EP0876496A1 - An expression system for anaerobic gene expression in higher plants - Google Patents
An expression system for anaerobic gene expression in higher plantsInfo
- Publication number
- EP0876496A1 EP0876496A1 EP96946241A EP96946241A EP0876496A1 EP 0876496 A1 EP0876496 A1 EP 0876496A1 EP 96946241 A EP96946241 A EP 96946241A EP 96946241 A EP96946241 A EP 96946241A EP 0876496 A1 EP0876496 A1 EP 0876496A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- expression system
- gene
- promoter
- expression
- gapc4
- 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.)
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Classifications
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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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
-
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8237—Externally regulated expression systems
-
- 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
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
Definitions
- the invention relates to an expression system for anaerobic gene expression in higher plants.
- a specific field of application of the invention is agriculture, in particular resistance breeding and increasing the performance of useful plants.
- the aim of the invention is to achieve the expression of resistance factors in useful plants and to generate corresponding transgenic plants.
- the object of the invention is to achieve the anaerobic expression of the T4 lysozyme gene in the potato and to generate corresponding transgenic plants.
- a specific task is to make potatoes resistant to phytopathogenic bacteria.
- the object of the invention is achieved by an expression system which comprises the promoter GapC4 or parts or variants of the promoter GapC4 and a gene to be expressed.
- genes for an antibacterial protein in particular for T4 lysozyme, resistance genes against viruses, nematodes, bacteria and fungi, genes with insecticidal activity, genes which increase glycolysis and genes which increase fermentation.
- the expression system is used according to the invention for anaerobic gene expression in higher plants. It is preferably used in crops such as potatoes, rice, cereals, corn, tomatoes, brassicaceae, legumes, cotton, sugar beet and carrots.
- the invention also relates to higher plants, preferably transgenic crop plants such as potatoes, rice, cereals, corn, tomatoes, brassicaceae, legumes, cotton, sugar beet and carrots, which contain the expression system according to the invention.
- transgenic potatoes which contain an expression system consisting of the promoter GapC4 and the gene for T4 lysozyme.
- the great advantage of the promoter GapC4 used according to the invention is that it has an induction profile which is extremely suitable for the purpose.
- the anaerobic expression reaches the strength of the 35S promoter of the cauliflower mosaic virus (35S CaMV), which is frequently used under aerobic conditions for the expression of foreign genes.
- the promoter is active in all tissues, such as flowers, leaves and roots. So far, the isolation of the GapC4 gene and the anaerobic induction of the GapC4 promoter in a transient expression system in maize suspension culture cells have been published (Kersanach et al., 1994, Nature 367: 387-389).
- the promoter is particularly active in the potato crop.
- plants can experience prolonged periods of great moisture, which can decrease the amount of moisture Plant does not survive available oxygen (Perata and Alpi, 1993, Plant Sei. 93, 1-17).
- the tolerance of plants to insufficient oxygen supply is quite different for individual species.
- the embryo in the rice caryopsis germinates without difficulty even under such conditions, whereas maize seedlings only survive for about 24 hours without oxygen.
- a general strategy for adapting higher plants to anaerobic conditions is to increase glycolysis and to activate fermentation processes.
- the genes involved in glycolysis and fermentation can be brought under the control of an anaerobically inducible promoter. These genes are then expressed when there is insufficient oxygen supply.
- GapC4 promoter reporter gene constructs are transformed into the potato. After infection of the transgenic potato with Erwinia carotovora and after incubation of tissues of the transgenic potato under anaerobic conditions, the reporter gene expression can be measured. The reporter gene can then be replaced in vitro by the T4 lyse enzyme gene and transformed into the potato. The transgenic potatoes are then examined for increased resistance to Erwinia carotovora.
- the reporter gene constructs were constructed as described below:
- Plasmids pUK443 and pUK444 which carry 785 and 461 base pairs of the GapC4 promoter, the first intron of the GapC4 gene and the ⁇ -glucuronidase reporter gene, were used for T-DNA constructs. In order to rule out possible negative effects of the GapC4 intron from maize in transgenic tobacco plants and potatoes, the intron was cut out from the plasmids pUK443 and pUK444 by restriction digestion with the enzymes Xhol and Wcol.
- the intronless plasmids pUK403 and pUK404 were generated.
- the promoter reporter gene fragments of these plasmids were cut out by a PvuII digest and cloned into the Smal site of pOCA28.
- the resulting pOCA28 derivatives, pUK4030, 4040 and 4041 carry the GapC4 promoter-reporter gene construct.
- the promoterless ⁇ -glucuronidase reporter gene was cloned into pOCA28.
