KR20010034796A - Novel insecticidal toxins from Xenorhabdus nematophilus and nucleic acid sequences coding therefor - Google Patents

Abstract

Novel isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus luminescens, which are expressed and produce novel insecticidal toxins Nucleic acid sequences are described herein. The invention also describes compositions and formulations containing pesticidal toxins that can control pests. The present invention also describes methods of making toxins, and methods of controlling insect pests using nucleotide sequences in microorganisms or imparting insect resistance, for example, in transformed plants.

Description

Novel insecticidal toxins from Xenorhabdus nematophilus and nucleic acid sequences coding therefor

The present invention relates to novel toxins from Xenorhabdus nematophyllus, Xenorhabdus poinarii and Photorhabdus luminescens, nucleic acid sequences expressed to produce toxins, and toxins. A method of making and a method of controlling insects using toxins and corresponding nucleic acid sequences.

Pests are a major cause of crop loss. In the United States alone, about $ 7.7 billion are lost each year due to invasion of various insects. In addition to the loss of crops, pests are also a burden to vegetable and fruit growers and ornamental producers, which are annoying to gardeners and homeowners.

Pests are controlled primarily by the strong application of chemical pesticides, which are active through the inhibition of insect growth, the prevention of insect nutrition or propagation, or the killing of insects. While good insect control can be achieved this way, these chemicals also sometimes affect other beneficial insects. Another problem resulting from the use of a wide range of chemical pesticides is the emergence of resistant insect strains. This is partially alleviated by various resistance treatment strategies, but the need for alternative pest control agents is increasing. Bacillus thuringiensis strains expressing insecticidal toxins such as biological insect control agents, for example, δ-endotoxin, are also applied with satisfactory results to provide substitutes or supplements for chemical insecticides. Recently, genes encoding some of these δ-endotoxins have been isolated, and their expression in heterologous hosts appears to provide another tool for the control of economically important pests. In particular, the expression of pesticidal toxins, eg, Bacillus suringiensis δ-endotoxins in transformed plants provides effective protection against selected pests, and the transformed plants expressing such toxins are commercialized to allow farmers to To reduce the application of chemical insect control agents. However, even in this case, the possibility of the development of resistance remains and only a few specific pests can be controlled. As a result, there remains a long-standing but unmet need to discover new and useful insect control agents that provide economic benefits to farmers and are environmentally acceptable.

The present invention addresses the long lasting need for novel insect control agents. There is a particular need for control agents that target economically important pests and efficiently control insect strains resistant to existing insect control agents. Also preferred are formulations that are applied to minimize the burden on the environment.

In the search for novel insect control agents, certain kinds of nematodes from the genus Heterorhabdus and Steinernema are of particular interest because of their insecticidal properties. They kill insect larvae, and their offspring feed on dead larvae. In fact, its pesticidal activity is due to live symbiotic bacteria in nematodes. These symbiotic bacteria are photoherhabdus in the case of heteroherhabdose and genorhabdus in the case of steinernema.

Nucleotide sequences isolated from Genohabdus nematophilus, and nucleotide sequences substantially similar thereto, are described that are highly toxic to expressed and economically important pests, particularly plant pests. The invention also relates to insecticidal toxins resulting from the expression of nucleotide sequences, and insecticidal toxins, which can inhibit the ability of the pest to survive, grow and reproduce, or limit insect-related damage or loss in crop plants. Compositions and formulations containing are described. The invention also relates to methods of making toxins and methods of controlling the insects or imparting insect resistance to transformed plants by using nucleotide sequences, for example in microorganisms, and toxins and compositions and formulations comprising toxins Methods of using are described, for example, toxins, compositions or formulations are applied to insect invasive areas, or to protect against insects by protecting them from harmful insect areas or plants.

The new toxin is very pesticidal against Plutella xylostella (cabbage moth), an economically important pest. The toxin can be used in many insect control strategies to provide maximum efficiency with minimal impact on the environment.

According to one embodiment, the invention provides a nucleotide selected from the group consisting of (a) 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. An isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the sequence, or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a) and which is expressed to produce one or more toxins active against insects to provide. In a specific example of this embodiment, the nucleotide sequence is a nucleotide sequence substantially similar to nucleotide sequences 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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; And isoencrypted. Preferably, the nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 11, 13 and 15. Most preferably, the nucleotide sequence comprises nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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. In another embodiment, the nucleotide sequence comprises about 3.0 kb DNA fragment contained in pCIB9369 (NRRL B-21883).

According to a preferred embodiment, the toxin resulting from the expression of the nucleic acid molecule of the present invention has activity against flutella xylostella.

In another embodiment, the present invention provides a nucleotide sequence selected from the group consisting of nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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; 20, 25, 30, 35, 40, 45, or 50 (preferably with the same sequence as the nucleotide portion of 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pairs of) 20) a nucleotide portion of a base pair and provides an isolated nucleic acid molecule that is expressed to produce one or more toxins that are active against insects.

The invention also provides a chimeric gene comprising a heterologous promoter sequence operably linked with a nucleic acid molecule of the invention. The present invention also provides a recombinant vector comprising a chimeric gene. Moreover, the present invention provides a host cell comprising such a chimeric gene. The host cell according to this aspect of the invention is a bacterial cell, yeast cell or plant cell, preferably it may be a plant cell. Moreover, the present invention provides a plant comprising such plant cells. Preferably, the plant is corn.

In another embodiment, the invention provides toxins produced by the expression of the DNA molecules of the invention. According to a preferred embodiment, the toxin of the present invention has activity against flutella xylostella.

In one embodiment, the toxin is NRRL Accession No. B-21883. Produced by E. coli strains.

In another embodiment, the 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.

Also provided is a composition comprising an insecticidal effective amount of the toxin of the invention. In another aspect, the invention provides a host cell comprising (a) a host cell comprising a chimeric gene comprising a heterologous promoter sequence operably linked to a nucleic acid molecule of the invention, and (b) one or more active against insects A method for producing a toxin active against insects is characterized by the expression of a toxin producing nucleic acid molecule in a cell.

In another aspect, the invention provides a method of producing an insect-tolerant plant, characterized by introducing into the plant a nucleic acid molecule of the invention that is expressible in the plant in an amount effective to control the insect. According to a preferred embodiment, the insect is flutella xylostella.

In another aspect, the invention provides a method of controlling insects, characterized in that it delivers an effective amount of the toxin according to the invention to the insect. According to a preferred embodiment, the insect is flutella xylostella. Preferably, the toxin is delivered orally to the insect.

Another aspect of the present invention provides a method for preparing a double stranded random polynucleotide comprising: (a) adding to the population of double stranded random fragments one or more single-chain or double-stranded oligonucleotides each comprising the same region and different regions for the double stranded template polynucleotide, and (b) ) An identity region where the resulting mixture of double-stranded random fragments and oligonucleotides is denatured into single-chain fragments, and (c) the resulting population of single-chain fragments, together with the polymerase, form a pair of annealed fragments, wherein The identity region is sufficient to incubate single member of the pair to initiate replication of the other member) under conditions that anneal single chain fragments to form mutated double stranded polynucleotides, and (d) the second and third steps are performed. Repeated at two or more additional cycles, the mixture obtained in the second stage of the further cycle is mutated from the third stage of the previous Mutations to the nucleic acid molecules according to the present invention to include cleaved double stranded polynucleotides, characterized in that additional cycles form additional mutagenic double stranded polynucleotides, resulting in cleavage into a population of double stranded random fragments of the desired size To provide a way to trigger.

Other aspects and advantages of the invention will be apparent to those skilled in the art from the description of the invention and from the study of non-limiting examples.

Justice

"Activity" of the toxin of the present invention means that the toxin acts as an orally active insect control agent and has a toxic effect or may disrupt or interfere with insect food intake, which will not kill or kill the insect. Can be. When the toxin of the present invention is delivered to an insect, the result is generally to kill the insect or to prevent the insect from eating a source that makes the toxin effective for the insect.

By "coupled / operably linked" is meant two nucleic acid sequences that are physically or functionally related. For example, a promoter or regulatory DNA sequence may be a DNA sequence that encodes an RNA or protein when the two sequences are operably linked or arranged so that the regulatory factor DNA sequence affects the expression level of the coding or structural DNA sequence. It is said to be "combined with".

A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked or combined with a nucleic acid sequence that encodes an mRNA or is expressed as a protein to regulate transcription or expression of the nucleic acid sequence to which the regulatory nucleic acid sequence is bound. . The regulatory nucleic acid sequence of the chimeric gene is not typically operatively linked to the originally identified linked nucleic acid sequence.

A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (eg, mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). Preferably, RNA is then translated in an organism to produce a protein.

"Controlling" an insect means, through a toxic effect, to prevent pests from surviving, growing, feeding and / or breeding, or limiting insect-related damage or loss in crop plants. "Controlling" an insect may or may not mean killing an insect, but preferably means killing an insect.

To "deliver" the toxin means that the toxin comes into contact with the insect, producing a toxic effect and controlling the insect. Toxins are recognized in many ways recognized, for example by ingestion by insects or in transformed plant expression, formulated protein composition (s), sprayable protein composition (s), bait matrix or other fields. It may be delivered orally by contact with an insect via a toxin delivery system.

As used herein, an “expression cassette” refers to a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, including a promoter operably linked with the nucleotide sequence of interest operably linked with the termination signal. . It also generally includes sequences necessary for proper translation of the nucleotide sequence. An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that one or more components thereof are heterologous to one or more other components thereof. Expression cassettes are also obtained in recombinant form, which may be natural but useful for heterologous expression. Generally, however, the expression cassette is heterologous to the host, ie the particular nucleic acid sequence of the expression cassette must not be naturally occurring in the host cell but must have been introduced by a transformation event into the host cell or into the ancestors of the host cell. The nucleotide sequence in the expression cassette can be expressed under the control of a constitutive promoter or inducible promoter that initiates transcription only when the host cell is exposed to a particular external stimulus. In the case of multicellular organisms (eg plants), the promoter may also be specific for a particular tissue or organ, or stage of development.

A "gene" is a defined region that is located within the genome and comprises, in addition to the coding nucleic acid sequences mentioned above, other primary regulatory nucleic acid sequences involved in the regulation of expression, ie, the transcription and translation of the coding portion. The gene may also include other 5 'and 3' non-toxin sequences and termination sequences. Another element that may be present is, for example, an intron.

A "gene of interest", when delivered to a plant, has desirable properties for the plant, such as antibiotic resistance, viral resistance, insect resistance, disease resistance or resistance to other pests, herbicide tolerance, improved nutritional value, industrial processes, or altered fertility. Genes confer improved performance. A "gene of interest" can also be one that is delivered to a plant to produce a commercially useful enzyme or metabolite.

A “heterologous” nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host cell into which it is introduced, and includes non-natural multiple copies of the native nucleic acid sequence.

A “homologous” nucleic acid sequence is a nucleic acid sequence naturally associated with the host cell into which it is introduced.

"Homologous recombination" is the interchange of nucleic acid fragments between homologous nucleic acid molecules.

"Pesticide" is preferably defined as a toxic biological activity capable of controlling insects by killing them.

The nucleic acid sequence is “isoencoded” with the 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.

A “isolated” nucleic acid molecule or an isolated enzyme is present by human hands, separated from its natural environment, and is therefore a nucleic acid molecule or enzyme that is not a natural product. Isolated nucleic acid molecules or enzymes may be present in purified form or may be present in a non-natural environment, eg, in a recombinant host cell.

A "nucleic acid molecule" or "nucleic acid sequence" is a linear segment of single-stranded or double-stranded DNA or RNA that can be isolated from a source. In the present invention, the nucleic acid molecule is preferably a segment of DNA. "ORF" means open reading frame.

"Plant" is a plant at all stages of development, in particular seed plants.

"Plant cells" are structural and physiological units of plants, including protoplasts and cell walls. Plant cells may be in the form of isolated single cells or cultured cells, or as part of higher tissue units, for example plant tissues, plant organs or whole plants.

"Plant cell culture" means a culture of plant units such as protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, embryos, knapsacks, conjugates and embryos at various stages of development.

"Plant material" refers to a leaf, stem, root, flower or part of a flower, fruit, pollen, egg cell, conjugate, seed, cutting, cell or tissue culture or other part or product of a plant.

A "plant organ" is a differentiated and visible part of a plant, for example roots, stems, leaves, buds or embryos.

As used herein, "plant tissue" refers to a group of plant cells organized into structural and functional units. Includes tissues of plants in plants or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and groups of plant cells organized into structural and / or functional units. The use of this term with or without the specific form of plant tissue described above or otherwise included by this definition is not intended to exclude other forms of plant tissue.

A "promoter" is a non-translated DNA sequence upstream of a coding region that contains a binding site for RNA polymerase II and initiates transcription of DNA. The promoter region also includes other elements that act as regulators of gene expression.

A "protoplasm" is a plant cell isolated in the absence of a cell wall or in the presence of only part of a cell wall.

A "regulatory element" refers to a sequence that is involved in regulating the expression of a nucleotide sequence. Regulatory elements include promoters and termination signals operably linked to the nucleotide sequence of interest. They also generally include the sequences necessary for proper translation of the nucleotide sequence.

In the broadest sense, as used herein for a nucleotide sequence, the term "substantially similar" refers to a nucleotide sequence that corresponds to a reference nucleotide sequence, wherein the corresponding sequence is, for example, related to polypeptide function. If only changes in amino acids that do not affect occur, they encode polypeptides having substantially the same structure and function as the polypeptides encoded by the reference nucleotide sequence. Preferably, substantially similar nucleotide sequences encode a polypeptide encoded by a reference nucleotide sequence. The percentage of identity between substantially similar nucleotide sequences and reference nucleotide sequences is preferably at least 80%, more preferably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably More than 99%. The nucleotide sequence "substantially similar" to the reference nucleotide sequence is more preferably washed when washed at 50 ° C. in 2 × SSC, 0.1% SDS in 50% in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA. Is more preferably 7% sodium dodecyl sulfate (SDS) when washed at 50 ° C. in 1 × SSC, 0.1% SDS at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ , 1 mM EDTA. When washing at 50 ° C. in 0.5 × SSC, 0.1% SDS at 50 ° C. in 0.5 M NaPO ₄ , 1 mM EDTA, preferably at 7 ° C. in sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 50 ° C. in 1 mM EDTA When washing at 50 ° C. in 0.1 × SSC, 0.1% SDS at more preferably 7% sodium dodecyl sulfate (SDS), 0.5 × NaM ₄ , 0.1 × SSC at 50 ° C., 65% in 0.1% SDS in 1 mM EDTA When washed at 캜, hybridization is made to the reference nucleotide sequence.

"Synthetic" refers to a nucleotide sequence that includes structural features not present in the native sequence. For example, artificial sequences similar to the G + C content and normal codon distribution of dicotyledonous and / or monocotyledonous genes are synthesized.

"Transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism. In particular, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest.

"Transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host or the nucleic acid molecule may also exist as an extrachromosomal molecule. Such extrachromosomal molecules can self replicate. Transformed cells, tissues or plants are understood to include the final product of the transformation process as well as their transformed progeny. A "non-transformed", "non-transgenic" or "non-recombinant" host is a bacterium that does not contain a wild type organism, eg, a heterologous nucleic acid molecule, or Means plants.

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 (Gln Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 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) and valine (Val; V ). In addition, (Xaa; X) represents all amino acids.

Brief description of the sequence in the sequence list

SEQ ID NO: 1 is the sequence of about 3.0 kb DNA fragment contained in the Genohabdus nematophilus clone pCIB9369, which comprises the following ORF at a specific nucleotide position.

Name Departure End

orf1 569 979

orf2 1045 2334

SEQ ID NO: 2 is the sequence of about 15kDa protein encoded by orf1 of clone pCIB9369.

SEQ ID NO: 3 is the sequence of about 47.7 kDa larval hormone esterase-like protein encoded by orf2 of clone pCIB9369.

SEQ ID NO: 4 is the DNA sequence of orf1 of genoahabdus nematophilus clone pCIB9381.

SEQ ID NO: 5 is the sequence of a protein encoded by orf1 of clone pCIB9381.

SEQ ID NO: 6 is the DNA sequence of orf2 of genorhavdus nematophyllus clone pCIB9381.

SEQ ID NO: 7 is the sequence of a larval hormone esterase-like protein encoded by orf2 of clone pCIB9381.

SEQ ID NO: 8 is the DNA sequence of orf1 of the Gennorhavdus poinary clone pCIB9354.

SEQ ID NO: 9 is the sequence of a protein encoded by orf1 of clone pCIB9354.

SEQ ID NO: 10 is the DNA sequence of orf2 of the Gennorhavdus poinary clone pCIB9354.

SEQ ID NO: 11 is the sequence of a larval hormone esterase-like protein encoded by orf2 of clone pCIB9354.

SEQ ID NO: 12 is the DNA sequence of orf1 of photorehabdus luminescence clone pCIB9383-21.

SEQ ID NO: 13 is the sequence of a protein encoded by orf1 of clone pCIB9383-21.

SEQ ID NO: 14 is the DNA sequence of orf2 of photorehabdus luminescence clone pCIB9383-21.

SEQ ID NO: 15 is the sequence of a larval hormone esterase-like protein encoded by orf2 of clone pCIB9383-21.

submission

The following materials were deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 Peoria North University Street 1815, Illinois, USA, under the terms of the Budapest Convention on the International Recognition of Microbial Deposits for the Patent Process. All restrictions on the availability of deposited materials are removed from modification upon granting the patent.

Clone Access Number Deposit Date

pCIB9369 NRRL B-21883 November 12, 1997

pCIB9354 NRRL B-30109 February 25, 1999

pCIB9381 NRRL B-30110 February 25, 1999

pCIB9383-21 NRRL B-30111 February 25, 1999

New Nucleic Acid Sequences Expressed to Produce Insecticidal Toxins

The present invention relates to nucleic acid sequences that are expressed to produce novel toxins, and to the production and use of the toxins to control pests. Nucleic acid sequences are isolated from Genohabdus nematophilus, Genohabdus poinari and Photorhabdus luminescence, members of the Enterobacteriaceae family. Gennorhabdus is a symbiotic bacterium of nematodes in Steinernema. Photorehabdus is a symbiotic bacteria of the nematode of the genus Heterorhabditis. Nematodes colonize insect larvae, kill them, and their offspring feed on dead larvae. Insecticidal activity is substantially produced by the symbiotic Zenorhavdus and Portorhavdus bacteria. We first separate nucleic acid sequences of the invention. Expression of the nucleic acid sequences of the present invention produces toxins that can be used to control Lepidopteran insects such as flutella xylostella (cabbage moth).

