WO2003064661A1 - Identification de genes d'organismes vegetaux, ces genes etant specifiques de caracteristiques - Google Patents

Identification de genes d'organismes vegetaux, ces genes etant specifiques de caracteristiques Download PDF

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Publication number
WO2003064661A1
WO2003064661A1 PCT/EP2003/000903 EP0300903W WO03064661A1 WO 2003064661 A1 WO2003064661 A1 WO 2003064661A1 EP 0300903 W EP0300903 W EP 0300903W WO 03064661 A1 WO03064661 A1 WO 03064661A1
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WIPO (PCT)
Prior art keywords
plant
donor
genetic
genes
receptor
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PCT/EP2003/000903
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German (de)
English (en)
Inventor
Ulrich Zimmermann
Rainer Hedrich
Original Assignee
Ulrich Zimmermann
Kaldenhoff Ralf
Rainer Hedrich
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 Ulrich Zimmermann, Kaldenhoff Ralf, Rainer Hedrich filed Critical Ulrich Zimmermann
Priority to EP03704493A priority Critical patent/EP1470230A1/fr
Publication of WO2003064661A1 publication Critical patent/WO2003064661A1/fr

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    • 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
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses

Definitions

  • the invention relates to methods for identifying characteristic-specific genes of plant organisms, in particular methods for identifying individual genes or groups of genes (gene clusters) which cause the occurrence of a certain characteristic of the plant organism, and applications of such methods, in particular for genetic modification of plant species with trait-specific genes of other plant species.
  • the invention also relates to applications of biochemical and genetic engineering processes, such as. B. Applications of heterologous fusion processes, marking processes and segregation processes in the implementation of the above. Identification procedures.
  • the invention also relates to a device for identifying feature-specific genes of plant organisms.
  • the species-specific genetic material of a plant causes the characteristics to be expressed and the characteristics to be passed on to subsequent generations.
  • Features whose occurrence is caused by individual genes or gene groups include, for example, the resistance of a plant to physical conditions (e.g. heat resistance, drought resistance), the adaptation to chemical conditions (e.g. ability to symbiosis) or the resistance to foreign, animal or plant organisms (e.g. resistance to plant viruses or toxic plants).
  • trait-specific genes also extends to attempts to express certain traits of wild plants in crop plants and thus to be able to use them better.
  • the transfer of the ability to produce active ingredients from wild to cultivated plants or from sea plants in land plants requires knowledge of the genes that trigger the active ingredient production in the respective plant.
  • systematic methods for identifying trait-specific genes in plants have so far not been available.
  • the object of the invention is to provide improved methods for identifying trait-specific genes which cause at least one predetermined trait to appear in a plant.
  • the methods according to the invention are to be distinguished in particular by a wide range of applications with a large number of different features.
  • the object of the invention is also to provide applications of the identification methods.
  • One object of the invention is, in particular, to modify plant organisms with trait-specific genes, this being done with increased effectiveness and improved expression of the desired trait than in conventional transgenic plants.
  • the object of the invention is also to provide devices for implementing the aforementioned methods.
  • An important aspect of the invention is to provide methods for identifying genes which cause the occurrence of at least one predetermined feature in a plant under investigation (hereinafter: donor plant), wherein genetic material (DNA) of the donor plant and a receptor Plant which does not have the predetermined characteristic, is connected by heterologous fusion and at least one hybrid plant is produced from the genetic hybrid material thus formed which shows the predetermined characteristic of the donor plant.
  • the genes sought, which are part of the genetic material of the donor plant, are determined from the genetic material of this at least one selected hybrid plant.
  • This technique has the advantage that the heterologous fusion and cultivation of the hybrid material creates a model system in which feature-specific genes of the donor plant are integrated into the genes of the receptor plant that are not specific for the feature sought, so that a simple genetic comparison of the genetic material of the at least one hybrid plant with the genetic material of the receptor plant, the feature-specific genes of the donor plant can be found.
  • This method can advantageously be carried out universally with pairs of donor and receptor plants which differ in relation to a specific plant characteristic.
  • the genes identified in plant organisms according to the invention are understood to mean at least one gene or more genes which are distributed on one or more chromosomes of a plant and have a natural, synthetic, plant or non-plant origin.
  • Color-specific genes are the single gene or the gene cluster, which are effective for the expression of a certain characteristic of the plant organism.
  • the characteristic-specific genes encode the substances required for the characteristic expression.
