EP0856057A2 - Polynucleotide and encoded protein useful in controlling of melolontha sp. scarabs - Google Patents

Polynucleotide and encoded protein useful in controlling of melolontha sp. scarabs

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Publication number
EP0856057A2
EP0856057A2 EP96945813A EP96945813A EP0856057A2 EP 0856057 A2 EP0856057 A2 EP 0856057A2 EP 96945813 A EP96945813 A EP 96945813A EP 96945813 A EP96945813 A EP 96945813A EP 0856057 A2 EP0856057 A2 EP 0856057A2
Authority
EP
European Patent Office
Prior art keywords
protein
polynucleotide
proteins
bacillus
cell
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Withdrawn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
EP96945813A
Other languages
German (de)
French (fr)
Inventor
Wolfgang Schnetter
Jiambing Zhang
Lutz Krieger
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.)
Syngenta Participations AG
Original Assignee
Novartis Erfindungen Verwaltungs GmbH
Ciba Geigy AG
Novartis AG
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Filing date
Publication date
Application filed by Novartis Erfindungen Verwaltungs GmbH, Ciba Geigy AG, Novartis AG filed Critical Novartis Erfindungen Verwaltungs GmbH
Publication of EP0856057A2 publication Critical patent/EP0856057A2/en
Withdrawn legal-status Critical Current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)
    • C12N15/8241—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
    • C12N15/8286—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 insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/825—Bacteria

Definitions

  • the invention relates to isolated polynucleotides and the proteins encoded by them, and their use for combating hawthorn beetles (Scarabaeidae).
  • the invention also relates to a method for producing these proteins
  • Greenhorn beetles such as the field cockchafer (Melolontha melolontha) or the forest cockchafer (Melolontha hippocastan ⁇ ) and especially their larvae (grubs) can cause great damage in agricultural and forestry crops. Since their control by chemical insecticides is difficult and environmentally harmful, efforts are being made to an increasing extent to control the multiplication and spread of these insects using biological agents. For example, EP 633936 A1 describes a method in which vegetative cells, spores or protein crystals of certain strains of Bacillus thurmgiensis are used to control hawthorn beetles. The effectiveness of this method is, however, effective not satisfying
  • B. popilliae is the causative agent of the so-called Milky Disease in larvae of cockchafer and other hawthorn beetles.
  • the larvae infected with the Bacillus have high concentrations of vegetative cells and sporangia of B. popilliae in their hamolymph, which cause a milky white discoloration of the grub
  • the bacterium B. popilliae is characterized, among other things, by the fact that it does not form catalase, most isolates are resistant to the antibiotic vancomycin and, during sporulation, form a conspicuous protein crystal, which is arranged inside the spindle-shaped sporangium next to the actual spore in its capacity as a pathogen B. popilliae has a high specificity for scarabaeids.
  • the B. popilliae subspecies isolated from different scarabaeid species differ in part considerably in their growth properties, the composition of the protein crystal and their plasmids
  • Bafer larvae are infected with B. popilliae by ingestion of the sp ⁇ rangia in the larvae, the spores germinate, and the vegetative bacterial cells penetrate through the intestinal epithelium and the basement membrane into the hamolymph and then multiply there over the next three to four weeks sporulate the B. popilliae cells, which ultimately leads to the death of the kafer larva
  • B. popilliae forms predominantly vegetative cells and only exceptionally spores under in vitro conditions.
  • WO 87 05 928 describes a method for obtaining the spores in vitro, in which the vegetative cells of B.
  • popilliae in one defined medium can be cultivated and finally stimulated to sporulation by adding a certain adjuvant, but even with this method only a sporulation rate of about 80% is achieved.
  • a considerable amount of equipment and personnel is consequently required and financial effort required, which makes this method economically unprofitable and therefore unsuitable for practice It is therefore an object of the present invention to provide biological agents for controlling scarabids which allow satisfactory control of these pests and which are technically simple and inexpensive to produce even in large quantities
  • polynucleotides which encode proteins which are identical or at least related to the crystal proteins which are characteristic of Bacillus popilliae, and which fully or partially or by way of substitution represent the nucleotide sequence shown in the sequence listing SEQ ID NO: 1 , Deletion, insertion and / or inversion of a nucleotide sequence derived therefrom or a nucleotide sequence which hybridizes wholly or partly therewith.
  • polynucleotides according to the invention themselves can either be obtained from a natural source or can also be produced synthetically or semi-synthetically.
  • telomeres are preferably present as a component of a recombinant DNA vector molecule which has the ability to express the crystal protein or protein crystal characteristic of Bacillus popilliae or a protein related thereto in a prokaryotic or eukaryotic cell.
  • This vector molecule has the advantage, inter alia, that it can be introduced into any cell, for example a commercially available, easy-to-cultivate bacterial culture of E. coli or Bacillus thuringiensis, subjected to a promoter which is already present in or incorporated into the cell and can be both replicated and expressed as Vector molecules are particularly suitable for plasmids derived from gram-positive bacteria
  • the use of transformed host cells which contain a polynucleotide according to the invention which is coupled to a promoter which is naturally contained in the host cell or as a result of a recombination.
  • Polynucleotide (s) are introduced into the host organism, ie into a microorganism, a virus, a protozoon, a plant cell or the like, for example by transformation, transduction or conjugation, integrated into the genetic material of these cells or viruses and brought to expression
  • Bacillus thunngiensis for example Bacillus thunngiensis subsp kurstaki, have proven to be particularly suitable host cells. Bacillus thunngiensis has been very well studied and it is comparatively easy to produce the crystal protein of B. popilliae in large amounts in this system
  • the protein (s) according to the invention can be used alone or in combination with at least one other substance as a biological insecticide for combating scarabids, ie for inhibiting feeding and / or killing adult and / or larval scarabids, in particular Melolontha - Types and species closely related to them.
  • substance here includes chemical and biological materials including microorganisms.
  • pathogens such as viruses, rickettsia, bacteria, fungi, microsporidia and others can result in an advantageous increase in the crystal toxin effect
  • the proteins according to the invention are used together with spores of Bacillus popilliae and / or Bacillus thunngiensis.
  • Bacillus sphaericus spores are also well suited
  • Another, also very advantageous variant provides that the proteins according to the invention are used in combination with cytolysing proteins and / or receptor proteins for the intestinal epithelium of scarabaeids, preferably in the form of fusion proteins
  • proteins according to the invention are used in combination with fungal spores
  • one or more of the polynucleotides according to the invention infiltrates (transforms) into a microorganism (e.g. a bacterium, virus, fungus or protozoan) or into a cell of an animal or plant cell culture Control and control of a promoter contained in this microorganism or in this cell, naturally or as a result of a recombination, preferably subject to regulation and expression.
  • a microorganism e.g. a bacterium, virus, fungus or protozoan
  • Control and control of a promoter contained in this microorganism or in this cell naturally or as a result of a recombination, preferably subject to regulation and expression.
  • the polynucleotide (s) is introduced into a bacterium of the Bacillus thunngiensis type
  • the invention also encompasses the possibility of transferring polynucleotides according to the invention into plants or parts of plants in order to protect them against scarab acid seizure.
  • it also includes the use of polynucleotides according to the invention for the production of transgenic plants with the property of a knee protein in all or some plant tissues to synthesize, which is similar or similar to the crystal protein characteristic of Bacillus popilliae, and which inhibits the feeding and / or killing of larval and / or adult Scarabaeids, in particular Melolontha species and closely related to them Species is suitable and / or also for inactivating and / or killing such soil organisms which damage plants and / or fungi and / or transmit diseases
  • a method for the production of plants or plant tissues or plant propagation material with recombined genetic material, which comprises a heterologous polynucleotide according to the invention, the expression of which leads to a protein which is similar or similar to the crystal protein which occurs naturally and characteristically in Bacillus popilliae.
  • This method according to the invention is characterized in that plant cells or plant tissue are transformed with a recombinant DNA which contains a polynucleotide according to the invention and additional regulatory nucleotide sequences which can bring about the stable integration and expression of the polynucleotide in the plant cells in that the plant or whose propagation material or both are then regenerated from the plant cells or the corresponding tissue transformed with the heterologous DNA, and that, if appropriate, this regenerated plant or its propagation material or both is biologically reproduced.
  • Example 1 The invention is explained in more detail below on the basis of exemplary embodiments Example 1:
  • a strain of Bacillus popilliae subsp melolonthae is isolated from white grubs of Melolontha melolontha.
  • the common DNA methods are used to prepare the total DNA material from the bacterial cells of this strain, on the one hand, the crystal protein is isolated, purified and its amino acid sequence (in a commercially available protein sequencer) is determined.
  • Corresponding oligonucleotides are synthesized for partial sequences from the two end regions of the protein in order to serve in a polymerase chain reaction (PCR) as a primer for the synthesis of a DNA probe for the crystal protein gene.
  • the PCR is carried out using the total DNA from Bacillus popilliae subsp melolonthae
  • the isolated crystal protein can also first be split into short fragments, then the amino acid sequence of some of these fragments determined and, by comparison with the amino acid sequence of the Cry II A protein from Bacillus thunngiensis known in the prior art, two fragments which are presumably from the two end regions selected of the protein.
  • the DNA sequences corresponding to these fragments are then provided as oligonucleotides and used as primers for the PCR synthesis
  • the product of the PCR is used as a probe for hybridization in the Southern blot.
  • a 5.3 kB Eco RI fragment of the Bacillus poptllae genome is identified.
  • the 5.3 kB Eco RI fragment is e.g. incorporated into the plasmid pBCSK + and cloned into the E. coli strain XL 1 Blue MRF '.
  • the 5.3 kB Eco RI fragment of the Bacillus popilliae genome obtained according to Example 1 is incorporated into a plasmid for gram-positive bacteria and introduced into bacterial cells of a crystal-free strain of Bacillus thunngiensis subsp kurstaki.
  • a plasmid is particularly suitable as a cloning vector for Bacillus thunngiensis known plasmid such as pHT304, pHT315 or pHT370, the preparation of which is described in the publication by O Arantes and D Lerecius in Gene, 148 (1991), pages 115-1 19. Reference is made to this publication in this respect and its content is hereby incorporated into the present description included
  • Recombined bacteria are multiplied or cloned and, if necessary, stimulated to synthesize the crystal protein. This is done by inducing spore formation, preferably simply by changing the culture conditions in a targeted manner. Then the protein release is induced, for example by changing the culture conditions again, by spore germination or autolysis of the sporangia The protein released is separated from the culture medium, purified and, if necessary, stored in a cool, dry and dark place until use
  • crystal protein according to the invention for controlling adult Scarabaeids of the species Melolontha melolontha.
  • Cockchafer grubs (Melolontha melolontha) were dug up in the field, kept individually in breeding vessels in the laboratory and fed with carrot slices. After 3 weeks, 10 animals were fed the protein according to the invention in an aqueous suspension with or without spores. Just a day later, the feed intake was greatly reduced, inhibited
  • the preparation is introduced into the soil, possibly in combination with other pathogens and preferably together with bait.