In clone pCIB9369 the nucleotide sequence of the invention is defined by a about 3.0 kb DNA fragment deposited under accession number NRRL B-21883 according to the Budapest Treaty for Patent Deposit. The sequence of such DNA fragments is set forth in SEQ ID NO: 1. Two open reading frames (ORFs) are present in SEQ ID NO: 1 (nucleotides 569-979 and nucleotides 1045-2334, respectively) encoding proteins of predicted sizes 15kDa and 47.7kDa (SEQ ID NOS: 2 and 3, respectively). The two ORFs are arranged in operon-like structures. Studies on known sequences that show homology to each individual ORF using the UWGCG Blast and Gap programs do not confirm significant suitability for ORF # 1, and ORF # 2 and Bacillus surringensis cry3A protein in the art A 21% identity was identified which was not significantly considered between. Gap analysis of the protein encoded by ORF # 2 of pCIB9369 by the Blast program confirmed 30.6% AA identity and 44.1% AA similarity with the larval hormone esterase-related protein [GenBank Accession No. 2921553; Henikoff et al., PNAS USA 89: 10915-10919 (1992). The nucleotide sequence of the present invention is also compared with the known Gennorhabdus nematophilus sequence encoding the insecticidal toxin toxb4 [WO 95/00647], but no significant homology is revealed. The 3.0 kb DNA fragment is also compared to the nucleotide sequence disclosed in WO 98/08388. Twenty-two sequences of 60 nucleotides (60mer) each from the 38.2kb DNA fragment described in WO 98/08388 are compared to the 3.0kb DNA fragment of the present invention using the UWGCG Gap program. The nucleotide sequence of the first 60mer starts at base 1 of the 38.2 kb DNA fragment and the other 60mers are located about 2.0 kb apart. Each of the 22 sequences as well as their complementary sequences are tested. The highest percent identity between the 3.0 kb DNA fragment of the invention and one of these 60mers is 53%, which is not significant homology. Five different DNA fragments of 38.2 kb sequence are also tested by Southern blot analysis for hybridization to the 3.0 kb fragment of the present invention. None of these show a positive hybridization signal.

The nucleotide sequences of pCIB9381, pCIB9354 and pCIB9383-21 also show two open reading frames in each of these clones. The nucleotide sequence of the two ORFs at each of pCIB9381 and pCIB9383-21 is very homologous to that at pCIB9369. Thus, the ORF # 2 proteins of pCIB9381 and pCIB9383-21 have essentially the same homology to the larvae 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 with the larval hormone esterase-related protein (29.2% AA identity and 42.2% AA similarity).

In a preferred embodiment, the invention comprises nucleotide sequences substantially similar to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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, It is expressed to produce insecticidal toxins. The invention also encompasses recombinant vectors comprising the nucleic acid sequences of the invention. In such vectors, the nucleic acid sequence is preferably contained in an expression cassette comprising regulatory elements for expressing the nucleotide sequence in a host cell capable of expressing the nucleotide sequence. Such regulatory elements generally include a promoter and a termination signal, and preferably also include elements that effectively translate a polypeptide encoded by a nucleic acid sequence of the invention. Vectors comprising nucleic acid sequences are generally used to amplify nucleic acid sequences of the invention in such host cells as they can be replicated in particular host cells, preferably as extrachromosomal molecules. In one embodiment, the host cells for such vectors are microorganisms such as bacteria, in particular E. coli. Coli. In other embodiments, the host cell for such a recombinant vector is endogenous or engraftment. Preferred host cells for such vectors are eukaryotic cells such as yeast, plant cells or insect cells. Plant cells (eg corn cells) are the most preferred host cells. In another preferred embodiment, such vectors are viral vectors and are used for the replication of nucleotide sequences in certain host cells, eg insect cells or plant cells. Recombinant vectors are also used to transform the nucleotide sequences of the invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells. In one embodiment, such host cell is a prokaryotic cell. In a preferred embodiment, such host cells are eukaryotic cells, for example yeast cells, insect cells or plant cells. In the most preferred embodiment, the host cell is a plant cell, eg corn cell.

Nucleotide sequences of the present invention can be isolated using the techniques disclosed in the following examples, or by PCR using the sequences described in the sequences listed as criteria for constructing PCR primers. For example, an oligonucleotide having a sequence of approximately first and last 20-25 consecutive nucleotides of orf1 of SEQ ID NO: 1 (eg, nucleotides 569-588 and 957-976 of SEQ ID NO: 1) may be supplied to a feed strain (Genorhavdus nema). It can be used as a PCR primer to amplify the orf1 coding sequence (nucleotides 569-976 of SEQ ID NO: 1) directly from Topophilus strain ATCC 19061. Other gene sequences of the invention can likewise be amplified by PCR using the ends of the coding sequences described in the sequences listed as reference for PCR primers from each feed strain.

In another preferred embodiment, the pesticidal toxin comprises one or more polypeptides encoded by the nucleotide sequence of the present invention. The molecular weight of the pesticidal toxin according to the present invention exceeds 6,000, as measured by size classification experiments. After treatment with protease K, only a minimal reduction in pesticidal activity is observed in insect biopsies, indicating that the insecticidal toxin is substantially resistant to protease K treatment. Insecticidal toxins retain pesticidal activity after storage at 22 ° C. or 4 ° C. for 2 weeks. They also maintain pesticidal activity after lyophilization and storage at 22 ° C. for 2 weeks. Insecticidal toxins are still active after incubation at 60 ° C. for 5 minutes, but they lose pesticidal activity after incubation at 100 ° C. or 80 ° C. for 5 minutes for 5 minutes.

In a further aspect, the nucleotide sequences of the invention can be modified by incorporating any mutation by techniques known as in vitro recombination or DNA replacement. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and US Pat. No. 5,605,793, which is incorporated herein by reference. Millions of mutant copies of nucleotide sequences are based on the original nucleotide sequences of the present invention and variants with improved properties are recovered, for example, increased pesticidal activity, enhanced stability, or a range of different specificities or target pests. . Such methods include forming a mutated double stranded polynucleotide from a templated double stranded polynucleotide comprising a nucleotide sequence of the present invention, wherein the templated double stranded polynucleotide is separated into double stranded random fragments of the desired size. Adding to the obtained population of double stranded random fragments one or more single-chain or double-stranded oligonucleotides each comprising the same region and different regions for the double stranded template nucleotides; Denaturing the obtained mixture of double stranded random fragments and oligonucleotides with single chain fragments; Single chain fragments in the identity region, wherein the resulting population of single chain fragments, together with the polymerase, form a pair of annealed fragments, wherein the identity region is sufficient for one member of the pair to initiate replication of the other member. Culturing under conditions of annealing to form mutated double-stranded polynucleotides; Repeating the second and third steps with two or more additional cycles, such that the mixture obtained in the second stage of the further cycle comprises a double-stranded polynucleotide mutagenesis from the third stage of the previous cycle, with the additional cycle further mutations To form the induced double-stranded polynucleotides. In a preferred embodiment, the concentration of a single species of double stranded random fragment in the population of double stranded random fragments is less than 1% of the total DNA weight. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 polynucleotides. In another preferred embodiment, the double stranded random fragment is about 5 bp to 5 kb in size. In a further preferred embodiment, the fourth step of the method is characterized by repeating the second and third steps at least ten times.

Expression of Nucleotide Sequences in Heterologous Microbial Hosts

As biological insect control agents, insecticidal toxins are produced by the expression of nucleotide sequences in heterologous host cells capable of expressing nucleotide sequences. In a first aspect, a Genorhavdus nematophilus, Genorhavdus poinary or photorhavdus luminescence cell is described which comprises a modification of at least one nucleotide sequence of the invention at its chromosomal location. Such modifications include mutations or deletions of the regulatory elements present, thus leading to altered expression of nucleotide sequences, or incorporation of novel regulatory elements that regulate the expression of nucleotide sequences. In another embodiment, an additional copy of one or more nucleotide sequences is inserted into a chromosome into a Genoahabdus nematophyllus, Genoahabdus poinary or photoheravdus luminescence or by extrachromosomal replication containing a nucleotide sequence. It adds by introduction of a molecule.

In other embodiments, one or more nucleotide sequences of the present invention are inserted into a suitable expression cassette comprising a promoter and a termination signal. Inducible promoters are used in which the expression of the nucleotide sequence is constitutive or in response to various types of stimuli that initiate transcription. In a preferred embodiment, the cell in which the toxin is expressed is a microorganism such as a virus, bacterium or fungus. In a preferred embodiment, the virus, eg, baculovirus, contains the nucleotide sequence of the present invention in its genome and contains a large amount of the corresponding pesticidal toxin after infection of a suitable eukaryotic cell suitable for viral replication and expression of the nucleotide sequence. Expression. The pesticidal toxin thus produced is used as an insecticide. Alternatively, baculoviruses engineered to include nucleotide sequences are used to infect insects in vivo and kill them by expression of pesticidal toxins or by a combination of viral infections and expression of insecticidal toxins.

Bacterial cells are also hosts for the expression of the nucleotide sequences of the invention. In a preferred embodiment, so-called endogenous non-pathogenic symbiotic bacteria, which can survive and replicate in plant tissues, or so-called ectopic non-pathogenic symbiotic bacteria, which can colonize the pilosphere or soil layer, are used. These bacteria are Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia (Erwinia), Flavoacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas Contains bacteria Symbiotic fungi, such as Trichoderma and Gliocladium, are also possible hosts for expressing the nucleotide sequences of the present invention for the same purpose.

These genetic engineering techniques are specific to differently available hosts and are known in the art. For example, using the expression vectors pKK223-3 and pKK223-2, E. coli. Heterologous genes in E. coli can be expressed as transcriptional or translational fusions behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest method is to insert the operons into a transcriptional fusion into a vector such as pKK223-3, which allows homologous ribosomal binding sites of heterologous genes to be used. Techniques for overexpression in Gram-positive species (eg Bacillus) are also known in the art and can be used in the present invention. See Quax et al. In: Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993). Alternative systems for overexpression, for example, depend on yeast vectors and include the use of Pichia, Saccharomyces and Kluyveromyces. Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud eds. American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12: 173-177 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990).

In another preferred embodiment, one or more of the described nucleotide sequences is transferred to Pseudomonas fluorescens strain CGA267356 having bioregulatory properties (as described in published applications EU 0 472 494 and WO 94/01561). Is expressed. In another preferred embodiment, the nucleotide sequences of the present invention are also delivered to Pseudomonas aureofaciens strains 30-84 with bioregulatory properties. Expression in heterologous bioregulatory strains requires the selection of a vector suitable for replication in the selected host and a suitable selection of promoter. Techniques for expression in gram negative and gram positive bacteria and fungi are well known in the art.

Expression of Nucleotide Sequences in Plant Tissues

In a particularly preferred embodiment, one or more pesticidal toxins of the invention are expressed in higher organisms, for example plants. In this case, the transformed plant expressing an effective amount of toxins protects itself from pests. When insects begin to feed these transformed plants, they also consume expressed toxins. This may prevent the insect from further biting into plant tissue, or may even harm or kill the insect. The nucleotide sequence of the invention is inserted into an expression cassette, which is then preferably integrated stably into the genome of the plant. In another preferred embodiment, the nucleotide sequence is included in a non-pathogenic self-replicating virus. Plants transformed according to the invention may be monocots or dicotyledons, corn, wheat, barley, rye, sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, radishes, spinach, asparagus , Onion, garlic, pepper, celery, pumpkin, zucchini, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry , Blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugar cane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, arabidopsis (Arabidopsis) and woody plants (eg, conifers and deciduous trees).

Once a preferred nucleotide sequence is transformed into a particular plant species, it can be propagated within this species or transferred to other variants of the same species, particularly including commercial varieties, using traditional breeding techniques.

The nucleotide sequence of the invention is preferably expressed in the transformed plant, thus biosynthesizing the corresponding toxin in the transformed plant. In this way, transformed plants with enhanced resistance to insects are produced. For its expression in transformed plants, the nucleotide sequences of the present invention may require alteration and optimization. In many cases genes from microbial organisms can be expressed at high levels without alteration in plants, but low expression in transformed plants can result from microbial nucleotide sequences with codons that are undesirable for plants. It is known in the art that all organisms specifically prefer codon use, and the codons of the nucleotide sequences described herein can be adapted to suit plant preferences, while maintaining the encoded amino acids. In addition, high expression in plants is best achieved from coding sequences having a GC content of at least about 35%, preferably at least about 45%, more preferably at least about 50%, most preferably at least about 60%. Microbial nucleotide sequences with low GC content can be poorly expressed in plants due to the presence of ATTTA motifs that can destabilize the message, and AATAAA motifs that can cause inadequate polyadenylation. Preferred gene sequences can be suitably expressed in both monocotyledonous and dicotyledonous species, but as the preferences differ, the sequence can be altered to cause specific codon preferences and GC content preferences of monocotyledonous or dicotyledonous plants. See Murray et al., Nucl. Acids Res. 17: 477-498 (1989). In addition, nucleotide sequences are screened for the presence of inappropriate junction sites that can cause message cleavage. All changes that are required to be carried out in the nucleotide sequence as described above are site directed mutagenesis, PCR, and published patent applications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol). And well known techniques of synthetic gene construction using the methods described in WO 93/07278 (Ciba-Geigy).

In order to effectively initiate translation, sequences adjacent to the starting methionine may require alteration. For example, they can be altered to include sequences known to be effective in the plant. Josh proposed a consensus suitable for plants (NAR 15: 6643-6653 (1987)), and Clontech proposed an additional consensus initiation factor (1993/1994 catalog, page 210). ). These consensus are suitable for use with the nucleotide sequences of the invention. The sequence comprises a nucleotide sequence up to and including ATG (without modifying the second amino acid), or up to GTC subsequent to ATG and with the possibility (with the possibility of modifying the second amino acid of the transformed gene). Incorporated into the construct.

Expression of the nucleotide sequence in the transformed plant is driven by a promoter that has been shown to be functional in the plant. The choice of promoter varies depending on the time and space requirements for expression and the target species. Thus, the expression of the nucleotide sequences of the present invention in leaves, ear, flowers (eg, inflorescences, cones, corn cobs, etc.), roots and / or seeds is preferred. In many cases, however, protection against one or more pests is sought, so expression is desirable in many tissues. Many promoters from dicotyledons have been shown to be effective in monocots, on the contrary ideally, dicotyledonous promoters are selected for expression in dicotyledonous plants and monocotyledonous promoters are selected for expression in monocotyledonous plants. However, there is no restriction on the origin of the selected promoter; To the extent they are effective in driving the expression of nucleotide sequences in preferred cells.

Preferred promoters expressed constitutively include promoters from genes encoding actin or ubiquitin and CaMV 35S and 19S promoters. Nucleotide sequences of the invention can also be expressed under the control of a chemically regulated promoter. Insecticidal toxins can thereby be synthesized only when the crop plants are treated with inducible chemicals. Preferred techniques for chemical induction of gene expression are described in detail in published application EP 0 332 104 (Ciba-Geigy) and US Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

A preferred category of promoter is one that is wound inducible. Many promoters are described that are expressed at the site of injury and at the site of plant pathogenic infection. Ideally, these promoters are only locally active at the site of infection, in which pesticidal toxins only accumulate in cells that need to synthesize pesticidal toxins that kill the invading pests. Preferred promoters of this kind include those described in the literature. 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 Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201 (1993).

Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific and flower specific. Promoters suitable for expression in green tissues include many that regulate genes involved in photosynthesis, many of which have been cloned from both monocotyledonous and dicotyledonous plants. Preferred promoters are corn PEPC promoters from the phosphoenol carboxylase gene. Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989). Preferred promoters for root specific expression are those described by de Framond. FEBS 290: 103-103 (1991); EP 0 452 269 to Ciba-Geigy. Preferred stem specific promoters are those described in US Pat. No. 5,625,136 (Ciba-Geigy), which drives the expression of maize trpA gene.

Particularly preferred embodiments of the invention are transformed plants which express one or more nucleotide sequences of the invention in a root-preferred or root-specific manner. A further preferred embodiment is a transformed plant which expresses the nucleotide sequence in a wound-induced or pathogen infection-induced manner.

In addition to the selection of a suitable promoter, constructs for the expression of pesticidal toxins in plants require that a suitable transcription terminator be attached downstream of the heterologous nucleotide sequence. Many such terminators are available and are known in the art (eg tm1 from CaMV, E9 from rbcS). Useful terminators known to function in plants can be used in the present invention.

Many other sequences can be incorporated into the expression cassettes described herein. These include sequences shown to enhance expression, such as intron sequences (eg from Adh1 and bronze1) and viral leader sequences (eg from TMV, MCMV and AMV).

This may be desirable for targeted expression of the nucleotide sequences of the invention on different cell loci in plants. In certain cases, positioning in the cytoplasm is preferred, while in other cases, positioning in certain intracellular organs may be desirable. Intracellular positioning of the transformed gene encoded enzyme is performed using techniques well known in the art. In general, DNA encoding a target peptide from known organelle-target gene products is engineered and fused upstream of the nucleotide sequence. Many such target sequences are known for chloroplasts and their function is shown in heterologous constructs. Expression of the nucleotide sequences of the invention is also targeted to the endoplasmic reticulum or the fear of the host cell. Techniques for doing this are well known in the art.

Suitable vectors for plant transformation are described elsewhere herein. For Agrobacterium-mediated transformation, a binary vector or a vector carrying one or more T-DNA border sequences is suitable, while for direct gene transfer any linear vector containing any construct of interest and containing only the construct of interest May be preferred. In the case of direct gene transfer, transformation or co-transformation using one DNA species can be used. Schocher et al. Biotechnology 4: 1093-1096 (1986)]. For direct gene transfer and Agrobacterium-mediated delivery, transformations are generally (but not necessarily) selective markers that can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (vesta). Is performed. Examples of such labels include neomycin phosphotransferases, hygromycin phosphotransferases, dihydrofolate reductases, pocifinotricin acetyltransferases, 2,2-dichloroproprioric acid dehalogenases, acetohydrides Oxyacid synthase, 5-enolpyrubil-succimate-phosphate synthase, haloarylnitrilease, protophorhyrigen oxidase, acetyl-coenzyme A carboxylase, dihydrophteroate synthase, chloramphenicol acetyl transfer Lase and β-glucuronidase. However, the selection of selective or selective markers for plant transformation is not critical to the present invention.

The recombinant DNA described above can be introduced into plant cells in a number of ways known in the art. One skilled in the art understands that the choice of method depends on the type of plant targeted for transformation. Suitable methods for transforming plant cells include microinjection (Crossway et al., BioTechniques 4: 320-334 (1986)); Electroporation [Riggs et al., Proc. Natl. Acad. Sci. USA 83: 5602-5606 (1986)], Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6: 915-921 (1984)); See also: Corn Transgenics (Ishda et al., Nature Biotechnology 14: 745-750 (June 1996)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984); Hayashimoto et al., Plant Physiol. 93: 857-863 (1990) (rice), and Agracetus, Inc., Madison, Wisconsin. And impact particle acceleration using a device available from Dupont, Inc. of Wilmington, Delaware, USA (Sanford et al., US Pat. No. 4,945,050 and McCabe et al., Biotechnology 6: 923-926). 1988). See also: Weissinger et al., Annual Rev. Genet. 22: 421-477 (1988); Sanford et al., Particulate Science and Technology 5: 27-37 (1987) (onion); Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplasts); Christou et al., Plant Physiol. 87: 671-674 (1988) (soybeans); McCabe et al., Bio / Technology 6: 923-926 (1988) (soy); Klein et al., Proc. Natl. Acad. Sci. USA, 84: 4305-4309 (1988) (corn); Klein et al., Bio / Technology 6: 559-563 (1988) (corn); Klein et al., Plant Physiol. 91: 440-444 (1988) (corn); Fromm et al., Bio / Technology 8: 833-839 (1990) (corn); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) (corn); Koziel et a., Biotechnology 11: 194-200 (1993) (corn); Shimamoto et al., Nature 338: 274-277 (1989) (rice); Christou et al., Biotechnology 9: 957-962 (1991) (rice); Datta et al., Bio / Technology 8: 736-740 (1990) (rice); European Patent Application EP 0 332 581 (Buff and other Pooideae); Vasil et al., Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al., Plant Physiol, 102: 1077-1084 (1993) (wheat); Wan et al., Plant Physiol. 104: 37-48 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89: 525-533 (1994) (barley); Umbeck et al., Bio / Technology 5: 263-266 (1987) (cotton); Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216 (Dec. 1993) (sorghum); Somers et al., Bio / Technology 10: 1589-1594 (1992, Dec) (oats); Torbert., Plant Cell Reports 14: 635-640 (1995) (oats); Weeks et al., Plant Physiol. 102: 1077-1084 (1993) (wheat); Chang et al., WO 94/13822 (wheat); And Nehra et al., The Plant Journal 5: 285-297 (1994) (wheat)]. A particularly preferred set of embodiments may be found in the literature for introducing recombinant DNA molecules into corn by microprojectile bombardment. 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). A further preferred embodiment is the protoplast transformation described in EP 0 292 435. Plant transformation can be performed with a single DNA species or multiple DNA species (ie, co-transformation), both of which are suitable for use as peroxidase coding sequences.

