JP2002512036A - A novel insecticidal toxin from Xenorhabdusnematophilus of the genus Xenorabdus and a nucleic acid sequence encoding the same - Google Patents

Abstract

  (57) [Summary] Disclosed herein are novel nucleic acid sequences isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens of the genus Xenorhabdus, whose expression results in a novel insecticidal toxin. The present invention also discloses compositions and formulations containing insecticidal toxins that can control insects. The invention further relates to a method for the production of toxins and to the use of nucleotide sequences in microorganisms, for example for controlling pest insects, or in transgenic plants for conferring insect resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to Xenorabdus nematophilus (Xenorh
abdus nematophilus), Xenorhabdus poinarii
) And Photolabdus luminescence (Photorhabd)
us luminescens), a nucleic acid sequence whose expression results in said toxin,
And methods of making and using toxins and corresponding nucleic acid sequences for controlling insects.

[0002] Insect pests are a major cause of crop damage. About $ 7.7 billion is lost annually in the United States alone due to infestations of insects of various genera. In addition to field crop damage, pests are a burden to vegetable and fruit growers, and to ornamental flower producers, and they are annoying to gardeners and homeowners. Pests can be controlled primarily by inhibiting insect growth, disrupting insect feeding or reproduction, or intensive application of chemical pesticides that act through the death of insects. Good insect control can be achieved in this way, but these chemicals can sometimes also affect other beneficial insects. Another problem resulting from the widespread use of chemical pesticides is the emergence of resistant insect varieties. This has been alleviated in part by various resistance management strategies, but the need for alternative pest control agents has increased. Bacillus thuringiensis strains of the genus Bacillus which express biological insect control agents, for example insecticidal toxins such as delta-endotoxin, have been applied with satisfactory results and are an alternative to chemical pesticides. Supplies or supplements. Recently, genes encoding some of these δ-endotoxins have been isolated and their expression in heterologous hosts provides another tool for the control of economically important pests. It is shown. In particular, expression of insecticidal toxins in transgenic plants, such as the delta-endotoxin of Bacillus thuringiensis of the genus Bacillus, provides effective protection against selected pests, and transgenic plants expressing such toxins have It has been commercialized and has reduced the need for farmers to apply chemical insect control agents. Furthermore, even in this case, the possibility that resistance develops remains, and only a few specific insect pests can be controlled. As a result, providing economic benefits to farmers,
And the need to find new and effective insect control agents that are environmentally acceptable has been long felt but has not yet been realized.

[0003] The present invention describes a longstanding need for new insect control agents. In particular, what is needed is a control agent that targets insect pests of economic importance and effectively controls insect strains that are resistant to existing insect control agents. In addition, agents that minimize the environmental impact of their application are desirable. In the search for new insect control agents, certain nematodes from the genus Heterorhabdus and the genus Steinernema of Steinernema are of particular interest because of their insecticidal properties. They kill insect larvae, and their children grow up in dead larvae. In fact, insecticidal activity is due to commensal bacteria that live in the nematode body. These commensal bacteria are Photorhabdus in the case of Heterorhabdus, and Xenorhabdus in the case of Steinernema.

The present invention relates to Xenorhabdus nematoph of the genus Xenorhabdus, the expression of which produces an insecticidal toxin which is very toxic to economically important pests, especially plant pests.
ilus, as well as nucleotide sequences substantially similar thereto. The present invention further includes insecticidal toxins due to expression of the nucleotide sequence and insecticidal toxins that can inhibit the ability of the pest to survive, grow or reproduce, or limit insect-related damage and damage in crop plants. It relates to compositions and formulations. The invention further relates to methods of producing toxins and methods of using nucleotide sequences, e.g., in microorganisms for controlling insects or in transgenic plants to confer insect resistance, and for example, in insect susceptible regions or Toxins and methods of using toxin-containing compositions and formulations to apply toxins, compositions or formulations to insect-infested areas to treat plants prophylactically or to provide protection or resistance to insect pests .

[0005] A new toxin is Plutella xylostella, an economically important pest.
ella xylostella (Pryllidae) is highly insecticidal. Toxins have been used in a number of insect control strategies and can produce maximum efficacy with minimal impact on the environment. According to one aspect, the invention relates to (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 an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the nucleotide sequence selected from the group consisting of SEQ ID NO: 14, or (b) a nucleotide sequence isocoding to the nucleotide sequence of (a), Provide a nucleic acid molecule whose expression results in at least one toxin active against insects. In one embodiment of this aspect, the nucleotide sequence is 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 Isocodes with a nucleotide sequence substantially similar to SEQ ID NO: 14. Preferably, the nucleotide sequence is nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-23 of SEQ ID NO: 1
34, 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 is
It 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 is SEQ ID NO: 1.
Nucleotides 569-979, 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. In another embodiment, the nucleotide sequence comprises an approximately 3.0 kb DNA fragment contained in pCIB9369 (NRRL B-21883).

According to the present invention, the toxin resulting from the expression of a nucleic acid molecule of the present invention is
Has activity against xylostella. In another aspect, the invention relates to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. 20, 25, 30, 35, 40, or 40, each having a sequence identical to a contiguous 20, 25, 30, 35, 40, 45 or 50 (preferably 20) base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: , 45 or 50 (preferably 20) base pair nucleotide moieties, the expression of which results in at least one toxin that is active against insects.

[0007] The invention also provides a chimeric gene comprising a heterologous promoter sequence operably linked to the nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector containing such a chimeric gene. Further, the present invention provides a host cell containing such a chimeric gene. The host cell of this aspect of the invention may be a bacterial cell, a yeast cell or a plant cell, preferably a plant cell. Further, the present invention provides a plant including such a plant cell. Preferably, the plant is maize.

[0008] In yet another aspect, the present invention provides a toxin produced by expression of a DNA molecule of the present invention. According to a preferred embodiment, the toxin of the present invention comprises Plutella x
Has activity against ylostella. In one embodiment, the toxin is produced by an E. coli strain designated NRRL accession number B-21883.

In another embodiment, the toxin of the invention comprises SEQ ID NOs: 2, 3, 5, 7, 9, 11, 1
An amino acid sequence selected from the group consisting of 3 and 15. Compositions comprising an insecticidal effective amount of a toxin of the invention are also provided. In another aspect, the invention relates to a method for producing a toxin that is active against insects, comprising: (a) a chimera which itself comprises a heterologous promoter sequence operably linked to a nucleic acid molecule of the invention. Providing a host cell containing the gene and (b) expressing the nucleic acid molecule in the cell to produce at least one toxin that is active against insects.

In yet another aspect, the present invention is a method of producing an insect resistant plant comprising introducing a nucleic acid molecule of the present invention into a plant, wherein the nucleic acid molecule is present in an amount effective to control insects. Methods are provided that are expressible in plants. According to a preferred embodiment, the insect is Plutella xylostella. In yet another aspect, the invention provides a method of controlling an insect, comprising delivering an effective amount of a toxin of the invention to the insect. According to a preferred embodiment, the insect is Plutella xylostella. Preferably, the toxin is delivered to the insect orally.

Yet another aspect of the invention is a method for mutating a nucleic acid molecule of the invention cleaved into a population of double-stranded random fragments of a desired size, comprising: )
Adding to the population of double-stranded random fragments one or more single or double-stranded oligonucleotides, each comprising a region of identical structure and a region of heterologous structure to the double-stranded template polynucleotide. (B) denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments, and (c) replicating the resulting population of single-stranded fragments with respect to one member of the pair to the other. Incubation with the polymerase under conditions that result in annealing of the single-stranded fragment in a region of the same structure sufficient to prime the pair of annealed fragments, thereby producing a mutated double-stranded polynucleotide. And (d) repeating the second and third steps at least two more times, wherein the mixture resulting from performing the second step one more time is a mixture of the previous third step Containing mutated double-stranded polynucleotide is the provision of a further method comprising the further resulting process when you run once mutated double-stranded polynucleotide.

