WO1999054472A1 - Nouvelles toxines insecticides issues de xenorhabdus nematophilus et sequences d'acides nucleiques codant ces toxines - Google Patents
Nouvelles toxines insecticides issues de xenorhabdus nematophilus et sequences d'acides nucleiques codant ces toxines Download PDFInfo
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- WO1999054472A1 WO1999054472A1 PCT/EP1999/002629 EP9902629W WO9954472A1 WO 1999054472 A1 WO1999054472 A1 WO 1999054472A1 EP 9902629 W EP9902629 W EP 9902629W WO 9954472 A1 WO9954472 A1 WO 9954472A1
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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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
-
- 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
-
- 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
Definitions
- Insect pests are mainly controlled by intensive applications of chemical insecticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or death of the insects. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects.
- Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management strategies, but there is an increasing need for alternative pest control agents.
- Biological insect control agents such as Bacillus thuringiensis strains expressing insecticidal toxins like ⁇ -endotoxins, have also been applied with satisfactory results, offering an alternative or a complement to chemical insecticides.
- the present invention addresses the long-standing need for novel insect control agents Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents Furthermore, agents whose application minimizes the burden on the environment are desirable
- the present invention is drawn to nucleotide sequences isolated from Xenorhabdus nematophilus, and nucleotide sequences substantially similar thereto, whose expression result in insecticidal toxins that are highly toxic to economically important pests, particularly plant pests
- the invention is further drawn to the insecticidal toxin resulting from the expression of the nucleotide sequence, and to compositions and formulations containing the insecticidal toxin, that are capable of inhibiting the ability of insect pests to survive, grow or reproduce, or of limiting insect-related damage or loss in crop plants
- the invention is further drawn to a method of making the toxin and to methods of using the nucleotide sequence, for example in microorganisms to control insects or in transgenic plants to confer insect resistance, and to a method of using the toxin, and compositions and formulations comprising the toxin, for example applying the toxin, composition or formulation to insect infested areas, or to prophylactically treat insect susceptible areas or plants to confer
- the novel toxin is highly insecticidal against Plutella xylostella (diamondback moth), an economically important insect pest.
- the toxin can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment
- the present invention provides an isolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO.1 , nucleotides 1045-2334 of SEQ ID NO:1 , SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO.8, SEQ ID NO:10, SEQ ID NO.12, and SEQ ID NO:14; or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said nucleic acid molecule results in - 3 -
- the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 569-979 of SEQ ID NO:1 , nucleotides 1045-2334 of SEQ ID NO:1 , SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
- the nucleotide sequence comprises nucleotides 569-979 of SEQ ID NO:1 , nucleotides 1045-2334 of SEQ ID NO:1 , SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
- the nucleotide sequence comprises the approximately 3.0 kb DNA fragment comprised in pCIB9369 (NRRL B-21883).
- the toxins resulting from expression of the nucleic acid molecules of the invention have activity against Plutella xylostella.
- the present invention provides an isolated nucleic acid molecule comprising a 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO:1 , nucleotides 1045-2334 of SEQ ID NO:1 , SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO: 14, wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.
- the present invention provides toxins produced by the expression of DNA molecules of the present invention.
- the toxins of the invention have activity against Plutella xylostella. - 4 -
- the toxins are produced by the E. co/ strain designated as NRRL accession number B-21883.
- a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 3, 5, 7, 9, 11 , 13, and 15.
- composition comprising an insecticidally effective amount of a toxin of the invention.
- the present invention provides a method of producing a toxin that is active against insects, comprising: (a) obtaining a host cell comprising a chimeric gene, which itself comprises a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention; and (b) expressing the nucleic acid molecule in the cell, which results in at least one toxin that is active against insects.
- the present invention provides a method of producing an insect- resistant plant, comprising introducing a nucleic acid molecule of the invention into the plant, wherein the nucleic acid molecule is expressible in the plant in an effective amount to control insects.
- the insects are Plutella xylostella.
- the present invention provides a method of controlling insects comprising delivering to the insects an effective amount of a toxin according to the present invention.
- the insects are Plutella xylostella.
- the toxin is delivered to the insects orally.
- Yet another aspect of the present invention is the provision of a method for mutagenizing a nucleic acid molecule according to the present invention, wherein the nucleic acid molecule has been cleaved into population of double-stranded random fragments of a desired size, comprising: (a) adding to the population of double-stranded random fragments one or more single- or double-stranded oligonucleotides, wherein the oligonucleotides each comprise an area of identity and an area of heterology to a double- stranded template polynucleotide; (b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; (c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of the single-stranded fragments at the areas of identity to form pairs of annealed fragments, the areas of identity being sufficient for one member of a pair
- Activity of the toxins of the invention is meant that the toxins function as orally active insect control agents, have a toxic effect, or are able to disrupt or deter insect feeding, which may or may not cause death of the insect.
- a toxin of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the toxin available to the insect.
- Associated with / operatively linked refer to two nucleic acid sequences that are related physically or functionally.
- a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
- a “chimeric gene” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulator nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence.
- the regulator nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.
- control insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage or loss in crop plants.
- To "control” insects may or may not mean killing the insects, although it preferably means killing the insects.
- a toxin means that the toxin comes in contact with an insect, resulting in toxic effect and control of the insect.
- the toxin can be delivered in many recognized ways, e.g., orally by ingestion by the insect or by contact with the insect via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.
- a “gene” is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
- a gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
- Gene of interest refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability.
- the “gene of interest” may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
- a "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
- a “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced. "Homologous recombination” is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.
- insects are defined as a toxic biological activity capable of controlling insects, preferably by killing them.
- a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
- nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
- An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
- nucleic acid molecule or “nucleic acid sequence” is a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source.
- the nucleic acid molecule is preferably a segment of DNA.
- ORF' means open reading frame.
- Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
- a "plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
- Plant tissue as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
- a "protoplast” is an isolated plant ceil without a cell wall or with only parts of the cell wall.
- Regulatory elements refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
- the percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
- a nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1 % SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1 % SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1 % SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50
- Synthetic refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic. - 9 -
- Transformation is a process for introducing heterologous nucleic acid into a host cell or organism.
- transformation means the stable integration of a DNA molecule into the genome of an organism of interest.
- Transformed / transgenic / recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
- the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
- Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
- non-transformed refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
- Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).
- Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y),
- SEQ ID NO:1 is the sequence of the approximately 3.0 kb DNA fragment comprised in Xenorhabdus nematophilus clone pCIB9369, which comprises the following ORFs at the specified nucleotide positions:
- SEQ ID NO:6 is the DNA sequence of orf2 of Xenorhabdus nematophilus clone pCIB9381.
- SEQ ID NO:7 is the sequence of the Juvenile Hormone Esterase-like protein encoded by orf2 of clone pCIB9381.
- SEQ ID NO:8 is the DNA sequence of orfl of Xenorhabdus poinarii clone pCIB9354.
- SEQ ID NO:9 is the sequence of the protein encoded by orf 1 of clone pCIB9354.
- SEQ ID NO:10 is the DNA sequence of orf2 of Xenorhabdus poinarii clone pCIB9354.