- pUK4030 and 4040 carry the reporter gene constructs with the promoter proximal to the right T-DNA border sequence. In pUK4041, the reporter gene is in the other orientation.
- the ⁇ -glucuronidae reporter gene can be removed from the plasmids pUK403 or 404 by restriction digestion and the T4 lysozyme gene can be cloned in its place.
- the T4 lysozyme gene controlled by the GapC4 promoter is cloned into an Agrobacterium T-DNA vector. After introduction into Agrobacterium tumefaciens, all constructs are transformed into the potato according to standard protocols (During et al., 1993, Plant J. 3, 587-598; Fladung, 1990, Plant Breeding 104, 295-304). Transgenic potatoes are selected with the help of an antibiotic resistance gene on the T-DNA and are based on expression of the ß-glucuronidase reporter gene
- REPLACEMENT BUTT (RULE 26) and the T4 lysozyme gene under anaerobic conditions and after infection with Erwinia carotovora.
- plant tissue is incubated in an airtight glass container (Merck) together with Anaerocult A (Merck) for at least 12 hours.
- Anaerocult A Merck
- fluorimetric GUS assay the plant material is homogenized and incubated with the ß-glucuronidase substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG) at 37 ° C. Fluorescence is quantified according to Jefferson et al., EMBO J. 6, 3901-3907 (1987) and protein concentrations are determined according to Bradford, Anal. Biochem. 7, 248-254 (1976).
- the intact anaerobically induced plant material is vacuum-filtered with a solution of 1 mM X-gluc (5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid) and incubated at 37 ° C. overnight.
- the chlorophyll is extracted with 70% ethanol (Jefferson see above).
- RNA filter In order to measure the expression of the T4 lysozyme gene, Northern blot analyzes are carried out after anaerobic incubation of the tissue with a T4 lysozyme-specific probe.
- Total RNA is isolated with the RNeasy Kit (Qiagen). The concentration of the RNA is determined photometrically and 10 micrograms of the RNA are placed in a lane on a 1% agarose gel which contains formaldehyde. After electrophoresis, the RNA from the gel is blotted with 0.05 N NaOH as a transfer buffer onto nitrocellulose or nylon membranes (Amersham) (Sambrook et al. See above). Labeling and hybridization of the T4 lysozyme-specific probe with the RNA filter is carried out according to standard conditions (Sambrook et al. See above, During et al., See above).
- REPLACEMENT BLA ⁇ (RULE 26) the tuber material under the macerated tissue is used.
- the infection test is carried out with a pathogenic strain of Erwinia carotovora ssp. atroseptica or ⁇ p. carotovora performed in plastic containers with the exclusion of air.
- Slices of a defined size are made from potato tubers and inoculated freshly cut with a defined number of bacterial cells in a small volume in the middle. Incubation takes place in plastic containers with a layer of water on the bottom on a soaked filter paper. Saturated air humidity is reached.
- the bacterial growth is tracked based on the tissue maceration and the resulting bacterial mucus.
- the resulting bacterial mucus creates an air-tight covering of the potato cells.
- the extent of the maceration is determined after a defined time.
- the relative decrease in susceptibility can be determined by comparison with control explants.
- eye cuttings infected with bacteria can be planted out and cultivated under moist conditions in the greenhouse. Silting up the earth leads to a lack of oxygen, which favors the multiplication of the bacteria. The number of emerged, healthy shoots is determined in comparison to control explants. The extent of the reduced susceptibility can thus be determined.
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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Ein Expressionssystem für die anaerobe Genexpression in höheren PflanzenAn expression system for anaerobic gene expression in higher plants
Beschreibungdescription
Die Erfindung betrifft ein Expressionssystem für die anaerobe Genexpression in höheren Pflanzen. Ein konkretes Anwendungsgebiet der Erfindung ist die Landwirtschaft, insbesondere die ResistenzZüchtung und die Steigerung der Leistungsfähigkeit von Nutzpflanzen.The invention relates to an expression system for anaerobic gene expression in higher plants. A specific field of application of the invention is agriculture, in particular resistance breeding and increasing the performance of useful plants.