[0012] Other aspects and advantages of the invention will be apparent to those skilled in the art from the following description and non-limiting examples of the invention. Definitions "Activity" of a toxin of the present invention means that the function of the toxin as an orally active insect control agent has a toxic effect or disrupts or prevents insect feeding, which may also cause insect death. I do. When the toxin of the invention is delivered to an insect, the result is typically the death of the insect, or the insect does not feed on a source that makes the toxin available to the insect.

“Associated / operably linked” refers to two nucleic acid sequences that are physically or functionally related. For example, two sequences are operably linked,
Alternatively, a promoter or regulatory DNA sequence is said to be "associated" with a DNA sequence that encodes an RNA or protein when the regulator DNA sequence is positioned to affect the level of expression of the coding or structural DNA sequence.

“Chimeric gene” refers to a promoter or regulatory nucleic acid sequence operable with a nucleic acid sequence that encodes mRNA or is expressed as a protein, such that the regulator nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid sequence. A recombinant nucleic acid sequence that is operably linked or associated. The regulator nucleic acid sequence of the chimeric gene is usually not operably linked to the associated nucleic acid sequence as found in nature.

“Coding sequence” refers to RNA, for example, mRNA, rRNA, tRNA, snR
A nucleic acid sequence that is transcribed into NA, sense RNA or antisense RNA. Preferably, the RNA is then translated in the organism to produce a protein. By "controlling" insects is meant inhibiting the ability of harmful insects to survive, grow, feed and / or reproduce by toxic effects, or limit insect-related damage or damage in crop plants. . "Controlling" an insect may or may not mean killing the insect, but preferably means killing the insect.

“Delivering” a toxin means that the toxin comes into contact with insects, which produces toxic effects and controls insects. Toxins can be produced by a number of known routes, for example, orally by ingestion by insects, or transgenic plant expression, formulated protein composition (s), sprayable protein composition (s), bait It can be delivered by contact with insects via a matrix or any other art-known toxin delivery system.

An “expression cassette,” as used herein, is a nucleic acid sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, operably associated with a stop codon. A nucleic acid sequence that includes a promoter operably linked to the nucleotide sequence to be linked is meant. It typically also includes sequences necessary for proper translation of the nucleotide sequence. The expression cassette containing the nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. The expression cassette may also be obtained in a recombinant form that is natural but useful for heterologous expression. However, typically, the expression cassette is heterologous with respect to the host, i.e.,
The particular nucleic acid sequence of the expression cassette is not naturally occurring in the host, and must have been introduced into the host cell or host cell ancestor by transformation events. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter, which initiates transcription only when the host cell is exposed to any particular external stimulus, or of an inducible promoter. In the case of multicellular organisms, such as plants, the promoter may further be specific for a particular tissue, organ or developmental stage.

A “gene” is a gene which is located in the genome and which, in addition to the coding sequences described above, is involved in the control of expression, in other words, other mainly regulatory nucleic acid sequences involved in the transcription and translation of the coding part. Is a limited area that includes The gene may also include other 5 'and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

“Gene of interest (of interest)” refers to any desired characteristic, such as antibiotic resistance, virus resistance, insect resistance, disease resistance or other pest resistance, herbicide tolerance when introduced into a plant. Refers to any gene that imparts improved nutritional value, improved performance in industrial processes, or altered fertility to a plant. The "gene of interest" can also be one that is transferred to a plant for the production of commercially valuable enzymes or metabolites in the plant.

A “heterologous” nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host cell into which it is introduced, for example, non-naturally occurring multiple copies of the native nucleic acid sequence. A "homologous" nucleic acid sequence is a nucleic acid sequence that is 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. "Insecticidal" means, preferably by killing insects,
It is defined as a toxic biological activity that can control them. A nucleic acid sequence "isocodes" with a reference nucleic acid sequence if the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

An “isolated” nucleic acid molecule or enzyme is a nucleic acid molecule or enzyme that exists by hand and is not a natural product of its original environment. An isolated nucleic acid molecule or enzyme can be in a purified form, or can be in a non-native environment, such as a recombinant host cell. A “nucleic acid molecule” or “nucleic acid sequence” is a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.

“ORF” means an open reading frame. A "plant" is any plant at any stage of development, especially a seed plant. "Plant cells" are the structural and physiological units of a plant, including protoplasts and cell walls. Plant cells may be in the form of isolated single cells or cultured cells, or may be present as part of a higher organizational unit, such as a plant tissue, plant organ or whole plant.

“Plant cell culture” refers to the culture of plant units, such as protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sac, zygotes and embryos at various stages of development. means. "Plant material" refers to a leaf, stem, root, flower or flower part, fruit, pollen, egg cell, zygote, seed, cut, cell or tissue culture, or any other part or product of a plant.

“Plant organs” are different and visually structured and differentiated parts of a plant, such as roots, stems, leaves, florets or embryos. “Plant tissue” as used herein refers to a group of plant cells organized into structural and functional units. In the plant kingdom or in culture
ure) plants. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures and any group of plant cells organized into structural and / or functional units. The use of this term, together with or in the absence of any particular type of plant tissue as described above or otherwise included in this definition, excludes any other type of plant tissue Not something.

A “promoter” contains a binding site for RNA polymerase II,
Untranslated DNA sequence upstream of the coding region that initiates DNA transcription. The promoter region may also include other elements that act as regulators of gene expression. "Protoplasts" are isolated plant cells that have no cell wall or have only a portion of the cell wall.

“Regulatory element” refers to a sequence involved in controlling the expression of a nucleotide sequence. Regulatory elements include a promoter operably linked to the nucleotide sequence and a stop codon. They typically include sequences necessary for proper translation of the nucleotide sequence. In its broadest sense, the term "substantially similar" as used herein in reference to a nucleotide sequence means a nucleotide sequence corresponding to a reference nucleotide sequence, in which case the corresponding sequence Is
Encode a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, for example, only those amino acid changes that do not affect polypeptide function occur. Desirably, a substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between substantially similar nucleotide sequences and the reference nucleotide sequence is desirably at least 80%, more desirably at least 85%.
, Preferably at least 90%, more preferably at least 95%, more preferably at least 99%. A nucleotide sequence "substantially similar" to the reference nucleotide sequence is obtained by washing in 2X SSC, 0.1% SDS at 50 ° C. while washing in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA. Hybridize with the reference nucleotide sequence at 50 ° C., more preferably at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, washing at 50 ° C. in 1 × SSC, 0.1% SDS. And more desirably in 0.5% SDS, 0.1% SDS at 50 ° C. while washing at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, preferably 0.1 × SSC
7% sodium dodecyl sulfate (SDS), 0.5M, washing at 50 ° C in 0.1% SDS
NaPO ₄ , 1 mM EDTA at 50 ° C., more preferably 65% in 0.1 × SSC, 0.1% SDS.
7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM ED while washing at
Hybridizes with the reference nucleotide sequence at 50 ° C. in TA.

“Synthetic” refers to a nucleotide sequence that includes structural features not present in the native sequence. For example, artificial sequences that more closely resemble the G + C content and normal codon distribution of dicot and / or monocot genes are said to be synthetic. “Transformation” is a method for introducing a heterologous nucleic acid into a host cell or organism. In particular, "transformation" refers to the stable integration of a DNA molecule into the genome of the organism.