- SEQ ID NO:1 1 is the sequence of the Juvenile Hormone Esterase-like protein encoded by orf2 of clone pCIB9354.
- SEQ ID NO:12 is the DNA sequence of orfl of Photorhabdus luminescens clone pCIB9383-21.
- SEQ ID NO:13 is the sequence of the protein encoded by orfl of clone pCIB9383-21.
- SEQ ID NO:14 is the DNA sequence of orf2 of Photorhabdus luminescens clone pCIB9383-21.
- SEQ ID NO:15 is the sequence of the Juvenile Hormone Esterase-like protein encoded by o ⁇ f2 of clone pCIB9383-21.
- nucleic acid sequences whose expression results in novel toxins, and to the making and using of the toxins to control insect pests.
- the nucleic acid sequences are isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens, members of the Enterobacteriaceae family.
- Xenorhabdus are symbiotic bacteria of nematodes of the genus Steinernema.
- Photorhabdus are symbiotic - 11 -
- the bacteria of nematodes of the genus Heterorhabditis colonize insect larva, kill them, and their offspring feed on the dead larvae.
- the insecticidal activity is actually produced by the symbiotic Xenorhabdus and Photorhabdus bacteria.
- the inventors are the first to isolate the nucleic acid sequences of the present invention.
- the expression of the nucleic acid sequences of the present invention results in toxins that can be used to control Lepidopteran insects such as Plutella xylostella (Diamondback Moth).
- a nucleotide sequence of the present invention in clone pCIB9369 is characterized by an approximately 3.0 kb DNA fragment deposited pursuant to the Budapest Treaty for Patent Deposits under Accession Number NRRL B-21883. The sequence of this DNA fragment is set forth in SEQ ID NO:1.
- Two open reading frames (ORF) are present in SEQ ID NO:1 (nucleotides 569-979 and nucleotides 1045-2334, respectively), coding for proteins of predicted sizes of 15 kDa and 47.7 kDa (SEQ ID NOs:2 and 3, respectively).
- the two ORFs are arranged in an operon-like structure.
- the nucleotide sequence of the present invention is also compared to known Xenorhabdus nematophilus sequences encoding the insecticidal toxin toxb4 (WO 95/00647), but no significant homology is found.
- the 3.0 kb DNA fragment is also compared to the nucleotide sequence published in WO 98/08388. Twenty-two sequences of 60 nucleotides each (60-mers) from the 38.2 kb DNA fragment described in WO 98/08388 are compared to the 3.0 kb DNA fragment of the present invention using the UWGCG Gap program.
- the nucleotide sequence of the first 60-mer starts at base 1 of the 38.2 kb DNA fragment and the other 60-mers are located at approximately 2.0 kb intervals. Each of the 22 sequences as well as their complementary sequences are tested. The highest percent of identity between the 3.0 kb DNA fragment of the present invention and one of these 60-mers is 53%, which is not a significant homology. Furthermore, five different DNA fragments of the 38.2 kb sequence are tested for hybridization to the 3.0 kb fragment of the present invention by Southern blot analysis. None of them reveal a positive hybridization signal. - 12 -
- the nucleotide sequences of pCIB9381 , pCIB9354, and pCIB9383-21 also reveal two open reading frames in each of these clones.
- the nucleotide sequences of the two ORFs in each of pCIB9381 and pCIB9383-21 are highly homologous to those in pCIB9369.
- the ORF #2 proteins of pCIB9381 and pCIB9383-21 have essentially the same homology to the juvenile hormone esterase-related protein as does the ORF #2 protein of pCIB9369.
- the invention encompasses a nucleotide sequence substantially similar to nucleotides 569-979 of SEQ ID NO:1 , nucleotides 1045-2334 of SEQ ID NO:1 , SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, whose expression results in an insecticidal toxin.
- the present invention also encompasses recombinant vectors comprising the nucleic acid sequences of this invention.
- host cells for such recombinant vectors are endophytes or epiphytes.
- a preferred host cell for such vectors is a eukaryotic cell, such as a yeast, a plant cell, or an insect cell.
- Plant cells such as maize cells are most preferred host cells, in another preferred embodiment, such vectors are viral vectors and are used for replication of the nucleotide sequences in particular host cells, e.g. insect cells or plant cells.
- Recombinant vectors are also used for transformation of the nucleotide sequences of this invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells.
- such host cells are prokaryotic cells.
- such host cells are eukaryotic cells, such as yeast cells, insect cells, or plant cells.
- the host cells are plant cells, such as maize cells.
- the insecticidal toxins comprise at least one polypeptide encoded by a nucleotide sequence of the invention.
- the molecular weight of an insecticidal toxin according to the invention is larger than 6,000, as determined by size fractionation experiments. After treatment with proteinase K, only a minimal decrease in insecticidal activity is observed in the insect bioassay, indicating that the insecticidal toxins are substantially resistant to proteinase K treatment.
- the insecticidal toxins retain their insecticidal activity after being stored at 22°C or at 4°C for 2 weeks. They also retains their insecticidal activity after being freeze dried and stored at 22°C for 2 weeks.
- the insecticidal toxins are also still active after incubation for 5 minutes at 60°C, but they loses their insecticidal activity after incubation for 5 minutes at 100°C or 80°C.
- the nucleotide sequences of the invention can be modified by incorporation of random mutations in a technique known as in-vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and US Patent 5,605,793, which are incorporated herein by reference. Millions of mutant copies of a nucleotide sequence are produced based on an original nucleotide sequence of this invention and variants with improved properties, such as increased insecticidal activity, enhanced stability, or different specificity or range of target insect pests are recovered.
- the method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising a nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of - 14 -
- the insecticidal toxins are produced by expression of the nucleotide sequences in heterologous host cells capable of expressing the nucleotide sequences.
- heterologous host cells capable of expressing the nucleotide sequences.
- Xenorhabdus nematophilus, Xenorhabdus poinarii, or Photorhabdus luminescens cells comprising modifications of at least one nucleotide sequence of this invention at its chromosomal location are described. Such modifications encompass mutations or deletions of existing regulatory elements, thus leading to altered expression of the nucleotide sequence, or the incorporation of new regulatory elements controlling the expression of the nucleotide sequence.
- additional copies of one or more of the nucleotide sequences are added to Xenorhabdus nematophilus, Xenorhabdus poinarii, or Photorhabdus luminescens cells either by insertion into the chromosome or by introduction of extrachromosomally replicating molecules containing the nucleotide sequences.
- At least one of the nucleotide sequences of the invention is inserted into an appropriate expression cassette, comprising a promoter and termination signals. Expression of the nucleotide sequence is constitutive, or an inducible promoter - 15 -
- the cell in which the toxin is expressed is a microorganism, such as a virus, a bacteria, or a fungus.
- a virus such as a baculovirus, contains a nucleotide sequence of the invention in its genome and expresses large amounts of the corresponding insecticidal toxin after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleotide sequence.
- the insecticidal toxin thus produced is used as an insecticidal agent.
- baculoviruses engineered to include the nucleotide sequence are used to infect insects in-vivo and kill them either by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.