Der Verlust an Erntegut durch Erkrankungen von Pflanzen ist ein weltweites Problem. Zum Beispiel führt die Erkrankung der Kartoffel an Knollennaßfäule (Verfaulen der Knolle) und Schwarzbeinigkeit (Verfaulen der unteren Stengelabschnitte), nach Infektion durch das phytopathogene Bakterium Erwinia carotovora , weltweit zu Ernteverlusten in einer geschätzten Höhe von 100 Millionen Dollar (Perombelon und Kelman, 1980, Ann. Rev. Phytopathol. , 18, 361-387). Es gibt eine Reihe von Arbeiten, die sich mit der gentechnischen Übertragung von Resistenzfaktoren auf Pflanzen beschäftigen (Lamb et al. , 1992, Bio/Technology, 10, 1436-1445; Hain und Fischer, 1994, Current Opinion in Biotechnology, 125-130; Zhu et al., 1994, Bio/Technology, 12, 807-812). Zur Resistenzsteigerung der Kartoffel gegenüber Erwinia carotovora wurde das T4 Lysozymgen des Bakteriophagen T4 in transgenen Kartoffeln exprimiert (During et al., 1993, Plant J. 3, 587-598).Loss of crop due to diseases of plants is a worldwide problem. For example, the potato's disease of wet bulb rot (rotting of the tuber) and black-leggedness (rotting of the lower stem sections), after infection by the phytopathogenic bacterium Erwinia carotovora, leads to an estimated total harvest loss of $ 100 million worldwide (Perombelon and Kelman, 1980, Ann Rev. Phytopathol., 18, 361-387). There are a number of papers dealing with the genetic engineering transfer of resistance factors to plants (Lamb et al., 1992, Bio / Technology, 10, 1436-1445; Hain and Fischer, 1994, Current Opinion in Biotechnology, 125-130 ; Zhu et al., 1994, Bio / Technology, 12, 807-812). To increase the resistance of the potato to Erwinia carotovora, the T4 lysozyme gene of the bacteriophage T4 was expressed in transgenic potatoes (During et al., 1993, Plant J. 3, 587-598).
Da sich bakterielle Erkrankungen von Pflanzen jedoch oft unter anaeroben Bedingungen ausbreiten, werden die bisher übertragenen Resistenzfaktoren für Pflanzen nur sehr eingeschränkt wirksam. Das gilt besonders für die oben erwähnte Erkrankung der Kartoffel an Knollennaßfäule und Schwarzbeinigkeit, da die Infektion durch Erwinia carotovora überwiegend unter anaeroben Bedingungen erfolgt. Verstärkt wird dieser Effekt noch durch die Bildung eines Schleims aus Bakterien und Abbauprodukten der pflanzlichen Zellwand. Für eine effektive Expression eines antibakteriellen Proteins unter optimalen Bedingungen ist deshalb die Steuerung deε entsprechenden Fremdgens durch einen unter diesen Bedingungen aktiven Promotor anzustreben.However, since bacterial diseases of plants often spread under anaerobic conditions, the resistance factors transmitted to plants have only a very limited effect. This applies particularly to the above-mentioned disease of the potato from wet rot and black-leggedness, since the infection by Erwinia carotovora mainly occurs under anaerobic conditions. This effect is reinforced by the formation of mucus from bacteria and degradation products of the plant cell wall. For An effective expression of an antibacterial protein under optimal conditions should therefore be aimed at controlling the corresponding foreign gene by means of a promoter active under these conditions.
Bisher wurden 3 anaerobe Promotoren in transgenen Pflanzen getestet: Dabei handelt es sich um den Adhl Promotor aus Mais, den Adh Promotor aus Arabidopεis thaliana und den GapC Promotor aus Arabidopsis thaliana . Der Adhl Promotor aus Mais wurde in Tabak und Reis untersucht (Ellis et al . , 1987, EMBO J. 6, 11-16; Kyozuka et al., 1991, Mol. Gen. Genet. 228, 40-48), der GapC Promotor aus Arabidopsis wurde in Tabak (Yang et al . , 1993, Plant Physiol. 101, 209-210), und der Adh Promotor aus Arabidopεis wurde in Arabidopsis selber untersucht (Dolferus et al., 1994, Plant Physiol. 105, 1075-1087). Dabei stellte sich heraus, daß alle Promotoren nur eine 2 bis 81 fache Induktion des Reportergens über Hintergrund vermitteln und nicht in allen Geweben aktiv sind.So far 3 anaerobic promoters have been tested in transgenic plants: These are the Adhl promoter from maize, the Adh promoter from Arabidopsis thaliana and the GapC promoter from Arabidopsis thaliana. The maize Adhl promoter was studied in tobacco and rice (Ellis et al., 1987, EMBO J. 6, 11-16; Kyozuka et al., 1991, Mol. Gen. Genet. 228, 40-48), the GapC Arabidopsis promoter was studied in tobacco (Yang et al., 1993, Plant Physiol. 101, 209-210), and the Arabidopsis adh promoter was investigated in Arabidopsis itself (Dolferus et al., 1994, Plant Physiol. 105, 1075- 1087). It was found that all promoters only mediate a 2 to 81-fold induction of the reporter gene via the background and are not active in all tissues.