“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 be present as an extrachromosomal molecule. Such extrachromosomal molecules can self-replicate. The transformed cell, tissue or plant is
It is understood that not only the final product of the transformation step, but also its transgenic progeny. A "non-transformed", "non-transgenic" or "non-recombinant" host refers to a wild-type organism, for example, a bacterium or plant that does not contain a heterologous nucleic acid molecule.

Nucleotides are designated by their base 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 (Prp; P), serine (S
er; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). Further, (Xaa; X) represents any amino acid.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING SEQ ID NO: 1 is the sequence of an approximately 3.0 kb DNA fragment contained in the Xenorhabdus nematophilus clone pCIB9369 containing the following ORFs at the indicated nucleotide positions: . Name start stop orf1 569 979 orf2 1045 2334 SEQ ID NO: 2 is the sequence of the 1515 kDa protein encoded by orf1 of clone pCIB9369.

SEQ ID NO: 3 is the sequence of a ホ ル モ ン 47.7 kDa juvenile hormone esterase-like protein encoded by orf2 of clone pCIB9369. SEQ ID NO: 4 is Xenorhabdus nematophilus
This is the DNA of orf1 of clone pCIB9381. SEQ ID NO: 5 is the sequence of the protein encoded by orf1 of clone pCIB9381.

[0033] SEQ ID NO: 6 is a copy of Xenorhabdus nematophil
us) DNA sequence encoded by orf2 of clone pCIB9381. SEQ ID NO: 7 is the sequence of the juvenile hormone esterase-like protein encoded by orf2 of clone pCIB9381. SEQ ID NO: 8 is the orf1 DNA of Xenorhabdus poinarii clone pCIB9354.

[0034] SEQ ID NO: 9 is the sequence of the protein encoded by orf1 of clone pCIB9354. SEQ ID NO: 10 is the DNA sequence of orf2 of Xenorhabdus poinarii clone pCIB9354. SEQ ID NO: 11 is the sequence of the juvenile hormone esterase-like protein encoded by orf2 of clone pCIB9354. SEQ ID NO: 12 is Photorhabdus luminesce
ns) DNA sequence of orf1 of clone pCIB9383-21.

[0035] SEQ ID NO: 13 is the sequence of the protein encoded by orf1 of clone pCIB9383-21. SEQ ID NO: 14 is Photorhabdus lumines
cens) DNA sequence of orf2 of clone pCIB9383-21. SEQ ID NO: 15 is the sequence of the juvenile hormone esterase-like protein encoded by orf2 of clone pCIB9383-21.

Deposits The following materials were obtained from the International Recognition of the Deposit of Microorganis
Under the terms of the Budapest Treaty on ms for the Purposes of Patent Procedure, Agricultural Research Service, Patent Culture Collection (NRRL), 1815
Deposited with North University Street, Peoria, Illinois 61604. All restrictions on the availability of the deposited material are removed at the time of grant and cannot be revoked.

Deposit date of clone number 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 1999 The present invention relates to nucleic acid sequences whose expression results in a novel toxin, and to the production and use of toxins for controlling pest insects. Nucleic acid sequences are isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens, a member of the Enterobacteriaceae family. Xenolabudus is a symbiotic bacterium of the nematodes of the genus Steinernema. Photolabdus is a nematode symbiotic bacterium of the genus Heterorhabditis. Nematodes colonize insect larvae and kill them, and their offspring feed on dead larvae. Insecticidal activity is actually produced by symbiotic xenolabdus and photolabdus bacteria. We are the first to isolate the nucleic acid sequences of the present invention. Expression of the nucleic acid sequences of the present invention may be performed by Lepidopteran insects, such as Plutel
This produces a toxin that can be used to control la xylostella.

The nucleotide sequence of the present invention in clone pCIB9369 is characterized by a DNA fragment of about 3.0 kb deposited according to the Budapest Treaty on Patent Deposit under accession number NRRL B-21883. The sequence of this DNA fragment is set forth in SEQ ID NO: 1. Two open reading frames (ORFs) are shown in SEQ ID NO: 1 (nucleotides 569-979 and 104).
5-2334) and encodes proteins of predicted size 15 kDa and 47.7 kDa (SEQ ID NOs: 2 and 3, respectively). The two ORFs are aligned in an operon-like structure. A search for known sequences showing homology to each individual ORF using the UWGCG Blast and Gap program did not reveal any significant matches for ORF # 1, and ORF # 2 and the Bacillus Bacillus thuringensis cry3A protein , But this is not considered to be significant in the art. Gap analysis of the protein encoded by ORF # 2 of pCIB9369 by the Blast program showed a 30.6% increase in juvenile hormone esterase related protein.
Identify% AA identity and 44.1% AA similarity (GenBank accession number 2921553; Heniko
ff et al., PNAS USA 89: 10915-10919 (1992)). The nucleotide sequence of the present invention comprises a known Xenorabdus nematophilus Xenorha which encodes the insecticidal toxin toxb4.
Comparison with the bdus nematophilus sequence (WO95 / 00647) also revealed no significant homology. The 3.0 kb DNA fragment is also compared to the nucleotide sequence published in WO98 / 08388. 60 nucleotides (60-nucleotide) from a 38.2 kb DNA fragment described in WO98 / 08388
mers) were converted using the UWGCG Gap program to the 3.0 kb DN of the invention.
Compare with fragment A. The nucleotide sequence of the first 60-mer starts at base 1 of the 38.2 kb DNA fragment, and the other 60-mers are located at about 2.0 kb intervals. Examine each of the 22 sequences, as well as their complementary sequences. The 3.0 kb DNA fragments of the present invention and their 60-m
The highest percent identity with one of the ers is 53%, which is not a significant homology. In addition, five different DNA fragments of the 38.2 kb sequence are tested for hybridization with the 3.0 kb fragment of the invention by Southern blot analysis. None of them reveal a positive hybridization signal.

The nucleotide sequences of pCIB9381, pCIB9354 and pCIB9383-21 also reveal two open reading frames in each of these clones. pCIB9381 and pCIB938
The nucleotide sequences of the two ORFs in each of 3-21 are highly homologous to those of pCIB9369. Therefore, the ORF # 2 protein of pCIB9381 and pCIB9383-21 has essentially the same homology to the juvenile hormone esterase-related protein, as does the ORF # 2 protein of pCIB9369. The nucleotide sequence of ORF # 1 of pCIB9354 is 77% identical to the nucleotide sequence of ORF # 1 of pCIB9369,
The nucleotide sequence of ORF # 2 of pCIB9354 is 79% identical to the nucleotide sequence of ORF # 2 of pCIB9369. The ORF # 2 protein of pCIB9354 also has homology to juvenile hormone esterase-related proteins (29.2% AA identity and 42.2% AA
Similarity).

In a preferred embodiment, the invention relates to SEQ ID NO: 1 whose expression results in an insecticidal toxin.
Nucleotides 569-979, 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. The present invention also includes a recombinant vector containing the nucleic acid sequence of the present invention. In such a vector, the nucleic acid sequence may preferably be included in an expression cassette that includes regulatory elements for expression of the nucleotide sequence in a host cell capable of expressing the nucleotide sequence. Such regulatory elements usually include a promoter and a stop codon, and preferably also include elements that enable efficient translation of a polypeptide encoded by a nucleic acid sequence of the present invention. Vectors containing nucleic acid sequences are usually capable of replication in particular host cells, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of the invention in host cells. In one embodiment, the host cell for such a vector is a microorganism, such as a bacterium, especially E. coli. In another embodiment, the host cell for such a recombinant vector is an endophytic or epiphytic plant. Preferred host cells for such vectors are eukaryotic cells, such as yeast, plant cells or insect cells. Plant cells, such as corn cells, are the most preferred host cells. In another preferred embodiment, such a vector is a viral vector and is used for the replication of a nucleotide sequence in a particular host cell, such as an insect cell or a plant cell. Recombinant vectors are also used for transforming the nucleotide sequences of the present invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells. In one embodiment, such a host cell is a prokaryotic cell. In a preferred embodiment, such host cells are eukaryotic cells, such as yeast cells, insect cells or plant cells. In a most preferred embodiment, the host cell is a plant cell, for example, a corn cell.