In another preferred embodiment, the nucleotide sequences of the invention are directly transformed into the chromatin genome. The main advantage of chromatin transformation is that the plastid can generally express bacterial genes without substantial alteration and that the plastid can express multiple open reading frames under the control of a single promoter. Chromosome transformation techniques are described in US Pat. Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT application WO 95/16783 and in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique of chloroplast transformation involves the region of cloned pigmented DNA flanked by a selective label with a gene of interest to a suitable target tissue, e.g., bioballistic or protoplast transformation (e.g. calcium chloride or PEG mediated transformation). It includes using. Adjacent regions of 1 to 1.5 kb, called target sequences, promote homologous recombination with the pigment genome, thus replacing or altering specific regions of the genetic material in the pigment body. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes that confer resistance to spectinomycin and / or streptomycin are used as selective markers for transformation. Svab, Z., Hajduklewicz P., and Maliga , P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M. and Maliga, P. (1992) Plant Cell 4, 39-45]. This produces a stable allogeneic transformant with a frequency of about once per 100 impacts of the target leaf. The presence of a cloning site between these labels forms a chromosomal target vector for introduction of a foreign gene (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in the frequency of transformation are obtained by replacing the recessive rRNA or r-protein antibiotic resistance gene with the bacterial aadA gene encoding the dominant selectable label, spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase [ See Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917. Previously, this label has been used successfully for high frequency transformation of the chromatin genome of the green alga Chlamydomonas reinhardtii. Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089. Other selective markers useful for chromatin transformation are known in the art and are included within the scope of the present invention. In general, about 15-20 cell division cycles are required to reach homochromosomal states after transformation. Chromosome expression, in which genes are inserted by homologous recombination in all copies of the thousands of template chromatin genomes present in each plant cell, has the advantage of enormous copy number over nuclear-expressed genes, resulting in 10% of the total soluble plant protein. Allow expression levels that can easily be exceeded. In a preferred embodiment, the nucleotide sequence of the present invention is inserted into a pigment target vector and transformed into the pigment genome of a preferred plant host. An allogeneic plant is obtained in the chromatin genome containing the nucleotide sequence of the present invention, and the nucleotide sequence can be highly expressed.

Formulation of Insecticide Composition

The invention also includes compositions comprising one or more pesticidal toxins of the invention. In order to effectively control pests, such compositions preferably contain a sufficient amount of toxins. This amount will vary depending on the crops to be protected, the specific pests that are targeted and environmental conditions (eg soil humidity, temperature or type). In a preferred embodiment, the composition comprising the pesticidal toxin comprises a host cell expressing the toxin without further purification. In another preferred embodiment, cells expressing insecticidal toxin are lyophilized before use as insecticide. In another embodiment, the pesticidal toxin is engineered to be secreted from the host cell. If purification of the toxin from the host cell in which the toxin is expressed is desired, several degrees of purification of the insecticidal toxin are performed.

The invention also includes the preparation of a composition comprising one or more pesticidal toxins of the invention, which is homogeneously mixed with one or more compounds or groups of compounds described herein. The present invention also relates to a method for treating a plant, characterized by applying the insecticidal toxin or a composition containing the insecticidal toxin to the plant. Insecticidal toxins may be applied in the form of a composition or plant to be treated in the crop area, using additional compounds simultaneously or sequentially. These compounds may be fertilizers or micronutrient donors or other agents that affect plant growth. They may also, if desired, of selective herbicides, insecticides, fungicides, fungicides, nematicides, chelators or species of these agents together with additional carriers, surfactants or application promoting aids commonly used in the field of formulations. It may be a mixture. Suitable carriers and auxiliaries are solid or liquid and may correspond to materials commonly used in formulation techniques such as natural or regenerated inorganic materials, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

A preferred method of applying the pesticidal toxin of the present invention is to spray the environment where pests live, such as soil, water or leaves of plants. The number of applications and the rate of application depends on the type and intensity affected by the pest. Insecticidal toxins can also penetrate the plant through the roots through the soil by impregnating parts of the plant with the liquid composition or by applying the compound to the soil in solid form, for example in the form of a soil (soil application) ( Systemic action). Pesticide toxins can also be applied to seeds by impregnating the seeds with liquid formulations containing pesticidal toxins or by applying them in solid formulations (applications). In certain cases, additional types of application are also possible, for example selective treatment of plant stems or shoots. Insecticidal toxins may also be provided as bait located above or below the ground.

Insecticidal toxins are used in unmodified form or preferably together with adjuvants commonly used in the formulation art, and therefore are known methods by means of emulsifying thickeners, coating pastes, direct spray or diluent solutions, diluent emulsions, Formulated as wettable powders, soluble powders, dispersants, granules, and inclusions in polymeric materials. As with the properties of the composition, the method of application, for example spraying, misting, dispersing, spraying or injecting, is selected according to the intended subject and the prevailing environment.

Formulations, compositions or preparations containing pesticidal toxins and, if necessary, solid or liquid auxiliaries are known methods, for example pesticidal toxins and extenders, for example solvents, solid carriers and, if necessary, surfactant compounds. Prepared by homogeneously mixing and / or grinding the (surfactant).

Suitable solvents include aromatic hydrocarbons, preferably fractions of 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 paraffin ), Alcohols and glycols and ethers and esters thereof, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strong 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.

For example, solid carriers used in dispersants and dispersible powders are generally natural inorganic fillers such as calsite, talc, kaolin, montmorillonite or attapulgite. In order to improve the physical properties, highly disperse silicic acid or highly dispersible absorbent polymers can be added. Suitable granular adsorptive carriers are in porous form, for example pumice, broken brick, sepiolite or bentonite; Suitable nonabsorbent carriers are materials such as calsite or sand. In addition, many pregranulated materials of inorganic or organic nature can be used, for example dolomite or ground plant residues.

Suitable surfactant compounds are nonionic, cationic and / or anionic surfactants with good emulsifying, dispersing and wetting properties. The term "surfactant" is also understood to include mixtures of surfactants. Suitable anionic surfactants can be water soluble soaps and water soluble synthetic surfactant compounds.

Suitable soaps are alkali metal salts, alkaline earth metal salts of higher fatty acids (chains of 10 to 22 carbon atoms) or unsubstituted or substituted ammonium salts, for example sodium or potassium salts of oleic acid or stearic acid, or for example coconut oil or Of natural fatty acid mixtures obtainable from resin oils. Fatty acid methyltaurine salts may also be used.

More frequently, however, so-called synthetic surfactants are used, in particular fatty acid sulfonates, fatty acid sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.

Fatty acid sulfonates or sulfates are generally present in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have alkyl radicals of 8 to 22 carbon atoms which also include alkyl moieties of alkyl radicals, for example, Sodium or calcium salt of lignonsulfonic acid, fatty acid alcohol sulfates obtained from dodecyl sulfate or from natural fatty acids. These compounds also include salts of sulfuric acid esters and sulfonic acids of fatty alcohol / ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain two sulfonic acid groups and one fatty acid radical having 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or naphthalenesulfonic acid / formaldehyde condensation products. Also salts of the corresponding phosphates, for example the phosphate esters of p-nonylphenol and adducts of ethylene oxide 4 to 14 mole.

Nonionic surfactants are preferably aliphatic or cycloaliphatic alcohols, or polyglycol ether derivatives of saturated or unsaturated fatty acids and alkylphenols, which derivatives contain 3 to 30 glycol ether groups and 8 to 20 glycols at the (aliphatic) hydrocarbon moiety. Carbon atoms and alkyl residues of alkylphenols contain 6 to 18 carbon atoms.

Further suitable nonionic surfactants are water soluble adducts of polyethylene oxide and polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycols containing 1 to 10 carbon atoms in the alkyl chain, with adducts of 20 to 250 ethylene It contains a glycol ether group and 10 to 100 propylene glycol ether groups. These compounds generally contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of nonionic surfactants are nonylphenolpolyethoxyethanol, castor oil polyglycol ether, polypropylene / polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable nonionic surfactants.

The cationic surfactant is preferably a quaternary ammonium salt having at least one C8-22 alkyl radical as N-substituent and having as lower substituents unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salt is preferably in the form of a halide, methylsulfate or ethylsulfate, for example stearyltrimethylammonium chloride or benzyldi (2-chloroethyl) ethylammonium bromide.

Surfactants commonly used in the formulation art are described, for example, in the literature. See, "McCutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents", Chemical Publishing Co., Inc. New York, 1980].

The invention is further described with reference to the following detailed examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless stated otherwise. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and described in the literature. See Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994): T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989); and by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984)].

A. Isolation of Nucleotide Sequences Expressed to Produce Toxins Active Against Species Insects

Example 1

Growth of Genoa Havdus and Fotor Havdous Strains

For insect bioassay, the following strains are grown in growth medium recommended by ATCC for 3 days at 25 ° C. in nutrient broth. To isolate DNA, the cultures are grown under the same conditions for 24 hours.

Genoahabdus nematophilus strain ATCC 19601

Genoahabdus nematophilus strain Ps1, USDA isolate

Genoahabdus poinary strain ATCC 49122

Photorehabdus luminescence strain Ps5, USDA isolate

Example 2

Insect bioassay

Flutella xylostella (Px) bioassay was performed in 50 μl of each E. coli. E. coli cultures are performed by aliquoting the solid artificial P. xylostella diets. Biever and Boldt, Annals of Entomological Society of America, 1971; Shelton, et al. J. Ent. Sci 26:17]. 4 ml of diet is poured into 1 ounce of clear plastic cup (Bioserve product # 9051). Five newborn P. xylostellas from a dietary laboratory colony are placed in a cup containing each diet and then covered with a white paper cover (Bioserve product # 9049). Ten larvae are assayed for concentrate. Trays of cups are placed in a lighter-dark cycle of 14:10 (hours) at 72 ° F. for 3 days in the incubator. Record the number of live larvae in each cup.

Example 3

Result of bioassay

Juicy from Genohabdus nematophilus strain ATCC 19601 provides 100% mortality against flutella xylostella (Px) in the insect bioassay of Example 2. The juices of each of Genohabdus nematophyllus strain Ps1, Genohabdus poinary strain ATCC 49122 and photorhavdus luminescence strain Ps5 were likewise fluteella xylostella (Px) in the insect bioassay of Example 2 Provides a 100% mortality rate for.

Example 4

Organization of the Cosmid Library

Total DNA is isolated from Gennorhabdus nematophilus strain ATCC 19061 by treatment of just grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg / ml lysozyme at 37 ° C. for 30 minutes. Protease K is added at a final concentration of 100 μg / ml in 0.5% SDS and incubated at 45 ° C. The solution becomes clear and very viscous. SDS concentration is increased to 1% and 300 mM NaCl and the same volume of phenol-chloroform-isoamyl alcohol are added. Samples are mixed gently for 5 minutes and centrifuged at 3K. Repeat this twice. The aqueous phase is then mixed with 0.7 volume of isopropanol and centrifuged. The DNA pellet is washed three times with 70% ethanol and gently resuspended in 0.5X TE. 6 μg of DNA is treated with 0.3 units of Sau3A / μg DNA in a volume of 100 μl at 37 ° C. for 3.5 minutes. The sample is then heated at 65 ° C. for 30 minutes to inactivate the enzyme and then incubated at 37 ° C. for 30 minutes with 2 units of bovine intestinal alkaline phosphatase. Samples are mixed with the same volume of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed, mixed with 0.7 volume of isopropanol and centrifuged. The pellet is resuspended in 0.5 × TE at a concentration of 100 ng / ml.

SuperCos cosmid vectors (Stratagene, La Jolla, Canada) are prepared as described by the supplier using the BamHI cloning site. SuperCos prepared at 100 ng / ml are ligated with X. nematophilus DNA previously cleaved with Sau3A at 6 ° C. overnight at a volume of 5 μl at a 2: 1 ratio. The ligation mixture is filled using Gigapack XL III (Stratagene) as described by the supplier. Charged phage is XL-1MR E. coli as described by the supplier. Infect with E. coli cells (Stratagene). Cosmid libraries are plated with 50 μg / ml kanamycin on L-agar and incubated at 37 ° C. for 16 hours. 500 colonies are spotted on a fresh L-kan plate at a density of 50 / plate. The cells are washed off with L broth, mixed with 20% glycerol and frozen at -80 ° C.

X. For a large genome of 4.2Mb of Nematophilus, 450 clones with an average size of 40 kb correspond to four times the coverage of the genome. Thus, selecting 450 clones gives a 99% chance of gene discovery.

Cosmid libraries are constructed in a similar manner from Gennorhabdus nematophyllus strain Ps1, Gennorhabdus poinary strain ATCC 49122, and photorhabdus luminescence strain Ps5.

Example 5

Cosmid bioassay and identification of clones with insecticidal activity

400 E. coli from each cosmid library. E. coli clones are screened by insect bioassay to produce clones with activity against flutella xylostella. Insecticidal cosmid clones are identified as pCIB9362 from Gennorhabdus nematophilus strain ATCC 19061. A 42 kb insecticidal cosmid clone from Genoahabdus nematophilus strain Ps1 was identified as pCIB9379. A 42 kb insecticidal cosmid clone from Gennorhabdus poinary strain ATCC 49122 was identified as pCIB9354. A 7 kb insecticidal cosmid clone from Photorehabdus luminescence strain Ps5 was identified as pCIB9383-21.

Example 6

Isolation of Subclones with Insecticidal Activity

Clone pCIB9362 is lysed with SaclI and 9 kb DNA fragments are isolated. This fragment is linked to a Bluescript digest using SacII. The linking mixture was transferred to DH5α E. coli as described in molecular cloning second edition (Sambrook et al.). Transform into E. coli cells. Transformation mixture is plated on L agar + 100 μg / ml ampicillin and incubated at 37 ° C. overnight. The isolated colonies are grown with 100 μg / ml ampicillin in L broth and the plasmid DNA is isolated by alkaline miniprep as described in the molecular cloning second edition. The 9 kb SacII clone was identified as pCIB9362-3 and provided 100% mortality when bioassayed against flutella xylostella. 3 μg of pCIB9362-3 is isolated, digested with 0.3 units of Sau3A / μg DNA at 37 ° C. for 4, 6 and 8 minutes and heated at 75 ° C. for 15 minutes. Samples are pooled, ligated into pUC19 previously cleaved with BamHI and treated with bovine intestinal alkaline phosphatase. The linkage is DH5α. E. coli cells are transformed and plated with Xgal / Amp on L agar as described in molecular cloning and grown at 37 ° C. overnight. White colonies are taken out and grown in L broth with 100 μg / ml ampicillin and plasmid DNA is isolated as described previously. DNA is lysed with EcoRI / HindIII and new restriction patterns are sequenced. Sequencing primers are ordered from Genosys Biotechnologies (Woodland, Texas, USA). Sequencing was performed using dideoxy chain-termination methods and was performed using Applied Biosystems Inc. Completed using a model 377 automated DNA sequencer (Foster City, Canada). Sequences are assembled using Sequencer 3.0 from Gene Codes Corporation, Ann Arbor, Michigan.

After identification of restriction sites and potential ORFs, pCIB9362-3 was cleaved with ClaI, 3.0 kb fragments were isolated and cloned into Bluescript, and DH5α. Transform into E. coli cells. The isolated colonies are grown as described above and the plasmid DNA is isolated by alkaline method. The subclone is identified as pCIB9369 and provides 100% mortality for flutella xylostella in bioassays. pCIB9369 was deposited on November 12, 1997 and was identified by NRRL Accession No. B-21883 at the USDA ARS Patent Culture Collection.

The 20 kb fragment identified as pCIB9381 is subcloned using NotI lysate from cosmid pCIB9379.

Example 7

Bioassay results

Insect bioassays are performed on pCIB9369 for several insects.

insect pCIB9369 Plutella xylostella Heliothis virescens Helioverpa zea Spodoptera exguia. C. elegans +++ nananana na = no activity + = significant proliferation inhibition ++ = greater than 40% but less than 100% mortality +++ = 100% mortality

These results indicate that the pesticidal toxin resulting from the expression of the 3.0 kb nucleotide sequence in pCIB9369 is very active against flutella xylostella.

Bioassays for pCIB9381, pCIB9354 and pCIB9383-21 also show high pesticidal activity against flutella xylostella.

Example 8

Size fraction of pesticidal activity

this. Genohabdus nematophyllus cosmid clone pCIB9369 and Stratagene Blue Script vector in E. coli host DH5 in medium supplemented with 50% terripic broth and 50% luria broth and supplemented with 50 μg / ml ampicillin Proliferate. Shake flask cultures (300 mL in 1000 mL baffle flasks) are grown at 37 ° C. overnight at 250 RPM. Cultures of each strain are centrifuged at 4 ° C. on a Sorvall GS-3 rotator at 7,000 RPM. The pelleted cells are resuspended in 30 ml of 50 mM NaCl, 25 mM Tris Base, pH 7.0. The concentrated cells are sonicated and digested by cooling on ice between cycles for about eight 10-second cycles using a Branson model 450 sonicator. The sonicates are centrifuged at 4 ° C. for 10 minutes at 6,000 RPM on a Sorvall SS34 rotator. The supernatant obtained is filtered through a 0.2 micron filter. Pellets from centrifuged sonicates are resuspended in 30 ml of 50 mM NaCl, 25 mM Tris Base, pH 7.0.

A 3 ml fraction of the filtrate is applied to a Bio-Rad Econo-Pac 10DG column previously equilibrated with 10 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. Discard the fluid collected during the sample load. Samples are dispensed by two successive additions of 4 mL of NaCl-tris equilibration buffer each. The first three fractions are stored for testing. The first fraction contains all of the materials having a molecular weight of at least about 6,000. Subsequent fractions contain materials with molecular weights less than 6,000.

Samples of the sonicated filtrate and pellets resuspended after sonication are tested using three fractions from a 10DG column for activity against P. xylostella neoplasia in surface contamination analysis. The filtered supernatant of the sonication and the fraction of the first column from the 9369 sample are very active against P. xylostella. The second and third fractions from the 9369 sample are not active. None of the samples from DH5a containing the Blue Script plasmid culture were active. These results suggest that the encoded pesticidal activity is greater than molecular weight 6,000 for the X. nematophilus clone pCIB9369 as well as homologues from pCIB9381, pCIB9354 and pCIB9383-21.

Example 9

Stability of Insecticidal Activity

300 ml of Luria gravy supplemented with 100 μg / ml ampicillin was inoculated with pCIB9369 and grown at 37 ° C. overnight. Samples are placed in sterile 15 ml screw cap tubes and stored at 22 ° C and 4 ° C. One sample is centrifuged, the supernatant is removed, lyophilized and stored at 22 ° C. These samples are stored for 2 weeks under these conditions and then bioassayed for Px. Lyophilized material is resuspended in the same volume as before the sample was lyophilized. All samples are resuspended by vortexing. The other sample is treated at 100 ° C. for 5 minutes.