- Bacterial cells are also hosts for the expression of the nucleotide sequences of the invention.
- non-pathogenic symbiotic bacteria which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used.
- Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas.
- Symbiotic fungi such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive nucleotide sequences for the same purpose.
- the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coll, either in transcriptional or translational fusion, behind the tac or trc promoter.
- the simplest procedure is to insert the operon into a vector such as pKK223- 3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used.
- Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al.
- At least one of the described nucleotide sequences is transferred to and expressed in Pseudomonas fluorescens strain CGA267356 (described in the published application EU 0 472 494 and in WO 94/01561 ) which has biocontrol characteristics.
- a nucleotide sequence of the invention is transferred to Pseudomonas aureofaciens strain 30-84 which also has biocontrol characteristics. Expression in heterologous biocontrol strains requires the selection of vectors appropriate for replication in the chosen host and a suitable choice of promoter. Techniques are well known in the art for expression in gram-negative and gram- positive bacteria and fungi.
- At least one of the insecticidal toxins of the invention is expressed in a higher organism, e.g., a plant.
- transgenic plants expressing effective amounts of the toxins protect themselves from insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed toxins. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect.
- a nucleotide sequence of the present invention is inserted into an expression cassette, which is then preferably stably integrated in the genome of said plant.
- the nucleotide sequence is included in a non-pathogenic self- replicating virus.
- a nucleotide sequence of this invention is preferably expressed in transgenic plants, thus causing the biosynthesis of the corresponding toxin in the transgenic plants. In this way, transgenic plants with enhanced resistance to insects are generated.
- the nucleotide sequences of the invention may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby.
- coding sequences that have at least 35% about GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%.
- Microbial nucleotide sequences which have low GC contents may express poorly in plants due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation.
- preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res.
- nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, and WO 93/07278 (to Ciba-Geigy).
- sequences adjacent to the initiating methionine may require modification.
- they can be modified by the inclusion of sequences known to be effective in plants.
- Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Ciontech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleotide sequences of this invention.
- the sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (whilst leaving the - 18 -
- expression of the nucleotide sequences in transgenic plants is driven by promoters shown to be functional in plants.
- the choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species.
- expression of the nucleotide sequences of this invention in leaves, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is preferred.
- inflorescences e.g. spikes, panicles, cobs, etc.
- dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
- monocotyledonous promoters for expression in monocotyledons.
- Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters.
- the nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the insecticidal toxins to be synthesized only when the crop plants are treated with the inducing chemicals.
- Preferred technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 (to Ciba- Geigy) and US patent 5,614,395.
- a preferred promoter for chemical induction is the tobacco PR-1a promoter.
- a preferred category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the insecticidal toxins only accumulate in cells which need to synthesize the insecticidal toxins to kill the invading insect pest.
- Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151 -158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Moiec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191 -201 (1993).
- tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green - 19 -
- a preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)).
- a preferred promoter for root specific expression is that described by de Framond (FEBS 290: 103-106 (1991 ); EP 0 452 269 to Ciba-Geigy).
- a preferred stem specific promoter is that described in US patent 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.
- transgenic plants expressing at least one of the nucleotide sequences of the invention in a root-preferred or root-specific fashion. Further preferred embodiments are transgenic plants expressing the nucleotide sequences in a wound-inducible or pathogen infection-inducible manner.
- constructions for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence.
- an appropriate transcription terminator e.g. tm1 from CaMV, E9 from rbcS. Any available terminator known to function in plants can be used in the context of this invention.
- sequences which have been shown to enhance expression such as intron sequences (e.g. from Adh1 and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).
- intron sequences e.g. from Adh1 and bronzel
- viral leader sequences e.g. from TMV, MCMV and AMV.
- nucleotide sequences of the present invention may be target expression of different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression of the nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host cells. Techniques to achieve this are well-known in the art.
- Vectors suitable for plant transformation are described elsewhere in this specification.
- binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be - 20 -
- transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
- transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta).
- markers examples include neomycin phosphotransf erase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2, 2-dichloroproprionic acid dehalogenase, acetohydroxyacid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, haloarylnitrilase, protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyl transferase, and ⁇ -glucuronidase.
- selectable or screenable marker for plant transformation is not, however, critical to the invention.
- a particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment can be found in Koziel et al., Biotechnology 11: 194-200 (1993), Hill et al., Euphytica 85:119-123 (1995) and Koziel et al., Annals of the New York Academy of Sciences 792:164-171 (1996).
- An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435. Transformation of plants can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with the peroxidase coding sequence.
- a nucleotide sequence of the present invention is directly transformed into the plastid genome.
- a major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451 ,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride etal. (1994) Proc. Natl. Acad. Sci. USA 91 , 7301-7305.
- Plastid expression in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear- expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein.
- a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
- Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
- a preferred method of applying insecticidal toxins of the present invention is by spraying to the environment hosting the insect pest like the soil, water, or foliage of plants.
- the number of applications and the rate of application depend on the type and intensity of infestation by the insect pest.
- the insecticidal toxins can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application).
- the insecticidal toxins may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing insecticidal toxins, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds.
- the insecticidal toxins can also be provided as bait located above or below the ground.
- insecticidal toxins are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances.
- the methods of application such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
- compositions or preparations containing the insecticidal toxins and, where appropriate, a solid or liquid adjuvant are prepared in known manner, for example by homogeneously mixing and/or grinding the insecticidal toxins with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
- extenders for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
- Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or water.
- aromatic hydrocarbons preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate,
- the solid carriers used e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite.
- Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand.
- a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
- Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.
- surfactants will also be understood as comprising mixtures of surfactants.
- Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.
- Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil.
- the fatty acid methyltaurin salts may also be used.
- so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
- the fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium - 25 -
- lignonsulfonic acid of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids.
- These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts.
- the sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms.
- Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.
- non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.
- non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol.
- Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
- the following strains are grown in nutrient broth at 25°C for 3 days in the growth media recommended by ATCC.
- DNA isolation the cultures are grown for 24 hr under the same conditions.
- Plutella xylostella (Px) bioassays are performed by aliquoting 50 ⁇ l of each E.coli culture on the solid artificial P. xylostella diet (Biever and Boldt, Annals of Entomological Society of American 971 ; Shelton, et al J. Ent. Sci 26:17). 4 ml of the diet is poured into 1 oz. clear plastic cups (Bioserve product #9051 ). 5 neonate P. xylostella from a diet adapted lab colony are placed in each diet containing cup and then covered with a white - 27 -
- Total DNA is isolated from Xenorhabdus nematophilus strain ATCC 19061 by treating freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg/ml lysozyme for 30 minutes at 37°C. Proteinase K is added to a final concentration of 100 ⁇ g/ml in 0.5% SDS and incubated at 45°C. The solution clears and becomes very viscous. The SDS concentration is increased to 1 % and 300 mM NaCI and equal volume of phenol- chloroform-isoamyl alcohol are added. The sample is gently mixed for 5 minutes and centrifuged at 3K. This is repeated twice.