The nucleotide sequences of the present invention can be isolated using the techniques described in the Examples below or by PCR using the sequences listed in the sequences listed as a basis for constructing PCR primers. . For example, an oligonucleotide having a sequence of approximately the first and last 20-25 contiguous nucleotides of orf1 of SEQ ID NO: 1 (eg, nucleotides 569-588 and 957-976 of SEQ ID NO: 1) can be obtained from a source strain (Xenolabdusnemato Filos Xenorhabdus nematophilus ATCC 19061) can be used as a PCR primer to amplify the orfl coding sequence (nucleotides 569-976 of SEQ ID NO: 1) directly. Other gene sequences of the invention can be similarly amplified by PCR from the respective source strains using the ends of the coding sequences listed in the sequences listed as basis for PCR primers.

[0042] In another preferred embodiment, the insecticidal toxin includes at least one polypeptide encoded by a nucleotide sequence of the present invention. The molecular weight of the insecticidal toxins of the invention is greater than 6,000, as determined by size fractionation experiments. After treatment with proteinase K, only a minimal reduction in insecticidal activity is observed in the insect bioassay, indicating that the insecticidal toxin is substantially resistant to proteinase K treatment. The insecticidal toxin retains its insecticidal activity after being stored at 22 ° C. or 4 ° C. for two weeks. They retain their insecticidal activity even after being lyophilized and stored at 22 ° C. for 2 weeks. Insecticidal toxins are active after 5 minutes of incubation at 60 ° C, but after 5 minutes of incubation at 100 ° C or 80 ° C, they lose their insecticidal activity.

In yet another embodiment, the nucleotide sequences of the present invention may be modified by in vitro recombination or by incorporating random mutations in a technique known as DNA shuffling. This technology is described in Stemmer et al., Nature 370: 389-391 (
1994) and US Patent No. 5,605,793, the contents of which are incorporated herein by reference. Numerous mutant copies of the nucleotide sequence have been generated based on the original nucleotide sequence of the invention and have improved properties,
For example, mutants exhibiting increased insecticidal activity, enhanced stability, or different specificities or target insect pest ranges are recovered. The method includes generating a mutated double-stranded polynucleotide from a template double-stranded polynucleotide that has been cleaved into a double-stranded random fragment of a desired size, including the nucleotide sequence of the present invention, The resulting population of double-stranded random fragments is then provided with one or more single or double-stranded oligonucleotides comprising a region of identical structure and a region of heterologous structure to the double-stranded template polynucleotide. Adding; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;
Annealing of the single stranded fragment at the same structural region where one member of the pair is sufficient to prime the other replicates to produce a pair of annealed fragments, thereby producing a mutated double stranded poly Incubate with the polymerase under conditions that produce nucleotides; and repeat the second and third steps at least two more times, wherein the mixture resulting from one more second step is a mixture of the previous third step Comprising the step of producing a mutated double-stranded polynucleotide which, when run once more, results in an additional mutated double-stranded polynucleotide. In a preferred embodiment, the concentration of a single double-stranded random fragment in the population of double-stranded random fragments is:
Less than 1% by weight of total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 polynucleotides. In yet another preferred embodiment, the size of the double-stranded random fragment is between about 5 bp and 5 kb.
In a further preferred embodiment, the fourth step of the method comprises repeating the second and third steps at least 10 times.

Expression of Nucleotide Sequences in Heterologous Microbial Hosts As biological insect control agents, insecticidal toxins are produced by expression of nucleotide sequences in heterologous host cells capable of expressing the nucleotide sequences. In a first embodiment, a Xenorhabdus nematophilus, Xenorhabdus poinarii or Photorhabdus lum comprising at least one modification of the nucleotide sequence of the invention at its chromosomal location.
Inescens cells are described. Such modifications include mutations or deletions of existing regulatory elements, thus resulting in altered expression of the nucleotide sequence, or the incorporation of new regulatory elements that control the expression of the nucleotide sequence. In another embodiment,
By insertion into the chromosome, or by introduction of an extrachromosomal replicating molecule containing the nucleotide sequence, one or more additional copies of the nucleotide sequence are added to the Xenorhab
It is added to dus nematophilus, Xenorhabdus poinarii or Photorhabdus luminescens cells.

In another embodiment, at least one nucleotide sequence of the invention is inserted into a suitable expression cassette that includes a promoter and a stop codon. Expression of the nucleotide sequence is constitutive, or an inducible promoter is used that responds to various types of stimuli to initiate transcription. In a preferred embodiment, the cells in which the toxin is expressed are microorganisms, such as viruses, bacteria, fungi. In a preferred embodiment, the virus, e.g., a baculovirus, contains a nucleotide sequence of the invention in its genome and, after infection of a suitable eukaryotic cell suitable for viral replication and expression of the nucleotide sequence, a large amount of the corresponding killing agent. Express insect toxins. The insecticide toxin thus produced is used as an insecticide. Alternatively, a baculovirus engineered to contain a nucleotide sequence can be used in vivo.
And used to kill them by expression of insecticidal toxins or by a combination of viral infection and expression of insecticidal toxins.

Bacterial cells are also hosts for expression of the nucleotide sequences of the present invention. In a preferred embodiment, a non-pathogenic commensal bacterium capable of surviving and replicating in plant tissue, a so-called endoparasitic plant, or a non-pathogenic commensal bacterium capable of colonizing the phylosphere or rhizosphere, a so-called epiphytic plant, is used. Such bacteria include Agrobacterium (
Genus Agrobacterium, genus Alcaligenes, azospirillum (Azos)
pirillum), Azotobacter, Bacillus,
Clavibacter genus, Enterobacter genus, Erwinia genus, Flavobacter genus, Klebsiella (
Klebsiella, Pseudomonas, Rhizobium
Bacteria of the genus, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium, are also possible hosts for expression of the nucleotide sequences of the invention for the same purpose.

The techniques for these genetic manipulations are specific for different available hosts,
Known in the art. For example, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli in a transcription or translation fusion behind a tac or trc promoter. For expression of an operon encoding multiple ORFs, the simplest approach is to insert the operon into a vector, such as pKK223-3, by transcriptional fusion, to enable the use of the cognate ribosome binding site of a heterologous gene. That is. Techniques for overexpression in Gram-positive species such as Bacillus are also known in the art and can be used in the context of the present invention (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 are, for example, by yeast vectors and include the use of Pichia, Pichia, Saccharomyces Saccharomyces and Kluyveromyces Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied mole).
cular genetics, Baltz, Hegeman, and Skatrud eds., American Society for M
icrobiology, Washington (1993); Dequin & Barre, Biotechnology 12: 173-17
7 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990)).