Processing result

22 ° C (2 weeks) ++

4 ° C (2 weeks) ++

Freeze Drying (2 weeks) ++

100 ℃ na for 5 minutes

na = no activity.

Example 10

Thermal inactivation of pesticidal activity

Measure the thermal stability of the toxin. this. Cultures of E. coli strain pCIB9369 (E. coli host DH5a containing 3.0 kb DNA of Genohabdus nematophyllus) are grown overnight in a 50:50 mixture of luria juice and terry juice. Cultures are grown in culture tubes on a tube roller at 37 ° C. 1 ml of each culture sample is placed in a 1.5 ml Eppendorf tube and placed at 60 ° C and 80 ° C. Samples are removed after 5 minutes and cooled to room temperature. This sample is analyzed for P. xylostella along with the untreated portion of the culture. 50 μl of sample is spread over the diet, dried and newborn larvae P. xylostella are applied to the surface. Analytes are incubated at room temperature for 5 days.

Untreated samples and samples treated at 60 ° C. resulted in 100% mortality. Samples treated only at 80 ° C. and dietary controls did not result in measurable killing rates.

Example 11

Protease treatment of pesticidal activity

this. Genohabdus nematophyllus cosmid clone pCIB9369 and Stratagene's Blue Script vector in E. coli host DH5 are grown in medium supplemented with 50% terripic juice and 50% luria juice and supplemented with 50 μg / ml ampicillin. Shake flask cultures (300 mL in 1000 mL baffle flasks) are grown at 37 ° C. overnight at 250 RPM. Cultures of each strain are centrifuged at 4 ° C. on a Sorvall GS-3 rotator at 7,000 RPM. The pelleted cells are resuspended in 30 ml of 50 mM NaCl, 25 mM Tris Base, pH 7.0. Concentrated cells are sonicated and digested by cooling on ice between cycles for about eight 10-second cycles using a Branson model 450 sonicator. The sonicates are centrifuged at 4 ° C. for 10 minutes at 6,000 RPM on a Sorvall SS34 rotator. The supernatant obtained is filtered through a 0.2 micron filter. 1 ml of supernatant sample is adjusted to 5 mM Ca ++ by adding CaCl ₂ . Protease K (Gibco BRL; Gaithersburg, MD) is added at 500 μg / ml. Samples are incubated at 37 ° C. for 2 and 24 hours. Control samples are prepared and incubated with addition of Ca ++ but not protease.

Sonicated filtrate from pCIB9369 samples incubated in the presence of pCIB9369 and 5 mM Ca ++ results in a 100% mortality rate against flutella xylostella neonatal larvae. PCIB9369 samples incubated with protease K in addition to Ca ++ show a slightly lower kill rate of about 90% for flutella.

Example 12

Sequence comparison with the protein sequence of cry3A and the protein sequence of the larval 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 with sequence in Genbank as examined by UWGCG Blast and Gap program. Gap analysis of the protein encoded by ORF # 2 of pCIB9369 by the Blast program confirms some insignificant homology (21% identity) to the Bacillus suringensis cry3A protein. However, Gap analysis of the protein encoded by ORF # 2 (SEQ ID NO: 3) of pCIB9369 by the Blast program confirms homology (30.6% AA identity and 44.1% AA similarity) to the larval hormone esterase-related protein. GenBank Accession No. 2921553; Henikoff et al., PNAS USA 89: 10915-10919 (1992).

The nucleotide sequences of pCIB9381, pCIB9354 and pCIB9383-21 also reveal two open reading frames for each of these clones. The nucleotide sequences of the two ORFs in each of pCIB9381 and pCIB9383-21 are at least 90% identical to those in pCIB9369. Thus, the ORF # 2 proteins of pCIB9381 and pCIB9383-21 have essentially the same homology to the larvae hormone esterase-related protein as 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 (29.2% AA identity and 42.2% AA similarity) to the larval hormone esterase-related protein.

Example 13

Sequence Comparison of Sequences from pCIB9369 and WO 98/08388

22 sequences of 60 nucleotides each (60mer) are derived from a 38.2 kb DNA fragment whose nucleotide sequence is described in WO 98/08388 and compared to the nucleotide sequence of pCIB9362-3 comprising pCIB9369. The first 60mer starts at base 1 of the 38.2 kb DNA fragment, while the other 60mers are located about 2 kb apart on the DNA fragment. Its location on a 38.2 kb DNA fragment is described below:

1-60; 2,041-2,100; 4,021-4,080; 6,001-6,060; 8,041-8,100; 10,021-10,080; 12,001-12,060; 14,041-14,100; 16,021-16,080; 18,001-18,060; 20,041-20,100; 22,021-22,080; 24,001-24,060; 26,041-26,100; 28,021-28,080; 30,001-30,060; 32,041-32,100; 34,021-34,080; 36,001-36,060; 38,041-38,100; 38,161-38,220.

Sequences are compared using the UWGCG Gap program and tested for each of the 22 60mer sequences as well as their complementarity sequences. The result of these arrangements is 53% highest identity, which is not considered significant homology in the art.

Example 14

Southern blot analysis using probes derived from WO 98/08388 sequences

Pairs of oligonucleotides are designed to amplify DNA fragments of the 38.2 kb DNA fragment disclosed in WO 98/08388. Oligonucleotides are ordered from Genosys Biotechnologies (Woodland, Texas, USA) and their positions in the 38.2 kb DNA fragment are indicated below. In addition, their sizes and the sizes of the amplified PCR fragments are described:

VK1046: position 20-40

VK1047: Position 2,078-2,100

PCR fragments amplified using VK1046 and VK1047: 2,080 bp

VK1048: Position 11,221-11,241

VK1049: Position 13,360-13,380

PCR fragments amplified using VK1048 and VK1049: 2,120 bp

VK1050: Position 26,581-26,601

VK1051: Position 28,537-28,560

PCR fragment amplified using VK1050 and VK1051: 1,979 bp

VK1052: Position 18,901-18,921

VK1053: Position 20,321-20,340

Size of PCR fragments amplified using VK1052 and VK1053: 1,439 bp

VK1054: Position 34,261-34,281

VK1055: Position 35,320-35,340 BP

PCR fragment amplified using VK1054 and VK1055: 1,079 bp

PCR reactions were completed using Perkin-Elmer 9600 Thermo-Cycler under the following conditions: 94 ° C., 2 minutes; Then at 94 ° C., 30 seconds; At 54 ° C., 30 seconds; 30 cycles at 72 ° C., 4 minutes. The sample contains 800 ng of Gennorhavdus nematophilus DNA, each pair of 0.1-0.5 μM oligonucleotides, 250 μM dNTP, 5U Taq polymerase and 1 × buffer (Perkin-Elmer) in a final volume of 100 μl. The completed reaction is precipitated in ethanol, resuspended in TE and placed on a 1% SeaPlaque (FMC, Rockland, USA) TBE gel. After electrophoresis, the fragments are stained with ethidium bromide and visualized with ultraviolet light and then cleaved from the gel. The gel pieces are melted at 65 ° C. and 10 μl aliquots are mixed with 10 μl of distilled water, boiled for 5 minutes and placed on ice. Next, 15 μl of random priming label buffer (GIBCO-BRL, Gaithersburg, MD), 6 μl dNTP mixture (in the absence of dCTP), 80 μCiα-dCT ³² P and 1 μl Klenow fragment are mixed. The labeling reaction is carried out at room temperature for 60 minutes. Samples are washed on a Nick column (Pharmacia Biotech) according to the supplier's recommendation. Boil the probe for 5 minutes and place on ice.

Southern blots are DNA derived from Genoahabdus nematophilus whole DNA, cosmids pCIB9362 and pCIB9363 (these cosmids overlap at least 25kb, both contain DNA fragments of pCIB9369; pCIB9362 is used for subcloning) , DNAs derived from the subclones pCIB9362-3 (9 kb SacIl fragment) and pCIB9369 (2.96 kb ClaI fragment) are cleaved with ClaI, SacII or HindIII. Cleavage reactions are loaded onto a 0.75% agarose TBE gel and electrophoresis is performed overnight. The picture is taken and treated as described by Bio-Rad to blot the gel into Zeta-Probe hybridization membrane. After blotting, the film is baked at 80 ° C. for 30 minutes. The membrane is then placed in 7% SDS, 250 mM sodium phosphate, pH 7.2 and incubated at 67 ° C. for 30 minutes. Fresh solution is added and equilibrated to 67 ° C., then the radioactive probe described above is added and hybridized overnight. Membranes are washed at 67 ° C. for 30 minutes at 2 × SSC, 0.5% SDS and then at 67 ° C. for 30 minutes at 0.5 × SSC, 0.5% SDS. The membrane is exposed to the film for 1 hour and 3 hours. The film is developed and the results show that the PCR probe from the WO 98/08388 sequence does not hybridize to the DNA of cosmid or to the DNA of the subclone described herein. But the strong hybridization signal X. Observed when using Nematophilus DNA.

These results are consistent with the results of the sequence comparison, indicating that the clone pCIB9369 is different from the nucleotide sequence described in WO 98/08388.

B. Expression of Nucleic Acid Sequences of the Invention in Heterologous Microbial Hosts

Suitable microorganisms for heterologous expression of the nucleotide sequences of the present invention are all microorganisms capable of colonizing a plant or soil layer. By themselves they come in contact with pests. They are Gram-negative microorganisms (eg Pseudomonas, Enterobacter and Serratia), Gram-positive microorganisms Bacillus and fungi Trichoderma, Gliocladium and Saccharomyces cerevisiae. It includes. Particularly preferred heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aureofaciens, Pseudomonas antiaru Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, Bacillus cereus, Trichoderma viride, Trichoderma herb Trinoderma harzianum, Gliocladium virens and Saccharomyces cerevisiae.

Example 19

this. Expression of nucleotide sequences in E. coli and other Gram-negative bacteria

Many genes were expressed in a heterologous manner in Gram-negative bacteria. The expression vector pKK223-3 (Pharmacia catalog # 27-4935-01) is E. coli. Allow expression in E. coli. This vector has a strong tac promoter regulated by the lac repressor and induced by IPTG. Brosius, J. et al., Proc. Natl. Acad. Sci. USA 81]. There are many other manifestations of this. It was developed for use in E. coli. The heat induced expression vector pP _L (Pharmacia # 27-4946-01) uses a tightly regulated bacteriophage λ promoter that allows for high levels of expression of the protein. The lac promoter provides other means of expression, but this promoter is not expressed at the same high level as the tac promoter. Adding a wide host range of replicons to these expression system vectors allows the expression of nucleotide sequences in closely related Gram-negative bacteria (eg, Pseudomonas, Enterobacter, Serratia and Erwinia). For example, pLRKD211 (Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820 (1984) contains a broad host range of replicon ori T that allows replication of many Gram-negative bacteria.

this. In E. coli, induction by IPTG is required for expression of the tac (ie trp-lac) promoter. When this same promoter (eg on a broad host range of plasmid pLRKD211) is introduced into Pseudomonas, it is constitutively active without being induced by IPTG. This trp-lac promoter is placed before the gene or operon of interest for expression in Pseudomonas or other closely related bacteria for constitutive expression of this gene. Thus, the nucleotide sequences that are expressed and produce pesticidal toxins are thus placed behind strong constitutive promoters and transferred to bacteria with plant or soil layer colonization properties, using these organisms as pesticides. Other possible promoters can be used for constitutive expression of nucleotide sequences in Gram-negative bacteria. These include, for example, the Pseudomonas regulatory genes gafA and lemA [WO 94/01561] and the Pseudomonas savastanoi JAA operon promoter [Gaffney et al., J. Bacteriol. 172: 5593-5601 (1990).

Example 20

Expression of nucleotide sequences in Gram-positive bacteria

Heterologous expression of nucleotide sequences in Gram-positive bacteria is another means of producing insecticidal toxins. Expression systems for Bacillus and Streptomyces are best characterized. Promoters for the erythromycin resistance gene (ermR) from Streptococcus pneumoniae are Gram-positive aerobic and anaerobic bacteria and also E. coli. It appears to be active in E. coli (Teueu-Cuot et al., Nucl Acids Res 18: 3660 (1990)). Additional antibiotic resistance promoters from the thiostrepton gene were used in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). Shuttle vector pHT3101 is also suitable for expression in Bacillus (Lereclus, FEMS Microbiol Lett 60: 211-218 (1989)). An important advantage of this approach is that many Gram-positive bacteria produce spores with a long shelf life that can be used in formulations that produce insecticides. Bacillus and Streptomyces species are aggressive colonizing bacteria of the soil.

Example 21

Expression of nucleotide sequences in fungi

Trichoderma harzianum and Gliocladium virens have been shown to provide varying levels of bioregulation in the field [US 5,165,928 and US 4,996,157, both Cornell Research Foundation]. Nucleotide sequences that are expressed to produce pesticidal toxins can be expressed in these fungi. This can be done in a number of ways well known in the art. One is the protoplast-mediated transformation of fungi by PEG or electroporation-mediated techniques. Alternatively, particle bombardment can be used to transform protoplasts or other fungal cells with the ability to develop into regenerated mature structures. The vector pAN7-1, originally developed for Aspergillus transformation and now widely used for fungal transformation, is described in Curragh et al., Mycol. Res. 97 (3): 313-317 (1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 56: 117-124 (1987)] are engineered to contain nucleotide sequences. These plasmids are E. coli adjacent to the Aspergillus nidulans gpd promoter and trpC terminator. Contains E. coli's hygromycin B resistance gene (Punt et al., Gene 56: 117-124 (1987)).

In a preferred embodiment, the nucleic acid sequences of the invention are expressed in yeast Saccharomyces cerevisiae. For example, two ORFs from pCIB9369, pCIB9381, pCIB9354 or pCIB9383 are cloned into separate vectors with GAL1 inducible promoter and CYC1 terminator, respectively. Each vector has ampicillin resistance and 2μ replicon. The vector preferably differs in yeast proliferation label. The constructs are transformed independently and together into S. cerevisiae. ORFs are expressed together and tested for protein expression and pesticidal activity.

C. Formulation of Insecticidal Toxin

Pesticide formulations are made using the active ingredient, which produces or instead of isolated toxins and comprises suspensions or concentrates of the cells described in the examples above. For example, E. coli expressing insecticidal toxin. E. coli cells can be used to control pests. The formulations are prepared in liquid or solid form and are described below.

Example 18

Liquid Formulations of Pesticide Compositions

In the following examples, the percentage of the composition is in weight.

1. Emulsifying Thickener a b c

Active ingredient 20% 40% 50%

Calcium Dodecylbenzenesulfonate 5% 8% 6%

Castor Oil Polyethylene Glycol 5%--

Ether (ethylene oxide 36mole)

Tributylphenol Polyethylene Glycol-12% 4%

Ether (ethylene oxide 30mole)

Cyclohexanone-15% 20%

Xylene Mixture 70% 25% 20%

Emulsions of the required concentration can be prepared by dilution with water from these thickeners.

2. Liquid a b c d

Active ingredient 80% 10% 5% 95%

Ethylene Glycol Monomethyl Ether 20%---

Polyethylene Glycol 400-70%--

N-methyl-2-pyrrolidone-20%--

Epoxidized Coconut Oil--1% 5%

Petroleum Distillate--94%-

(Boiling range 160-190 ℃)

These solutions are suitable for application in the form of trace drops.

3. Granules a b

Active ingredient 5% 10%

Kaolin 94%-

Highly dispersible silicic acid 1%-

Aterpulzite-90%

The active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier and the solvent is then evaporated off in vacuo.

4. Dispersant a b

Active ingredient 2% 5%

Highly dispersible silicic acid 1% 5%

Talc 97%-

Kaolin-90%

Ready to use dispersants are obtained by intimate mixing of the active ingredient with the carrier.

Example 19

Solid Formulations of Insecticide Composition

In the following examples, the percentage of the composition is in weight.

1.wetting powder a b c

Active ingredient 20% 60% 75%

Sodium Lignosulfonate 5% 5%-

Sodium Lauryl Sulfate 3%-5%

Sodium Diisobutylnaphthalene Sulfonate-6% 10%

Octylphenol Polyethylene Glycol Ether-2%-

(Ethylene oxide 7-8mole)

Highly dispersible silicic acid 5% 27% 10%

Kaolin 67%--

The active ingredient is thoroughly mixed with the adjuvant and the mixture is thoroughly ground in a suitable mill to dilute with water to obtain a wettable powder which can provide a suspension of the desired concentration.

2. Emulsifying Thickener

10% active ingredient

Octylphenol polyethylene glycol ether 3%

(Ethylene oxide 4-5mole)

Calcium Dodecylbenzenesulfonate 3%

Castor Oil Polyglycol Ether 4%

(Ethylene oxide 36mole)

Cyclohexanone 30%

Xylene Mixture 50%

Emulsions of the required concentration can be obtained by dilution with water from this thickener.

3. Dispersant a b

Active ingredient 5% 8%

Talc 95%-

Kaolin-92%

Ready-to-use dispersants are obtained by mixing the active ingredient with the carrier and grinding the mixture in a suitable mill.

4. Extruded Granule

10% active ingredient

2% sodium lignosulfonate

Carboxymethylcellulose 1%

Kaolin 87%

The active ingredient is mixed, triturated with adjuvants and the mixture is then wetted with water. The mixture is extruded and then dried in an air stream.

5. Epidermal Granules

Active ingredient 3%

Polyethylene Glycol 200 3%

Kaolin 94%

The finely divided active ingredient is applied homogeneously in a mixer to kaolin moistened with polyethylene glycol. Non-dispersible epidermal granules are obtained in this way.

6. Suspension Thickener

40% active ingredient

Ethylene Glycol 10%

Nonylphenol Polyethylene Glycol 6%

(Ethylene oxide 15mole)

10% sodium lignosulfonate

Carboxymethylcellulose 1%

37% formaldehyde aqueous solution 0.2%

0.8% silicone oil in 75% aqueous emulsion

32% of water

The finely divided active ingredient can be closely mixed with the adjuvant to obtain a suspension thickener, from which it can be diluted with water to obtain a suspension of the desired concentration.

The pesticide formulations described above are applied in an amount controlled by pesticidal toxins, according to methods well known in the art.

D. Expression of nucleotide sequences in transformed plants

The nucleic acid sequences described herein can be incorporated into plant cells using conventional recombinant DNA techniques. In general, this involves inserting a coding sequence of the invention into an expression system in which the coding sequence is heterologous (ie, not generally present) using standard cloning methods known in the art. The vector contains elements necessary for the transcription and translation of the inserted protein-coding sequence. Many vector systems known in the art can be used, for example plasmids, bacteriophage viruses and other altered viruses. Suitable vectors include viral vectors (eg, lambda vector systems lambda gtl1, lambda gtl0 and Charon 4); Plasmid vectors (eg, pBl121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII); And other similar systems, but is not limited thereto. The components of the expression system can also be altered to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications can be used. The expression system described herein can be used to substantially transform crop plant cells under suitable conditions. The transformed cells can be regenerated into whole plants so that the nucleotide sequences of the invention can confer insect resistance to the transformed plants.

Example 22

Modification of coding sequences and contiguous sequences

The nucleotide sequences described herein can be modified for expression in a transformed plant host. Host plants that express nucleotide sequences and produce insecticidal toxins in the cells of the host plant are more resistant to insect attacks and are therefore better equipped to reduce the crop losses associated with these attacks.