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Wood Science & Technology (AREA)
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  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
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Abstract

Isolated polynucleotides are disclosed, as well as the proteins coded thereby, their use for controlling common cockchafers (Scarabaeidae )and a process for producing these proteins. The polynucleotides have the nucleotide sequence corresponding to the sequence protocol SEQ. ID. NO. 1 in whole or in part or a nucleotide sequence related thereto and derived therefrom by substitution, deletion, insertion and/or inversion or a nucleotide sequence capable of completely or partially hybridising therewith. These polynucleotides code for proteins that are identical or at least related to the Bacillus popilliae characteristic crystal proteins and which are useful to inhibit feeding and/or to kill Scarabaeidae adults and/or larvae, in particular of the Melolontha species and of species closely related thereto.

Description

POLYNUKLEOTIDE UND DIE DURCH DIESE KODIERTEN PROTEINE , GEEIGNET ZUR BEKÄMPFUNG VON BLATTHORNKAFERNPOLYNUCLEOTIDES AND THE PROTEINS CODED BY THESE, SUITABLE FOR COMBATING BLADE HORN BEETLE
Beschreibungdescription
Die Erfindung betrifft isolierte Polynukleotide und die durch diese kodierten Proteine, sowie deren Verwendung zur Bekämpfung von Blatthornkafern (Scarabaeidae) Außerdem betrifft die Erfindung ein Verfahren zur Herstellung dieser ProteineThe invention relates to isolated polynucleotides and the proteins encoded by them, and their use for combating hawthorn beetles (Scarabaeidae). The invention also relates to a method for producing these proteins
Blatthornkafer wie der Feldmaikafer (Melolontha melolontha) oder der Waldmaikafer (Melolontha hippocastanϊ) und insbesondere deren Larven (Engerlinge) können große Schaden in land- und forstwirtschaftlichen Kulturen anrichten Da ihre Bekämpfung mittels chemischer Insektizide schwierig und für die Umwelt bedenklich ist, werden verstärkt Bemühungen unternommen, die Vermehrung und Ausbreitung dieser Insekten mit biologischen Mitteln zu kontrollieren So ist beispielsweise in der EP 633936 AI ein V£ifahren beschrieben, bei dem vegetative Zellen, Sporen oder Proteinkristalle bestimmter Stamme von Bacillus thurmgiensis zur Bekämpfung von Blatthornkafern eingesetzt Die Wirksamkeit dieses Verfahrens ist allerdings nicht befriedigendGreenhorn beetles such as the field cockchafer (Melolontha melolontha) or the forest cockchafer (Melolontha hippocastanϊ) and especially their larvae (grubs) can cause great damage in agricultural and forestry crops. Since their control by chemical insecticides is difficult and environmentally harmful, efforts are being made to an increasing extent to control the multiplication and spread of these insects using biological agents. For example, EP 633936 A1 describes a method in which vegetative cells, spores or protein crystals of certain strains of Bacillus thurmgiensis are used to control hawthorn beetles. The effectiveness of this method is, however, effective not satisfying
Aus der WO 8705928 ist der Vorschlag bekannt, Sporen von Bacillus popilliae (B. popilliae) zur biologischen Bekämpfung von Scarabaeidenlarven einzusetzenFrom WO 8705928 the proposal is known to use spores of Bacillus popilliae (B. popilliae) for the biological control of Scarabid larvae
B. popilliae ist der Erreger der sogenannten Milky Disease bei Larven von Maikäfern und anderer Blatthornkafer Die mit dem Bacillus infizierten Larven weisen in ihrer Hamolymphe hohe Konzentrationen an vegetativen Zellen und Sporangien von B. popilliae auf, die eine milchig weiße Verfärbung des Engerlings bewirkenB. popilliae is the causative agent of the so-called Milky Disease in larvae of cockchafer and other hawthorn beetles. The larvae infected with the Bacillus have high concentrations of vegetative cells and sporangia of B. popilliae in their hamolymph, which cause a milky white discoloration of the grub
B popilliae als Verursacher der Milky Disease wurde erstmals von Dutky beim Japankafer (Popillia japonica) in den USA beschrieben (in Journal of Agricultural Research 61 (1940) S 57-68, "Two new sporeforming bacteπa causing milky disease of the Japanese beetle") und spater von Hurpin und Vago sowie von Wille auch in Engerlingen des Feldmaikafers (Melolontha melolontha) nachgewiesen (B Hurpin und C Vago in Entomophaga 3 (1958) S. 285-330, "Les maladies du hanneton commun (Melolontha melolontha L (Col , Scarabaeidae)" , H Wille in Mitteilungen. Schweizerische Entomologische Gesellschaft 29 ( 1956) S.271 -282 "Bacillus fribourgensis n sp , Erreger einer "milky disease" im Engerling von Melolontha melolontha L ")B popilliae as the cause of the Milky Disease was first described by Dutky in the Japanese beetle (Popillia japonica) in the USA (in Journal of Agricultural Research 61 (1940) S 57-68, "Two new sporeforming bacteπa causing milky disease of the Japanese beetle") and later by Hurpin and Vago as well as by Wille also in grubs of Feldmaikafer (Melolontha melolontha) (B Hurpin and C Vago in Entomophaga 3 (1958) pp. 285-330, "Les maladies du hanneton commun (Melolontha melolontha L (Col, Scarabaeidae)", H Will in Notices. Swiss Entomological Society 29 (1956) pp. 271-282 "Bacillus friborgensis n sp, pathogen of a "milky disease" in the white grubs of Melolontha melolontha L ")
Das Bakterium B. popilliae ist u a dadurch charakterisiert, daß es keine Katalase bildet, die meisten Isolate gegen das Antibiotikum Vancomycin resistent sind und wahrend der Sporulation einen auffälligen Proteinkristall bilden, der innerhalb des spindelförmigen Sporangiums neben der eigentlichen Spore angeordnet ist In seiner Eigenschaft als Pathogen für Scarabaeiden weist B. popilliae eine hohe Spezifität auf Die aus verschiedenen Scarabaeidenarten isolierten B. popilliae Subspecies unterscheiden sich zum Teil erheblich in ihren Wachstumseigenschaften, der Zusammensetzung des Proteinkristalls sowie ihren PlasmidenThe bacterium B. popilliae is characterized, among other things, by the fact that it does not form catalase, most isolates are resistant to the antibiotic vancomycin and, during sporulation, form a conspicuous protein crystal, which is arranged inside the spindle-shaped sporangium next to the actual spore in its capacity as a pathogen B. popilliae has a high specificity for scarabaeids. The B. popilliae subspecies isolated from different scarabaeid species differ in part considerably in their growth properties, the composition of the protein crystal and their plasmids
Die Infizierung der Kaferlarven mit B. popilliae erfolgt durch perorale Aufnahme der Spρrangien Im Darm der Larven keimen die Sporen aus, und die vegetativen Bakterienzellen dringen durch das Darmepithel und die Basalmembran in die Hamolymphe ein und vermehren sich dort wahrend der folgenden drei bis vier Wochen Dann sporulieren die B. popilliae -Zellen, was letztendlich zum Tod der Kaferlarve führtBafer larvae are infected with B. popilliae by ingestion of the spρrangia in the larvae, the spores germinate, and the vegetative bacterial cells penetrate through the intestinal epithelium and the basement membrane into the hamolymph and then multiply there over the next three to four weeks sporulate the B. popilliae cells, which ultimately leads to the death of the kafer larva