Ziel der Erfindung ist es, die Expression von Resistenz¬ faktoren in Nutzpflanzen zu erreichen und entsprechende transgene Pflanzen zu erzeugen. Der Erfindung liegt die Aufgabe zugrunde, die anaerobe Expression des T4 Lysozymgens in der Kartoffel zu erreichen und entsprechende transgene Pfanzen zu erzeugen. Eine spezifische Aufgabe besteht darin, Kartoffeln gegenüber phytopathogenen Bakterien resistent zu machen.The aim of the invention is to achieve the expression of resistance factors in useful plants and to generate corresponding transgenic plants. The object of the invention is to achieve the anaerobic expression of the T4 lysozyme gene in the potato and to generate corresponding transgenic plants. A specific task is to make potatoes resistant to phytopathogenic bacteria.
Die Aufgabe der Erfindung wird durch ein Expressionssystem gelöst, das den Promotor GapC4 oder Teile oder Varianten des Promotors GapC4 und ein zu exprimierendes Gen umfaßt.The object of the invention is achieved by an expression system which comprises the promoter GapC4 or parts or variants of the promoter GapC4 and a gene to be expressed.
Gemäß der Erfindung handelt es um Gene für ein antibakterielles Protein, insbesondere für T4-Lysozym, um Resistenzgene gegen Viren, Nematoden, Bakterien und Pilze, Gene mit insektizider Wirkung, glykolyse-steigernde Gene und gärungssteigernde Gene. Das Expressionssystem wird erfindungsgemäß für die anaerobe Genexpression in höheren Pflanzen angewendet. Bevorzugt kommt es in Kulturpflanzen wie Kartoffeln, Reis, Getreide, Mais, Tomaten, Brassicaceen, Leguminosen, Baumwolle, Zuckerrübe und Möhren zur Anwendung.According to the invention, there are genes for an antibacterial protein, in particular for T4 lysozyme, resistance genes against viruses, nematodes, bacteria and fungi, genes with insecticidal activity, genes which increase glycolysis and genes which increase fermentation. The expression system is used according to the invention for anaerobic gene expression in higher plants. It is preferably used in crops such as potatoes, rice, cereals, corn, tomatoes, brassicaceae, legumes, cotton, sugar beet and carrots.
Die Erfindung betrifft auch höhere Pflanzen, vorzugsweise transgene Kulturpflanzen wie Kartoffeln, Reis, Getreide, Mais, Tomaten, Brassicaceen, Leguminosen, Baumwolle, Zuckerrübe und Möhren, die das erfindungsgemäße Expressionssystem enthalten.The invention also relates to higher plants, preferably transgenic crop plants such as potatoes, rice, cereals, corn, tomatoes, brassicaceae, legumes, cotton, sugar beet and carrots, which contain the expression system according to the invention.
Von besonderer Bedeutung sind transgene Kartoffeln, die ein Expressionssystem aus Promotor GapC4 und dem Gen für T4- Lysozym enthalten.Of particular importance are transgenic potatoes which contain an expression system consisting of the promoter GapC4 and the gene for T4 lysozyme.
Der große Vorteil des erfindungsgemäß eingesetzten Promotors GapC4 (GenBank Acceεsion Nr. L40803) besteht darin, daß er ein für die Zielstellung hervorragend geeignetes Induktionsprofil hat. Die anaerobe Expression erreicht die Stärke des unter aeroben Bedingungen für die Expression von Fremdgenen häufig verwendeten 35S Promotors des Blumenkohl Mosaikvirus (35S CaMV) . Weiterhin ist der Promotor in allen Geweben, wie Blüte, Blatt und Wurzel, aktiv. Bisher wurde die Isolierung des GapC4 Gens sowie die anaerobe Induktion des GapC4 Promotors in einem transienten Expresεionεsystem in Mais Suspensionskulturzellen veröffentlicht (Kersanach et al., 1994, Nature 367:387-389).The great advantage of the promoter GapC4 used according to the invention (GenBank Acquisition No. L40803) is that it has an induction profile which is extremely suitable for the purpose. The anaerobic expression reaches the strength of the 35S promoter of the cauliflower mosaic virus (35S CaMV), which is frequently used under aerobic conditions for the expression of foreign genes. Furthermore, the promoter is active in all tissues, such as flowers, leaves and roots. So far, the isolation of the GapC4 gene and the anaerobic induction of the GapC4 promoter in a transient expression system in maize suspension culture cells have been published (Kersanach et al., 1994, Nature 367: 387-389).