Gene expression transformed in a plant of a gene derived from a microbial source may require modification of the gene to carry out and optimize its expression in the plant. In particular, bacterial ORFs that encode separate enzymes but are encoded by the same transcript in native microorganisms are best expressed on separate transcripts in plants. To accomplish this, each microbial ORF is isolated separately and cloned into a cassette providing a plant promoter sequence at the 5 'end of the ORF and a plant transcription terminator at the 3' end of the ORF. An isolated ORF sequence preferably comprises an initiating ATG codon and a terminating STOP codon, but may comprise additional sequences in addition to the initiating ATG and STOP codons. In addition, ORFs can be end-cut, but still retain the required activity; Particularly for long ORFs, end-cutting modifications retaining activity may be desirable for expression in the transformed organism. "Plant promoter" and "plant transcription terminator" refer to promoters and transcription terminators that function in plant cells. This includes promoters and transcription terminators that can be derived from non-plant sources such as viruses (eg, cauliflower mosaic virus).

In certain cases, modifications to the ORF coding sequence and the contiguous sequence are not necessary. It is sufficient to isolate the fragment containing the ORF of interest and insert it downstream of the plant promoter. For example, Gaffney et al., Science 261: 754-756 (1993), do not modify the coding sequence in successful regulation of the CaMV 35S promoter and CaMV tml terminator in transformed plants, but still upstream of the attached ATG. The Pseudomonas nahG gene was expressed with the x bp of the Pseudomonas gene and the y bp downstream of the STOP codon still attached to the nahG ORF. Preferably few contiguous microbial sequences remain unattached upstream of the ATG and downstream of the STOP codon. In practice, such constructs may depend on the utility of the restriction site.

In other cases, expression of genes derived from microbial sources can cause problems in expression. These problems are well characterized in the art and are particularly common with genes derived from specific sources (eg Bacillus). These problems can be applied to the nucleotide sequences of the present invention, and modifications of these genes can be carried out using techniques that are now well known in the art. The following problems can occur.

1. Using Codons

Preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the use of codons in cloned microbial ORFs for use in plant genes (and in specific genes from target plants) can identify codons in the ORF that are preferably changed. In general, plant evolution tends to strongly favor nucleotides C and G at the third base position of the monocotyledonous, whereas dicots frequently use nucleotides A or T at this position. By modifying the gene to introduce the desired codon usage to a particular target transformed species, many of the problems described below for the GC / AT content and inappropriate splicing are overcome.

2. GC / AT content

Plant genes generally have a GC content of at least 35%. ORF sequences rich in A and T nucleotides can cause many problems in plants. First of all, it is believed that the motif of ATTTA causes destabilization of the message and is found at the 3 'end of many short-lived mRNAs. Second, it is believed that the generation of polyadenylation signals (eg AATAAA) at inappropriate locations in the message results in premature cleavage of transcription. Monocots can also recognize AT-rich sequences as splicing sites (see below).

3. Sequences Adjacent to Initiating Methionine

Plants differ from microorganisms in that the message does not have a defined ribosomal binding site. Rather, it is believed that ribosomes slide in search of the first useful ATG attached to the 5 'end of the message and initiating decryption. However, certain nucleotides adjacent to ATG are preferred and it is believed that the expression of microbial genes can be enhanced by the inclusion of eukaryotic consensus translational initiation factors in ATG. Clontech, 1993/1994 catalog, page 210, which is incorporated herein by reference, discloses that E. One sequence has been proposed as a consensus translation initiation factor for the expression of the E. coli uidA gene. In addition, Joshi, NAR 15: 6643-6653 (1987), incorporated herein by reference, compares many plant sequences adjacent to ATG and proposes another consensus sequence. In situations where difficulties arise in the expression of microbial ORFs in plants, one of these sequences can be included in the starting ATG to improve translation. In such cases, the last three nucleotides of the consensus may not be suitable for inclusion in the modified sequence due to modification of the second AA residue. Preferred sequences adjacent to the starting methionine can differ between different plant species. Investigating 14 maize genes located in the GenBank database gives the following results:

Position before initiation ATG in 14 maize genes

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1

C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0

A 2 3 1 4 3 2 3 7 2 3

G 6 3 6 0 6 5 4 6 1 5

This analysis can be performed on the desired plant species in which the nucleotide sequence is incorporated and the sequence adjacent to the ATG is modified to incorporate the desired nucleotide.

4. Elimination of Unsuitable Splicing Sites

Genes cloned from non-plant sources and not optimized for expression in plants are also motifs that are recognized as 5 'or 3' splicing sites in plants and are cleaved, thus producing end-cut or deleted messages. It may contain. These sites can be removed using techniques well known in the art.

Techniques for modifying coding sequences and contiguous sequences are well known in the art. If the initial expression of the microbial ORF is low and deemed suitable for changing the sequence as described above, the construction of the synthetic gene can be performed according to methods well known in the art. These are described, for example, in published patent specifications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol) and WO 93/07278 (Ciba-Geigy), all of which are herein disclosed. Cited for reference. In most cases, it is desirable to use an transient assay protocol (well known in the art) to analyze the expression of the gene construct before delivery to the transformed plant.

Example 23

Construction of Plant Expression Cassettes

The coding sequence intended for expression in the transformed plant is first assembled in an expression cassette behind a suitable promoter that is expressible in the plant. The expression cassette may also include additional sequences necessary or selected for expression of the transformed gene. Such sequences include, but are not limited to, exogenous sequences that enhance expression such as transcription terminators, introns, essential sequences, and sequences intended for targeting of gene products to particular intracellular organ and cell compartments. These expression cassettes can then be readily delivered to the plant transformation vectors described below. The following is a description of the various components of a representative expression cassette.

1. Promoter

The selection of the promoter used in the expression cassette determines the spatial and temporal expression pattern of the transformed gene in the transformed plant. The selected promoter expresses the transformed gene in a specific cell type (e.g. leaf epidermal cells, lobe cells, root cortical cells) or in a specific tissue or organ (e.g. root, leaf or flower), and the selection results in the accumulation of gene products Affects the preferred position of. Alternatively, the selected promoter can drive the expression of a gene under many induction conditions. Promoters vary in their strength, the ability to promote transcription. Depending on the host cell system used, one of many suitable promoters can be used, including the original promoter of the gene. The following are non-limiting examples of promoters that can be used in expression cassettes.

a. Constitutive expression, ubiquitin promoter

Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from many species for use in transformed plants. Sunflower-Binet et al, Plant Science 79: 87-94 (1991); Corn-Christensen et al. Plant Molec. Biol. 12: 619-632 (1989); And Arabidopsis-Norris et al., Plant Mol. Biol. 21: 895-906 (1993). Corn ubiquitin promoters have been developed in transformed monocotyledonous systems and their sequences and vectors constructed for monocotyledonous transformation are described in patent publication EP 0 342 926 (Lubrizol), which is incorporated herein by reference. Taylor et al., Plant Cell Rep. 12: 491-495 (1993)] A vector (pAHC25) containing its high activity in cell suspensions of many monocots is described when introduced via the corn ubiquitin promoter and the first intron and 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 transformed plants of monocots and dicotyledons. Suitable vectors are derivatives of pAHC25 or the transformation vectors described herein modified by introducing a suitable ubiquitin promoter and / or intron sequence.

b. Constitutive expression, CaMV 35S promoter

The construction of the plasmid pCGN1761 is described in published patent application EP 0 392 225 (Example 23), which is incorporated herein by reference. pCGN1761 contains a "double" CaMV 35S promoter and a tml transcription terminator with the only EcoRI site between the promoter and terminator and has a pUC-type backbone. Derivatives of pCGN1761 with modified polylinkers comprising NotI and XhoI sites in addition to EcoRl sites are constructed. This derivative is named pCGN1761ENX. pCGN1761ENX is useful for cloning a cDNA sequence or coding sequence (including microbial ORF sequences) in its polylinker to express it under the control of a 35S promoter in transformed plants. The entire 35S promoter-coding sequence-tml terminator cassette of this construct was cleaved into a 5 'promoter by the HindIII, SphI, SalI and XbaI sites and into a 3' terminator by the XbaI, BamHI and BglI sites and then It can be delivered to a transformation vector as described. In addition, double 35S promoter fragments can be removed by 5 ′ cleavage by HindIII, SphI, SalI, XbaI or PstI, and 3 ′ cleavage by polylinker restriction sites (EcoRI, NotI or XhoI) for replacement with other promoters. have. If desired, modifications around the cloning site can be made by introduction of sequences that can enhance translation. This is particularly useful where overexpression is desired. For example, pCGN1761ENX can be modified by optimization of the translational initiation site described in Example 37 of US Pat. No. 5,639,949, which is incorporated herein by reference.

c. Constitutive expression, actin promoter

It is known that several isoforms of actin are expressed in most cell types, with the result that the actin promoter is an excellent choice for constitutive promoters. In particular, promoters from the rice ActI gene have been cloned and characterized. McElroy et al. Plant Cell 2: 163-171 (1990). The 1.3 kb fragment of the promoter was found to contain the regulatory elements required for expression in rice protoplasts. In addition, many expression vectors based on the ActI promoter have been specifically constructed for use in monocots. McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991). They incorporate ActI-intron 1, Adhl 5 'contiguous sequences and sequences from Adhl-intron 1 (from corn alcohol dehydrogenase gene) and CaMV 35S promoter. The vector showing the highest expression is a fusion of 35S and ActI introns or ActI 5 'contiguous sequences and ActI introns. Expression is enhanced by optimization of the sequence around the starting ATG (of the GUS reporter gene). See McElroy et al., Mol. Gen. Genet. 231: 150-160 (1991) can be readily modified for gene expression and is particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments are removed from McElroy constructs and used to replace the dual 35S promoter in pCGN1761ENX, which is then useful for insertion of specific gene sequences. The fusion gene so constructed can then be transferred to a suitable transformation vector. In a different report, it was found that the rice ActI promoter with the first intron also indicated high expression in cultured barley cells. Chibbar et al. Plant Cell Rep. 12: 506-509 (1993).

d. Inducible Expression, PR-1 Promoter

In pCGN1761ENX the dual 35S promoter can be replaced with other selected promoters resulting in moderately high expression levels. For example, one of the chemically controllable promoters described in US Pat. No. 5,614,395 can replace a dual 35S promoter. The selected promoter is preferably cleaved by a restriction enzyme from its source, or can be PCR-amplified using primers having suitable terminal restriction sites. If PCR-amplification is performed, the promoter should be resequencing to clone the amplified promoter in the target vector and then check for amplification errors. Chemically / pathogen-controlled tobacco PR-1a promoter was cleaved from plasmid pCIB1004 [see Example 21 of EP 0 332 104, incorporated herein by reference, for construction] and delivered to plasmid pCGN1761ENX [Uknes et al., 1992] do. pCIB1004 is cleaved with NcoI and terminally smoothed by treatment of the 3 ′ overhang of the obtained linear fragment with T4 DNA polymerase. The fragment is then cleaved with HindIII and the resulting PR-1a promoter-containing fragment is gel purified and cloned into pCGN1761ENX, of which 35S promoter has been removed. This is done by cleaving with XhoI, end-smoothing with T4 polymerase, then cleaving with HindIII and separating the large vector-terminator containing the fragment from which the pCIB1004 promoter fragment is cloned. It uses the pCGN1761ENX derivative with the PR-1a promoter and tml terminator and the unique EcoRI and NotI sites to produce an interfering polylinker. The selected coding sequence can be inserted into this vector and the fusion product (ie, promoter-gene-terminator) can subsequently be transferred to the selected transformation vector, including those defined below. Many chemical modulators can be used to express coding sequences selected in plants transformed according to the invention, including the benzothiadiazole, isnicotinic acid and salicylic acid compounds described in US Pat. Nos. 5,523,311 and 5,614,395.

e. Inducible Expression, Ethanol-Induced Promoter

Promoters inducible by particular alcohols or ketones, for example ethanol, may also be used to confer inducible expression of the coding sequences of the invention. Such promoters are, for example, alcA gene promoters from Aspergillus nidulans. See, eg, Caddick et al. (1988) Nat. Biotechnol. 16: 177-180]. In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by AlcR transcription factors in the presence of chemical inducers. For the present invention, at least a 35S promoter [Caddick et al. (1988) Nat. Biotechnol. 16: 177-180, the CAT coding sequence in the plasmid palcA: CAT comprising the alcA gene promoter sequence fused to form an expression cassette with the coding sequence under the control of the alcA gene promoter. This is done using methods well known in the art.

f. Inducible expression, glucocorticoid-inducible promoter

Induction of expression of the nucleic acid sequences of the present invention using systems based on steroid hormones is also contemplated. For example, glucocorticoid-mediated induction systems are used (Aoyama and Chua (1997) The Plant Journal 11: 605-612), and gene expression is glucocorticoids, eg synthetic glucocorticoids, preferably Dexamethasone is derived by applying a concentration, preferably in the range from 0.1 to 1 mM, more preferably in the range from 10 to 100 mM. For the present invention, the luciferase gene sequence is replaced by the nucleic acid sequence of the present invention to form an expression cassette having the nucleic acid sequence of the present invention under the control of six copies of the GAL4 upstream activating sequence fused to a 35S minimal promoter. This is done using methods well known in the art. The interaction factor is the hormone-binding domain of the rat glucocorticoid receptor [Picard et al. (1988) Cell 54: 1073-1080], the activating domain of the herpes virus protein VP16 [Trijenberg et al. (1988) Genes Devel. 2: 718-729], Gee4 DNA-binding domain [Keegan et al. (1986) Science 231: 699-704. Expression of the fusion protein is controlled by promoters suitable for expression in the plants known in the art or described herein. This expression cassette is also included in a plant comprising the nucleic acid sequence of the invention fused to a 6xGAL4 / minimal promoter. Thus, tissue- or organ-specificity of the fusion protein is obtained to induce inducible tissue- or organ-specificity of the insecticidal toxin.

g. Root Specific Expression

Another pattern of gene expression is root expression. Suitable root promoters are described by de Framond (FEBS 290: 103-106 (1991)) and published patent application EP 0 452 269, which is incorporated herein by reference. This promoter is transferred into a vector (eg pCGN1761ENX) suitable for insertion of the selected gene and subsequent delivery of the entire promoter-gene-terminator cassette into the transformation vector of interest.

h. Wound-induced promoter

Wound-induced promoters may also be suitable for gene expression. Many promoters have been described [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)], all of which are suitable for use in the present invention. Logemann et al. Describe the 5 'upstream sequence of the dicotyledonous potato wunI gene. Xu et al. Show that the wound-inducing promoter from dicotyledonous potato (pin2) is active in monocotyledonous rice. Rohrmeier & Lehle also describes the cloning of maize Wipl cDNA that can be wound induced and used to isolate cognate promoters using standard techniques. Similarly, Firek et al. And Warner et al. Describe wound-derived genes from the monocotyledonous Asparagus officinalis, which are expressed at local wounds and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes of the present invention, and used for expression of these genes at plant wound sites.

i. Pith-preferred expression

Patent application WO 93/07278, incorporated herein by reference, describes the isolation of maize trpA genes that are preferentially expressed in pith cells. Gene sequences and promoters are shown extending from transcription initiation to -1726 bp. Using standard molecular biology techniques, this promoter or part thereof can be delivered to a vector such as pCGN1761 (where it can replace the 35S promoter) and used to drive expression of foreign genes in a number-preferred manner. can do. In fact, fragments containing a water-preferred promoter or portion thereof can be delivered to a vector and modified for use in the transformed plant.

j. Leaf-specific expression

Corn genes encoding phosphoenol carboxylase (PEPC) have been described in the literature (Hudspeth & Grula (Plant Molec. Biol. 12: 576-589 (1989)) .. Using standard molecular biology techniques, The promoter for this gene can be used to drive expression of the gene in a leaf-specific method in transformed plants.

k. Pollen-specific manifestations

WO 93/07278 describes the isolation of maize calcium-dependent protein kinase (CDPK) expressed in pollen cells. Gene sequences and promoters extend up to 1400 bp from the onset of transcription. Using standard molecular biology techniques, this promoter or part thereof can be delivered to a vector such as pCGN1761 (where it can replace the 35S promoter) and used to pollute the nucleic acid sequence of the invention in a pollen-specific manner. Expression can be driven.

2. Warrior Terminator

Many transcription terminators are useful for use in expression cassettes. They are involved in the termination of transcription after the transformed gene and its correct polyadenylation. Suitable transcription terminators are known to function in plants and include CaMV 35S terminators, tml terminators, nopaline synthase terminators and pea rboS E9 terminators. They can be used for both monocotyledonous and dicotyledonous plants. In addition, native transcription terminators of genes can be used.

3. Sequences for Enhancing or Controlling Expression

Many sequences have been found to enhance gene expression from within the transcription unit and these sequences can be used with the genes of the invention to increase the expression of transformed plants.

Many intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, introns of the maize Adhl gene have been found to significantly enhance the expression of wild-type genes under their cognate promoter when introduced into maize cells. Intron 1 has been found to be particularly effective and to enhance expression in fusion constructs of the chloramphenicol acetyltransferase gene. Callis et al. Genes Develop. 1: 1183-1200 (1987). In the same experimental system, introns from the corn bronze 1 gene have a similar effect in enhancing expression. Intron sequences have been commonly incorporated into plant transformation vectors, generally within non-translating leader sequences.

Many non-translating leader sequences derived from viruses are also known to enhance expression and they are particularly useful for dicotyledonous cells. In particular, leader sequences from tobacco mosaic virus (TMV, "W sequence"), maize gastric yellow spot virus (MCMV) and alfalfa mosaic virus (AMV) have been shown to be useful for enhancing expression. Gallie et. al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990).

4. Targeting Gene Products Into Cells

Many mechanisms for targeting gene products are known to exist in plants, and the sequences regulating the function of these mechanisms have been characterized in some detail. For example, targeting of gene products to chloroplasts is regulated by signal sequences found at the amino terminal ends of many proteins that degrade during chloroplast uptake to yield mature proteins. See, eg, Comai et al. J. Biol. Chem. 263: 15104-15109 (1988). These signal sequences can be fused to heterologous gene products to effect uptake of the heterologous products into chloroplasts. Van den Broeck, et al. Nature 313: 358-363 (1985). DNA encoding a suitable signal sequence can be isolated from the 5 'end of the cDNA encoding the RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein and many other proteins known to be located in the chloroplast. In Example 37 of 5,639,949, entitled "Expression With Chloroplast Targeting".

Other gene products are located in other intracellular organs (eg mitochondria and peroxysomes). See, eg, Unger et al. Plant Molec. Biol. 13: 411-418 (1989). CDNAs encoding these products can also be engineered to validate targeting of these heterologous gene products to these intracellular organelles. An example of such a sequence is the aspartate amino transferase isoform specific for nuclear-encoding ATPase and mitochondria. Targeting cellular protein bodies are described in the literature. Rogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985).

In addition, sequences are defined that result in the targeting of gene products to other cell compartments. The amino terminal sequence is involved in ER, targeting to apoplasts, and extracellular secretion from erupted cells (Kohehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, the amino terminal sequence, along with the carboxy terminal sequence, is involved in the fear targeting of the gene product. Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990).

Suitable targeting sequences described above can be fused to the transformed gene sequence of interest to direct the transformed gene product to an organelle or cell compartment. To target the chloroplast, the chloroplast signal sequence from, for example, the RUBISCO gene, CAB gene, EPSP synthase gene or GS2 gene is fused to the frame for the amino terminal ATG of the transformed gene. The signal sequence selected should comprise a known cleavage site and the constructed fusion should take into account the amino acids following the cleavage site necessary for cleavage. In certain cases, this requirement may be met by the addition of a few amino acids between the cleavage site and the transformed gene ATG, or by replacement of a specific amino acid in the transformed gene sequence. Fusions constructed for chloroplast uptake can be tested for chloroplast uptake by in vitro detoxification of in vitro transcribed constructs followed by in vitro chloroplast uptake using the techniques disclosed in the literature. 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). Such construction techniques are well known in the art and are equally applicable to mitochondria and peroxysomes.