Im Gegensatz zu anderen Bacillus-AΛen bildet B. popilliae jedoch unter in vitro Bedingungen überwiegend vegetativen Zellen und nur ausnahmsweise Sporen Die WO 87 05 928 beschreibt zwar ein Verfahren zur Gewinnung der Sporen in vitro, bei dem die vegetativen Zellen von B. popilliae in einem definierten Medium kultiviert werden und schließlich durch Zugabe eines bestimmten Adjuvans zur Sporulation angeregt werden, aber auch mit dieser Methode wird nur eine Sporulationsrate von etwa 80% erreicht Um ausreichende Mengen an infektiösem Sporenmaterial für eine biologische Bekämpfung zu gewinnen, ist infolgedessen ein betrachtlicher apparativer, personeller und finanzieller Aufwand erforderlich, der diese Methode wirtschaftlich unrentabel und damit für die Praxis ungeeignet macht Aufgabe der vorliegenden Erfindung ist es daher, biologische Mittel zur Bekämpfung von Scarabaeiden bereitzustellen, die eine befriedigende Kontrolle dieser Schädlinge zulassen, und die technisch einfach und kostengünstig auch in großtechnischen Mengen herstellbar sindIn contrast to other Bacillus plants, B. popilliae forms predominantly vegetative cells and only exceptionally spores under in vitro conditions. WO 87 05 928 describes a method for obtaining the spores in vitro, in which the vegetative cells of B. popilliae in one defined medium can be cultivated and finally stimulated to sporulation by adding a certain adjuvant, but even with this method only a sporulation rate of about 80% is achieved.To obtain sufficient amounts of infectious spore material for biological control, a considerable amount of equipment and personnel is consequently required and financial effort required, which makes this method economically unprofitable and therefore unsuitable for practice It is therefore an object of the present invention to provide biological agents for controlling scarabids which allow satisfactory control of these pests and which are technically simple and inexpensive to produce even in large quantities
Diese Aufgabe wird durch die Bereitstellung von Polynukleotiden gelost, die Proteine codieren, die mit den für Bacillus popilliae charakteristischen Kristallproteinen identisch oder zumindest verwandt sind, und die die im Sequenzprotokoll SEQ ID NO: 1 dargestellte Nukleotidsequenz ganz oder teilweise oder eine damit verwandte, durch Substitution, Deletion, Insertion und/oder Inversion davon abgeleitete Nukleotidsequenz oder eine ganz oder teilweise damit hybridisierende Nukleotidsequenz aufweisen.This object is achieved by the provision of polynucleotides which encode proteins which are identical or at least related to the crystal proteins which are characteristic of Bacillus popilliae, and which fully or partially or by way of substitution represent the nucleotide sequence shown in the sequence listing SEQ ID NO: 1 , Deletion, insertion and / or inversion of a nucleotide sequence derived therefrom or a nucleotide sequence which hybridizes wholly or partly therewith.
Es wurde namlich überraschenderweise festgestellt, daß der Proteinkristall aus B. popilliae maßgeblich für eine Fraßhemmung bei Scarabaeidenlarven verantwortlich ist, und daß dieser Proteinkristall bei adulten Tieren nach oraler Verabreichung zu deren Tod fuhrt Durch die Bereitstellung der erfindungsgemäßen Polynukleotide ist es erstmals möglich, diese B. popilliae - Proteinkristalle in praktisch unbegrenzten Mengen, d h insbesondere auch im großbiotechnischen Umfang zu gewinnen bzw herzustellenSurprisingly, it was found that the protein crystal from B. popilliae is largely responsible for inhibiting feeding in scarab larvae and that this protein crystal in adult animals leads to their death after oral administration. By providing the polynucleotides according to the invention it is possible for the first time to popilliae - protein crystals in practically unlimited quantities, ie in particular also to be obtained or produced on a large scale
Die erfindungsgemäßen Polynukleotide selbst können entweder aus einer natürlichen Quelle gewonnen oder auch synthetisch oder halbsynthetisch hergestellt werden.The polynucleotides according to the invention themselves can either be obtained from a natural source or can also be produced synthetically or semi-synthetically.
Sie liegen vorzugsweise als Bestandteil eines rekombinanten DNS-Vektormolekül vor, das die Fähigkeit zur Expression des bzw. eines für Bacillus popilliae charakteristischen Kristallproteins bzw. Proteinkristalls oder eines damit verwandten Proteins in einer prokaryontischen oder eukaryontischen Zelle aufweist Diese Vektormolekul hat u.a. den Vorteil, daß es in eine beliebige Zelle, beispielsweise einer handelsübliche, leicht zu kultivierenden Bakterienkultur von E. coli oder Bacillus thuringiensis eingeschleust, einem in der Zelle bereits vorhandenen oder miteingeschleusten Promotor unterworfen und sowohl repliziert als auch zur Expression gebracht werden kann Als Vektormolekule sind besonders aus gram-positiven Bakterien stammende Plasmide geeignetThey are preferably present as a component of a recombinant DNA vector molecule which has the ability to express the crystal protein or protein crystal characteristic of Bacillus popilliae or a protein related thereto in a prokaryotic or eukaryotic cell. This vector molecule has the advantage, inter alia, that it can be introduced into any cell, for example a commercially available, easy-to-cultivate bacterial culture of E. coli or Bacillus thuringiensis, subjected to a promoter which is already present in or incorporated into the cell and can be both replicated and expressed as Vector molecules are particularly suitable for plasmids derived from gram-positive bacteria
Zur biotechnologischen Gewinnung der von den erfindungsgemaßen Polynukleotiden kodierten Kristallproteinen wird der Einsatz von transformierten Wirtszellen vorgeschlagen, die ein erfindungsgemaßes Polynukleotid enthalten, das mit einem Promotor gekoppelt ist, der in der Wirtszelle natürlicherweise oder als Folgen einer Rekombination enthalten ist Das bzw die erfindungsgemaße(n) Polynukleotid(e) werden in den Wirtsorganimus, d h in einen Mikroorganismus, einen Virus, ein Protozoon, eine Pflanzenzelle o a eingeschleust, z B durch Transformation, Transduktion oder Konjugation, in das genetische Material dieser Zellen bzw Viren integriert und zur Expression gebrachtFor the biotechnological production of the crystal proteins coded by the polynucleotides according to the invention, the use of transformed host cells is proposed which contain a polynucleotide according to the invention which is coupled to a promoter which is naturally contained in the host cell or as a result of a recombination. Polynucleotide (s) are introduced into the host organism, ie into a microorganism, a virus, a protozoon, a plant cell or the like, for example by transformation, transduction or conjugation, integrated into the genetic material of these cells or viruses and brought to expression
Als besonders geeignete Wirtszellen haben sich die vegetativen Zellen von Bacillus thunngiensis, z B Bacillus thunngiensis subsp kurstaki erwiesen Bacillus thunngiensis ist sehr gut untersucht und es ist in diesem System vergleichsweise einfach, das Kristallprotein von B. popilliae auch in großen Mengen herzustellenThe vegetative cells of Bacillus thunngiensis, for example Bacillus thunngiensis subsp kurstaki, have proven to be particularly suitable host cells. Bacillus thunngiensis has been very well studied and it is comparatively easy to produce the crystal protein of B. popilliae in large amounts in this system
Das bzw die erfindungsgemaße(n) Protein(e) kann/können allein oder in Kombination mit wenigstens einer anderen Substanzen als biologisches Insektizid zur Bekämpfung von Scarabaeiden, d h zur Hemmung der Fraßtatigkeit und/oder Abtötung von adulten und/oder larvalen Scarabaeiden, insbesondere Melolontha-Arten und mit diesen nah verwandte Arten eingesetzt werden Der Begriff "Substanz" umfaßt hier chemische und biologische Materialien einschließlich Mikroorganismen Durch die Kombination mit anderen Pathogenen wie Viren, Rickettsien, Bakterien, Pilzen, Microsporidien u a kann eine vorteilhafte Steigerung der Kristalltoxin- Wirkung herbeigeführt werdenThe protein (s) according to the invention can be used alone or in combination with at least one other substance as a biological insecticide for combating scarabids, ie for inhibiting feeding and / or killing adult and / or larval scarabids, in particular Melolontha - Types and species closely related to them. The term "substance" here includes chemical and biological materials including microorganisms. The combination with other pathogens such as viruses, rickettsia, bacteria, fungi, microsporidia and others can result in an advantageous increase in the crystal toxin effect