Überraschenderweise ist der Promotor besonders in der Kulturpflanze Kartoffel aktiv.Surprisingly, the promoter is particularly active in the potato crop.
Weitere Anwendungsmöglichkeiten der Erfindung: Ein weiteres Problem sind besonders in feuchtem Klima zeitweise Überflutungen von Feldern, die zu Ernteausfällen führen können. Als aerobe Organismen können Pflanzen längere Perioden großer Feuchtigkeit, die zur Abnahme des für die Pflanze verfügbaren Sauerstoffs führt, nicht überleben (Perata und Alpi, 1993, Plant Sei. 93, 1-17). Die Toleranz von Pflanzen gegenüber unzureichender Sauerstoffversorgung ist für einzelne Arten recht unterschiedlich. So keimt der Embryo in der Reis-Karyopse auch unter solchen Bedingungen ohne Schwierigkeiten, wohingegen Maiskeimlinge nur ca. 24 Stunden ohne Sauerstoff überleben. Eine generelle Anpasεungsstrategie höherer Pflanzen an anaerobe Bedingungen ist die Steigerung der Glykolyse sowie das Anschalten von Gärvorgängen. Um die Toleranz von Pflanzen gegenüber unzureichender SauerstoffVersorgung zu erhöhen, können die Gene, die bei der Glykolyse sowie bei der Gärung beteiligt sind unter die Kontrolle eines anaerob induzierbaren Promotors gebracht werden. Diese Gene werden dann bei unzureichender SauerstoffVersorgung exprimiert.Further possible applications of the invention: Another problem, especially in damp climates, is occasional flooding of fields, which can lead to crop failures. As an aerobic organism, plants can experience prolonged periods of great moisture, which can decrease the amount of moisture Plant does not survive available oxygen (Perata and Alpi, 1993, Plant Sei. 93, 1-17). The tolerance of plants to insufficient oxygen supply is quite different for individual species. The embryo in the rice caryopsis germinates without difficulty even under such conditions, whereas maize seedlings only survive for about 24 hours without oxygen. A general strategy for adapting higher plants to anaerobic conditions is to increase glycolysis and to activate fermentation processes. In order to increase the tolerance of plants to inadequate oxygen supply, the genes involved in glycolysis and fermentation can be brought under the control of an anaerobically inducible promoter. These genes are then expressed when there is insufficient oxygen supply.
Die Erfindung wird nachfolgend an Ausführungsbeispielen näher erläutert.The invention is explained in more detail below using exemplary embodiments.
AusführungsbeispielEmbodiment
Um zu untersuchen, ob der GapC4 Promotor bei der Kartoffel anaerob, sowie nach Infektion mit Erwinia carotovora induziert wird, werden GapC4 Promotor- Reportergenkonstrukte in die Kartoffel transformiert. Nach Infektion der transgenen Kartoffel mit Erwinia carotovora , sowie nach Inkubation von Geweben der transgenen Kartoffel unter anaeroben Bedingungen kann die Reportergenexpression gemessen werden. Das Reportergen kann dann in vitro durch das T4 Lyεozymgen ersetzt werden und in die Kartoffel transformiert werden. Die transgenen Kartoffeln werden anschließend auf erhöhte Resistenz gegen Erwinia carotovora untersucht.In order to investigate whether the GapC4 promoter is induced anaerobically in the potato and after infection with Erwinia carotovora, GapC4 promoter reporter gene constructs are transformed into the potato. After infection of the transgenic potato with Erwinia carotovora and after incubation of tissues of the transgenic potato under anaerobic conditions, the reporter gene expression can be measured. The reporter gene can then be replaced in vitro by the T4 lyse enzyme gene and transformed into the potato. The transgenic potatoes are then examined for increased resistance to Erwinia carotovora.
Die Reportergenkonstrukte wurden wie im folgenden beschrieben konstruiert:The reporter gene constructs were constructed as described below:
Alle Agrobakterium T-DNA Konstrukte baεieren auf dem binärenAll Agrobacterium T-DNA constructs are based on the binary
Vektor pOCA28 (Honma et al . , 1993, Proc. Natl. Acad. Sei.Vector pOCA28 (Honma et al., 1993, Proc. Natl. Acad. Sci.