The mechanisms described above for cell targeting can be used with heterologous promoters as well as with their cognate promoters to perform specific cell-targeting objectives under the transcriptional control of promoters having expression patterns different from those of the promoter from which the targeting signal is induced. Can be.

Example 24

Construction of Plant Transformation Vectors

Many transformation vectors useful for plant transformation are known to experts in the field of plant transformation, and genes suitable for the present invention can be used with such vectors. The choice of vector depends on the desired transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers commonly used for transformation include the nptII gene conferring resistance to kanamycin and related antibiotics (Messing & Vierrra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983), the bar gene conferring resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)], hph gene confers resistance to antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), dhfr confers resistance to metatrexate Genes (Bourouis et al., EMBO J. 2 (7): 1099-1104 (1983)), and EPSPS genes conferring resistance to glyphosate (US Pat. Nos. 4,940,935 and 5,188,642). do.

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are useful for transformation using Agrobacterium tumefasion. They generally carry one or more T-DNA border sequences and are described in pBlN19 [Bevan, Nucl. Acids Res. (1984) and vectors such as pXYZ. Below, the construction of two representative vectors suitable for Agrobacterium transformation are described.

a. pCIB200 and pCIB2001

Binary vectors pCIB200 and pCIB2001 are used in the construction of recombinant vectors for use with Agrobacterium and consist of the following methods. pTJS75kan is described in pTJS75 [Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)] by dissolving NarI to cleave the tetracycline-resistant gene, followed by NPTII (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)] to form AccI fragments from pUC4K. The XhoI linker is linked to the EcoRV fragment of pCIB7 containing the left and right T-DNA boundaries, the plant selective nos / nptII chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)) , XhoI-dissolved fragment is cloned into SalI-dissolved pTJS75kan to form pCIB200 (see EP 0 332 104, Example 19). pCIB200 contains the following polylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI and SalI. pCIB2001 is a derivative of pCIB200 formed by insertion into a polylinker of additional restriction sites. The only restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI and StuI. In addition to containing these unique restriction sites, pCIB2001 also contains plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, E. coli. It has RK2-induced trfA function and OriT and Oriv function from RK2 for migration between E. coli and other hosts. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing their own regulatory signals.

b. pCIB10 and its hygromycin selective derivatives

The binary vector pCIB10 contains genes encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the broad host range plasmid pRK252. To replicate in both E. coli and Agrobacterium. Its construction is described in the literature (Rothstein et al., Gene 53: 153-161 (1987)). Many derivatives of pCIB10 are constructed that incorporate the gene for hygromycin B phosphotransferase described in the literature (Gritz et al., Gene 25: 179-188 (1983)). These derivatives allow the selection of transformed plant cells for hygromycin alone (pCIB743) or for hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors Suitable for Non-Agrobacterium Transformation

Transformations that do not use Agrobacterium tumefacience exclude the requirement for T-DNA sequences in the selected transformation vector, and the vectors lacking these sequences are then as described above containing T-DNA sequences. Can be used in addition to the vector. Agrobacterium-free transformation techniques include particle bombardment, protoplast uptake (eg PEG and electroporation) and microinjection. The choice of vector depends mainly on the preferred choice for the species to be transformed. Below, the construction of representative vectors suitable for non-Agrobacterium transformation is described.

a. pCIB3064

pCIB3064 is a pUC derived vector suitable for direct gene transfer techniques with selection by the herbicide vasta (or phosphinothricin). Plasmid pCIB246 is E. coli. CaMV 35S promoters and CaMV 35S transcriptional terminators in fusions effective for the E. coli GUS gene and are described in published PCT application WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5 'to the start. These sites are mutated in a manner that removes ATG and generates restriction sites SspI and PvuII using standard PCR techniques. The novel restriction sites are 96 and 37 bp from the only SalI site and 101 and 42 bp from the substantial starting site. The derivative of pCIB246 obtained is named pCIB3025. The GUS gene is then cleaved with SalI and SacI to cleavage from pCIB3025, and the ends are blunted and reconnected to produce plasmid pCIB3060. Plasmid pJIT82 was obtained from the John Innes Center, Norwich, USA, cleaved a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes and inserted into the HpaI site of pCIB3060. Thompson et al. EMBO J 6: 2519-2523 (1987). It produces pCIB3064 comprising a bar gene under 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 unique sites SphI, PstI, HindIII and BamHI. do. This vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

b. pSOG19 and pSOG35

pSOG35 is a selective marker that confers resistance to methotrexate. Transformation vector using E. coli gene dihydrofolate reductase (DFR). PCR is used to amplify 18 bp of the GUS non-toxin leader sequence from 35S promoter (-800 bp), maize Adhl gene (-550 bp) and intron 6 and pSOG10. this. The 250 bp fragment encoding the coli dihydrofolate reductase type II gene is also amplified by PCR, and these two PCR fragments are SacI- from pB1221 (Clontech) containing the pUC19 vector backbone and the nopaline synthase terminator. Assembled into PstI fragments. Assembly of these fragments produces a pSOG19 containing the 35S promoter fused with an intron 6 sequence, a GUS leader, a DHFR gene and a nopaline synthase terminator. In pSOG19, the GUS leader is replaced with a leader sequence from maize gastric disease spot virus (MCMV) to produce the vector pSOG35. pSOG19 and pSOG35 have pUC genes for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites useful for cloning foreign material.

3. Vectors Suitable for Chloroplast Transformation

For expression of the nucleotide sequence of the present invention in plant plastids, the plastiform 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 chromatin transformation and selection of transformants for spectinomycin resistance. In addition, the nucleotide sequence is inserted into pPH143 to replace the aadH gene. In this case, transformation is selected for resistance to PROTOX inhibitory factors.

Example 25

Transformation

Once the nucleotide sequence of the invention is cloned into the expression system, it is transformed into plant cells. Methods for transforming and regenerating plants are well known in the art. For example, Ti plasmid vectors have been used for direct DNA uptake, liposomes, electroporation, microinjection and microprojection methods as well as delivery of foreign DNA. In addition, bacteria of the genus Agrobacterium can be used to transform plant cells. The following describes not only representative techniques for transforming dicotyledonous and monocotyledonous plants, but also representative chromatin transformation techniques.

1. Transformation of Dicotyledonous Plants

Transformation techniques for dicotyledonous plants are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium technology involves the direct uptake of exogenous genetic material by protoplast cells. This can be done by PEG or electroporation mediated absorption, particle bombardment-mediated delivery or microinjection. Examples of these techniques are described in the literature. 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 et al., Nature 327: 70-73 (1987). In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for the transformation of dicotyledonous plants due to its high transformation efficacy and its wide availability to many different species. Agrobacterium transformation is generally suitable for Agrobacterium strains (e.g. pCIB200 and pCIB2001) that may rely on supplementation of vir genes carried on the Ti plasmid or chromosome co-resident with the host Agrobacterium strains. ClB542 strain) (Ukness et al. Plant Cell 5: 159-169 (1993)], which includes the delivery of a binary vector (eg pCIB200 or pCIB2001) that carries foreign DNA of interest. Delivery of the recombinant binary vector to Agrobacterium is a bacterium that carries the recombinant binary vector. E. coli, a helper E. capable of carrying a plasmid such as pPK2013 and moving the recombinant binary vector to a target Agrobacterium strain. E. coli strains are used by the parental mating method. Alternatively, the recombinant binary vector can be delivered to Agrobacterium by DNA transformation. Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988).

Transformation of the target plant species by recombinant Agrobacterium generally involves coculture of the graft from the plant with Agrobacterium, according to protocols well known in the art. Transformed tissue is regenerated on a selective medium that carries antibiotic or herbicide resistant labels present between the binary plasmid T-DNA boundaries.

Another approach to transforming plant cells into genes involves driving inert or biologically active particles in plant tissues and cells. These techniques are all described in US Pat. Nos. 4,945,050, 5,036,006 and 5,100,792 to Sanford et al. In general, the method involves propelling inert, biologically active particles under conditions effective to penetrate into and incorporate into the cell's outer surface. When inert particles are used, the vectors can be introduced into the cells by applying the particles with a vector containing the desired gene. In addition, the target cell is surrounded by the vector to allow the vector to be transported along the particle to the cell. Biologically active particles (eg, anhydrous yeast cells, anhydrous bacteria, or bacteriophages, each containing DNA to be introduced) can also be propagated into plant cell tissue.

2. Transformation of monocots

Transformation of most monocot species is now routine. Preferred techniques include direct gene transfer to protoplasts using PEG or electroporation techniques and include particle bombardment into cellular tissue. Transformation can be performed using a single DNA species or multiple DNA species (ie co-transformation), both of which are suitable for use in the present invention. Co-transformation can have the advantage of avoiding complete vector construction and producing transformed plants with non-binding positions for the genes of interest and genes of the selective marker, thus eliminating the selective marker in subsequent production, which Preferred. The disadvantage of using co-transformation, however, is that less than 100% of the assembly of separate DNA species into the genome is made by Schocher et al. Biotechnology 4: 1093-1096 (1986)].

In patent applications EP 0 292 435, EP 0 392 225 and WO 93/07278, preparation of callus and protoplasts from selected homologous lines of corn, transformation of protoplasts using PEG or electroporation And techniques for regeneration of corn plants from transformed protoplasts. Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Fromm et al., Biotechnology 8: 833-839 (1990), describe the transformation of A188-induced corn strains using particle bombardment. The technology for this is disclosed. In addition, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) disclose a technique for the transformation of selected homologs of maize by particle bombardment. This technique uses a PDS-1000He Biolistics device for impacting immature corn embryos and 1.5-2.5 mm long immature corns cut from corn ears 14-15 days after pollination.

Transformation of rice can also be performed by direct gene transfer techniques using protoplasts or particle bombardment. Protoplast-mediated transformation has been described for the Japonica type and Indica type. Zhang et 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 of these types can be conventionally transformed using particle bombardment. See Christou et al. Biotechnology 9: 957-962 (1991). WO 93/21335 also describes a technique for the transformation of rice by electroporation.

Patent application EP 0 332 581 describes a technique for the production, transformation and regeneration of Pooideae protoplasts. These techniques transform Dactylis and wheat. In addition, wheat transformation uses particle bombardment with cells of type C long term reproducible callus (Vasil et al., Biotechnology 10: 667-674 (1992)) and immature embryos and immature embryo-derived callus. The particle bombardment method of Vasil et al., Biotechnology 11: 1553-1558 (1993) and Weeks et al., Plant Physiol. 102: 1077-1084 (1993). However, a preferred technique for wheat transformation involves the transformation of wheat by particle bombardment of immature embryos and involves a high sucrose or high maltose step prior to gene transfer. Prior to impact, any number of embryos (0.75-1 mm long) were subjected to 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg / l 2,4 to induce somatic embryos. Plated on MS medium containing -D, which progresses to cancerous phase. On the selected day of shock, the embryos are removed from the induction medium and placed on osmoticum (ie, induction medium containing sucrose or maltose added at a preferred concentration, generally 15%). Embryos are template separated for 2-3 hours and then shocked. Twenty embryos per target plate are common but not important. Suitable gene-carrying plasmids (eg pCIB3064 or pSG35) are precipitated onto gold particles of μm size using standard methods. Each embryo plate is launched into a DuPont Biolistics ^R helium device using a burst pressure of about 1000 psi and a standard 80 mesh screen. After the impact, the embryos are placed back in the dark to recover for about 24 hours (still on Osmoticum). After 24 hours, embryos are removed from Osmoticum and placed back on the residual induction medium about one month before they are regenerated. After about one month, embryonic grafts with embryonic callus developed were regenerated medium (MS + 1 mg) containing additional suitable selection agents (10 mg / l Vaster for pCIB3064 and 2 mg / l methotrexate for pSOG35). / l NAA, 5 mg / l GA). After about one month, the developed shoots are transferred to a large sterile container known as "GA7s" containing half-concentration MS, 2% sucrose and the same concentration of the selection agent.

Transformation of monocots using Agrobacterium is also described. See WO 94/00977 and US Pat. No. 5,591,616, incorporated herein by reference.

3. Transformation of Chromosome

Seeds of Nicotiana tabacum cv 'Xanthi nc' are germinated at 7 per plate in 1 "annular arrangements on T agar medium and 12-14 days after sowing are essentially as described in the literature [see : Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917] Impact with 1 μm tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145. Seeds were incubated for 2 days after the leaf was excised on T medium, and 500 μg / ml spectinomycin dihydrochloride (350-500 μmol photons / m ² / s) off-axis with bright light (350-500 μmol photons / m ² / s) On plates of RMOP medium (Sigma, St. Louis) (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) 3 to 8 weeks after impact. Resistant shoots that appear under the leaves turned into white on the same selective medium Roast, callus is formed, second shoot is isolated and subcloned Complete isolation of transformed chromatin genome copy (homoplasmicity) in independent subclones is analyzed by Southern blot standard techniques Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor.BamHI / EcoRI-cleaved whole cell DNA [Mettler, IJ (1987) Plant Mol Biol Reporter 5, 346-349] on a 1% tris-borate (TBE) agarose gel, transferred to a nylon membrane (Ammersham), and 0.7 kb BamHI / HindIII DNA fragment from pC8 containing the rps7 / 12 chromatin target sequence portion Probe with a ³² P-labeled random prime DNA sequence corresponding to Allogeneic shoots were grown on spectinomycin-containing MS / IBA medium [McBride, KE et al. (1994) PNAS 91, 7301-7305, aseptically treated to take root and transfer to the greenhouse.

E. Crossing and Seed Production

Example 26

mating

Plants obtained by transformation with the nucleic acid sequences of the invention can be a wide variety of plant species, including those of monocotyledonous and dicotyledonous plants. However, the plants used in the method of the present invention are preferably selected from the list of agricultural crops which are important for agricultural management as described above. Expression of the genes of the invention, along with other properties important for production and quality, can be incorporated into plant strains via crosses. Crossing approaches and techniques are known in the art. See, eg, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986)].

The genetic properties engineered into the transformed seeds and plants described above are inherited by sexual reproduction or asexual growth, and thus can be maintained and propagated in progeny plants. Maintenance and propagation generally leads to the use of known agricultural methods developed for specific purposes, such as tilling, sowing or harvesting. Specialized methods such as hydroponics or greenhouse technology can also be applied. Growing crops are susceptible to attack and damage caused by insects or infections, as well as competition by weed plants, so that methods for controlling weeds, plant diseases, insects, nematodes, and other adverse conditions that improve yield are performed. do. These include the application of agrochemicals such as herbicides, fungicides, spores, nematicides, growth regulators, ripening agents and insecticides, as well as mechanical methods such as tilling of soil or removal of weeds and infected plants.

Advantageous genetic properties of the transformed plants and seeds according to the invention can further be used in plant mating, which may include improved properties such as resistance to pests, herbicides or stress, improved nutritional value, increased yield, or collapse or It is aimed at the development of plants with an improved structure which reduces the loss from folding. Many mating steps are characterized by appropriately defined human intervention, such as the selection of the lines to be crosscrossed, the indication of the parental lines, or the selection of suitable offspring plants. Depending on the desired properties, different breeding methods are taken. Related arts are well known in the art and include, but are not limited to, hybrid crosses, winter crosses, back crosses, multiline crosses, variant combinations, cross-cross hybrids, dimerity techniques, and the like. Hybridization techniques also include the infertility of plants from which male or female infertility plants are obtained by mechanical, chemical or biochemical methods. The heterogeneous moisture of male infertility plants with different lines of pollen ensures that the male infertility genome, which is not a fertility plant, consistently obtains the characteristics of both parental lines. Thus, for example, improved plants and plants for transformed seeds and plants according to the present invention which increase the efficacy of conventional methods such as herbicides or pesticide treatment or eliminate the need for such methods due to their altered genetic properties. Can be used to cross lines. Alternatively, new crops with improved stress resistance can be obtained, and due to their optimized genetic “apparatus”, produce a higher quality harvest product than products that do not tolerate significant reverse development conditions.

Example 27

Seed production

In seed production, the germination quality and uniformity of the seed harvested and sold by the farmer is not important, whereas the germination quality and uniformity of the seed is an essential product property. Since it is difficult to keep crops free from other crops and weed seeds, to control seed-bearing diseases, and to produce seeds with good germination, the implementation of very extensive and suitably defined seed production can lead to the growth, management and It has been developed by seed producers with experience in the field of sales. Therefore, it is common for farmers to purchase certified seeds that meet certain quality standards instead of using seeds harvested from their own crops. Proliferative substances used as seeds are usually treated with a protective coating comprising herbicides, insecticides, fungicides, fungicides, nematicides, fungicides or mixtures thereof. Commonly used protective coatings include compounds such as captan, carboxycin, thiram (TMTD ^R ), metallaxyl (Apron ^R ) and pyrimifos-methyl (Actellic ^R ). If desired, these compounds can be formulated with additional carriers, surfactants or application-promoting adjuvants commonly used in the formulation art to protect against damage caused by bacterial, fungal or animal pests. The protective coating can be applied by impregnating the proliferative material with the liquid formulation or by coating the combined wet or anhydrous formulation. Other methods of application are also possible, such as treatment directed at the shoots or fruits.

A further aspect of the present invention is to provide a novel agricultural method, such as the method exemplified above, using a transformed plant, transformed plant material or transformed seed according to the invention.

The seed may be provided in a bag, container or container made of a suitable filling material, where the bag or container may be sealed to contain the seed. Bags, containers or containers can be designed for short or long term storage of seeds or both. Examples of suitable filler materials include paper, such as kraft paper, hard or soft plastic or other polymeric materials, glass or metal. Preferably the bag, container or container consists of multiple layers of the same or different kinds of filler material. In one embodiment, a bag, container or container is provided to exclude or limit water and moisture upon contact with the seed. In one example, the bag, container or container is sealed, for example, sealed to prevent the ingress of water or moisture. In another embodiment, the moisture absorbent material is disposed between or adjacent to the filler material layer. In another embodiment, the bag, container or container, or constituent fill material is treated to limit, inhibit or prevent disease, contamination or other adverse effects upon storage or transportation of the seed. Examples of such treatments are, for example, sterilization by chemical means or radiation exposure. Seeds of transformed plants comprising the genes of the invention expressed in plants transformed at higher levels than in wild-type plants by the present invention, together with suitable carriers, for their use to confer broad disease resistance to plants A commercial bag is constructed that includes the indication label.

The embodiments described above are exemplary. These techniques of the present invention will enable those skilled in the art to secure many variations of the present invention. All such obvious and foreseeable modifications are included within the scope of the appended claims.