  • the features investigated according to the invention include all types of properties of plant organisms which can be determined phenotypically and / or by molecular substance analysis, by means of which the respective organism differs from other plants.
  • the hybrid material formed by the heterologous fusion is selectively cultivated.
  • selective cultivation cell division and growth take place under selectively acting conditions in such a way that only certain parts of the genetic donor material are retained.
  • the selective cultivation has the advantage that the desired feature-specific genes of the donor plant can be identified with increased speed, effectiveness and target accuracy.
  • the selective cultivation takes place, for example, in an environment with physical or chemical conditions to which only the donor plant, but not the receptor plant, is resistant. Accordingly, hybrid material, which essentially consists of the genetic material of the receptor plant, can be excluded from further cultivation.
  • a higher selectivity is achieved by subjecting the genetic material of the donor plant to a genetic modification in which previously known resistance genes are introduced into at least one chromosome or chromosome section of the donor plant.
  • a genetic modification in which previously known resistance genes are introduced into at least one chromosome or chromosome section of the donor plant.
  • the feature-specific genes can also be localized by labeling chromosomes or chromosome sections with specific marker substances (e.g. fluorescent substances).
  • specific marker substances e.g. fluorescent substances.
  • the selection can also be carried out using promoter-reporter genes.
  • feature-specific gene clusters can be determined, which may be distributed over several donor chromosomes.
  • An important application of the invention is the isolation and transfer of the trait-specific genes found to useful plants so that they express the desired trait. Since feature-specific gene clusters can be determined with the method according to the invention, the generation of transgenic crop plants according to the invention is considerably more effective than conventional cloning techniques, in which one has concentrated on individual genes.
  • the invention also relates to a device which is designed to carry out the method according to the invention and comprises a device for providing genetic donor and receptor material, a fusion device, a cultivation device and a device for genetic analysis.
  • a device for providing genetic donor and receptor material a device for providing genetic donor and receptor material
  • a fusion device a device for providing genetic donor and receptor material
  • a cultivation device a device for genetic analysis.
  • An advantage of the invention is that the device according to the invention can be fully automated.
  • the individual components can be formed with facilities available per se from biotechnology and, in particular, fluid microsystem technology. Further advantages and details of the invention will become apparent from the following description of the accompanying drawings. Show it:
  • FIG. 2 shows a schematic illustration of various embodiments of the method according to the invention for identifying feature-specific genes.
  • FIG. 1 schematically shows the first steps 100, 200 of the method according to the invention, namely the provision of genetic donor and receptor materials and their heterologous fusion 300.
  • the provision of genetic material comprises the known steps of obtaining and possibly modifying cell material donor plant D and receptor plant R. Details of the provision are described below with reference to FIG. 2.
  • the schematically illustrated donor plant D represents the plant organism in whose genetic material (donor material) characteristic-specific genes are to be identified.
  • the donor material comprises, for example, the five chromosomes C D which are schematically illustrated in the right partial image.
  • Individual genes or gene segments that are effective for the expression of predetermined features of the donor plant D are exemplified on the donor chromosomes.
  • the different patterns of the highlighted genes or gene segments relate to different characteristics. For a specific characteristic, for example, a single gene T 3 (on the chromosome C D (3 ), a gene segment Ti, T 4 (e.g. on C D , ⁇ or C D , 4 ), two gene segments T 2 , T 5 on different chromosomes (e.g.
  • the gene segments T 2 and T 5 jointly encode a characteristic of the plant D.
  • characteristic-specific genes can be identified in relation to one characteristic or in parallel in relation to several characteristics. In the latter case, however, this requires a sufficiently large distinguishability of the characteristics during cultivation.
  • the donor plant D interesting features of the donor plant D are, for example, the ability to symbiosis (mycorrhizability, nodule formation), the ability to produce certain substances, such as. As flavorings, oils, active ingredients, polymer compounds (z. B. alginates) and substances that are important for metabolism or transport processes, or resistance to environmental conditions, such as. B. drought resistance, water resistance, heat resistance, cold resistance, mechanical stability of the plant architecture, salt resistance, resistance to foreign organisms or the like.
  • such plants are preferably used as donor plants that have relatively few chromosomes (e.g. fewer than seven different chromosomes). This has the advantage of a higher accuracy and effectiveness of the method.
  • alfalfa (with four different chromosomes) is used to identify the ability to symbiosis or to test the production of the active ingredient nicotine tobacco (with six different chromosomes) as a donor plant.
  • Any plant organism which does not have the property of interest of the donor plant can generally be used as the receptor plant R.