In einer bevorzugten Ausführungsform der Erfindung werden die erfindungsgemaßen Proteine zusammen mit Sporen von Bacillus popilliae und/oder Bacillus thunngiensis, eingesetzt. Die Sporen von Bacillus sphaericus sind ebenfalls gut geeignet Eine andere, ebenfalls sehr vorteilhafte Variante sieht vor, daß die erfindungsgemaßen Proteine in Kombination mit cytolysierenden Proteinen und/oder Rezeptorproteinen für das Darmepithel von Scarabaeiden, vorzugsweise in Form von Fusionsproteinen, eingesetzt werdenIn a preferred embodiment of the invention, the proteins according to the invention are used together with spores of Bacillus popilliae and / or Bacillus thunngiensis. Bacillus sphaericus spores are also well suited Another, also very advantageous variant provides that the proteins according to the invention are used in combination with cytolysing proteins and / or receptor proteins for the intestinal epithelium of scarabaeids, preferably in the form of fusion proteins
Noch eine andere Variante sieht vor, daß die erfindungsgemaßen Proteine in Kombination mit Pilzsporen eingesetzt werdenYet another variant provides that the proteins according to the invention are used in combination with fungal spores
Es besteht auch die Möglichkeit, das bzw die erfindungsgemaße(n) Protein(e), allein oder in Kombination mit wenigstens einer anderen Substanz, zur Bekämpfung von solchen Bodenorganismen einzusetzen, die Pflanzen und/oder Pilze schadigen und/oder Krankheiten übertragen Die Bekämpfung umfaßt die Inaktivierung, insbesondere die Hemmung der Fraßtatigkeit, und/oder die Abtötung der betreffenden BodenorganismenIt is also possible to use the protein (s) according to the invention, alone or in combination with at least one other substance, to combat those soil organisms which damage plants and / or fungi and / or transmit diseases the inactivation, in particular the inhibition of feeding, and / or the killing of the soil organisms in question
Zur Gewinnung bzw Erzeugung der erfindungsgemaßen Proteine ist ein Verfahren vorgeschlagen, bei dem ein oder mehrere der erfindungsgemaßen Polynukleotide in einen Mikroorganismus (z B ein Bakterium, Virus, Pilz oder Protozoon), oder eine Zelle einer tierischen oder pflanzlichen Zellkultur einschleust (transformiert), der Kontrolle und Steuerung eines in diesem Mikroorganismus bzw dieser Zelle natürlicherweise oder als Folge einer/der Rekombination enthaltenen, vorzugsweise reguberbaren Promotors unterwirft und zur Expression bringt. In einer besonders bevorzugten Ausführungsvariante dieses Verfahrens wird das (die) Polynukleotid(e) in ein Bakterium der Art Bacillus thunngiensis eingeschleustTo obtain or produce the proteins according to the invention, a method is proposed in which one or more of the polynucleotides according to the invention infiltrates (transforms) into a microorganism (e.g. a bacterium, virus, fungus or protozoan) or into a cell of an animal or plant cell culture Control and control of a promoter contained in this microorganism or in this cell, naturally or as a result of a recombination, preferably subject to regulation and expression. In a particularly preferred embodiment of this method, the polynucleotide (s) is introduced into a bacterium of the Bacillus thunngiensis type
Die Erfindung umfaßt auch die Möglichkeit, erfindungsgemaße Polynukleotide in Pflanzen bzw Pflanzenteile zu übertragen, um diese gegen Scarabaeidenfraß zu schützen Das heißt mit anderen Worten sie umfaßt auch die Verwendung von erfindungsgemaßen Polynukleotiden zur Erzeugung transgener Pflanzen mit der Eigenschaft, in allen oder einigen Pflanzengeweben ein Kπstallprotein zu synthetisieren, das dem für Bacillus popilliae charakteristischen Kristallprotem gleicht oder ahnlich ist, und das zur Hemmung der Fraßtatigkeit und/oder Abtötung von larvalen und/oder adulten Scarabaeiden, insbesondere Melolontha-Arten und mit diesen nah verwandte Arten, geeignet ist und/oder- auch zur Inaktivierung und/oder Abtötung von solchen Bodenorganismen, die Pflanzen und/oder Pilze schädigen und/oder Krankheiten übertragenThe invention also encompasses the possibility of transferring polynucleotides according to the invention into plants or parts of plants in order to protect them against scarab acid seizure. In other words, it also includes the use of polynucleotides according to the invention for the production of transgenic plants with the property of a knee protein in all or some plant tissues to synthesize, which is similar or similar to the crystal protein characteristic of Bacillus popilliae, and which inhibits the feeding and / or killing of larval and / or adult Scarabaeids, in particular Melolontha species and closely related to them Species is suitable and / or also for inactivating and / or killing such soil organisms which damage plants and / or fungi and / or transmit diseases
Zu diesem Zweck ist ein Verfahren vorgeschlagen zur Erzeugung von Pflanzen oder Pflanzengeweben oder Pflanzenvermehrungsmaterial mit rekombiniertem genetischem Material, das ein erfindungsgemäßes heterologes Polynukleotid umfaßt, dessen Exprimierung zu einem Protein führt, das dem in Bacillus popilliae natürlicher- und charakteristischerweise vorkommenden Kristallprotein gleicht oder ahnlich ist. Diese erfindungsgemäße Verfahren zeichnet sich dadurch aus, daß man Pflanzenzellen oder Pflanzengewebe mit einer rekombinanten DNA transformiert, die ein erfindungsgemäßes Polynukleotid enthält und zusätzlich regulatorische Nukleotid Sequenzen, welche die stabile Integration und die Exprimierung des Polynukleotids in den Pflanzenzellen bewirken können, daß man die Pflanze oder deren Vermehrungsmaterial oder beides dann aus den mit der heterologen DNA transformierten Pflanzenzeüen oder dem entsprechenden Gewebe regeneriert, und daß man gegebenenfalls diese regenerierte Pflanze oder ihr Vermehrungsmaterial oder beides biologisch vervielfältigt.For this purpose, a method is proposed for the production of plants or plant tissues or plant propagation material with recombined genetic material, which comprises a heterologous polynucleotide according to the invention, the expression of which leads to a protein which is similar or similar to the crystal protein which occurs naturally and characteristically in Bacillus popilliae. This method according to the invention is characterized in that plant cells or plant tissue are transformed with a recombinant DNA which contains a polynucleotide according to the invention and additional regulatory nucleotide sequences which can bring about the stable integration and expression of the polynucleotide in the plant cells in that the plant or whose propagation material or both are then regenerated from the plant cells or the corresponding tissue transformed with the heterologous DNA, and that, if appropriate, this regenerated plant or its propagation material or both is biologically reproduced.
Die mit diesem Verfahren oder auf andere Weise erzeugten, gentechnisch transformierten Zellen mit einer stabil in ihr Genom integrierten (rekombinierten) heterologen DNA, die ein erfindungsgemäßes Polynukleotid enthält, welches ein Protein kodiert, das dem in Bacillus popilliae natürlicher- und charakteristischerweise vorkommenden Kristallprotein gleich oder ähnlich ist, und wobei dieses Proteins unter der Steuerung, eines von den Polymerasen der Zellen erkannten, in den Zellen natürlicherweise oder als Folge einer/der Rekombination enthaltenen Promotors exprimiert wird, sind ebenfalls Gegenstand der Erfindung .The genetically transformed cells produced by this method or in another way with a heterologous DNA which is stably integrated (recombined) in their genome and which contains a polynucleotide according to the invention which encodes a protein which is identical to or characteristic of the crystal protein which occurs naturally and characteristically in Bacillus popilliae is similar, and wherein this protein, under the control of one recognized by the polymerases of the cells, is naturally expressed in the cells or as a result of a / the recombination contained promoter are also the subject of the invention.
Die Erfindung wird im folgenden anhand von Ausführungsbeispielen naher erläutert Beispiel 1:The invention is explained in more detail below on the basis of exemplary embodiments Example 1:
Herstellung eines erfindungsgemaßen PolynukleotidsProduction of a polynucleotide according to the invention