In another preferred embodiment, Pseudomonas fluorescens CG of Pseudomonas spp. Wherein at least one of the nucleotide sequences described has bioregulatory characteristics.
It is transferred into the A267356 strain (described in published applications EU0472494 and WO94 / 01561) and expressed there. In another preferred embodiment, the nucleotide sequence of the present invention is transferred to strain Pseudomonas aureofaciens 30-84, which also has biocontrol characteristics. Expression in a heterologous control strain requires the selection of a suitable vector for replication in the selected host and the appropriate selection of a 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 Tissue In a particularly preferred embodiment, at least one of the insecticidal toxins of the invention is expressed in higher organisms, such as plants. In this case, the transgenic plant expressing an effective amount of the toxin will protect itself from pests. When an insect begins to eat such a transgenic plant, it also consumes the expressed toxin. This may cause the insects to hesitate to further bite the plant tissue, or even harm or kill the insects. The nucleotide sequence of the invention is inserted into an expression cassette,
It is then preferably stably integrated into the genome of said plant. In another preferred embodiment, the nucleotide sequence is contained in a non-pathogenic self-replicating virus. Plants transformed according to the present invention are monocotyledons or dicotyledons, such as corn, wheat, barley, rye, sweet potato, kidney beans, peas, chicory, lettuce, cabbage, cauliflower, Broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, toenas, pumpkin, asa, zucchini, apple,
Pear, Karin, Melon, Plum, Cherry, Peach, Nectarine, Apricot, Strawberry, Grape, Raspberry, Blackberry, Pineapple, Avocado, Papaya, Mango, Banana, Soybean, Tomato, Sugar Sorghum, Sugarcane, Sugar beet, Sunflower, Rapeseed , Clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis Arabidopsis and woody plants such as, but not limited to, conifers and deciduous trees.

Once the desired nucleotide sequence has been transformed in a particular plant species, it can be propagated in that species using traditional breeding techniques, or other variants of the same species, for example, commercially available Can be transferred during varieties. The nucleotide sequences of the present invention are preferably expressed in transgenic plants, thus resulting in the biosynthesis of the corresponding toxin in the transgenic plants. In this way, transgenic plants are generated that exhibit enhanced resistance to insects. For their expression in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. In many cases, genes from microorganisms can be expressed at high levels in plants without modification, but low expression in transgenic plants is due to microbial nucleotide sequences with unfavorable codons in plants. obtain. All organisms have specific preferences for codon usage,
It is known in the art that the codons of the nucleotide sequences described herein can be altered to suit plant preferences while retaining the amino acids encoded thereby. In addition, high expression in a plant can result in a coding sequence having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than about 50%, and most preferably greater than about 60%. Best achieved from Microbial nucleotide sequences having a low GC content may cause ATTT
It can be poorly expressed in plants due to the presence of the A motif, as well as the AATAAA motif, which can cause inappropriate polyadenylation. Preferred gene sequences can be expressed properly in both monocotyledonous and dicotyledonous plant species, but sequences have been shown to differ in their selection (Murray et al., Nucl. Acids Res. 17 : 477-4
98 (1989)) and can be modified to cause specific codon and GC content selection in monocots or dicots. In addition, nucleotide sequences are screened for the presence of illegitimate splice sites that can cause truncation of the message. All changes that need to be made within nucleotides such as those described above are published patent applications EP0385962 (Monsanto), EP0359472 (Lubrizol) and WO93.
/ 07278 (Ciba-Geigy) Site-directed mutagenesis, PC
This is done using well-known techniques of R and synthetic gene construction.

For efficient initiation of translation, the sequence adjacent to the initiation methionine requires modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi suggests proper consensus for plants (NAR 15:66
43-6653 (1987)), and Clontech suggest yet another consensus translation initiator (catalog 1993/1994, page 210). These consensuses are suitable for use with the nucleotide sequences of the present invention. The sequence may be up to and including the ATG (but leaving the unmodified second amino acid), or
The GTC after G, including up to and including the potential to modify the second amino acid of the transgene, is incorporated into the construct.

[0052] Expression of the nucleotide sequence in the transgenic plant is driven by a promoter that has been shown to be functional in the plant. The choice of promoter will also depend on the temporal and spatial requirements for expression, and also on the target species.
Thus, leaves, ears, inflorescences (eg, spikes, panicles
), Cobs, etc.), roots, and / or saplings are preferred. However, in many cases protection against more than one type of pest is sought, and expression in multiple tissues is therefore desirable. A number of promoters from dicots have been shown to be operable in monocots and vice versa, but ideally, dicot promoters are preferred for expression in dicots. And a monocot promoter is selected for expression in monocots. However, there is no restriction on the source of the selected promoter. In driving the expression of the nucleotide sequences in the desired cells, it is sufficient that they be operable.

Preferred constitutively expressed promoters include promoters from genes encoding actin or ubiquitin, and the CaMV 35S and 19S promoters. The nucleotide sequences of the present invention can also be expressed under the control of a chemically regulated promoter. This allows insecticidal toxins to be synthesized only when the crop plants are treated with the derived chemical. Preferred techniques for chemical induction of gene expression are detailed in published application EP0332104 (Ciba-Geigy) and US Pat. No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0054] A preferred category of promoters are those that are wound inducible. At the wound site,
A number of promoters that are also expressed at sites of phytopathogenic infection have been described. Ideally, such a promoter should only be locally active at the site of infection, in which case the insecticidal toxin would need to synthesize an insecticidal toxin to kill the invading insect pest. It only accumulates in some cells. Preferred promoters of this type include those described below: Stanford
et al., Mol. Gen. Genet. 215: 200-208 (1989); Xu et al., Plant Molec. B
iol. 22: 573-588 (1993); Logemann et al., Plant Cell 1: 151-158 (1989); R
ohmeler & Lehle, Plant Molec. Biol. 22: 783-792 (1993); Firek et al. Pl
ant Molec. Biol. 22: 129-142 (1993); and Warner et al. Plant J. 3: 191-2.
01 (1993).

Preferred tissue-specific expression patterns include green tissue-specific, root-specific, stem-specific and flower-specific patterns. Suitable promoters for expression in green tissue include many that regulate genes involved in photosynthesis, many of which have been cloned from both monocots and dicots. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxylate gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989).
)). A preferred promoter for root-specific expression is Framond (FEBS 290: 103-
106 (1991); EP0452269 (Ciba-Geigy)). Preferred stem-specific promoters are those described in U.S. Patent No. (Ciba-Geigy), which drives expression of the maize trpA gene.

A particularly preferred embodiment of the present invention is a transgenic plant expressing at least one of the nucleotide sequences according to the invention in a root-selective or root-specific manner. A further preferred embodiment is a transgenic plant that expresses the nucleotide sequence in a wound-inducing or pathogen-inducing manner. In addition to selecting an appropriate promoter, constructs for expression of insecticidal toxins in plants require an appropriate transcription terminator that is linked downstream of the heterologous nucleotide sequence. Several such terminators are available and known in the art (eg, tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of the present invention.

[0057] A number of other sequences can be incorporated into the expression cassettes described in the present invention. These are sequences that have been shown to enhance expression, such as intron sequences (
For example, from Adh1 and bronze1) and viral leader sequences (eg, TMV,
From MCMV and AMV). It is preferred that different cell localizations in the plant are targeted for expression of the nucleotide sequences of the invention. In some cases localization in the cytosol is preferred, while in other cases localization in some subcellular organelles is preferred. Subcellular localization of the transgene encoding enzyme is performed using techniques well known in the art. Typically, DNA encoding a target peptide from a known organelle targeting gene product is engineered and fused upstream of the nucleotide sequence. Many such target sequences are known for chloroplasts and have shown their function in heterologous constructs. Expression of the nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuole of a host cell. Techniques for accomplishing this are well known in the art.