  • Plants whose genome is already known are preferably used. This owns Advantages in the analysis of the genetic material of cultivated hybrids (see below) and the identification of donor genes.
  • receptor plants are preferably used whose genetic engineering and biotechnological handling have already been developed and are known.
  • the genetic material of the receptor plant shows no genes or gene clusters on the chromosomes C R which are identical to the characteristic-specific genes of the donor plant D.
  • Arabidopsis with 10 chromosomes, for example, is used as the receptor plant R.
  • heterologous fusion 300 of cells of the donor and receptor plants D, R many hybrids Hi, H 2 , H 3 ... are formed, the genetic material of which is illustrated schematically in the lower part of FIG. 1.
  • the fusion results initially differ in the distribution of parts of the donor and receptor chromosomes or the complete chromosomes in the fused hybrid chromosomes C H.
  • all the chromosomes of the fusion partners are contained in the cells of the hybrid material.
  • the position of feature-specific donor genes in chromosomes of the hybrid material is generally changed.
  • An essential feature of the invention consists in the subsequent cultivation of the hybrid cells, possibly with the application of a selection pressure, which is described in more detail below with reference to FIG. 2.
  • Stable organisms form two groups, which on the one hand more and more resemble the donor plant and on the other hand more and more the receptor plant.
  • Each group comprises two subgroups, each formed by organisms that are similar to one of the fusion partners and have characteristics of the other fusion partner.
  • These hybrids are also referred to below as model plants.
  • model plants contain the genetic receptor material and of the donor material only the individual gene or the gene cluster that is characteristic of the desired trait.
  • the result of the cultivation can also result in several model plants whose genetic properties are compared with one another for the final identification of the characteristic-specific genes.
  • FIG. 2 schematically illustrates identification methods according to the invention with various exemplary embodiments.
  • the individual boxes indicate procedural steps that are mandatory (solid lines) or optional (dashed lines).
  • the provision 100, 200 of genetic donor and receptor materials initially comprises in each case the selection 110 of a suitable donor D and receptor R. This selection is made according to the principles explained above with reference to FIG. 1.
  • cell material is obtained from the donor and receptor plants by methods known per se. Depending on the application, certain parts of plants are used to obtain cell material.
  • parts (organs) of the donor plant are used which express the desired feature at all or particularly strongly. With these parts it can be expected that the genes sought will be expressed. Root parts are preferably used to determine genes which are effective, for example, for nodule formation.
  • the cell material of the donor and receptor plants is each subjected to protoplasting 140.
  • Protoplastization is used to obtain donor and receptor protoplasts.
  • Protoplasting 140 is also carried out according to methods known per se.
  • the protoplasts are then isolated. This is preferably done according to known protocols using washing and enzyme solutions.
  • a large number (for example several 1000) of donor and receptor protoplasts are present, which are then subjected to the heterologous Fusion 300.
  • heterologous fusion for hybrid formation has the particular advantage that the species limit is exceeded when genetic material is mixed.
  • the sought-after characteristic can be transferred to an organism that essentially resembles the receptor plant R and can thus be recorded with high selectivity.
  • all methods known per se for heterologous fusion in particular electrically, chemically or virally triggered fusions, can be used. Further division and regeneration of protoplasts that have not been fused can be prevented by iodoacetate treatment. A phy- sical or chemical separation (filtering) of unfused protoplasts.
  • the heterologous fusion 300 there are, for example, a few 1000 hybrid cells which are subsequently subjected to the cultivation 400.
  • hybrid growth 410 takes place first. Already in the context of hybrid growth 410, not all chromosomes are evenly inherited in all hybrids. There is natural chromosome segregation. Hybrid growth 410 takes place, for example, under growth conditions which are customary per se.
  • the hybrid growth 410 is followed by a vitality test 420, which according to a first embodiment of the invention is carried out without a selection pressure.
  • the vitality test 420 checks whether the grown hybrids are still viable and show, for example, metabolism.
  • a substance analysis test 430 of hybrid cells or, after regeneration 440 of hybrid plants, a phenotype test 450 follows.
  • the substance analysis test 430 includes, for example, a metabolism analysis and / or a proteome analysis of the grown hybrids.
  • the metabolism analysis determines whether the hybrid cell under consideration produces secondary substances that correspond to the characteristic sought.
  • a proteome analysis it is correspondingly determined whether the hybrid cell under consideration produces proteins that are characteristic of the desired feature.
  • Hybrid cells that fail molecular genetic test 430 are removed.
  • a regeneration 460 follows to the complete hybrid plant, followed by the phenotype test 450.
