenforschung filed Critical Institut fuer Pflanzengenetik und Kulturpflanzenforschung
Publication of EP1268831A2 publication Critical patent/EP1268831A2/fr
Withdrawn legal-status Critical Current

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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • 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
    • C12N9/90—Isomerases (5.)

Definitions

  • the invention relates to a process for the production of legumes with an increased protein content in the seed with a prolonged seed holding period by introducing recombinant DNA molecules.
  • the recombinant DNA molecules are introduced into the plant by means of a transformation system, wherein they comprise a plant-specific DNA sequence which is expressed in plants and whose gene product in the seed is a protein with the enzymatic activity of an ADP-glucose pyrophosphorylase (AGP) and / or a plastid phosphoglucomutase (pPGM) inhibits and / or whereby a sucrose transporter gene is overexpressed and, if necessary, the regulatory sequence of a seed-specific promoter in legumes. Furthermore, at least one selection marker gene is transferred separately, which is then removed again. The plants are selected which have an increased protein content and a longer duration of seed training.
  • AGP ADP-glucose pyrophosphorylase
  • pPGM plastid phosphoglucomutase
  • Pea seeds synthesize starch and proteins from the precursors sucrose and amino acids. This requires coordinated regulation of the two metabolic pathways. Starch and protein content have an inverse relationship to one another. A number of peas (shriveled seed mutants) have a block in the starch biosynthetic pathway and thus reduced starch contents. At the same time, the protein content is increased (Perez et al. 1993, Boutin et al. 1998). These mutants differ in the relative contents of albumins and 11s and 7s globulins. The proportion of albumin, which has a higher proportion of essential amino acids, is often increased.
  • the mutants relate, for example, to sucrose synthase, ADP-glucose pyrophosphorylase (AGP), or branching enzyme.
  • AGP ADP-glucose pyrophosphorylase
  • An interesting enzyme is plastid phosphoglucomutase (pPGM). It catalyzes the reaction Glc-lP to Glc-6-P in the amyloplasts. This is a metabolically important branch between the starch biosynthetic pathway (Glc-lP) and the oxidative pentose phosphate cycle (Glc-6-P). The latter is important for the synthesis of aromatic amino acids and provides reduction equivalents.
  • pPGM catalyzes an equilibrium reaction, which can be shifted from equilibrium in vivo and limit the rate of starch biosynthesis (Hattenbach et al. 1999, Tetlow et al. 1998). Mutant peas in which pPGM has failed have almost starch-free seeds. However, the starch synthesis in the maternal parts of the plant and thus the yield is obviously impaired (Harrison et al. 1998). The accumulation of storage material in semen depends on the number of cells in the embryo / endosperm, the duration of the cell division phase, the speed of semen filling and the duration of semen filling (Hanson 1991). Seed growth and yield components are therefore controlled at the level of seed growth rate and seed filling duration (Egli 1994).
  • Seeds are big because they either grow fast or long.
  • the seed growth rate is dependent on assimilate availability, intake and metabolism. For example, large seeds have a high rate. Wholesomeness, however, is not a yield-determining parameter, but is reciprocal to the number of seeds (Egli, 1998). Magnificent genotypes from the outset have fewer ovules or the flowers / pod shedding is higher.
  • the semen filling time is correlated with the yield, a longer semen filling period also brings more yield.
  • the cell size is obviously important.
  • the invention is therefore based on the object of genetically modifying the starch and storage protein ratio and the protein quality in legumes, in particular in grain peas.
  • certain plant genes are to be expressed in antisense or sense orientation; the transgenic plant should not contain foreign genes.
  • Another goal is to further develop the transformation technology under the aspect of direct application for the breeder.
  • recombinant DNA sequences of plant genes are introduced into the seeds of the plant by means of a transformation system.
  • the recombinant DNA sequences comprise a DNA sequence which can be expressed in the plants and whose gene product in seeds is a protein with the enzymatic activity an ADP-glucose pyrophosphorylase (AGP) and / or a plastid phosphoglucomutase (pPGM) and / or a sucrose transporter gene is overexpressed.
  • AGP ADP-glucose pyrophosphorylase
  • pPGM plastid phosphoglucomutase
  • sucrose transporter gene sucrose transporter gene
  • they may contain the regulatory sequence of a seed-specific promoter in legumes.
  • the method is further characterized in that at least one selection marker gene is transferred independently, which is then removed again in the 1st generation. Plants are selected that have an increased protein content and a longer duration of seed training (see Fig. 2).