<110> Novartis AG

<120> NOVEL INSECTICIDAL TOXINS FROM XENORHABDUS NEMATOPHILUS AND

NUCLEIC ACID SEQUENCES CODING THEREFOR

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atcgatgtga cggcagagta tttttattcc tgtaaactga cgacaatgca tttctaagat 60

atcaatataa taatgataaa tttattgatc atatatctgt tatattttga ttgaaaatta 120

ttgaatatac ctcttgtact aaattcatta catttttttt actttaaaca acattaaatt 180

cacacataat acagcttaaa tataacatgt gatatatatt atgattataa aaaacattaa 240

aataaataat acgccacata tattaacaat atctaattac tgatgatact attttctgag 300

tatatataaa tcttaaagaa aataattatt ttttatattt cacatcaatt taaaatctgc 360

ttagaatgcc ccccggcatc acaagaaaac aaaatcattc aagtaataca atagagttaa 420

atttaaaaat aacatgtata acaaaataca tagacaatta tacatgtaaa tgacagacaa 480

ctgacaaaac atagcaaaaa aacgccttaa atattaaggt atcaaaacaa tatatcagac 540

tatcttaaat ctaataggag aatccctc atg att aca ata cat atc agt ggt 592

Met Ile Thr Ile His Ile Ser Gly

1 5

ggt agt gta aca att aat aac aat ata gta aca gaa act gat gtc caa 640

Gly Ser Val Thr Ile Asn Asn Asn Ile Val Thr Glu Thr Asp Val Gln

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aat aca ccc gct tca gcg cct tta tca att act aat ttt agg gat atg 688

Asn Thr Pro Ala Ser Ala Pro Leu Ser Ile Thr Asn Phe Arg Asp Met

25 30 35 40

aca ata gaa cct cat tca tct gtt gag gcg ata aga acc gat aca ccg 736

Thr Ile Glu Pro His Ser Ser Val Glu Ala Ile Arg Thr Asp Thr Pro

45 50 55

att att cct gaa tca cga cca aat tac tat gtt gct aat tct ggc ccg 784

Ile Ile Pro Glu Ser Arg Pro Asn Tyr Tyr Val Ala Asn Ser Gly Pro

60 65 70

gcc tca tca gtc aga gct gtt ttc tat tgg tcc cac tct ttt aca tca 832

Ala Ser Ser Val Arg Ala Val Phe Tyr Trp Ser His Ser Phe Thr Ser

75 80 85

gaa tgg ttt gaa tct tcc tct att att gta aaa gca ggc gaa gac gga 880

Glu Trp Phe Glu Ser Ser Ser Ile Val Lys Ala Gly Glu Asp Gly

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gtc tta cat tca ccg ggt aat tct tta tat tac agc aag gtt gta att 928

Val Leu His Ser Pro Gly Asn Ser Leu Tyr Tyr Ser Lys Val Val Ile

105 110 115 120

tat aac gat aca gac aaa cgt gct ttt gtt acc ggc tac aat cta taa 976

Tyr Asn Asp Thr Asp Lys Arg Ala Phe Val Thr Gly Tyr Asn Leu

125 130 135

taac gcagaaatac aatccatatt tccaatgaat ttcaaataac atccttaagg 1030

caagaaacaa aatc atg aat aat gaa ccg atg aat act aat gaa tca 1077

Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser

1 5 10

caa gct tca gag ata gta ccc tca atg aat gaa tct ata tta gca gca 1125

Gln Ala Ser Glu Ile Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala

15 20 25

cct tat tca att tct aca cct aat tat gaa tgg gat atg tca tca ata 1173

Pro Tyr Ser Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile

30 35 40

ata aaa gat gct att att ggt ggt ata ggc ttt att cct ggt ccg ggc 1221

Ile Lys Asp Ala Ile Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly

45 50 55

tca gca ata tca ttt ttg tta ggg tta ttt tgg cca caa caa acc gac 1269

Ser Ala Ile Ser Phe Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp

60 65 70 75

aat act tgg gag caa att ctc caa aaa gta gaa caa atg atc gag caa 1317

Asn Thr Trp Glu Gln Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln

80 85 90

gcc aat ctc aaa act att caa gga ata ttg aac ggc gat ata caa gaa 1365

Ala Asn Leu Lys Thr Ile Gln Gly Ile Leu Asn Gly Asp Ile Gln Glu

95 100 105

att aaa ggc aaa atg gaa cat gtg caa ttc atg cta gaa tcc tca cct 1413

Ile Lys Gly Lys Met Glu His Val Gln Phe Met Leu Glu Ser Ser Pro

110 115 120

ggc act caa gaa agc cat gac gca tac atg ttt ctg gcg aga tat ctg 1461

Gly Thr Gln Glu Ser His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu

125 130 135

gtc agt ata gac gaa aaa ttc aag tct ttt gat aac aaa aca aat tat 1509

Val Ser Ile Asp Glu Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr

140 145 150 155

caa att ctt ccc atg tat acc aat acg att atg tta caa gcc cct tat 1557

Gln Ile Leu Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr

160 165 170

tgg aaa atg ggt ata gag aga aaa gat gag atc aaa cta aca gat ata 1605

Trp Lys Met Gly Ile Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile

175 180 185

gaa gtt aat gaa tta aaa gag ctg ata gga aaa tta tct acc agc gcc 1653

Glu Val Asn Glu Leu Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala

190 195 200

gat aaa tat att cat gat gtc tat act cgt gaa tat gat aat gcg atg 1701

Asp Lys Tyr Ile His Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met

205 210 215

aac act tca aca gca gca aat atc acc aat aat tta tta tct gta aga 1749

Asn Thr Ser Thr Ala Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg

220 225 230 235

ggc tat tgt tta tta cat ggt tta gaa tgt ctc gaa gtc att aac cat 1797

Gly Tyr Cys Leu Leu His Gly Leu Glu Cys Leu Glu Val Ile Asn His

240 245 250

ata caa aat aat agc ctt gag caa agt ttt tat cct aaa act atc agc 1845

Ile Gln Asn Asn Ser Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser

255 260 265

tac tcc acc gta ttc gat cgc cag aca aat aaa aca agg gtt caa gcc 1893

Tyr Ser Thr Val Phe Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala

270 275 280

ctg aca gaa gac gat caa atg caa gag cca ttc aag cct gct tta att 1941

Leu Thr Glu Asp Asp Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile

285 290 295

aat ggg aag tac aac aaa ata aaa tca ttg att ggg tat gta caa aga 1989

Asn Gly Lys Tyr Asn Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg

300 305 310 315

atc gga aac gca ccc aga gtt gga ggc att aaa gtc aca ttt gca aac 2037

Ile Gly Asn Ala Pro Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn

320 325 330

gat gca tct tat acc ctc ggt aca gta act tca gaa gta aac tca att 2085

Asp Ala Ser Tyr Thr Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile

335 340 345

gaa ctg aat gac agc gtt ata acc agc ctg gaa gta tgg gga aat ggc 2133

Glu Leu Asn Asp Ser Val Ile Thr Ser Leu Glu Val Trp Gly Asn Gly

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gct att gat gag gca ttc ttt aca tta agt gac gga cgt caa ttt agg 2181

Ala Ile Asp Glu Ala Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg

365 370 375

ctt ggc caa cgc tat gcc agt aac tat aga aaa tat gct gtc gat aac 2229

Leu Gly Gln Arg Tyr Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn

380 385 390 395

cac tat att tca gga ttg tac tta gcc agt gat gaa cct tca ttg gca 2277

His Tyr Ile Ser Gly Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala

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Gly Gln Ala Ala Gly Ile Ala Val Ser Tyr His Met Ile Ala Asp Lys

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aaa tca tagtattaa caggcttctg atttcagact aagcaagtaa gcggatcttc 2380

Lys ser

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Met Ile Thr Ile His Ile Ser Gly Gly Ser Val Thr Ile Asn Asn Asn

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Ile Val Thr Glu Thr Asp Val Gln Asn Thr Pro Ala Ser Ala Pro Leu

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Ser Ile Thr Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val

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Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn

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Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe

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Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser

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Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile

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Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser

20 25 30

Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Lys Asp Ala Ile

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Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe

50 55 60

Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln

65 70 75 80

Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln Ala Asn Leu Lys Thr

85 90 95

Ile Gln Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly Lys Met

100 105 110

Glu His Val Gln Phe Met Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser

115 120 125

His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu

130 135 140

Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met

145 150 155 160

Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile

165 170 175

Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile Glu Val Asn Glu Leu

180 185 190

Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His

195 200 205

Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala

210 215 220

Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu

225 230 235 240

His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser

245 250 255

Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe

260 265 270

Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp

275 280 285

Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn

290 295 300

Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro

305 310 315 320

Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr

325 330 335

Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile Glu Leu Asn Asp Ser

340 345 350

Val Ile Thr Ser Leu Glu Val Trp Gly Asn Gly Ala Ile Asp Glu Ala

355 360 365

Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr

370 375 380

Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn His Tyr Ile Ser Gly

385 390 395 400

Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly

405 410 415

Ile Ala Val Ser Tyr His Met Ile Ala Asp Lys Lys Ser

420 425

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Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn

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Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu

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Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val

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Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn

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Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe

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Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile

85 90 95

att gta aaa gca ggc gaa gac gga gtc tta cat tca ccg ggt aat tct 336

Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser

100 105 110

tta tat tac agc aag gtt gta att tat aac gat aca gac aaa cgt gct 384

Leu Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala

115 120 125

ttt gtt acc ggc tac aat cta taa 408

Phe Val Thr Gly Tyr Asn Leu

130 135

<210> 5

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<212> PRT

<213> Xenorhabdus nematophilus

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Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn

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Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu

20 25 30

Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val

35 40 45

Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn

50 55 60

Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe

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Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile

85 90 95

Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser

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115 120 125

Phe Val Thr Gly Tyr Asn Leu

130 135

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Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Val Ser Glu Ile

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Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser

20 25 30

aca cct aat tat gaa tgg gat atg tca tca ata ata aaa gat gcc att 144

Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Lys Asp Ala Ile

35 40 45

att ggt ggt ata ggc ttt att cct ggt ccg ggc tca gca ata tca ttt 192

Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe

50 55 60

ttg tta ggg tta ttt tgg cca caa caa acc gac aat act tgg gag caa 240

Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln

65 70 75 80

att ctc caa aaa gta gaa caa atg atc gag caa gcc aat ctc aaa act 288

Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln Ala Asn Leu Lys Thr

85 90 95

att caa gga ata ttg aac ggc gat ata caa gaa att aaa ggc aaa atg 336

Ile Gln Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly Lys Met

100 105 110

gaa cat gtg caa ttc atg cta gaa tcc tca cct ggc act caa gaa agc 384

Glu His Val Gln Phe Met Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser

115 120 125

cat gac gca tac atg ttt ctg gcg aga tat ctg gtc agt ata gac gaa 432

His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu

130 135 140

aaa ttc aag tct ttt gat aac aaa aca aat tat caa att ctt ccc atg 480

Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met

145 150 155 160

tat acc aat acg att atg tta caa gcc cct tat tgg aaa atg ggt ata 528

Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile

165 170 175

gag aga aaa gat gag ata aaa cta aca gat ata gaa gtt aat gaa tta 576

Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile Glu Val Asn Glu Leu

180 185 190

aaa gag ctg ata gga aaa tta tct acc agc gcc gat aaa tat att cat 624

Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His

195 200 205

gat gtc tat act cgt gaa tat gat aat gcg atg aac act tca aca gca 672

Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala

210 215 220

gca aat atc acc aat aat tta tta tct gta aga ggc tat tgt tta tta 720

Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu

225 230 235 240

cat ggt tta gaa tgt ctc gaa gtc att aac cat ata caa aat aat agc 768

His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser

245 250 255

ctt gag caa agt ttt tat cct aaa act atc agc tac tcc acc gta ttc 816

Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe

260 265 270

gat cgc cag aca aat aaa aca agg gtt caa gcc ctg aca gaa gac gat 864

Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp

275 280 285

caa atg caa gag cca ttc aag cct gct tta att aat ggg aag tac aac 912

Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn

290 295 300

aaa ata aaa tca ttg att ggg tat gta caa aga atc gga aac gca ccc 960

Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro

305 310 315 320

aga gtt gga ggc att aaa gtc aca ttt gca aac gat gca tct tat acc 1008

Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr

325 330 335

ctc ggt aca gta act tca gaa gta aac tca att gaa ctg aat gac agc 1056

Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile Glu Leu Asn Asp Ser

340 345 350

gtt ata acc agc ctg gaa gta tgg gga aat ggc gct gtt gat gag gca 1104

Val Ile Thr Ser Leu Glu Val Trp Gly Asn Gly Ala Val Asp Glu Ala

355 360 365

ttc ttt aca tta agt gac gga cgt caa ttt agg ctt ggc caa cgc tat 1152

Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr

370 375 380

gcc agt aac tat aga aaa tat gct gtc gat aac cac tat att tca gga 1200

Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn His Tyr Ile Ser Gly

385 390 395 400

ttg tac tta gcc agt gat gaa cct tca ttg gca ggt caa gca gca ggc 1248

Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly

405 410 415

att gca gtt tca tac cat atg ata gct gat aaa aaa tca tag 1290

Ile Ala Val Ser Tyr His Met Ile Ala Asp Lys Lys Ser

420 425

<210> 7

<211> 429

<212> PRT

<213> Xenorhabdus nematophilus

<400> 7

Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Val Ser Glu Ile

1 5 10 15

Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser

20 25 30

Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Lys Asp Ala Ile

35 40 45

Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe

50 55 60

Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln

65 70 75 80

Ile Leu Gln Lys Val Glu Gln Met Ile Glu Gln Ala Asn Leu Lys Thr

85 90 95

Ile Gln Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly Lys Met

100 105 110

Glu His Val Gln Phe Met Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser

115 120 125

His Asp Ala Tyr Met Phe Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu

130 135 140

Lys Phe Lys Ser Phe Asp Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met

145 150 155 160

Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile

165 170 175

Glu Arg Lys Asp Glu Ile Lys Leu Thr Asp Ile Glu Val Asn Glu Leu

180 185 190

Lys Glu Leu Ile Gly Lys Leu Ser Thr Ser Ala Asp Lys Tyr Ile His

195 200 205

Asp Val Tyr Thr Arg Glu Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala

210 215 220

Ala Asn Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu

225 230 235 240

His Gly Leu Glu Cys Leu Glu Val Ile Asn His Ile Gln Asn Asn Ser

245 250 255

Leu Glu Gln Ser Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe

260 265 270

Asp Arg Gln Thr Asn Lys Thr Arg Val Gln Ala Leu Thr Glu Asp Asp

275 280 285

Gln Met Gln Glu Pro Phe Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn

290 295 300

Lys Ile Lys Ser Leu Ile Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro

305 310 315 320

Arg Val Gly Gly Ile Lys Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr

325 330 335

Leu Gly Thr Val Thr Ser Glu Val Asn Ser Ile Glu Leu Asn Asp Ser

340 345 350

Val Ile Thr Ser Leu Glu Val Trp Gly Asn Gly Ala Val Asp Glu Ala

355 360 365

Phe Phe Thr Leu Ser Asp Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr

370 375 380

Ala Ser Asn Tyr Arg Lys Tyr Ala Val Asp Asn His Tyr Ile Ser Gly

385 390 395 400

Leu Tyr Leu Ala Ser Asp Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly

405 410 415

Ile Ala Val Ser Tyr His Met Ile Ala Asp Lys Lys Ser

420 425

<210> 8

<211> 408

<212> DNA

<213> Xenorhabdus poinarii

<220>

<221> CDS

<222> (1) .. (405)

<223> orf1 of pCIB9354

<400> 8

atg atc aca atc aat atc agt ggt ggt aat gta aca att aat aac aat 48

Met Ile Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn

1 5 10 15

atc agt tca gta acg gat atc caa aaa ccc ctt gat gca gaa ccc ctc 96

Ile Ser Ser Val Thr Asp Ile Gln Lys Pro Leu Asp Ala Glu Pro Leu

20 25 30

tca gtc acg aat tat aga gat ctg aca ata gag ccg cac tca tct att 144

Ser Val Thr Asn Tyr Arg Asp Leu Thr Ile Glu Pro His Ser Ser Ile

35 40 45

caa gca gac aga acg gac acc ccc att att cct gaa aca cgc cct gat 192

Gln Ala Asp Arg Thr Asp Thr Pro Ile Ile Pro Glu Thr Arg Pro Asp

50 55 60

tat tat atc gct aac tca ggc cct gct tca tca gtc aaa gct gtg ttt 240

Tyr Tyr Ile Ala Asn Ser Gly Pro Ala Ser Ser Val Lys Ala Val Phe

65 70 75 80

tat tgg tcg cat tcg ttt aca tcg gaa tgg ttc gag tat tca tct atc 288

Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Tyr Ser Ser Ile

85 90 95

acg gta aaa gca gga gaa gat gga ata tta aaa tca ccg agt aat gct 336

Thr Val Lys Ala Gly Glu Asp Gly Ile Leu Lys Ser Pro Ser Asn Ala

100 105 110

gta tat tac agt aaa gta gtc att tat aat gat aca gat aag cgg gct 384

Val Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala

115 120 125

ttt gtg act gga tat aac atg taa 408

Phe Val Thr Gly Tyr Asn Met

130 135

<210> 9

<211> 135

<212> PRT

<213> Xenorhabdus poinarii

<400> 9

Met Ile Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn

1 5 10 15

Ile Ser Ser Val Thr Asp Ile Gln Lys Pro Leu Asp Ala Glu Pro Leu

20 25 30

Ser Val Thr Asn Tyr Arg Asp Leu Thr Ile Glu Pro His Ser Ser Ile

35 40 45

Gln Ala Asp Arg Thr Asp Thr Pro Ile Ile Pro Glu Thr Arg Pro Asp

50 55 60

Tyr Tyr Ile Ala Asn Ser Gly Pro Ala Ser Ser Val Lys Ala Val Phe

65 70 75 80

Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Tyr Ser Ser Ile

85 90 95

Thr Val Lys Ala Gly Glu Asp Gly Ile Leu Lys Ser Pro Ser Asn Ala

100 105 110

Val Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala

115 120 125

Phe Val Thr Gly Tyr Asn Met

130 135

<210> 10

<211> 1056

<212> DNA

<213> Xenorhabdus poinarii

<220>

<221> CDS

<222> (1) .. (1053)