Vectors suitable for plant transformation are described elsewhere herein.
For Agrobacterium-mediated transformation, a binary vector or a vector carrying at least one T-DNA border sequence is suitable, whereas for direct gene transfer any vector is suitable and only the construct is used. Containing linear DNA is preferred. For direct gene transfer, transformation with a single DNA species or co-transformation may be used (Schocher et al., Biotechnology
4: 1093-1096 (1986)). For both direct and Agrobacterium-mediated transfer, transformation is usually (but not always)
Attempts are made with selectable markers that can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (basta basta). Examples of such markers are neomycin phosphotransferase,
Hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2,2-dichloropropionate dehalogenase, acetohydroxy acid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, haloaryl nitrilase, protopolylinogen oxidase, acetyl -Coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyltransferase and β-
Glucuronidase. However, the selection of a selectable or screenable marker for plant transformation is not critical to the invention.

[0059] The recombinant DNA can be introduced into plant cells by a number of methods known in the art. One skilled in the art will understand that the choice of method will depend on the type of plant targeted for transformation. Suitable methods for transforming plant cells include microinjection (Crossway et al., Bio Techniques 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 (1988); For corn transformation, see Ishida et al.
al., Nature Biotechnology 14: 745-750 (June 1996)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984); Hayashimoto et al., P.
lant Physiol. 93: 857-863 (1990) (rice)) and Agracetus, Inc., Madis
on, Wisconsin and ballistic particle acceleration using equipment available from Dupont, Inc., Wilmington, Delaware (eg, Sanford et al., US Pat. No. 4,945,050; and McCabe et al., Biotechnology 6: 923-926). 1988) Weissinger et al.
., Annual Rev. Genet. 22: 421-477 (1988); Sanford et al., Particulate Sc.
ience and Technology 5: 27-37 (1987) (onion); Svab et al., Proc. Nat.
l. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al.,
Plant Physiol. 87: 671-674 (1988) (soybean); McCabe et al., Bio / Technolog.
y 6: 923-926 (1988) (soybean); Klein et al., Proc. Natl. Acad. Sci. USA
85: 4305-4309 (1988) (maize); Klein et al., Bio / Technology 6: 559.
-563 (1988) (maize); Klein et al., Plant Physiol. 91: 440-444 (1
988) (maize); Fromm et al., Bio / Technology 8: 833-839 (1990); and Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) (maize); Koz
iel et al., Biotechnology 11: 194-200 (1993) (maize); Shimamoto
et al., Nature 338: 274-277 (1989) (rice); Christou et al., Biotechnolo
gy 9: 957-962 (1991) (rice); Datta et al., Bio / Technology 8: 736-740 (19
90) (rice); European Patent Application No. 0 332 581 (Camogaya 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 P.
hysiol. 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: 11212-112.
16 (Dec. 1993) (sorghum); Somers et al., Bio / Technology 10: 1589.
-1594 (Dec. 1992) (oat); Torbert et al., Plant Cell Reports 14:
635-640 (1995) (oat); Weeks et al., Plant Physiol. 102: 1077-1084.
(1993) (wheat); Chang et al., WO94 / 13822 (wheat), and Nehra et.
al., The Plant Journal 5: 285-297 (1994) (see also wheat)). A particularly preferred set of embodiments for introducing recombinant DNA molecules into corn by microinjection bombardment is described in Koziel et al., Biotechnology 11:19.
4-200 (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 a protoplast transformation method for maize as disclosed in EP 0 292 435. Plant transformation has been attempted using single or multiple DNA species (ie, co-transformation),
And both of these techniques are suitable for use with peroxidase coding sequences.

[0060] In another preferred embodiment, the nucleotide sequence of the invention is transformed directly into the plastid genome. The main advantage of plastid transformation is that plastids can generally express bacterial genes without substantial modification, and that plastids can express multiple open reading frames under the control of a single promoter. . Plastid transformation techniques are described extensively in: US Patent No. 5,451,513.
Nos. 5,545,817 and 5,545,818, and PCT application WO 95/16783, and McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. Basic techniques for chloroplast transformation include, for example, using biolistics or protoplast transformation (eg, calcium chloride or PEG-mediated transformation) to remove regions of the cloned plastid DNA flanking the selectable marker. Transfection into a suitable target tissue together with the gene. A flanking region of 1-1.5 kb, called the targeting region, facilitates homologous recombination with the plastid genome, thus allowing for the replacement or modification of certain regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes that confer resistance to spectinomycin and / or streptomycin are used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P. et al. , and Ma
liga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, JM,
and Maliga, P. (1992) Plant Cell 4, 39-45). This is about 1/100 of the target leaf
At the frequency of shock, stable homoplasmic transformation occurred. The presence of a cloning site between these markers allowed the construction of a plastid targeting vector for the introduction of foreign genes (Staub, JM, and Maliga, P. (1993) EMBO
J. 12, 601-606). Substantial increase in transformation frequency is due to the replacement of the recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxin aminoglycoside-3'-adenyltransferase. (Svab, Z., and Maliga, P. (1993) Pr
oc. Natl. Acad. Sci. USA 90, 913-917). Heretofore, this marker has been successfully used for high frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids
Res. 19: 4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present invention. Typically, about 15-20 cell division cycles are required after transformation to reach the homoplastid state. Plastid expression, in which the gene is inserted into all thousands of copies of the circular plastid genome present in each plant cell by homologous recombination, is 10% of the total soluble plant protein
In order to allow expression levels that can easily be over 100%, a huge copy number, an advantage over nuclear expressed genes, is utilized. In a preferred embodiment, the nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of the desired plant host. Plants that are genetically homologous to the plastid genome containing the nucleotide sequence of the present invention are obtained and selectively enable high expression of the nucleotide sequence.

Formulation of Insecticidal Compositions The present invention also includes compositions containing at least one insecticidal toxin of the present invention. In order to effectively control pests, such compositions preferably contain a sufficient amount of toxin. Such amounts will vary depending on the crop to be protected, the particular pest targeted and on environmental conditions such as humidity, temperature or soil type. In a preferred embodiment, compositions containing insecticidal toxins include host cells that express the toxin without further purification. In another preferred embodiment, cells expressing insecticidal toxins are lyophilized prior to their use as insecticides. In another embodiment, the insecticidal toxin is engineered to be secreted from the host cell. If it is desired to purify the toxins from the host cells in which they are expressed, varying degrees of purification of the insecticidal toxins are achieved.

The present invention further encompasses the preparation of a composition containing at least one insecticidal toxin of the present invention that is intimately mixed with one or more compounds or compounds described herein. I do. The present invention also relates to a method of treating a plant, comprising applying the insecticidal toxin or a composition containing the insecticidal toxin to the plant. The insecticidal toxin is applied to the crop area in the form of a composition or to the plant to be treated, simultaneously or sequentially with yet another compound. These compounds can be fertilizers or micronutrient donors or other preparations that affect plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of some of these preparations, and, if desired, are common in the formulation industry. It can be used together with further carriers, surfactants or application-promoting adjuvants commonly used in the art. Suitable carriers and adjuvants can be solid or liquid, and can be any of the substances commonly used in the art of formulation, such as natural or regenerated inorganic materials, solvents, dispersants, wetting agents, tackifiers,
It may correspond to a binder or a fertilizer.

A preferred method of applying the insecticidal toxins of the present invention is by spraying the environment harboring the pests, such as soil, water or foliage of plants. The frequency and rate of application depends on the type and extent of erosion by the pests. Insecticidal toxins cause roots to penetrate through the soil by impregnating the location of the plant with the liquid composition (systemic action) or by applying the compound to the soil in solid form, for example in particulate form (soil application). Can penetrate plants through. The insecticidal toxins can be applied to the seeds by impregnating the seeds with a liquid formulation containing the insecticidal toxins, or by coating them with a solid formulation (coating). In special cases, further types of application are also conceivable, for example selective treatment of plant stems or buds. The insecticidal toxins may also be provided as baits located above or below the ground.