ERSATZBUπ(REGEL26) USA 90, 6242-6246; Olszewski et ai . , 1988, Nucleic Acids Res 16, 10765-10782). Für T-DNA Konstrukte wurden Plasmide pUK443 und pUK444, die 785 bzw. 461 Basenpaare des GapC4 Promotors, das erste Intron des GapC4 Gens sowie das ß-Glucuronidase Reportergen tragen, eingesetzt. Um mögliche negative Effekte des GapC4 Intronε aus Mais in transgenen Tabakpflanzen und Kartoffeln auszuschließen, wurde das Intron durch Restriktionsverdau mit den Enzymen Xhol und Wcol aus den Plasmiden pUK443 und pUK444 herausgeschnitten. Nach einer Auffüllrekation der durch Restriktionsverdau entstandenen Enden wurden die intronlosen Plasmide pUK403 und pUK404 erzeugt. Die Promotor-Reportergenfragmente dieser Plasmide wurden durch einen PvuII Verdau herausgeschnitten und in die Smal Schnittstelle von pOCA28 kloniert. Die daraus resultierenden pOCA28 Derivate, pUK4030, 4040 und 4041 tragen das GapC4 Promotor-Reportergenkonstrukt. Als Kontrolle wurde das promotorlose ß-Glucuronidase Reportergen in pOCA28 kloniert. pUK4030 und 4040 tragen die Reportergenkonstrukte mit dem Promotor proximal zur rechten T-DNA Bordersequenz. In pUK4041 liegt das Reportergen in der anderen Orientierung vor.REPLACEMENT BUIP (RULE 26) USA 90, 6242-6246; Olszewski et ai. , 1988, Nucleic Acids Res 16, 10765-10782). Plasmids pUK443 and pUK444, which carry 785 and 461 base pairs of the GapC4 promoter, the first intron of the GapC4 gene and the β-glucuronidase reporter gene, were used for T-DNA constructs. In order to rule out possible negative effects of the GapC4 intron from maize in transgenic tobacco plants and potatoes, the intron was cut out from the plasmids pUK443 and pUK444 by restriction digestion with the enzymes Xhol and Wcol. After filling up the ends resulting from restriction digestion, the intronless plasmids pUK403 and pUK404 were generated. The promoter reporter gene fragments of these plasmids were cut out by a PvuII digest and cloned into the Smal site of pOCA28. The resulting pOCA28 derivatives, pUK4030, 4040 and 4041 carry the GapC4 promoter-reporter gene construct. As a control, the promoterless β-glucuronidase reporter gene was cloned into pOCA28. pUK4030 and 4040 carry the reporter gene constructs with the promoter proximal to the right T-DNA border sequence. In pUK4041, the reporter gene is in the other orientation.
Alle rekombinanten DNA Techniken werden nach Standardprotokollen durchgeführt (Sambrook et al . , 1989, Molecular cloning. A laboratory manual. New York: Cold Spring Harbor Laboratory Press) .All recombinant DNA techniques are carried out according to standard protocols (Sambrook et al., 1989, Molecular cloning. A laboratory manual. New York: Cold Spring Harbor Laboratory Press).
Für die Klonierung des T4-Lysozymgens, kann das ß- Glucuronidaεe Reportergen aus den Plasmiden pUK403 oder 404 durch Restriktionsverdau entfernt und an seine Stelle das T4- Lysozymgen hineinkloniert werden. Das durch den GapC4 Promotor kontrollierte T4 Lysozymgen wird in einen Agrobakterium T-DNA Vektor umkloniert. Alle Konstrukte werden nach Einführung in Agrobacterium tumefaciens gemäß Standardprotokollen in die Kartoffel transformiert (During et al., 1993, Plant J. 3, 587-598; Fladung, 1990, Plant Breeding 104, 295-304). Transgene Kartoffeln werden mit Hilfe eines Antibiotikaresistenzgens auf der T-DNA selektiert und werden auf Expression des eingeführten ß-Glucuronidase ReportergensFor the cloning of the T4 lysozyme gene, the β-glucuronidae reporter gene can be removed from the plasmids pUK403 or 404 by restriction digestion and the T4 lysozyme gene can be cloned in its place. The T4 lysozyme gene controlled by the GapC4 promoter is cloned into an Agrobacterium T-DNA vector. After introduction into Agrobacterium tumefaciens, all constructs are transformed into the potato according to standard protocols (During et al., 1993, Plant J. 3, 587-598; Fladung, 1990, Plant Breeding 104, 295-304). Transgenic potatoes are selected with the help of an antibiotic resistance gene on the T-DNA and are based on expression of the ß-glucuronidase reporter gene
ERSATZBUTT(REGEL26) und des T4 Lysozymgens unter anaeroben Bedingungen sowie nach Infektion mit Erwinia carotovora untersucht.REPLACEMENT BUTT (RULE 26) and the T4 lysozyme gene under anaerobic conditions and after infection with Erwinia carotovora.