- the aqueous phase is then mixed with 0.7 volumes isopropanol and centrifuged.
- the DNA pellet is washed three times with 70% ethanol and gently resuspended in 0.5X TE.
- 6 ⁇ g of DNA are treated with 0.3 unit of Sau3A per ⁇ g of DNA at 37°C for 3.5 minutes in a volume of 100 ⁇ l.
- the sample is then heated for 30 minutes at 65°C to inactivate the enzyme, then incubated with 2 units of calf intestinal alkaline phosphatase for 30 minutes at 37°C.
- the sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol and centrifuged.
- the aqueous phase is removed and mixed with 0.7 volumes isopropanol and centrifuged.
- the pellet is resuspended in 0.5X TE at a concentration of 100 ng/ml.
- SuperCos cosmid vector (Stratagene, La Jolla, CA) is prepared as described by the supplier utilizing the BamHI cloning site. Prepared SuperCos at 100 ⁇ g/ml is ligated with the X. nematophilus DNA previously digested with Sau3A at a ratio of 2:1 in a 5 ⁇ l volume - 28 -
- the ligation mixture is packaged using Gigapack XL III (Stratagene) as described by the supplier.
- Packaged phages are infected into XL-1 MR E coli cells (Stratagene) as described by the supplier.
- the cosmid library is plated on L-agar with 50 ⁇ g/ml kanamycin and incubated 16 hours at 37°C. 500 colonies are patched to fresh L-kan plates at a density of 50/plate. The cells are washed off with L broth and mixed with 20% glycerol and frozen at -80°C.
- Cosmid libraries from Xenorhabdus nematophilus strain Ps1 , Xenorhabdus poinarii strain ATCC 49122, and Photorhabdus luminescens strain Ps5 are constructed in a like manner.
- E.coli clones from each cosmid library are screened by insect bioassay yielding clones with activity against Plutella xylostella.
- An insecticidal cosmid clone from Xenorhabdus nematophilus strain ATCC 19061 is identified as pCIB9362.
- a 42 kb insecticidal cosmid clone from Xenorhabdus nematophilus strain Ps1 is identified as pCIB9379.
- a 42 kb insecticidal cosmid clone from Xenorhabdus poinarii strain ATCC 49122 is identified as pCIB9354.
- a 7 kb insecticidal cosmid clone from Photorhabdus luminescens strain Ps5 is identified as pCIB9383-21.
- Clone pCIB9362 is digested with Sacll and a 9 kb DNA fragment is isolated. This fragment is iigated into Bluescript cut with Sacll.
- the ligation mixture is transformed into DH5 ⁇ E. coli cells as described in Molecular Cloning, second edition (Sambrook et al.).
- the transformation mixture is plated on L agar plus 100 ⁇ g/ml ampillicin and incubated at 37°C overnight. Isolated colonies are grown up in L broth with 100 ⁇ g/ml ampillicin and plasmid DNA isolated by alkaline minipreps as described in Molecular Cloning, second edition.
- the 9 kb Sacll clone is identified as pCIB9362-3 and gives 100% mortality when bioassayed - 29 -
- 3 ⁇ g of pCIB9362-3 is isolated, digested with 0.3 unit of Sau3A per ⁇ g DNA for 4 , 6 and 8 min. at 37°C and heated at 75°C for 15 min.
- Samples are pooled and ligated into pUC19 previously digested with BamHI and treated with calf intestinal alkaline phosphatase.
- the ligation is transformed into DH5 ⁇ E coli cells, plated on L agar with Xgal/Amp as described in Molecular Cloning and grown overnight at 37°C. White colonies are picked and grown up in L broth with 100 ⁇ g/ml ampicillin and plasmid DNA is isolated as previously described.
- Sequencing primers are ordered from Genosys Biotechnologies (Woodlands, Tx). Sequencing is performed using the dideoxy chain-termination method and is completed using Applied Biosystems Inc. model 377 automated DNA sequencer (Foster City, CA). The sequence is assembled using Sequencher 3.0 from Gene Codes Corporation ( Ann Arbor, Michgan).
- a 20 kb fragment identified as pCIB9381 is subcloned from cosmid pCIB9379 using a Notl digest.
- Insect Bioassays are conducted for pCIB9369 for a variety of insects.
- Bioassays for pCIB9381 , pCIB9354, and pCIB9383-21 also show high insecticidal activity against Plutella xylostella.
- Xenorhabdus nematophilus cosmid clone pCIB9369 and Stratagene's Blue Script vector in E coli host DH5 are grown in media consisting of 50% Terrific broth and 50% Luria broth, supplemented with 50 ⁇ g/ml of ampicilin.
- Shake flask cultures 300 ml in 1000 ml baffled flasks ) are grown at 250 RPM, overnight at 37°C. Cultures of each strain are centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4°C. The pelleted cells are resuspended in 30 ml of 50mM NaCI, 25 mM Tris base, pH 7.0.
- the 3 ml fractions of the filtrates are applied to Bio-Rad Econo-Pac 10DG columns that had been previously equilibrated with 10 ml of 50mM NaCI, 25 mM Tris base, pH 7.0. The flow through collected during sample loading is discarded. The samples are fractionated with two subsequent additions of 4 ml each of the NaCI - Tris equilibration buffer. The first three fractions are saved for testing. The first fraction should contain all material above about 6,000 mol. wt. The subsequent fractions should contain material smaller than 6,000 mol. wt.
- the heat stability of the toxin is determined. Overnight cultures of the E coli strain pCIB9369 (E. coli host DH5a, carrying the 3.0 kb DNA of Xenorhabdus nematophilus ) are grown in a 50:50 mixture of Luria broth and terrific broth. Cultures are grown at 37°C in culture tubes on a tube roller. Samples of one ml each of the culture are placed in a 1.5 ml eppendorf tube and placed at 60°C, 80°C. The samples are removed after five minutes and allowed to cool to room temperature. This sample along with an untreated portion of the culture is assayed on P. xylostella. 50 ⁇ l of sample of sample is spread on diet, allowed to dry and neonate larvae P. xylostella applied to the surface. The assay is incubated for 5 days at room temperature.
- the untreated sample and the sample treated at 60°C cause 100% mortality.
- the sample treated at 80°C and a diet alone control do not cause observable mortality. - 32 -
- Example 11 Protease Treatment of the Insecticidal Activity
- Xenorhabdus nematophilus cosmid clone pCIB9369 and Stratagene's Blue Script vector in E coli host DH5 are grown in media consisting of 50% Terrific broth and 50% Luria broth, supplemented with 50 ⁇ g/ml of ampicilin.
- Shake flask cultures 300 ml in 1000 ml baffled flasks ) are grown at 250 RPM, overnight at 37°C. Cultures of each strain are centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4°C. The pelleted cells are resuspended in 30 ml of 50mM NaCI, 25 mM Tris base, pH 7.0.
- the concentrated cells are disrupted by sonication using a Branson Model 450 Sonicator for approximately eight 10 second cycles with cooling on ice between cycles.
- the sonicates are centrifuged in a Sorvall SS34 rotor at 6,000 RPM for 10 minutes at 4°C.