  • transgenic plants were preferably produced, which express in particular the small subunit of ADP-glucose pyrophosphorylase (AGPC, Weber et al. 1995), a key enzyme in starch biosynthesis, in an antisense orientation, preferably under the control of a seed-specific promoter, in particular the Legumin B4 promoter.
  • ADP-glucose pyrophosphorylase ADP-glucose pyrophosphorylase
  • the transgenic lines have a 10-15% higher protein content, the starch biosynthetic pathway is downregulated and the distribution of carbon is increased in favor of storage protein biosynthesis.
  • the seed filling time an important yield parameter, is extended by changing the seed-water ratio. Plants are produced which do not contain the DNA coupled to the marker gene and preferably integrate them at different loci and consequently enable the marker gene to be segregated.
  • the presented method can basically be applied to seeds of all crop types that store starch.
  • VfAGPC x 76940 Weber et al, Planta 195, 352-361.
  • the legumin B4 promoter (LeB4) and the sucrose binding protein promoter (SBP) preferably act as seed-specific promoters.
  • the invention is exemplified on the basis of the production of transgenic plant lines which express ADP-glucose pyrophosphorylase in antisense orientation (AGP-as), 1. Constructs
  • plasmid which contains only one selection marker (e.g. bar gene) and c) a plasmid which contains both, AGP-as and selection marker.
  • a construct was also transformed in parallel that contains both useful gene and selection marker on one plasmid.
  • the stable lines are produced by selfing and then selecting the seeds by hand. This is relatively easy because the AGJ * antisense seeds have a shriveled phenotype and are therefore easy to distinguish from the round and smooth-seeded wild type seeds.
  • Seed quality characteristics i) C / N ratio, ii) total sulfur content, iii) relative composition of the seeds with regard to protein classes (globulins / albumins) as well as starch (structure), amylose to amylopectin ratio, soluble sugars and lipids.
  • an antisense LXoression of the plastid phosphoglucomutase was carried out for the production of transgenic plant lines, preferably under the control of the LeB4 or the SBP promoter.
  • the effects similar to those of the AGP 'antisense plants were achieved with the antisense plants, the starch content being reduced even further.
  • a further variant of the invention for the production of transgenic plant lines with a prolonged seed training period consists in expressing a gene construct for a sucrose transporter in sense orientation. Seed growth requires cell expansion and an important determinant for this is the availability of water. Its inflow is dependent on a high osmotic gradient between symplasts and apoplasts.
  • a sucrose transporter was overexpressed under the control of the LeB4 promoter, which resulted in an increase in sucrose content, which in turn led to an increase in seed ripening time, which is a parameter that correlates with yield.
  • the starch biosynthesis is not affected.
  • the cDNA sequence of the Viciafaba sucrose transport protein 1 (VfSUTl) was preferred Gens (Weber et al. 1997) under control of the LeB4 promoter for transformation.
  • the conventional transformation mediated by agrobacteria could be expanded in such a way that selection and useful genes are transmitted independently of one another on different binary vectors.
  • the selection marker e.g. bar gene
  • the selection marker which is initially necessary for the transformation but is undesirable for the application can be selected.
  • Vicia narbonensis in which both the AGPC transcripts and the AGP enzyme activity in stage 7 cotyledons are reduced by 80 to 95% (Vicia seed development was divided into stages 1-7, Borisjuk et al 1995).
  • sucrose is increased twice. Dry transgenic seeds are wrinkled, but have an unchanged dry weight and germinate normally.
  • starch in ripe seeds is reduced by about 10-30%.
  • the extractable proteins of the albumin and globulin fractions are significantly higher. Dry seeds therefore have a significantly higher total nitrogen content, but interestingly enough, the total carbon is not reduced.
  • transgenic embryos have significantly higher fresh weights and a larger cell size. Seed ripening is extended by about 3-4 days. A histological analysis showed that transgenic storage parenchyma cells have a lower degree of differentiation compared to the wild type of the same age. However, this difference is eliminated in the ripe seeds. A change in starch biosynthesis may also affect the semen development program.
  • Glucose is reduced by up to 95%.
  • Hexose-P and Fru 1, 6-P 2 are significantly higher, but not 3PGA, PEP and Pyr.
  • Carbon assimilates are apparently redistributed into soluble sugars and proteins.
  • the seed ripening and thus the seed filling period is significantly extended, which is obviously triggered by the higher sucrose content.
  • Sucrose on the one hand induces storage-associated gene expression and influences the carbon distribution.
  • it increases the osmotic gradient between cytoplasm and apoplast, causing water to flow in and the cells to expand. In this way, the storage metabolism metabolism can be maintained.
  • the efficiency for transgenic Ro plants is about 3-4%.