  • This process involves the phenotype test 450 checked whether the regenerated hybrid plants resemble the receptor plants and thus represent the sought-after model plants.
  • the regeneration takes place according to known protocols for tissue and plant development from a cell culture. Furthermore, it can be checked whether the characteristic found in test 430 is also pronounced in the phenotype.
  • the vitality test 420 is followed by a regeneration 440 of complete hybrid plants, of which the surviving plants are subjected to the phenotype test 450 to determine the desired feature.
  • the phenotype test 450 checks the characteristics searched for for the first time. For example, resistance to environmental conditions or the production of active substances is tested. The plants which positively show the characteristic are also checked whether they resemble the receptor plants and thus represent the model plants sought.
  • an interaction test is carried out on each surviving hybrid plant.
  • the test can advantageously be carried out automatically, for example, using available image processing methods.
  • the genetic material of the at least one model plant is analyzed, which the cultivation 400 by means of the substance analysis test 430 with subsequent regeneration 460 or regeneration 440, in each case with subsequent phenotype test 450, is successful has gone through positively.
  • the genes of the model plants that are not contained in the genetic material of the receptor and are identified as feature-specific genes are determined. The identification is based on the comparison of the gene maps of the receptor and the model plants. If several model plants are found, their gene maps can be compared with one another and the characteristic-specific genes sought can be identified from the correlation.
  • bioinformatic evaluation of the identified genes, utilization of the model plants used, sequencing of the identified genes with a transfer to other plants and / or further analyzes of the hybrid plants found can take place.
  • the gene identification according to the invention according to modified embodiments is carried out as follows using a marking technique and / or by applying selection pressure.
  • a genetic modification 120 of the donor plants or a marking 160 of the chromosomes or chromosome sections in the protoplasts takes place when the genetic donor material is provided.
  • the conventional fluorescent markers are installed.
  • FISH technique the positions of the identified genes on the original donor chromosomes can be determined in the analysis 500 after determining the model plant.
  • the genetic donor material is specifically changed as part of the genetic modification 120.
  • at least one known gene is introduced into the genetic donor material, which brings about an antibiotic or herbicide resistance of the modified donor. For example, basta or kanamycin resistance is introduced.
  • a donor plant with e.g. B.
  • each chromosome there is preferably a specific modification of each chromosome in such a way that the resistance genes added to different chromosomes cause different resistances.
  • the vitality test 470 encompasses cultivation with exertion of a selection pressure, for example by culturing in the presence of at least one antibiotic or herbicide.
  • a selection pressure for example by culturing in the presence of at least one antibiotic or herbicide.
  • the hybrid cells can be divided into a number of resistance groups which corresponds to the number of chromosomes or chromosome segments which were contained in the genetic donor material.
  • the cells in each resistance group contain donor genetic material from a common donor chromosome.
  • the above-mentioned tests 430 and 450 then follow, each connected to the regenerations 460 and 440.
  • chromosome identification 500 it is possible without further measurement simply by assigning surviving mo- dell plants to one of the resistance groups the localization of the identified genes on one or more donor chromosomes or chromosome sections are given.
  • the method according to the invention is carried out with transgenic receptor plants.
  • promoter genes are inserted into its genetic material in combination with resistance genes.
  • the promoter genes are selected so that they activate the associated resistance genes based on the expression of a certain plant characteristic. For example, genes are known which activate a plant after mycorrhization. This enables selection based on promoter-reporter genes, which has the advantage of quick identification of the feature-specific genes.
  • a specific resistance gene is activated with the promoter gene, provided that the desired feature is formed by the cell. The tests are carried out accordingly under conditions that only the cells or plants with the activated resistance gene survive.
  • a chromosome segregation 150 of the genetic donor material can be provided.
  • the donor material can be divided into chromosome sections in order to increase the selectivity of the gene identification.
  • the chromosome segregation 150 is carried out using techniques known per se, e.g. B. using UV or X-rays or chemical fragmentation.
  • the identified genes can be sequenced with a transfer to other plants be provided.
  • Applications generally exist in the transfer of characteristics from wild or cultivated plants to cultivated plants, from sea plants to land plants and / or between plants in different geographical regions or from different site conditions. Examples include the transfer of active ingredient production from wild medicinal plants to useful plants or the alginate production of algae on land plants, or the transfer of rapid plant growth from economically inferior woods to precious woods.
  • the determined model plants themselves can continue to be used as useful plants.