Aus Engerlingen von Melolontha melolontha wird ein Stamm von Bacillus popilliae subsp melolonthae isoliert. Aus den Bakterienzellen dieses Stammes wird mit den gängigen Verfahren zum einen das Gesamt-DNA-Material präpariert, zum anderen wird das Kristallprotein isoliert, aufgereinigt und seine Aminosauresequenz (in einem handelsüblichen Protein- Sequenator) bestimmt. Zu Teilsequenzen aus den beiden Endbereichen des Proteins werden korrespondierende Oligonukleotide synthetisiert um in einer Polymerase-Kettenreaktion (PCR) als Primer zur Synthese einer DNA-Sonde für das Kristallprotein-Gen zu dienen. Die PCR wird unter Einsatz der Gesamt-DNA aus Bacillus popilliae subsp melolonthae durchgeführtA strain of Bacillus popilliae subsp melolonthae is isolated from white grubs of Melolontha melolontha. The common DNA methods are used to prepare the total DNA material from the bacterial cells of this strain, on the one hand, the crystal protein is isolated, purified and its amino acid sequence (in a commercially available protein sequencer) is determined. Corresponding oligonucleotides are synthesized for partial sequences from the two end regions of the protein in order to serve in a polymerase chain reaction (PCR) as a primer for the synthesis of a DNA probe for the crystal protein gene. The PCR is carried out using the total DNA from Bacillus popilliae subsp melolonthae
Alternativ kann man das isolierte Kristallprotein auch zunächst in kurze Fragmente spalten, dann die Aminosauresequenz einiger dieser Fragmente bestimmen und durch Vergleich mit der im Stand der Technik bekannten Aminosauresequenz des Cry II A - Proteins aus Bacillus thunngiensis zwei Fragmente auswählen, die mutmaßlich aus den beiden Endbereichen des Proteins stammen. Die zu diesen Fragmenten korrespondierenden DNA-Sequenzen werden dann als Oligonukleotide bereitgestellt und als Primer für die PCR- Synthese eingesetztAlternatively, the isolated crystal protein can also first be split into short fragments, then the amino acid sequence of some of these fragments determined and, by comparison with the amino acid sequence of the Cry II A protein from Bacillus thunngiensis known in the prior art, two fragments which are presumably from the two end regions selected of the protein. The DNA sequences corresponding to these fragments are then provided as oligonucleotides and used as primers for the PCR synthesis
Das Produkt der PCR wird als Sonde für eine Hybridisierung im Southern Blot eingesetzt Dabei wird ein 5,3 kB Eco RI Fragment des Bacillus poptllae-Genoms identifiziert Das 5,3 kB Eco RI Fragment wird z.B. in das Plasmid pBCSK+ eingebaut und in dem E. coli Stamm XL 1 Blue MRF' cloniert.The product of the PCR is used as a probe for hybridization in the Southern blot. A 5.3 kB Eco RI fragment of the Bacillus poptllae genome is identified. The 5.3 kB Eco RI fragment is e.g. incorporated into the plasmid pBCSK + and cloned into the E. coli strain XL 1 Blue MRF '.
Die Sequenzierung dieses 5,3 kB Eco RI Fragment, z B mittels der Kettenabbruch- Methode nach Sanger, unter Verwendung handelsüblicher Sequenasetestsysteme, z.B. eines T7 Sequencing Kits, führt zu dem im Sequenzprotokoll SEQ ID NO 1 dargestellten Ergebnis Beispiel 2:The sequencing of this 5.3 kB Eco RI fragment, for example using the Sanger chain termination method, using commercially available sequenase test systems, for example a T7 sequencing kit, leads to the result shown in the sequence listing SEQ ID NO 1 Example 2:
Gentechnologische Herstellung eines erfindungsgemaßen KristallproteinsGenetic engineering production of a crystal protein according to the invention
Das gemäß Beispiel 1 gewonnene 5,3 kB Eco RI Fragment des Bacillus popilliae- Genoms wird in ein Plasmid für gram-positive Bakterien eingebaut und in Bakterienzellen eines kristallfreien Stammes von Bacillus thunngiensis subsp kurstaki eingeschleust Als Plasmid eignet sich besonders ein als Klonierungsvektor für Bacillus thunngiensis bekanntes Plasmid wie pHT304, pHT315 oder pHT370, deren Herstellung in der Publikation von O Arantes und D Lerecius in Gene, 148 (1991), S 115-1 19, beschrieben ist Es wird insoweit Bezug auf diese Publikation genommen und ihr Inhalt hiermit in die vorliegende Beschreibung mit aufgenommenThe 5.3 kB Eco RI fragment of the Bacillus popilliae genome obtained according to Example 1 is incorporated into a plasmid for gram-positive bacteria and introduced into bacterial cells of a crystal-free strain of Bacillus thunngiensis subsp kurstaki. A plasmid is particularly suitable as a cloning vector for Bacillus thunngiensis known plasmid such as pHT304, pHT315 or pHT370, the preparation of which is described in the publication by O Arantes and D Lerecius in Gene, 148 (1991), pages 115-1 19. Reference is made to this publication in this respect and its content is hereby incorporated into the present description included
Rekombinierte Bakterien werden vermehrt bzw kloniert und bei Bedarf zur Synthese des Kristallproteins angeregt Die geschieht durch Induzierung der Sporenbildung, vorzugsweise einfach dadurch, daß die Kulturbedingungen gezielt verändert werden Dann wird die Proteinfreisetzung induziert, indem beispielsweise durch erneute Veränderung der Kulturbedingungen die Sporenkeimung bzw Autolyse der Sporangien ausgelost wird Das freigesetzte Protein wird aus dem Kulturmedium abgetrennt, gereinigt und gegebenenfalls bis zur Verwendung kühl, trocken und dunkel gelagertRecombined bacteria are multiplied or cloned and, if necessary, stimulated to synthesize the crystal protein. This is done by inducing spore formation, preferably simply by changing the culture conditions in a targeted manner. Then the protein release is induced, for example by changing the culture conditions again, by spore germination or autolysis of the sporangia The protein released is separated from the culture medium, purified and, if necessary, stored in a cool, dry and dark place until use
Beispiel 3:Example 3:
Verwendung des erfindungsgemäßen Kristallproteins zur Bekämpfung von adulten Scarabaeiden der Art Melolontha melolontha.Use of the crystal protein according to the invention for controlling adult Scarabaeids of the species Melolontha melolontha.
Adulte Maikäfer (Melolontha melolontha) wurden 10 Tage unter naturgetreuen Bedingungen in Käfigen gehalten Nach 2 Tagen wurde 20 Tieren eine wäßrigen Suspension des erfindungsgemaßen Proteins verabreicht Die Futteraufnahme war sofort gehemmt Bereits nach 4 Tagen waren 12 Tiere (= 60%) tot In der Praxis wird das Präparat auf die Nahrungspflanzen der Käfer aufgesprüht Beispiel 4:Adult cockchafer (Melolontha melolontha) were kept in cages for 10 days under lifelike conditions. After 2 days, an aqueous suspension of the protein according to the invention was administered to 20 animals. Feed intake was immediately inhibited. After just 4 days, 12 animals (= 60%) were dead the preparation is sprayed on the food plants of the beetles Example 4:
Verwendung des erfindungsgemäßen Kristallproteins zur Bekämpfung vonUse of the crystal protein according to the invention for combating
Scarabaeiden-Larven (Engerlingen) der Art Melolontha melolontha.Scarabaeid larvae of the species Melolontha melolontha.
Engerlinge der Maikäfer (Melolontha melolontha) wurden im Freiland ausgegraben, im Labor einzeln in Zuchtgefäßen gehalten und mit Karottenscheiben gefüttert. Nach 3 Wochen wurde 10 Tieren das erfindungsgemäße Protein in einer wäßrigen Suspension mit oder ohne Sporen verfüttert. Schon ein Tag später war die Futteraufhahme stark vermindert, gehemmtCockchafer grubs (Melolontha melolontha) were dug up in the field, kept individually in breeding vessels in the laboratory and fed with carrot slices. After 3 weeks, 10 animals were fed the protein according to the invention in an aqueous suspension with or without spores. Just a day later, the feed intake was greatly reduced, inhibited
In der Praxis wird das Präparat, eventuell in Kombination mit anderen Pathogenen und vorzugsweise zusammen mit Ködern in den Boden eingebracht. In practice, the preparation is introduced into the soil, possibly in combination with other pathogens and preferably together with bait.