Zur anaeroben Induktion wird pflanzliches Gewebe in einem luftdichten Glasbehälter (Merck) zusammen mit Anaerocult A (Merck) für mindestens 12 Stunden inkubiert. Für den fluorimetrischen GUS assay wird das Pflanzenmaterial homogenisiert und mit dem ß-Glucuronidase Substrat 4- Methylumbelliferyl-ß-D-Glucuronid (MUG) bei 37 °C inkubiert. Quantifizierungen der Fluoreszenz wird nach Jefferson et al., EMBO J. 6, 3901-3907 (1987) durchgeführt und Proteinkon¬ zentrationen werden nach Bradford, Anal. Biochem. 7, 248-254 (1976) bestimmt. Um die Gewebespezifität der Reportergenexpression zu messen wird das intakte anaerob induzierte Pflanzenmaterial mit einer Lösung 1 mM X-Gluc (5- Bromo-4-Chloro-3-Indolyl-ß-D-Glucuronεäure) vakuuminfiltriert und über Nacht bei 37 °C inkubiert. Zur besseren Sichtbarmachung der Anfarbung wird das Chlorophyll mit 70 % Ethanol extrahiert (Jefferson s.o.).For anaerobic induction, plant tissue is incubated in an airtight glass container (Merck) together with Anaerocult A (Merck) for at least 12 hours. For the fluorimetric GUS assay, the plant material is homogenized and incubated with the ß-glucuronidase substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG) at 37 ° C. Fluorescence is quantified according to Jefferson et al., EMBO J. 6, 3901-3907 (1987) and protein concentrations are determined according to Bradford, Anal. Biochem. 7, 248-254 (1976). In order to measure the tissue specificity of reporter gene expression, the intact anaerobically induced plant material is vacuum-filtered with a solution of 1 mM X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) and incubated at 37 ° C. overnight. To make the coloring more visible, the chlorophyll is extracted with 70% ethanol (Jefferson see above).
Um die Expression des T4 Lysozymgens zu messen, werden nach anaerober Inkubation des Gewebes Northern blot Analysen mit einer T4-Lysozym spezifischen Sonde durchgeführt. Gesamt RNA wird mit dem RNeasy Kit isoliert (Qiagen) . Die Konzentration der RNA wird photometrisch bestimmt und 10 Microgramm der RNA wird in eine Spur auf ein 1 % Agarosegel gegeben, welches Formaldehyd enthält. Nach Elektrophorese wird die RNA aus dem Gel mit 0,05 N NaOH als Transferpuffer auf Nitrozellulose oder Nylonmembranen (Amersham) geblottet (Sambrook et al. s.o.). Markierung und Hybridisierung der T4 Lysozym spezifischen Sonde mit dem RNA Filter erfolgt nach Standardbedingungen (Sambrook et al . s.o., During et al., s.o. ) .In order to measure the expression of the T4 lysozyme gene, Northern blot analyzes are carried out after anaerobic incubation of the tissue with a T4 lysozyme-specific probe. Total RNA is isolated with the RNeasy Kit (Qiagen). The concentration of the RNA is determined photometrically and 10 micrograms of the RNA are placed in a lane on a 1% agarose gel which contains formaldehyde. After electrophoresis, the RNA from the gel is blotted with 0.05 N NaOH as a transfer buffer onto nitrocellulose or nylon membranes (Amersham) (Sambrook et al. See above). Labeling and hybridization of the T4 lysozyme-specific probe with the RNA filter is carried out according to standard conditions (Sambrook et al. See above, During et al., See above).
Nach Bestätigung der anaeroben Induktion des Reportergens sowie des T4-Lysozymgens wird untersucht, ob beide Gene auch nach Infektion mit dem phytopathogenen Bakterium Erwinia carotovora induziert werden, bzw. ob das T4 Lysozym-Gen unter der Kontrolle des GapC4-Promotors so aktiviert wird, daß es Resistenz vermittelt. Die Induktion des Reportergens und des T4 Lysozym-Gens wird wie oben beschrieben bestimmt. DafürAfter confirmation of the anaerobic induction of the reporter gene and of the T4 lysozyme gene, it is examined whether both genes are also induced after infection with the phytopathogenic bacterium Erwinia carotovora or whether the T4 lysozyme gene is activated under the control of the GapC4 promoter in such a way that it conveys resistance. The induction of the reporter gene and the T4 lysozyme gene is determined as described above. Therefore
ERSATZBLAπ(REGEL26) wird das unter dem mazerierten Gewebe liegende Knollenmaterial genutzt.REPLACEMENT BLAπ (RULE 26) the tuber material under the macerated tissue is used.