The insecticidal toxins are preferably used in unmodified form, together with adjuvants conventionally used in the formulation industry, and are therefore emulsified concentrates, coatable pastes, directly sprayable or Formulated in dilutable solutions, diluted emulsions, wettable powders, soluble powders, dusts, granules, and encapsulated, for example, in polymeric materials.
As with the nature of the composition, the method of application, such as spraying, atomizing, dusting, dusting or pouring, is chosen according to the intended purpose and the general environment.

Formulations, compositions or formulations containing the insecticide toxin, and, if appropriate, solid or liquid adjuvants, can be prepared in a known manner, eg, by incorporating the insecticide toxin with a bulking agent, eg, a solvent, a solid carrier, And, where appropriate, a surfactant compound (surfactant);
It is prepared by mixing and / or grinding homogeneously. Suitable solvents include aromatic hydrocarbons, preferably fractions having 8 to 12 carbon atoms, such as xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffin, alcohols And glycols and their ethers and esters 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 dimethylformamide, and epoxidized vegetable oils For example, epoxidized coconut oil or soybean oil, or water.

For example, the solid carriers used in dusts and dispersible powders are usually natural inorganic fillers, such as calcite, talc, kaolin, montmorillonite or attapulgite. Highly dispersed silica or highly dispersed absorbent polymers can be added to improve physical properties. Suitable granulated adsorptive carriers are of the porous type, for example pumice, crushed brick, sepiolite or bentonite, and suitable non-sorbing carriers are for example substances such as calcite or sand. In addition, a vast number of inorganic or organic pre-granulated substances, such as dolomite or ground plant residues, can be used.

Suitable surfactant compounds are nonionic, cationic and / or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" is also understood to include mixtures of surfactants. Suitable cationic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are alkali metal salts, alkaline earth metal salts, or unsubstituted or substituted ammonium salts of higher fatty acids (chains with 10 to 22 carbon atoms), such as oleic acid or stearic acid, or, for example, coconut oil or animal oil. Sodium or potassium salts of natural fatty acid mixtures obtained from oils. Fatty acid methyltaurine salts can also be used.

However, more often, so-called synthetic surfactants, in particular fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylaryl sulfonates are used. The fatty sulfonate or sulfate is usually in the form of an alkali metal salt, an alkaline earth metal salt, or an unsubstituted or substituted ammonium salt, and has an alkyl group having 8 to 22 carbon atoms including the alkyl portion of the alkyl group. , For example,
For example, the sodium or calcium salts of lignone sulfonic acid, dodecyl sulfonate, or fatty alcohol sulfate obtained from natural fatty acids.
These compounds also include the sulfuric acid esters of fatty alcohol / ethylene oxide adducts and the salts of sulfonic acids. The sulfonated benzimidazole derivative preferably contains two sulfonic acid groups and one fatty acid group having 8 to 22 carbon atoms. Examples of alkylaryl sulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzene sulfonic acid, dibutyl naphthalene sulfonic acid, or other rensulfonic acid / formaldehyde condensation products. Also suitable are corresponding phosphates, for example salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 mol of ethylene oxide.

The nonionic surfactant is preferably a polyglycol ether derivative of an aliphatic or cycloaliphatic alcohol, or of a saturated or unsaturated fatty acid and an alkyl phenol, said derivative comprising 3 to 30 glycols. Ether group and 8
It contains -20 carbon atoms in the (aliphatic) hydrocarbon portion and 6-18 carbon atoms in the alkyl portion of the alkylphenol.

Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with polyethylene glycol, ethylenediamine propylene glycol and alkyl polypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, The adduct contains from 20 to 250 ethylene glycol ether groups and from 10 to 100 propylene glycol ether groups. These compounds are usually
Contains 5 ethylene glycol units / propylene glycol units.

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

The cationic surfactant preferably has at least one C 8 as an N substituent.
Quaternary ammonium salts having an alkyl group of -C22 and a lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl group as another substituent. The salt is preferably in the form of a halide, methylsulphonate or ethylsulphate, for example stearyltrimethylammonium chloride or benzyldi (2-chloroethyl) ethylammonium bromide.

Surfactants commonly used in the formulation industry include, for example, “McCutcheon's Detergents
and Emulsifiers Annual, ”MC Publishing Corp. Ringwood, New Jersey, 1979
And Sisely and Wood, “Encyclopedia of Surface Active Agents,” Chem
ical Publishing Co., Inc. New York, 1980. Examples The invention will be further described with reference to the following detailed examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise indicated. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in the following references: Ausubel (e
d.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc.
(1994); T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY (1989); and TJ Silhavy, ML Berman, and LW Enquist, Experimen
ts with Gene Fusions, Cols Spring Harbor Laboratory, Cold Spring Harbor,
NY (1984).

A. Isolation of a nucleotide sequence whose expression results in a toxin active against lepidopteran insects The following strains are grown in nutrient broth: Cultures are grown for 24 hours under the same conditions for DNA isolation.

Xenorhabdus nematophilus ATCC 19061 strain Xenorhabdus nematophilus Ps1 strain, USDA isolate Xenorhabdus poinarii ATCC49122 strain Photorhabdus luminescens Ps5 strain, USDA isolate By conversion, Konaga P
Perform lutella xylostella (Px) bioassay (Biever and Boldt, Anna
ls of Entomological Society of America, 1971; Shelton et al., J. Ent. Sc
i. 26:17). Add 4 ml of feed to a 1 oz clear plastic cup (Bioserve product #
9051). Five new moth larvae from a feed-adapted laboratory colony are placed in each feed-containing cup and then covered with a white paper lid (Bioserve product # 9049).
Ten larvae are assayed per concentration. Cup tray at 72 # F for 3 days, 14:10
(Time) Light: Put in incubator in dark cycle. Next, the number of live larvae in each cup is recorded.

Example 3 Bioassay Results Broth of Xenorhabdus nematophilus strain ATCC19061 shows 100% mortality against Plutella xylostella (Px) in the insect bioassay of Example 2. Each of the broths of Xenorhabdus nematophilus Ps1, Xenorhabdus poinarii ATCC49122 and Photorhabdus luminescens Ps5 also shows 100% mortality against Plutella xylostella (Px) in the insect bioassay of Example 2.

Example 4 Construction of a Cosmid Library Freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA were added at 2 mg / m
Total DNA is isolated by treatment with 1 lysozyme for 30 minutes at 37 ° C. Add proteinase K to a final concentration of 100 μg / ml in 0.5% SDS and incubate at 45 ° C. The solution becomes clear and sticky. Raise the SDS concentration to 1% and add 300 mM NaCl and an equal volume of phenol-chloroform-isoamyl alcohol. Mix the sample gently for 5 minutes and centrifuge at 3K. This is repeated twice.
The aqueous phase is then mixed with 0.7 volumes of isopropanol and centrifuged. Wash the DNA pellet three times with 70% ethanol and gently resuspend in 0.5 × TE. 6 μg of D
NA is treated with 0.3 units of Sau3A / 1 μg of DNA in a volume of 100 μl at 37 ° C. for 3.5 minutes. The specimen is then heated at 65 ° C. for 30 minutes to inactivate the enzyme, and then incubated with 2 units of calf intestinal alkaline phosphatase at 37 ° C. for 30 minutes. The specimen is mixed with an equal volume of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed, mixed with 0.7 volumes of isopropanol and centrifuged. Resuspend the pellet at a concentration of 100 ng / ml in 0.5XTE.