Claims

A n s p r ü c h eExpectations
1 Isoliertes Polynukleotid, das ein Protein codiert, welches mit einem in Bacillus popilliae natürlicherweise vorkommenden Kristallprotein identisch oder verwandt ist, und das entweder die im Sequenzprotokoll SEQ ED NO: 1 dargestellten Nukleotidsequenz oder einer Teilsequenz hiervon, oder eine durch Substitution, Deletion, Insertion und/oder Inversion hiervon abgeleitete Nukleotidsequenz oder eine ganz oder teilweise hiermit hybridisierende Nukleotidsequenz aufweist.1 Isolated polynucleotide which encodes a protein which is identical or related to a crystal protein naturally occurring in Bacillus popilliae, and which either contains the nucleotide sequence shown in the sequence listing SEQ ED NO: 1 or a partial sequence thereof, or one by substitution, deletion, insertion and / or has an inversion of a nucleotide sequence derived therefrom or a nucleotide sequence which hybridizes wholly or partly therewith.
2 Isoliertes Polynukleotid nach Anspruch 1 , dadurch gekennzeichnet, daß das Polynukleotid aus einer natürlichen Quelle gewonnen oder synthetisch oder halbsynthetisch hergestellt ist.2. Isolated polynucleotide according to claim 1, characterized in that the polynucleotide is obtained from a natural source or is produced synthetically or semi-synthetically.
3._ Rekombinantes DNS- Vektormolekul, das ein Polynukleotid nach Anspruch 1 oder 2 umfaßt, und das die Fähigkeit zur Expression des/eines für Bacillus popilliae charakteristischen Kristallproteins bzw. Proteinkristalls oder eines damit verwandten Proteins bzw. Proteinkristalls in einer prokaryontischen oder eukaryontischen Zelle aufweist3._ Recombinant DNA vector molecule which comprises a polynucleotide according to claim 1 or 2 and which has the ability to express the crystal protein or protein crystal characteristic of Bacillus popilliae or a protein or protein crystal related thereto in a prokaryotic or eukaryotic cell
4. Rekombinantes DNS- Vektormolekül nach Anspruch 3, dadurch gekennzeichnet, daß das Vektormolekül ein in gram-positiven Bakterien vorkommendes Plasmid ist.4. Recombinant DNA vector molecule according to claim 3, characterized in that the vector molecule is a plasmid occurring in gram-positive bacteria.
5. Transformierte Wirtszelle, die ein Polynukleotid nach Anspruch 1 oder 2 enthält, das mit einem in der Wirtszelle natürlicherweise oder als Folgen einer Rekombination enthaltenen Promotor gekoppelt ist, und die die Fähigkeit zur Expression dieses Polynukleotids und Synthetisierung eines Kristallproteins, das dem in Bacillus popilliae charakteristischerweise vorhandenen Kristallprotein gleicht oder ähnlich ist Transformierte Wirtszelle nach Anspruch 5, dadurch gekennzeichnet, daß die Wirtszelle Bacillus thunngiensis, vorzugsweise Bacillus thunngiensis subsp kurstaki ist5. A transformed host cell which contains a polynucleotide according to claim 1 or 2 which is coupled to a promoter contained in the host cell naturally or as a result of recombination, and which has the ability to express this polynucleotide and synthesize a crystal protein which is similar to that in Bacillus popilliae Characteristically present crystal protein is the same or similar Transformed host cell according to claim 5, characterized in that the host cell is Bacillus thunngiensis, preferably Bacillus thunngiensis subsp kurstaki
Isoliertes Protein, das von einer Nukleotidsequenz nach Anspruch 1 kodiert ist und/oder Teile eines solchen ProteinsIsolated protein encoded by a nucleotide sequence according to claim 1 and / or parts of such a protein
Isoliertes Protein, das die im Sequenzprotokoll SEQ ID NO 2 dargestellte Aminosauresequenz oder eine damit verwandte, durch Substitution, Deletion, Insertion und/oder Inversion davon ableitbare Aminosauresequenz ganz oder teilweise umfaßtIsolated protein which comprises, in whole or in part, the amino acid sequence shown in the sequence listing SEQ ID NO 2 or a related amino acid sequence which can be derived by substitution, deletion, insertion and / or inversion
Verwendung von Proteinen nach Anspruch 7 oder 8, allein oder in Kombination mit wenigstens einer anderen Substanz einschließlich Mikroorganismen, zur Hemmung der Fraßtatigkeit und/oder Abtötung von adulten und/oder larvalen Scarabaeiden, insbesondere Melolontha- Arten und mit diesen nah verwandte ArtenUse of proteins according to claim 7 or 8, alone or in combination with at least one other substance including microorganisms, for inhibiting the feeding and / or killing of adult and / or larval Scarabaeiden, in particular Melolontha species and species closely related to them
Verwendung von Proteinen nach Anspruch 7 oder 8, allein oder in Kombination mit wenigstens einer anderen Substanz einschließlich Mikroorganismen, zur Inaktivierung, insbesondere Hemmung der Fraßtatigkeit, und/oder zur Abtötung von Pflanzen und/oder Pilze schädigende und/oder Krankheiten übertragenden BodenorganismenUse of proteins according to claim 7 or 8, alone or in combination with at least one other substance, including microorganisms, for inactivating, in particular inhibiting, predation and / or for killing plants and / or fungi-damaging and / or disease-transmitting soil organisms
Verwendung nach Anspruch 9 oder 10, dadurch gekennzeichnet, daß als (eine der) andere(n) Substanz(en) Bakteriensporen, vorzugsweise Sporen von Bacillus popilliae und/oder Bacillus thunngiensis eingesetzt werdenUse according to claim 9 or 10, characterized in that bacterial spores, preferably spores of Bacillus popilliae and / or Bacillus thunngiensis, are used as one of the other substance (s)