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Abstract

Procédé d'identification de gènes spécifiques de caractéristiques, qui provoquent chez une plante donneur (D) l'apparition d'au moins une caractéristique prédéterminée. Au moins une plante modèle qui possède la caractéristique prédéterminée est produite par fusion hétérologue et culture à partir de matériel génétique de la plante donneur (D) et d'une plante récepteur (R) qui ne possède pas la caractéristique prédéterminée. Les gènes recherchés, spécifiques de caractéristiques, qui constituent une partie du matériel génétique de la plante donneur (D) sont déterminés à partir du matériel génétique de la plante modèle..
PCT/EP2003/000903 2002-01-30 2003-01-29 Identification de genes d'organismes vegetaux, ces genes etant specifiques de caracteristiques WO2003064661A1 (fr)

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EP03704493A EP1470230A1 (fr) 2002-01-30 2003-01-29 Identification de genes d'organismes vegetaux, ces genes etant specifiques de caracteristiques

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DE10203627.6 2002-01-30
DE10203627A DE10203627A1 (de) 2002-01-30 2002-01-30 Identifizierung von merkmalspezifischen Genen pflanzlicher Organismen

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5332408A (en) * 1992-08-13 1994-07-26 Lakeside Biotechnology, Inc. Methods and reagents for backcross breeding of plants
WO2000007434A1 (fr) * 1998-08-05 2000-02-17 Mary Wilkes Eubanks Nouvelles matieres genetiques destinees a etre transmises au mais
WO2000060089A1 (fr) * 1999-04-07 2000-10-12 Mendel Biotechnology, Inc. Procede de reproduction d'un trait genetique
WO2000070019A2 (fr) * 1999-05-17 2000-11-23 Icon Genetics, Inc. Procede de transformation vegetale rapide independante de la variete
WO2001011020A1 (fr) * 1999-08-05 2001-02-15 Icon Genetics, Inc. Procede de fabrication de chromosomes artificiels de plantes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5332408A (en) * 1992-08-13 1994-07-26 Lakeside Biotechnology, Inc. Methods and reagents for backcross breeding of plants
WO2000007434A1 (fr) * 1998-08-05 2000-02-17 Mary Wilkes Eubanks Nouvelles matieres genetiques destinees a etre transmises au mais
WO2000060089A1 (fr) * 1999-04-07 2000-10-12 Mendel Biotechnology, Inc. Procede de reproduction d'un trait genetique
WO2000070019A2 (fr) * 1999-05-17 2000-11-23 Icon Genetics, Inc. Procede de transformation vegetale rapide independante de la variete
WO2001011020A1 (fr) * 1999-08-05 2001-02-15 Icon Genetics, Inc. Procede de fabrication de chromosomes artificiels de plantes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GLEBA Y Y: "CELLULAR GENETIC ENGINEERING A NEW TECHNOLOGY IN PLANT BREEDING?", IMPACT OF SCIENCE ON SOCIETY (ENGLISH EDITION), vol. 36, no. 2, 1986, pages 165 - 174, XP009010522, ISSN: 0019-2872 *
KOSHINSKY H A ET AL: "Cre-lox site-specific recombination between Arabidopsis and tobacco chromosomes", PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 23, no. 6, September 2000 (2000-09-01), pages 715 - 722, XP002232337, ISSN: 0960-7412 *

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DE10203627A1 (de) 2003-07-31
EP1470230A1 (fr) 2004-10-27

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