- the resultant supernatants are filtered through a 0.2 ⁇ filter.
- One ml samples of the supernatants are adjusted to 5mM Ca++ with the addition of CaCI 2 .
- Protease K (Gibco BRL; Gaithersburg, MD ) is added to 500 ⁇ g / ml. Samples are incubated for 2 and 24 hr at 37°C. Control samples are prepared and incubated with added Ca++ but no protease.
- the sonicate filtrate from pCIB9369 and the pCIB9369 samples incubated in the presence of 5mM Ca++ produce 100% mortality on Plutella xylostella neonate larvae.
- the pCIB9369 samples incubated with protease K in addition to Ca++ show slightly less mortality, approximately 90% mortality on Plutella.
- Example 12 Sequence Comparisons with the Protein Sequence of cry3A and the Protein Sequence of a Juvenile Hormone Esterase
- the nucleotide sequence of pCIB9369 (SEQ ID NO:1) reveals two open reading frames (ORFs) at nucleotides 569-976 and 1045-2334.
- ORF #1 has no homology to any sequences in Genbank after a search with the UWGCG Blast and Gap programs.
- a Gap analysis of the protein encoded by ORF #2 of pCIB9369 by the Blast program identifies some insignificant homology (21 % identity) to Bacillus thuringensis cry3A protein.
- the nucleotide sequences of pCIB9381 , pCIB9354, and pCIB9383-21 also reveal two open reading frames in each of these clones.
- the nucleotide sequences of the two ORFs in each of pCIB9381 and pCIB9383-21 are all over 90% identical to those in pCIB9369.
- the ORF #2 proteins of pCIB9381 and pCIB9383-21 have essentially the same homology to the juvenile hormone esterase-related protein as does the ORF #2 protein of pCIB9369.
- the nucleotide sequence of ORF #1 of pCIB9354 is 77% identical to the nucleotide sequence of ORF #1 of pCIB9369, and the nucleotide sequence of ORF #2 of pCIB9354 is 79% identical to the nucleotide sequence of ORF #2 of pCIB9369.
- the ORF #2 protein of pCIB9354 also has homology to the juvenile hormone esterase-related protein (29.2% AA identity and 42.2% AA similarity).
- Twenty-two sequences of 60 nucleotides each (60-mers) are derived from the 38.2 kb DNA fragment whose nucleotide sequence is described in WO 98/08388 and are compared to the nucleotide sequence of pCIB9362-3, which comprises pCIB9369.
- the first 60-mer starts at base 1 in the 38.2 kb DNA fragment, while the other 60-mers are located at approximately 2 kb intervals on the DNA fragment.
- Their positions on the 38.2 kb DNA fragment are listed below:
- Pairs of oligonucleotides are designed to amplify DNA fragments of the 38.2 kb DNA fragment published in WO 98/08388.
- the oligonucleotides are ordered from Genosys Biotechnologies (The Woodlands, Texas) and their positions in the 38.2 kb DNA fragment are indicated below. Also listed are their and the sizes of the amplified PCR fragments: - 34 -
- VK1046 positions 20-40
- VK1047 positions 2,078-2,100
- VK1048 positions 11 ,221 -11 ,241
- VK1049 positions 13,360-13,380 Size of the PCR fragment amplified using VK1048 and VK1049: 2,120 bp
- VK1050 positions 26,581 -26,601
- VK1051 positions 28,537-28,560
- VK1052 positions 18,901 -18,921
- VK1053 positions 20,321 -20,340 Size of the PCR fragment amplified using VK1052 and VK1053: 1 ,439 bp
- VK1054 positions 34,261 -34,281
- VK1055 positions 35,320-35,340 BP
- the PCR reactions are completed using a Perkin-Elmer 9600 Thermo-Cycler with the following conditions: 94°C, 2 min.; then 30 cycles at 94°C, 30 sec; 54°C, 30 sec; 72°C, 4 min.
- the samples contain 800 ng of Xenorhabdus nematophilus DNA, 0.1 -0.5 ⁇ M of each pair of oligonucleotides, 250 ⁇ M dNTP, 5U Taq Polymerase and 1 X buffer (Perkin-Elmer) in a final volume of 100 ⁇ l.
- the completed reactions are precipitated in ethanol, resuspended in TE and loaded on a 1 % SeaPlaque (FMC, Rockland, Maine) TBE gel.
- the fragments are cut out from the gel after ethidium bromide staining and visualization under UV light.
- the gel slices are melted at 65°C and 10 ⁇ l aliquots are mixed with 10 ⁇ l distilled water, boiled for 5 min. and placed on ice. Then, 15 ⁇ l of Random Priming label buffer ( GIBCO-BRL, Gaithersburg, MD), 6 ⁇ l dNTP mix (without dCTP) , 80 ⁇ Ci ⁇ -dCT 32 P and 1 ⁇ l Klenow are mixed.
- the labeling reaction is carried out during 60 min. at room temperature. The samples are cleaned up on Nick columns (Pharmacia Biotech) - 35 -
- the probes are boiled for 5 min. and placed on ice.
- a Southern blot is performed by digesting Xenorhabdus nematophilus total DNA, DNA derived from cosmids pCIB9362 and pCIB9363 (these cosmids overlap over 25 kb and both contain the DNA fragment of pCIB9369; pCIB9362 was used for subcloning), DNA derived from subclones pCIB9362-3 (9 kb Sacll fragment) and pCIB9369 (2.96 kb Clal fragment), digested with Clal, Sacll or Hindlll. The digestion reactions are loaded on a 0.75% agarose TBE gel and run overnight.
- the film is developed and the results show that the PCR probes from the WO 98/08388 sequence do not hybridize to the DNA of the cosmids or the DNA of the subclones described in this invention. However, a strong hybridization signal is observed with X. nematophilus DNA.
- heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, - 36 -
- Example 19 Expression of the Nucleotide Sequences in E. coli and Other Gram-Negative Bacteria
- Expression vector pKK223-3 (Pharmacia catalogue # 27-4935-01 ) allows expression in E. coli. This vector has a strong tac promoter (Brosius, J. ef al., Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A number of other expression systems have been developed for use in E. coli.
- _ (Pharmacia #27-4946-01 ) uses a tightly regulated bacteriophage ⁇ promoter which allows for high level expression of proteins.
- the lac promoter provides another means of expression but the promoter is not expressed at such high levels as the rac promoter.
- expression of the nucleotide sequence in closely related gram negative- bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia is possible.
- pLRKD21 1 Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820 (1984) contains the broad host range replicon ori T which allows replication in many gram-negative bacteria.
- trp-lac tac (i.e. trp-lac) promoter.
- this same promoter e.g. on wide-host range plasmid pLRKD211
- This trp- lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacterium for the purposes of the constitutive expression of such a gene.
- a nucleotide sequence whose expression results in an insecticidal toxin can therefore be placed behind a strong constitutive promoter, transferred to a bacterium which has plant or rhizosphere colonizing properties turning this organism to an insecticidal agent.