Verwendung nach Anspruch 9 oder 10, dadurch gekennzeichnet, daß als (eine der) andere(n) Substanz(en) Pilzsporen eingesetzt werden Verwendung nach Anspruch 9 oder 10, dadurch gekennzeichnet, daß als eine (der) andere Substanz(en) cytolysierende Proteine und/oder Rezeptorproteine für das Darmepithel von Scarabaeiden eingesetzt werden, vorzugsweise in Form von Fusionsproteinen mit Proteinen nach Anspruch 7 oder 8Use according to claim 9 or 10, characterized in that fungal spores are used as (one of the) other substance (s) Use according to claim 9 or 10, characterized in that cytolytic proteins and / or receptor proteins for the intestinal epithelium of scarabaeids are used as another substance (s), preferably in the form of fusion proteins with proteins according to claim 7 or 8
Verfahren zur Herstellung von Proteinen nach Anspruch 7 oder 8, dadurch gekennzeichnet, daß man ein oder mehrere Polynukleotide nach Anspruch 1 in einen Mikroorganismus (z.B ein Bakterium, Virus, Pilz oder Protozoon), oder eine Zelle einer tierischen oder pflanzlichen Zellkultur einschleust (transformiert), der Kontrolle und Steuerung eines in dem Mikroorganismus bzw der Zelle natürlicherweise oder als Folge einer/der Rekombination enthaltenen, vorzugsweise regulierbaren Promotors unterwirft und zur Expression bringtProcess for the production of proteins according to claim 7 or 8, characterized in that one or more polynucleotides according to claim 1 are introduced (transformed) into a microorganism (for example a bacterium, virus, fungus or protozoon) or a cell of an animal or plant cell culture. , the control and control of a, preferably regulable promoter contained in the microorganism or the cell naturally or as a result of a / the recombination and brings to expression
Verfahren nach Anspruch 14, dadurch gekennzeichnet, daß das (die) Polynukleotid(e) in ein Bakterium der Art Bacillus thunngiensis eingeschleust wirdA method according to claim 14, characterized in that the polynucleotide (s) is introduced into a bacterium of the Bacillus thunngiensis type
Verwendung von Polynukleotiden nach Anspruch 1 zur Erzeugung transgener Pflanzen mit der Eigenschaft, in allen oder einigen Pflanzengeweben einen Kristallprotein zu synthetisieren, das dem für Bacillus popilliae charakteristischen Kristallprotein gleicht oder ahnlich ist, und das zur Hemmung der Fraßtatigkeit und/oder Abtötung von adulten und/oder larvalen Scarabaeiden, insbesondere Melolontha- Arten und mit diesen nah verwandte Arten geeignet istUse of polynucleotides according to claim 1 for the production of transgenic plants with the property of synthesizing in all or some plant tissues a crystal protein which is similar or similar to the crystal protein characteristic of Bacillus popilliae and which is for the inhibition of feeding and / or killing of adult and / or or larval scarabaeids, in particular Melolontha species and species closely related to them
Gentechnisch transformierte Zelle mit einer stabil in ihr Genom integrierten (rekombinierten) heterologen DNA, die ein Polynukleotid nach Anspruch 1 umfaßt, welches ein Kristallprotein kodiert, das dem in Bacillus popilliae naturlicher- und charakteristischerweise vorkommenden Kristallprotein gleicht oder ähnlich ist, wobei die Exprimierung dieses Proteins unter der Steuerung eines von den Polymerasen der Zelle erkannten, in der Zelle natürlicherweise oder als Folge einer/der Rekombination enthaltenen Promotors erfolgt I 0Genetically transformed cell with a heterologous DNA stably integrated into its genome (recombined), which comprises a polynucleotide according to claim 1, which encodes a crystal protein which is similar or similar to the crystal protein naturally and characteristically found in Bacillus popilliae, the expression of this protein under the control of a promoter recognized by the polymerases of the cell, contained in the cell naturally or as a result of / the recombination I 0
Verfahren zur Erzeugung von Pflanze oder Pflanzengeweben oderProcess for the production of plants or plant tissues or
Pflar-zenvermehrungsmaterial mit rekombiniertem genetischem Material, das ein heterologes Polynukleotid nach Anspruch 1 umfaßt, dessen Exprimierung zu einem Protein führt, das dem in Bacillus popilliae naturlicher- und charakteristischerweise vorkommenden Kristallprotein gleicht oder ähnlich ist, dadurch gekennzeichnet, daß man Zellen oder Gewebe dieser Pflanzen mit einer rekombinanten DNA transformiert, die ein Polynukleotid nach Anspruch 1 und regulatorische Nukleotidsequenzen enthalt, welche die stabile Integration und die Exprimierung des Polynukleotids in der(den) Pflanzenzellen bewirken können, die Pflanze oder deren Vermehrungsmaterial oder beides dann aus der(den) mit der heterologen DNA transformierten Pflanzenzelle(n) oder dem entsprechenden Gewebe regeneriert, und gegebenenfalls diese regenerierte Pflanze oder ihr Vermehrungsmaterial oder beides biologisch vervielfältigt Plant propagation material with recombined genetic material, which comprises a heterologous polynucleotide according to claim 1, the expression of which leads to a protein which is similar or similar to the crystal protein which is naturally and characteristically found in Bacillus popilliae, characterized in that cells or tissues of these plants transformed with a recombinant DNA containing a polynucleotide according to claim 1 and regulatory nucleotide sequences which can effect the stable integration and expression of the polynucleotide in the plant cell (s), the plant or its propagation material or both from or with the heterologous DNA transformed plant cell (s) or the corresponding tissue regenerated, and optionally this regenerated plant or its propagation material or both biologically reproduced
EP96945813A 1995-10-18 1996-10-17 Polynucleotide and encoded protein useful in controlling of melolontha sp. scarabs Withdrawn EP0856057A2 (en)