<223> JHE-like orf2 of pCIB9354

<400> 10

atg aat aat agt cca atg aat gat cag tta tca aca gcg cct tat tca 48

Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser

1 5 10 15

att tcg aca ccc aat tat gaa tgg gat atg tca tca atc ata aaa gat 96

Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp

20 25 30

gcc att atc ggt ggc ata gga ttt att ccc gga cca ggc cct gca atc 144

Ala Ile Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Pro Ala Ile

35 40 45

tct ttt tta tta gga ctg ttc tgg cca caa cag aca gac aat acc tgg 192

Ser Phe Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp

50 55 60

gat caa atc ctc caa aaa atc gaa caa atg ata gaa gaa gcg aat tta 240

Asp Gln Ile Leu Gln Lys Ile Glu Gln Met Ile Glu Glu Ala Asn Leu

65 70 75 80

aaa acc att aaa ggt ata tta aat gga gat ata caa gaa att aaa gga 288

Lys Thr Ile Lys Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly

85 90 95

aaa atg gac cat gtg aaa tct atg cta gag aat tct cct ggc agc cag 336

Lys Met Asp His Val Lys Ser Met Leu Glu Asn Ser Pro Gly Ser Gln

100 105 110

gaa agc cat gat gct tat atg ttt ctg gca agg ttt ttg gtc agt att 384

Glu Ser His Asp Ala Tyr Met Phe Leu Ala Arg Phe Leu Val Ser Ile

115 120 125

gat gaa aaa ttc aaa tct ttc gat gat aga aca aat tat caa att ctt 432

Asp Glu Lys Phe Lys Ser Phe Asp Asp Arg Thr Asn Tyr Gln Ile Leu

130 135 140

ccc atg tac acg aat aca att atg tta caa gcg cct tat tgg aaa atg 480

Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met

145 150 155 160

ggc atc gaa aag aaa gag gat atc ggt tta acc gat att gaa gtt ggt 528

Gly Ile Glu Lys Lys Glu Asp Ile Gly Leu Thr Asp Ile Glu Val Gly

165 170 175

gaa tta aaa gaa ctt atc gat aaa tta tat act aaa tca tat gat tat 576

Glu Leu Lys Glu Leu Ile Asp Lys Leu Tyr Thr Lys Ser Tyr Asp Tyr

180 185 190

atc aat aat acg tat aat cgt gaa tat aat aat gca atc aat acg tca 624

Ile Asn Asn Thr Tyr Asn Arg Glu Tyr Asn Asn Ala Ile Asn Thr Ser

195 200 205

acc gca gag agt atc acc aat aat tta ttg tct gtc aga gga tat tgt 672

Thr Ala Glu Ser Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys

210 215 220

tta tta cat ggt tgt gaa tgc ctt gaa gtt att gcg cat ata caa aac 720

Leu Leu His Gly Cys Glu Cys Leu Glu Val Ile Ala His Ile Gln Asn

225 230 235 240

aat agt ctt gat aaa ggc ttc tac cct aaa acg atc agc tat tcg agt 768

Asn Ser Leu Asp Lys Gly Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Ser

245 250 255

gtt ttc gat cgt cct aca aac aaa atg aga att cag gcg ctt aca gaa 816

Val Phe Asp Arg Pro Thr Asn Lys Met Arg Ile Gln Ala Leu Thr Glu

260 265 270

gat gac caa atg caa gaa ccg ttc aaa cct tct ttc gtc aat ggt caa 864

Asp Asp Gln Met Gln Glu Pro Phe Lys Pro Ser Phe Val Asn Gly Gln

275 280 285

tat aat aaa ata aaa tca ttg gag ggt tat gtc aca agg atc ggc aat 912

Tyr Asn Lys Ile Lys Ser Leu Glu Gly Tyr Val Thr Arg Ile Gly Asn

290 295 300

gcc ccc cga gtc ggc gga att aaa atc aca ttt gaa aac aac gca tct 960

Ala Pro Arg Val Gly Gly Ile Lys Ile Thr Phe Glu Asn Asn Ala Ser

305 310 315 320

tat act ctt ggc act gta act tca gaa aca acc tct att gaa ctc aat 1008

Tyr Thr Leu Gly Thr Val Thr Ser Glu Thr Thr Ser Ile Glu Leu Asn

325 330 335

gag agt gtt ata acc agc ata gaa gtg tgg gga gag tgg tgc cgt 1053

Glu Ser Val Ile Thr Ser Ile Glu Val Trp Gly Glu Trp Cys Arg

340 345 350

tga 1056

<210> 11

<211> 351

<212> PRT

<213> Xenorhabdus poinarii

<400> 11

Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser

1 5 10 15

Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp

20 25 30

Ala Ile Ile Gly Gly Ile Gly Phe Ile Pro Gly Pro Gly Pro Ala Ile

35 40 45

Ser Phe Leu Leu Gly Leu Phe Trp Pro Gln Gln Thr Asp Asn Thr Trp

50 55 60

Asp Gln Ile Leu Gln Lys Ile Glu Gln Met Ile Glu Glu Ala Asn Leu

65 70 75 80

Lys Thr Ile Lys Gly Ile Leu Asn Gly Asp Ile Gln Glu Ile Lys Gly

85 90 95

Lys Met Asp His Val Lys Ser Met Leu Glu Asn Ser Pro Gly Ser Gln

100 105 110

Glu Ser His Asp Ala Tyr Met Phe Leu Ala Arg Phe Leu Val Ser Ile

115 120 125

Asp Glu Lys Phe Lys Ser Phe Asp Asp Arg Thr Asn Tyr Gln Ile Leu

130 135 140

Pro Met Tyr Thr Asn Thr Ile Met Leu Gln Ala Pro Tyr Trp Lys Met

145 150 155 160

Gly Ile Glu Lys Lys Glu Asp Ile Gly Leu Thr Asp Ile Glu Val Gly

165 170 175

Glu Leu Lys Glu Leu Ile Asp Lys Leu Tyr Thr Lys Ser Tyr Asp Tyr

180 185 190

Ile Asn Asn Thr Tyr Asn Arg Glu Tyr Asn Asn Ala Ile Asn Thr Ser

195 200 205

Thr Ala Glu Ser Ile Thr Asn Asn Leu Leu Ser Val Arg Gly Tyr Cys

210 215 220

Leu Leu His Gly Cys Glu Cys Leu Glu Val Ile Ala His Ile Gln Asn

225 230 235 240

Asn Ser Leu Asp Lys Gly Phe Tyr Pro Lys Thr Ile Ser Tyr Ser Ser

245 250 255

Val Phe Asp Arg Pro Thr Asn Lys Met Arg Ile Gln Ala Leu Thr Glu

260 265 270

Asp Asp Gln Met Gln Glu Pro Phe Lys Pro Ser Phe Val Asn Gly Gln

275 280 285

Tyr Asn Lys Ile Lys Ser Leu Glu Gly Tyr Val Thr Arg Ile Gly Asn

290 295 300

Ala Pro Arg Val Gly Gly Ile Lys Ile Thr Phe Glu Asn Asn Ala Ser

305 310 315 320

Tyr Thr Leu Gly Thr Val Thr Ser Glu Thr Thr Ser Ile Glu Leu Asn

325 330 335

Glu Ser Val Ile Thr Ser Ile Glu Val Trp Gly Glu Trp Cys Arg

340 345 350

<210> 12

<211> 408

<212> DNA

<213> Photorhabdus luminescens

<220>

<221> CDS

<222> (1) .. (405)

<223> orf1 of pCIB9383-21

<400> 12

atg att aca atc aat atc act ggt gat aat gta aga gtt aat aac aat 48

Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn

1 5 10 15

ata gca aca gaa acc gac ctc caa aat aca cct gct tca gca ccc tta 96

Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu

20 25 30

tca att att aat ttt agg gat atg aca ata gaa cct cat tca tct gtt 144

Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val

35 40 45

gag gcg ata aga acc gat aca ccg att att cct gaa tca cga cca aat 192

Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn

50 55 60

tac tat gtt gct aat tct ggc ccg gcc tca tca gtc aga gct gtt ttc 240

Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe

65 70 75 80

tat tgg tcc cac tct ttt aca tca gaa tgg ttt gaa tct tcc tct att 288

Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile

85 90 95

att gta aaa gca ggc gaa gac gga gtc tta cat tca ccg ggt aat tct 336

Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser

100 105 110

tta tat tac agc aag gtt gta att tat aac gat aca gac aaa cgt gct 384

Leu Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala

115 120 125

ttt gtt acc ggc tac aat cta taa 408

Phe Val Thr Gly Tyr Asn Leu

130 135

<210> 13

<211> 135

<212> PRT

<213> Photorhabdus luminescens

<400> 13

Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn

1 5 10 15

Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu

20 25 30

Ser Ile Ile Asn Phe Arg Asp Met Thr Ile Glu Pro His Ser Ser Val

35 40 45

Glu Ala Ile Arg Thr Asp Thr Pro Ile Ile Pro Glu Ser Arg Pro Asn

50 55 60

Tyr Tyr Val Ala Asn Ser Gly Pro Ala Ser Ser Val Arg Ala Val Phe

65 70 75 80

Tyr Trp Ser His Ser Phe Thr Ser Glu Trp Phe Glu Ser Ser Ser Ile

85 90 95

Ile Val Lys Ala Gly Glu Asp Gly Val Leu His Ser Pro Gly Asn Ser

100 105 110

Leu Tyr Tyr Ser Lys Val Val Ile Tyr Asn Asp Thr Asp Lys Arg Ala

115 120 125

Phe Val Thr Gly Tyr Asn Leu

130 135

<210> 14

<211> 1320

<212> DNA

<213> Photorhabdus luminescens

<220>

<221> CDS

<222> (1) .. (1317)

<223> JHE-like orf2 of pCIB9383-21

<400> 14

atg aat aat gaa ccg atg aat act aat gaa tca caa gct tca gag ata 48

Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile

1 5 10 15

gta ccc tca atg aat gaa tct ata tta aat gaa tct ata tta aat gaa 96

Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu

20 25 30

tct ata tta gca gca cct tat tca att tct aca cct aat tat gaa tgg 144

Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser Thr Pro Asn Tyr Glu Trp

35 40 45

gat atg tca tca ata ata aaa gat gcc att att ggt ggt ata ggc ttt 192

Asp Met Ser Ser Ile Ile Lys Asp Ala Ile Ile Gly Gly Ile Gly Phe

50 55 60

att cct ggt ccg ggc tca gca ata tca ttt ttg tta ggg tta ttt tgg 240

Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe Leu Leu Gly Leu Phe Trp

65 70 75 80

cca caa caa acc gac aat act tgg gag caa att ctc caa aaa gta gaa 288

Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln Ile Leu Gln Lys Val Glu

85 90 95

caa atg atc gag caa gcc aat ctc aaa act att caa gga ata ttg aac 336

Gln Met Ile Glu Gln Ala Asn Leu Lys Thr Ile Gln Gly Ile Leu Asn

100 105 110

ggc gat ata caa gaa att aaa ggc aaa atg gaa cat gtg caa ttc atg 384

Gly Asp Ile Gln Glu Ile Lys Gly Lys Met Glu His Val Gln Phe Met

115 120 125

cta gaa tcc tca cct ggc act caa gaa agc cat gac gca tac atg ttt 432

Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser His Asp Ala Tyr Met Phe

130 135 140

ctg gcg aga tat ctg gtc agt ata gac gaa aaa ttc aag tct ttt gat 480

Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu Lys Phe Lys Ser Phe Asp

145 150 155 160

aac aaa aca aat tat caa att ctt ccc atg tat acc aat acg att atg 528

Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met Tyr Thr Asn Thr Ile Met

165 170 175

tta caa gcc cct tat tgg aaa atg ggt ata gag aga aaa gat gag ata 576

Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile Glu Arg Lys Asp Glu Ile

180 185 190

aaa cta aca gat ata gaa gtt aat gaa tta aaa gag ctg ata gga aaa 624

Lys Leu Thr Asp Ile Glu Val Asn Glu Leu Lys Glu Leu Ile Gly Lys

195 200 205

tta tct acc agc gcc gat aaa tat att cat gat gtc tat act cgt gaa 672

Leu Ser Thr Ser Ala Asp Lys Tyr Ile His Asp Val Tyr Thr Arg Glu

210 215 220

tat gat aat gcg atg aac act tca aca gca gca aat atc acc aat aat 720

Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala Ala Asn Ile Thr Asn Asn

225 230 235 240

tta tta tct gta aga ggc tat tgt tta tta cat ggt tta gaa tgt ctc 768

Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu His Gly Leu Glu Cys Leu

245 250 255

gaa gtc att aac cat ata caa aat aat agc ctt gag caa agt ttt tat 816

Glu Val Ile Asn His Ile Gln Asn Asn Ser Leu Glu Gln Ser Phe Tyr

260 265 270

cct aaa act atc agc tac tcc acc gta ttc gat cgc cag aca aat aaa 864

Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe Asp Arg Gln Thr Asn Lys

275 280 285

aca agg gtt caa gcc ctg aca gaa gac gat caa atg caa gag cca ttc 912

Thr Arg Val Gln Ala Leu Thr Glu Asp Asp Gln Met Gln Glu Pro Phe

290 295 300

aag cct gct tta att aat ggg aag tac aac aaa ata aaa tca ttg att 960

Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn Lys Ile Lys Ser Leu Ile

305 310 315 320

ggg tat gta caa aga atc gga aac gca ccc aga gtt gga ggc att aaa 1008

Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro Arg Val Gly Gly Ile Lys

325 330 335

gtc aca ttt gca aac gat gca tct tat acc ctc ggt aca gta act tca 1056

Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr Leu Gly Thr Val Thr Ser

340 345 350

gaa gta aac tca att gaa ctg aat gac agc gtt ata acc agc ctg gaa 1104

Glu Val Asn Ser Ile Glu Leu Asn Asp Ser Val Ile Thr Ser Leu Glu

355 360 365

gta tgg gga aat ggc gct gtt gat gag gca ttc ttt aca tta agt gac 1152

Val Trp Gly Asn Gly Ala Val Asp Glu Ala Phe Phe Thr Leu Ser Asp

370 375 380

gga cgt caa ttt agg ctt ggc caa cgc tat gcc agt aac tat aga aaa 1200

Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr Ala Ser Asn Tyr Arg Lys

385 390 395 400

tat gct gtc gat aac cac tat att tca gga ttg tac tta gcc agt gat 1248

Tyr Ala Val Asp Asn His Tyr Ile Ser Gly Leu Tyr Leu Ala Ser Asp

405 410 415

gaa cct tca ttg gca ggt caa gca gca ggc att gca gtt tca tac cat 1296

Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly Ile Ala Val Ser Tyr His

420 425 430

atg ata gct gat aaa aaa tca tag 1320

Met Ile Ala Asp Lys Lys Ser

435

<210> 15

<211> 439

<212> PRT

<213> Photorhabdus luminescens

<400> 15

Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile

1 5 10 15

Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu

20 25 30

Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser Thr Pro Asn Tyr Glu Trp

35 40 45

Asp Met Ser Ser Ile Ile Lys Asp Ala Ile Ile Gly Gly Ile Gly Phe

50 55 60

Ile Pro Gly Pro Gly Ser Ala Ile Ser Phe Leu Leu Gly Leu Phe Trp

65 70 75 80

Pro Gln Gln Thr Asp Asn Thr Trp Glu Gln Ile Leu Gln Lys Val Glu

85 90 95

Gln Met Ile Glu Gln Ala Asn Leu Lys Thr Ile Gln Gly Ile Leu Asn

100 105 110

Gly Asp Ile Gln Glu Ile Lys Gly Lys Met Glu His Val Gln Phe Met

115 120 125

Leu Glu Ser Ser Pro Gly Thr Gln Glu Ser His Asp Ala Tyr Met Phe

130 135 140

Leu Ala Arg Tyr Leu Val Ser Ile Asp Glu Lys Phe Lys Ser Phe Asp

145 150 155 160

Asn Lys Thr Asn Tyr Gln Ile Leu Pro Met Tyr Thr Asn Thr Ile Met

165 170 175

Leu Gln Ala Pro Tyr Trp Lys Met Gly Ile Glu Arg Lys Asp Glu Ile

180 185 190

Lys Leu Thr Asp Ile Glu Val Asn Glu Leu Lys Glu Leu Ile Gly Lys

195 200 205

Leu Ser Thr Ser Ala Asp Lys Tyr Ile His Asp Val Tyr Thr Arg Glu

210 215 220

Tyr Asp Asn Ala Met Asn Thr Ser Thr Ala Ala Asn Ile Thr Asn Asn

225 230 235 240

Leu Leu Ser Val Arg Gly Tyr Cys Leu Leu His Gly Leu Glu Cys Leu

245 250 255

Glu Val Ile Asn His Ile Gln Asn Asn Ser Leu Glu Gln Ser Phe Tyr

260 265 270

Pro Lys Thr Ile Ser Tyr Ser Thr Val Phe Asp Arg Gln Thr Asn Lys

275 280 285

Thr Arg Val Gln Ala Leu Thr Glu Asp Asp Gln Met Gln Glu Pro Phe

290 295 300

Lys Pro Ala Leu Ile Asn Gly Lys Tyr Asn Lys Ile Lys Ser Leu Ile

305 310 315 320

Gly Tyr Val Gln Arg Ile Gly Asn Ala Pro Arg Val Gly Gly Ile Lys

325 330 335

Val Thr Phe Ala Asn Asp Ala Ser Tyr Thr Leu Gly Thr Val Thr Ser

340 345 350

Glu Val Asn Ser Ile Glu Leu Asn Asp Ser Val Ile Thr Ser Leu Glu

355 360 365

Val Trp Gly Asn Gly Ala Val Asp Glu Ala Phe Phe Thr Leu Ser Asp

370 375 380

Gly Arg Gln Phe Arg Leu Gly Gln Arg Tyr Ala Ser Asn Tyr Arg Lys

385 390 395 400

Tyr Ala Val Asp Asn His Tyr Ile Ser Gly Leu Tyr Leu Ala Ser Asp

405 410 415

Glu Pro Ser Leu Ala Gly Gln Ala Ala Gly Ile Ala Val Ser Tyr His

420 425 430

Met Ile Ala Asp Lys Lys Ser

435

Claims (34)

(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, 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) An isolated nucleic acid molecule comprising a nucleotide sequence isocoding with the nucleotide sequence of (a) and producing one or more toxins expressed and active against insects. The nucleotide sequence of claim 1 wherein the nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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; Isolated nucleic acid molecule encoded. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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 isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 11, 13, and 15. 3. The isolated nucleic acid molecule of claim 1, wherein 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 isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is substantially similar to nucleotides 569-979, SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 12 of SEQ ID NO: 1. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9, or SEQ ID NO: 13. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence is substantially similar to nucleotides 1045-2334, SEQ ID NO: 6, SEQ ID NO: 10, or SEQ ID NO: 14 of SEQ ID NO: 1. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence encodes the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15. The isolated nucleic acid molecule of claim 1, wherein the nucleotide sequence comprises about 3.0 kb DNA fragment contained in pCIB9369 (NRRL B-21883). The isolated nucleic acid molecule of claim 1, wherein the toxin is active against Flutella xylostella. A nucleotide portion of 20 consecutive base pairs of a nucleotide sequence selected from the group consisting of nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334, 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; An isolated nucleic acid molecule comprising a nucleotide moiety of 20 base pairs of identical sequence and expressed to produce one or more toxins that are active against insects. A chimeric gene comprising a heterologous promoter sequence operably linked to the nucleic acid molecule of claim 1. A recombinant vector comprising the chimeric gene of claim 13. A host cell comprising the chimeric gene of claim 13. The host cell of claim 15 which is a bacterial cell. The host cell of claim 15 which is a yeast cell. The host cell of claim 15, which is a plant cell. A plant comprising the plant cell of claim 18. The plant of claim 19 which is corn. Toxin produced by the expression of a DNA molecule according to claim 1. The toxin of claim 21, wherein the toxin is active against flutella xylostella. 22. The method of claim 21, which is NRRL Accession No. B-21883. Toxin produced by E. coli strains. The toxin of claim 21 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 11, 13 and 15. The toxin of claim 24 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 13. The toxin of claim 24 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11 and 15. A composition comprising an effective amount of a pesticide according to claim 21. (a) obtaining a host cell according to claim 15, (b) expressing a nucleic acid molecule in a cell to produce at least one toxin active against the insect, wherein the toxin active against the insect is produced. A method for producing an insect-tolerant plant, characterized by introducing into the plant a nucleic acid molecule according to claim 1 or 12 which is expressible in an insect control effective amount in the plant. 30. The method of claim 29, wherein the insect is a flutella xylosteline. A method for controlling insects, characterized in that it delivers an effective amount of the toxin according to claim 21 to the insect. 32. The method of claim 31, wherein the insect is a flutella xylosteline. 33. The method of claim 32, wherein the toxin is orally delivered to the insect. (a) adding to the population of double-stranded random fragments one or more single-chain or double-stranded oligonucleotides each comprising the same and different regions for the double-stranded template polynucleotide, (b) denaturing the obtained mixture of double stranded random fragments and oligonucleotides with single stranded fragments, (c) in the identity region, where the resulting population of single-chain fragments, together with the polymerase, form a pair of annealed fragments, wherein the identity region is sufficient for one member of the pair to initiate replication of another member; Culture under conditions of annealing single chain fragments to form mutated double-stranded polynucleotides, (d) repeating the second and third steps with two or more additional cycles, such that the mixture obtained in the second stage of the further cycle comprises a double-stranded polynucleotide mutagenesis from the third stage of the previous cycle, To form additional mutagenesis double-stranded polynucleotides, 13. A method of causing mutation of a nucleic acid molecule according to claim 1 or 12 such that the nucleic acid molecule is cleaved into a population of double stranded random fragments of desired size.