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

Abstract

L'invention concerne un procédé de production de légumineuses, à plus haute teneur en protéines dans les semences et présentant une plus longue durée de remplissage du grain, par l'introduction de molécules d'ADN recombinant. Les molécules d'ADN recombinant sont introduites dans la plante à l'aide d'un système de transformation et présentent une séquence d'ADN propre aux plantes, exprimée dans les plantes et dont le produit génétique inhibe dans la semence une protéine avec l'activité enzymatique d'une ADP-Glucose Pyrophosphorylase (AGP) et/ou d'une phosphoglucomutase plastide (pPGM) et, éventuellement, la séquence régulatrice d'un promoteur spécifique à la semence dans les légumineuses. De plus, au moins un gène marqueur de sélection est transmis séparément puis à nouveau retiré. Les plantes présentant une teneur en protéines supérieure et une durée de remplissage du grain plus longue sont sélectionnées.
EP01940107A 2000-03-31 2001-03-23 Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures Withdrawn EP1268831A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10015989 2000-03-31
DE10015989 2000-03-31
PCT/DE2001/001208 WO2001075128A2 (fr) 2000-03-31 2001-03-23 Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures

Publications (1)

Publication Number Publication Date
EP1268831A2 true EP1268831A2 (fr) 2003-01-02

Family

ID=7637081

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01940107A Withdrawn EP1268831A2 (fr) 2000-03-31 2001-03-23 Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures

Country Status (5)

Country Link
EP (1) EP1268831A2 (fr)
AR (1) AR027750A1 (fr)
AU (1) AU7382001A (fr)
DE (1) DE10115762A1 (fr)
WO (1) WO2001075128A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7323560B2 (en) 2000-07-17 2008-01-29 E.I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
US7250557B2 (en) 2000-07-17 2007-07-31 E. I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5457274A (en) * 1992-05-15 1995-10-10 Research Corporation Technologies Pink-flowered, high protein soybean plants and methods for their production
US5498831A (en) * 1993-07-23 1996-03-12 Dna Plant Technology Corporation Pea ADP-glucose pyrophosphorylase subunit genes and their uses
DE4439748A1 (de) * 1994-10-31 1996-05-02 Inst Genbiologische Forschung Verfahren zur Veränderung des Blühverhaltens bei Pflanzen
NZ333674A (en) * 1996-07-09 2000-09-29 Unilever Plc Process for increasing the sucrose content of pea seeds
AUPO930597A0 (en) * 1997-09-19 1997-10-09 Commonwealth Scientific And Industrial Research Organisation Method for altering seed compostion
WO1999029161A1 (fr) * 1997-12-08 1999-06-17 Seminis Vegetable Seeds, Inc. Variete sans amidon de pisum sativum, a teneur elevee en saccharose
WO1999036551A1 (fr) * 1998-01-15 1999-07-22 E.I. Du Pont De Nemours And Company Homologues de phosphoglucomutase vegetale
AU3474899A (en) * 1998-04-09 1999-11-01 E.I. Du Pont De Nemours And Company Sucrose transporters from plants
EP1144663A1 (fr) * 1999-01-19 2001-10-17 Unilever Plc Procede permettant d'accroitre la teneur en oxydant soluble dans l'eau dans des pois recoltes mecaniquement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0175128A3 *

Also Published As

Publication number Publication date
WO2001075128A3 (fr) 2002-04-04
AU7382001A (en) 2001-10-15
DE10115762A1 (de) 2001-12-06
AR027750A1 (es) 2003-04-09
WO2001075128A2 (fr) 2001-10-11

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