- Other possible promoters can be used for the constitutive expression of the nucleotide sequence in gram-negative bacteria. These include, for example, the promoter from the Pseudomonas regulatory genes gafA and lemA (WO 94/01561 ) and the Pseudomonas savastanoi IAA operon promoter (Gaffney et al., J. Bacteriol. 172: 5593-5601 (1990). - 37 -
- Trichoderma harzianum and Gliocladium virens have been shown to provide varying levels of biocontrol in the field (US 5,165,928 and US 4,996,157, both to Cornell Research Foundation)
- a nucleotide sequence whose expression results in an insecticidal toxin could be expressed in such a fungus. This could be accomplished by a number of ways which are well known in the art One is protoplast-mediated transformation of the fungus by PEG or electroporation-mediated techniques.
- This plasmid contains the E coli the hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter and the f ⁇ C terminator (Punt et al., Gene 56: 117-124 (1987)).
- nucleic acid sequences of the invention are expressed in the yeast Saccharomyces cerevisiae.
- Example 18 Liquid Formulation of Insecticidal Compositions
- Emulsions of any required concentration can be produced from such concentrates by dilution with water. 39 -
- the active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier, and the solvent is subsequently evaporated off in vacuo.
- Ready-to-use dusts are obtained by intimately mixing the carriers with the active ingredient.
- the active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.
- Emulsions of any required concentration can be obtained from this concentrate by dilution with water.
- Ready-to-use dusts are obtained by mixing the active ingredient with the carriers, and grinding the mixture in a suitable mill. - 41 -
- the active ingredient is mixed and ground with the adjuvants, and the mixture is subsequently moistened with water.
- the mixture is extruded and then dried in a stream of air.
- the finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desire concentration can be obtained by dilution with water.
- Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems ⁇ gtH , ⁇ gtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems.
- the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
- the expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the nucleotide sequence of the invention confer insect resistance to the transgenic plants.
- nucleotide sequences described in this application can be modified for expression in transgenic plant hosts.
- a host plant expressing the nucleotide sequences and which produces the insecticidal toxins in its cells has enhanced resistance to insect attack and is thus better equipped to withstand crop losses associated with such attack.
- each microbial ORF is isolated individually and cloned within a cassette which provides a plant promoter sequence at the 5' end of the ORF and a plant transcriptional terminator at the 3' end of the ORF.
- the isolated ORF sequence preferably includes the initiating ATG codon and the terminating STOP codon but may include additional sequence beyond the initiating ATG and the STOP codon.
- the ORF may be truncated, but still retain the required activity; for particularly long ORFs, truncated versions which retain activity may be preferable for expression in transgenic organisms.
- plant promoter and “plant transcriptional terminator” it is intended to mean promoters and transcriptional terminators which operate within plant cells. This includes promoters and transcription terminators which may be derived from non-plant sources such as viruses (an example is the Cauliflower Mosaic Virus).
- modification to the ORF coding sequences and adjacent sequence is not required. It is sufficient to isolate a fragment containing the ORF of interest and to insert it downstream of a plant promoter.
- Gaffney et al. (Science 261 : 754- 756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants under the control of the CaMV 35S promoter and the CaMV tml terminator successfully without modification of the coding sequence and with x bp of the Pseudomonas gene upstream of the ATG still attached, and y bp downstream of the STOP codon still attached to the nahG ORF.
- Preferably as little adjacent microbial sequence should be left attached upstream of the ATG and downstream of the STOP codon. In practice, such construction may depend on the availability of restriction sites.
- genes derived from microbial sources may provide problems in expression. These problems have been well characterized in the art and are particularly common with genes derived from certain sources such as Bacillus. These problems may apply to the nucleotide sequence of this invention and the modification of these genes can be undertaken using techniques now well known in the art. The following problems may be encountered:
- the preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) will enable an identification of the codons within the ORF which should preferably be changed. Typically - 44 -
- Plant genes typically have a GC content of more than 35%.
- ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of messages and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice sites (see below).
- Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that ribosomes attach to the 5' end of the message and scan for the first available ATG at which to start translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG.
- Clontech (1993/1994 catalog, page 210, incorporated herein by reference) have suggested one sequence as a consensus translation initiator for the expression of the E coli uidA gene in plants.
- Genes cloned from non-plant sources and not optimized for expression in plants may also contain motifs which may be recognized in plants as 5' or 3' splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques well known in the art.
- the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non- limiting examples of promoters that may be used in expression cassettes.
- Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991); maize - Christensen et al. Plant Molec. Biol. 12: 619- 632 (1989); and Arabidopsis - Norris et al., Plant Mol. Biol. 21 :895-906 (1993)).
- the maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor et al.
- pAHC25 a vector that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
- the Arabidopsis ubiquitin promoter is ideal for use with the nucleotide sequences of the present invention.
- the ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons.
- Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences. - 47 -
- pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
- a derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX.
- the double 35S promoter fragment can be removed by 5' excision with Hindlll, SphI, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
- modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired.
- pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference.
- actin promoter is a good choice for a constitutive promoter.
- the promoter from the rice ActI gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)).
- a 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
- numerous expression vectors based on the ActI promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 23J . : 150-160 (1991 )).
- the promoter expression cassettes described by McElroy et al. can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter- containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors.
- the rice ActI promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).
- the double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels.
- one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395 may replace the double 35S promoter.
- the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector.
- the chemically/pathogen regulatable tobacco PR- l a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992).
- pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
- Trie fragment is then cleaved with Hindlll and the resultant PR-1 a promoter- containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed.
- a promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention.
- a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat. Biotechnol 16:177-180).
- the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
- the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter are replaced by a coding sequence of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.
- glucocorticoid- mediated induction system is used (Aoyama and Chua (1997) The Plant Journal 11 : 605- 612) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to 1 mM, more preferably from 10mM to 100mM.
- the luciferase gene sequences are replaced by a nucleic acid sequence of the invention to form an expression cassette having a nucleic acid sequence of the invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter.
- the trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al. (1986) Science 231 : 699-704) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al. (1988) Genes Devel.
- fusion protein fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080).
- the expression of the fusion protein is controlled by any promoter suitable for expression in plants known in - 50 -
- This expression cassette is also comprised in the plant comprising a nucleic acid sequence of the invention fused to the 6xGAL4/minimal promoter.
- tissue- or organ-specificity of the fusion protein is achieved leading to inducible tissue- or organ-specificity of the insecticidal toxin.
- a suitable root promoter is described by de Framond (FEBS 290: 103-106 (1991 )) and also in the published patent application EP 0 452 269, which is herein incorporated by reference. This promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.
- Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151 -158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191 -201 (1993)) and all are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene.
- Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al. have described a wound- induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.
- this promoter, or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner.
- fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
- a maize gene encoding phosphoenol carboxylase has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
- WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells.
- CDPK calcium-dependent protein kinase
- the gene sequence and promoter extend up to 1400 bp from the start of transcription.
- this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a nucleic acid sequence of the invention in a pollen-specific manner.
- transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation.
- Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons, in addition, a gene's native transcription terminator may be used.
- DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled “Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.
- sequences have been characterized which cause the targeting of gene products to other cell compartments.
- Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
- the transgene product By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment.
- chloroplast targeting for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene.
- the signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence.
- Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081 -1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
- vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
- pclB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner.
- pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol.
- Xhol linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the Xhol- - 55 -
- pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail.
- pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul.
- pCIB2001 in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacte ⁇ um-me ⁇ iale ⁇ transformation, the RK2-derived tdA function for mobilization between E. coli and other hosts, and the Or/Tand Or/V functions also from RK2.
- the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
- the binary vector pCIBI O contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives of pCIBI O are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
- Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for no - Agrobacterium transformation is described.
- pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin).
- the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
- the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
- the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
- the resultant derivative of pCIB246 is designated pCIB3025.
- the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
- the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Sma/ fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al.
- This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E coli) and a polylinker with the unique sites SphI, Pstl, Hindlll, and BamHI.
- This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
- pSOG35 is a transformation vector that utilizes the E coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
- DFR E coli gene dihydrofolate reductase
- PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E.
- plastid transformation vector pPH143 (WO 97/32011 , example 36) is used.
- the nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence.
- This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance.
- the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
- nucleic acid sequence of the invention Once a nucleic acid sequence of the invention has been cloned into an expression system, it is transformed into a plant cell.
- Methods for transformation and regeneration of plants are well known in the art.
- Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro- injection, and microprojectiies.
- bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.
- Transformation techniques for dicotyledons are well known in the art and include Agrobacfe ⁇ um-based techniques and techniques that do not require Agrobacterium.
- Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986), and Klein ef al., Nature 327: 70-73 (1987).
- the transformed cells are regenerated to whole plants using standard techniques known in the art.
- .grobacter/ ' um-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many - 58 -
- Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)).
- the binary vector carrying the foreign DNA of interest e.g. pCIB200 or pCIB2001
- an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)).
- the transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E coli carrying the recombinant binary vector, a helper E coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
- the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
- Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T- DNA borders.
- Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells.
- This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al.
- this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
- the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
- the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
- Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
- Transformation of most monocotyledon species has now also become routine.
- Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co- - 59 -
- Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts.
- Gordon-Kamm et al. Plant Cell 2: 603-618 (1990)
- Fromm et al. Biotechnology 8: 833-839 (1990)
- WO 93/07278 and Koziel et al. describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
- Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
- Protoplast-mediated transformation has been described for apon/ca-types and Indica-types (Zhang ef al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou ef al. Biotechnology 9: 957-962 (1991 )).
- WO 93/21335 describes techniques for the transformation of rice via electroporation.
- Patent Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology H: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high - 60 -
- any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
- MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
- embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical.
- An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures.
- Each plate of embryos is shot with the DuPont Biolistics® helium device using a burst pressure of -1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration.
- the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35).
- regeneration medium MS + 1 mg/liter NAA, 5 mg/liter GA
- selection agent 10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35.
- GA7s sterile containers which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
- Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in a 1" circular array on T agar medium and bombarded 12-14 days after sowing with 1 ⁇ m tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maiiga, P. (1993) PNAS 90, 913-917).
- Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 ⁇ mol photons/m /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 ⁇ g/ml spectinomycin dihydrochloride (Sigma, St. Louis, MO). Resistant - 61 -
- the plants obtained via tranformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra.
- the expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981 ); Crop Breeding, Wood D. R.
- the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation - 62 -
- Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
- the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
- different breeding measures are taken.
- the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
- Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means.
- Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
- the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties.
- new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
- germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important.
- seed production In seed production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important.
- Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.
- Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD ® ), methalaxyl (Apron ® ), and pirimiphos-methyl (Actellic ® ). If desired, these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
- the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
- the seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds.
- the bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.
- a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
- the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type.
- the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed.
- the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering.
- water absorbent materials are placed between or adjacent to packaging material layers.
- a vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
- An example of such treatment is sterilization, for example by chemical means or by exposure to radiation.
- Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.
- the microorganism identified under I. above was accompanied by:
- microorganism identified under I. above was received toy this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of requeet for conversion) .
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- Genetics & Genomics (AREA)
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- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Insects & Arthropods (AREA)
- Pest Control & Pesticides (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99917999A EP1082434A1 (fr) | 1998-04-21 | 1999-04-19 | NOUVELLES TOXINES INSECTICIDES ISSUES DE $i(XENORHABDUS NEMATOPHILUS) ET SEQUENCES D'ACIDES NUCLEIQUES CODANT CES TOXINES |
IL13907599A IL139075A0 (en) | 1998-04-21 | 1999-04-19 | Novel insecticidal toxins from xenorhabdus nematophilus and nucleic acid sequences coding therefor |
CA002326067A CA2326067A1 (fr) | 1998-04-21 | 1999-04-19 | Nouvelles toxines insecticides issues de xenorhabdus nematophilus et sequences d'acides nucleiques codant ces toxines |
PL99343565A PL343565A1 (en) | 1998-04-21 | 1999-04-19 | Novel insecticidal toxins from xenorhabdus nematophilus |
BR9909856-3A BR9909856A (pt) | 1998-04-21 | 1999-04-19 | Toxinas inseticidas oriundas de xenorhabdus nematophilus e sequências de ácidos nucléicos que codificam para as mesmas |
JP2000544804A JP2002512036A (ja) | 1998-04-21 | 1999-04-19 | キセノラブドゥス属のXenorhabdusnematophilusからの新規殺昆虫性毒素およびそれをコードする核酸配列 |
HU0101697A HUP0101697A3 (en) | 1998-04-21 | 1999-04-19 | Novel insecticidal toxins from xenorhabdus nematophilus and nucleic acid sequences coding therefor |
AU36073/99A AU3607399A (en) | 1998-04-21 | 1999-04-19 | Novel insecticidal toxins from (xenorhabdus nematophilus) and nucleic acid sequences coding therefor |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6398298A | 1998-04-21 | 1998-04-21 | |
US09/063,982 | 1998-04-21 | ||
US12350099P | 1999-03-09 | 1999-03-09 | |
US60/123,500 | 1999-03-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999054472A1 true WO1999054472A1 (fr) | 1999-10-28 |
Family
ID=26744021
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/002629 WO1999054472A1 (fr) | 1998-04-21 | 1999-04-19 | Nouvelles toxines insecticides issues de xenorhabdus nematophilus et sequences d'acides nucleiques codant ces toxines |
Country Status (13)
Country | Link |
---|---|
EP (1) | EP1082434A1 (fr) |
JP (1) | JP2002512036A (fr) |
KR (1) | KR100649909B1 (fr) |
CN (1) | CN100368544C (fr) |
AU (1) | AU3607399A (fr) |
BR (1) | BR9909856A (fr) |
CA (1) | CA2326067A1 (fr) |
HU (1) | HUP0101697A3 (fr) |
ID (1) | ID26556A (fr) |
IL (1) | IL139075A0 (fr) |
PL (1) | PL343565A1 (fr) |
TR (2) | TR200102111T2 (fr) |
WO (1) | WO1999054472A1 (fr) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003087377A1 (fr) * | 2002-04-17 | 2003-10-23 | Institut Pasteur | Proteines insecticides photorabdus luminescens |
WO2003087144A2 (fr) * | 2002-04-17 | 2003-10-23 | Institut Pasteur | Proteines insecticides photorabdus luminescens |
WO2004067727A2 (fr) * | 2003-01-21 | 2004-08-12 | Dow Agrosciences Llc | Melange et appariement de proteines tc pour lutter contre les ravageurs |
US7071386B2 (en) | 2003-01-21 | 2006-07-04 | Dow Agrosciences Llc | Xenorhabdus TC gene for pest control |
EP2019115A2 (fr) | 2002-06-28 | 2009-01-28 | Dow AgroSciences LLC | Protéines actives du point de vue pesticide et polynucléotides pouvant être obtenus à partir d'espèces de Paenibacillus |
US8777011B2 (en) | 2001-12-21 | 2014-07-15 | Novartis Ag | Capsule package with moisture barrier |
CN112368296A (zh) * | 2018-09-25 | 2021-02-12 | 孟山都技术公司 | 新型昆虫抑制性蛋白 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20020031137A (ko) * | 2002-03-28 | 2002-04-26 | 임학태 | 제초제 저항성 형질이 도입된 형질전환 브로콜리와 그에의해 생산된 일대교잡종 브로콜리의 순도검정에 이용 |
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-
1999
- 1999-04-19 KR KR1020007011670A patent/KR100649909B1/ko not_active IP Right Cessation
- 1999-04-19 AU AU36073/99A patent/AU3607399A/en not_active Abandoned
- 1999-04-19 HU HU0101697A patent/HUP0101697A3/hu unknown
- 1999-04-19 CN CNB998066885A patent/CN100368544C/zh not_active Expired - Fee Related
- 1999-04-19 TR TR2001/02111T patent/TR200102111T2/xx unknown
- 1999-04-19 BR BR9909856-3A patent/BR9909856A/pt not_active Application Discontinuation
- 1999-04-19 JP JP2000544804A patent/JP2002512036A/ja not_active Withdrawn
- 1999-04-19 CA CA002326067A patent/CA2326067A1/fr not_active Abandoned
- 1999-04-19 TR TR2000/03086T patent/TR200003086T2/xx unknown
- 1999-04-19 IL IL13907599A patent/IL139075A0/xx unknown
- 1999-04-19 ID IDW20002108A patent/ID26556A/id unknown
- 1999-04-19 PL PL99343565A patent/PL343565A1/xx unknown
- 1999-04-19 WO PCT/EP1999/002629 patent/WO1999054472A1/fr active IP Right Grant
- 1999-04-19 EP EP99917999A patent/EP1082434A1/fr not_active Withdrawn
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WO1993003154A1 (fr) * | 1991-08-02 | 1993-02-18 | Mycogen Corporation | Nouveaux microorganismes et insecticide |
WO1995000647A1 (fr) * | 1993-06-25 | 1995-01-05 | Commonwealth Scientific And Industrial Research Organisation | Gene de toxine issu du xenorhabdus nematophilus |
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Cited By (19)
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US8777011B2 (en) | 2001-12-21 | 2014-07-15 | Novartis Ag | Capsule package with moisture barrier |
WO2003087377A1 (fr) * | 2002-04-17 | 2003-10-23 | Institut Pasteur | Proteines insecticides photorabdus luminescens |
WO2003087144A2 (fr) * | 2002-04-17 | 2003-10-23 | Institut Pasteur | Proteines insecticides photorabdus luminescens |
FR2838749A1 (fr) * | 2002-04-17 | 2003-10-24 | Pasteur Institut | Proteines insecticides photorhabdus luminescens |
FR2838750A1 (fr) * | 2002-04-17 | 2003-10-24 | Pasteur Institut | Proteines insecticides photorhabdus luminescens |
WO2003087144A3 (fr) * | 2002-04-17 | 2004-04-08 | Pasteur Institut | Proteines insecticides photorabdus luminescens |
EP2019115A2 (fr) | 2002-06-28 | 2009-01-28 | Dow AgroSciences LLC | Protéines actives du point de vue pesticide et polynucléotides pouvant être obtenus à partir d'espèces de Paenibacillus |
US7902334B2 (en) | 2002-06-28 | 2011-03-08 | Dow Agrosciences Llc | Pesticidally active proteins and polynucleotides obtainable from Paenibacillus species |
US7071386B2 (en) | 2003-01-21 | 2006-07-04 | Dow Agrosciences Llc | Xenorhabdus TC gene for pest control |
WO2004067727A3 (fr) * | 2003-01-21 | 2005-03-17 | Dow Agrosciences Llc | Melange et appariement de proteines tc pour lutter contre les ravageurs |
US7491698B2 (en) | 2003-01-21 | 2009-02-17 | Dow Agrosciences Llc | Mixing and matching TC proteins for pest control |
US7517956B2 (en) | 2003-01-21 | 2009-04-14 | Dow Agrosciences Llc | Xenorhabdus TC proteins and genes for pest control |
US7709623B2 (en) | 2003-01-21 | 2010-05-04 | Dow Agrosciences Llc | Xenorhabdus TC proteins and genes for pest control |
US8084418B2 (en) | 2003-01-21 | 2011-12-27 | Dow Agrosciences Llc | Methods of inhibiting insects by treatment with a complex comprising a Photorhabdus insecticidal protein and one or two Xenorhabdus enhancer proteins |
WO2004067727A2 (fr) * | 2003-01-21 | 2004-08-12 | Dow Agrosciences Llc | Melange et appariement de proteines tc pour lutter contre les ravageurs |
CN112368296A (zh) * | 2018-09-25 | 2021-02-12 | 孟山都技术公司 | 新型昆虫抑制性蛋白 |
US11512324B2 (en) * | 2018-09-25 | 2022-11-29 | Monsanto Technology, Llc | Insect inhibitory proteins |
EP3855921A4 (fr) * | 2018-09-25 | 2022-11-30 | Monsanto Technology LLC | Nouvelles protéines insecticides |
US11987800B2 (en) | 2018-09-25 | 2024-05-21 | Monsanto Technology Llc | Insect inhibitory proteins |
Also Published As
Publication number | Publication date |
---|---|
TR200102111T2 (tr) | 2001-11-21 |
CN1303432A (zh) | 2001-07-11 |
HUP0101697A2 (hu) | 2001-09-28 |
KR100649909B1 (ko) | 2006-11-27 |
PL343565A1 (en) | 2001-08-27 |
JP2002512036A (ja) | 2002-04-23 |
AU3607399A (en) | 1999-11-08 |
BR9909856A (pt) | 2000-12-19 |
CA2326067A1 (fr) | 1999-10-28 |
KR20010034796A (ko) | 2001-04-25 |
TR200003086T2 (tr) | 2001-03-21 |
CN100368544C (zh) | 2008-02-13 |
ID26556A (id) | 2001-01-18 |
EP1082434A1 (fr) | 2001-03-14 |
HUP0101697A3 (en) | 2003-03-28 |
IL139075A0 (en) | 2001-11-25 |
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