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DE19540223 1995-10-18
DE19540223 1995-10-18
PCT/DE1996/001979 WO1997014798A2 (en) 1995-10-18 1996-10-17 Polynucleotides and proteins coded thereby useful for controlling melolontha sp. cockchafers

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US7033993B2 (en) 2001-04-13 2006-04-25 Dainippon Ink And Chemicals, Inc. Polypeptide having larvae growth inhibiting or insecticidal effect on scarabaeidae insects
EP1277763B1 (en) * 2001-07-04 2006-12-20 Dainippon Ink And Chemicals, Inc. Polypeptides having larvae growth inhibiting or insecticidal effect on scarabaeidae insects and polynucleotides encoding the same
US7364728B2 (en) * 2004-03-01 2008-04-29 Phyllom Llc Recombinant organisms producing insect toxins and methods for constructing same
US10743535B2 (en) 2017-08-18 2020-08-18 H&K Solutions Llc Insecticide for flight-capable pests

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US4824671A (en) * 1986-03-24 1989-04-25 Reuter Laboratories, Inc. In vitro method for producing infective bacterial spores and spore-containing insecticidal compositions
US5185148A (en) * 1991-12-16 1993-02-09 Mycogen Corporation Process for controlling scarab pests with Bacillus thuringiensis isolates

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US6204057B1 (en) 2001-03-20
JPH11513563A (en) 1999-11-24
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DE19642729C2 (en) 1999-04-15

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