Der Infektionsteεt wird mit einem pathogenen Stamm von Erwinia carotovora ssp . atroseptica oder εεp. carotovora in Plastikbehältern unter Luftabschluß durchgeführt. Aus Kartoffelknollen werden Scheiben definierter Größe hergestellt und in frisch geschnittenem Zustand mit einer definierten Anzahl von Bakterienzellen in einem kleinen Volumen in der Mitte inokuliert. Die Inkubation erfolgt in Plastikbehältern mit einer Wasεerschicht auf dem Boden auf einem durchnäßten Filterpapier. Dadurch wird gesättigte Luftfeuchtigkeit erreicht. Das Bakterienwachstum wird anhand der Gewebemazeration und des entstehenden Bakterienschleims nachverfolgt. Durch den sich bildenen Bakterienschleim erfolgt eine luftabschließende Abdeckung der Kartoffelzellen. In Abhängigkeit von der Inokulumsdichte wird nach einer definierten Zeit das Ausmaß der Mazeration bestimmt. Durch Vergleich mit Kontrollexplantaten kann der relative Rückgang der Suszeptibilität festgestellt werden.The infection test is carried out with a pathogenic strain of Erwinia carotovora ssp. atroseptica or εεp. carotovora performed in plastic containers with the exclusion of air. Slices of a defined size are made from potato tubers and inoculated freshly cut with a defined number of bacterial cells in a small volume in the middle. Incubation takes place in plastic containers with a layer of water on the bottom on a soaked filter paper. Saturated air humidity is reached. The bacterial growth is tracked based on the tissue maceration and the resulting bacterial mucus. The resulting bacterial mucus creates an air-tight covering of the potato cells. Depending on the inoculum density, the extent of the maceration is determined after a defined time. The relative decrease in susceptibility can be determined by comparison with control explants.
Alternativ können mit Bakterien infizierte Augenstecklinge unter feuchten Bedingungen im Gewächshaus ausgepflanzt und kultiviert werden. Durch Verschlammung der Erde kommt es zu Sauerstoffarmut, die die Vermehrung der Bakterien begünstigt. Die Anzahl aufgelaufener, gesunder Sprosse wird im Vergleich zu Kontrollexplantaten bestimmt. Dadurch ist das Ausmaß der reduzierten Suszeptibilität bestimmbar. Alternatively, eye cuttings infected with bacteria can be planted out and cultivated under moist conditions in the greenhouse. Silting up the earth leads to a lack of oxygen, which favors the multiplication of the bacteria. The number of emerged, healthy shoots is determined in comparison to control explants. The extent of the reduced susceptibility can thus be determined.
Claims
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| Application Number | Priority Date | Filing Date | Title |
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| DE19547272A DE19547272C1 (en) | 1995-12-19 | 1995-12-19 | Expression system for anaerobic gene expression in plants |
| DE19547272 | 1995-12-19 | ||
| PCT/DE1996/002428 WO1997022707A1 (en) | 1995-12-19 | 1996-12-17 | An expression system for anaerobic gene expression in higher plants |
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| EP0876496A1 true EP0876496A1 (en) | 1998-11-11 |
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| EP96946241A Withdrawn EP0876496A1 (en) | 1995-12-19 | 1996-12-17 | An expression system for anaerobic gene expression in higher plants |
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| US (1) | US6194201B1 (en) |
| EP (1) | EP0876496A1 (en) |
| JP (1) | JP2000515361A (en) |
| AU (1) | AU724617B2 (en) |
| CA (1) | CA2246299A1 (en) |
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| DE19956272B4 (en) * | 1999-11-23 | 2004-09-16 | Mpb Cologne Gmbh Molecular Plant & Protein Biotechnology | Process for the controlled post-harvest production of proteins in host organisms |
| DE10024740A1 (en) * | 2000-05-19 | 2001-11-29 | Mpb Cologne Gmbh Molecular Pla | Process for the controlled elimination of desired DNA sequences in host organisms |
| DE10038573A1 (en) * | 2000-08-03 | 2002-02-21 | Mpb Cologne Gmbh Molecular Pla | Procedure for selection on host cells with eliminated DNA sequences |
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| DE3926390A1 (en) * | 1989-08-10 | 1991-02-14 | Bayer Ag | USE OF LYSOCYME GENES IN RESISTANCE GROWTH PLANTS |
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| AU724617B2 (en) | 2000-09-28 |
| US6194201B1 (en) | 2001-02-27 |
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| NZ330738A (en) | 2000-05-26 |
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