Using the BamHI cloning site, the SuperCos Super
Prepare Cos cosmid vector (Stratagene, La Jolla, CA). Supercos, prepared at 100 ng / ml, is ligated with X. nematophilus DNA previously digested with Sau3A at a ratio of 2: 1 in a volume of 5 μl at 6 ° C. overnight. Gigapack as described by the supplier
The ligation mixture is packaged using XL III (Stratagene). Infect the packaged phage into XL-1MR E. coli cells (Stratagene) as described by the supplier. The cosmid library is plated on L-agar containing 50 μg / ml kanamycin and incubated at 37 ° C. for 16 hours. Collect 500 colonies on a fresh L-kan plate at a density of 50 colonies / plate. The cells are washed off with L broth, mixed with 20% glycerol and frozen at -80 ° C.

In the case of the 4.2 Mb large genome of X. nematophilus, it has an average size of 40 Kb
450 clones correspond to a 4-fold coverage of the genome. Thus, screening of 450 clones should find every gene with a 99% probability. Xenorhabdus nematophilus Ps1 strain, Xenorhabdus poinarii ATCC49122 strain and
A cosmid library from Photorhabdus luminescens Ps5 strain is constructed in a similar manner.

Example 5: Results of Cosmid Bioassay and Identification of Clones with Insecticidal Activity 400 E. coli clones from each cosmid library are screened by an insect bioassay that yields clones with activity against Plutella xylostella. An insecticidal cosmid clone from Xenorhabdus nematophilus ATCC 19061 is identified as pCIB9362. A 42 kb insecticidal cosmid clone from Xenorhabdus nematophilus strain Ps1 is identified as pCIB9379. Xenorhabdus
The 42 kb insecticidal cosmid clone from poinarii ATCC49122 strain is identified as pCIB9354. A 7 kb insecticidal cosmid clone from Photorhabdus luminescens strain Ps5 is identified as pCIB9383-21.

Example 6: Isolation of a subclone with insecticidal activity Clone pCIB9362 is digested with SacII and a 9 kb DNA fragment is isolated. This fragment is ligated to Bluescript cut with SacII. The ligation mixture is subjected to Molecular Cloning
(2nd edition, Sambrook et al.) In DH5α E. coli cells. The transformation mixture is plated on L agar + 100 μg / ml ampicillin and incubated at 37 ° C. overnight. Isolated colonies were picked with 100 μg / ml ampicillin and Molecul
Grow in L-broth containing plasmid DNA isolated by alkaline minipreps as described in ar Cloning second edition. The 9 kb SacII clone was identified as pCIB9362-3 and shows 100% mortality in a bioassay for P. coni. 3 μg of pCIB9362-3 was isolated and 37 units were obtained using 0.3 units of 1 μg of Sau3A / DNA.
Digest for 4, 6 and 8 minutes at 0 ° C and heat at 75 ° C for 15 minutes. Specimens are pooled and Ba
Ligation to pUC19 previously digested with mHI and treatment with calf intestinal alkaline phosphatase. The ligations are transformed into DH5α E. coli cells, plated on L agar containing Xgal / Amp as described in Molecular Cloning 2nd edition and grown at 37 ° C. overnight. White colonies are picked, grown in L-broth containing 100 μg / ml ampicillin and plasmid DNA and isolated as before. EcoR DNA
Digest with I / HindIII and sequence the new restriction pattern. Sequencing primers are ordered from Genosys Biotechnologies (Woodlands, TX). Perform sequencing using the chain terminator method and use Applied Biosystems
Sequencing is completed using an Inc. 377 type automatic DNA sequencer (Foster City, CA). The sequence is assembled using Sequencer 3.0 from Gene Codes Corporation (Ann Arbor, Michgan).

After identification of the restriction sites and possible ORFs, pCIB9362-3 was digested with ClaI and 3.
The 0 kb fragment is isolated, cloned in Bluescript and transformed in DH5α E. coli cells. The isolated colonies are grown as described above, and the plasmid DNA is isolated by the alkaline method. The subclone was identified as pCIB9369 and shows 100% mortality against P. coni in a bioassay. pCIB9369 has been deposited with the USDA ARS Patented Culture Collection on November 12, 1997 and is identified as NRRL B-21883.

The 20 kb fragment identified as CIB9381 is subcloned from cosmid pCIB9379 using NotI digestion. Example 7: Bioassay results An insect bioassay is performed on pCIB9369 on various insects.

[0084]

[Table 1]

[0085] These results indicate that the insecticidal toxin resulting from expression of the 3.0 kb nucleotide sequence in pCIB9369 is highly active against the moth Plutella xylostella. Bioassays for pCIB9381, pCIB9354 and pCIB9383-21 also show high insecticidal activity against Japanese moth.

Example 8: Size fractionation of insecticidal activity Xenorhabdus nematophilus cosmid clone pCIB9369 and S in E. coli host DH5
The tratagene's Blue Script vector is grown in medium containing 50% Terrific broth and 50% luria broth and supplemented with 50 μg / ml ampicillin. Shake flask cultures (300 ml in 1000 ml baffled flasks) are grown overnight at 37 ° C. at 250 RPM. Cultures of each strain are centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4 ° C. Resuspend the pelleted cells in 30 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. Using a Branson Model 450 sonicator, the enriched cells are crushed by sonicating eight times for about 10 seconds while cooling on ice between cycles. The sonicates are centrifuged in a Sorvall SS34 rotor at 6,000 RPM for 10 minutes at 4 ° C. The resulting supernatant is filtered through a 0.2μ filter. The pellet from the centrifuged sonicate is resuspended in 30 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0.

The 3 ml fraction of the filtrate is applied to a Bio-Rad Econo-Pac 10DG column pre-equilibrated with 10 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. Discard the flow-through collected during sample loading. 4 ml each of NaCl-Tris equilibration buffer
Separate the sample while adding it twice. The first three fractions are set aside for testing. The first fraction should contain all substances greater than about 6,000 weight mol. Subsequent fractions should contain less than 6,000 wmol.

A sample of the sonicated filtrate and the resuspended pellet after sonication, together with the three fractions from the 10DG column, are tested for activity on the newborn moth larvae in a surface contamination assay. The sonicated product from the 9369 specimen and the filtered supernatant of the first column fraction are highly active with Conger. The second and third column fractions from 9369 are not active. None of the specimens from DH5a using the Blue Script plasmid culture have any activity. These results indicate that Xenorhabdus nematophilus clone pCI
The insecticidal activity encoded for B9369, and homologs from pCIB9381, pCIB9354 and pCIB9383-21, suggests a molecular weight greater than 6,000.

Example 9 Stability of Insecticidal Activity 300 ml of Luria Broth supplemented with 100 μg / ml ampicillin are inoculated into pCIB9369 and grown at 37 ° C. overnight. Place the sample in a sterile 15 ml screw cap tube at 22 ° C and 4 ° C.
Store at ° C. One specimen is centrifuged, the supernatant is removed, lyophilized and stored at 22 ° C. After storing these specimens under these conditions for two weeks, bioassays are performed on Conger. The lyophilized material is resuspended in the same volume as before lyophilizing the specimen. Resuspend all samples with agitation. Another sample is processed at 100 ° C. for 5 minutes. Treatment Result 22 ° C. (2 weeks) ++ 4 ° C. (2 weeks) ++ Lyophilization (2 weeks) ++ 5 minutes at 100 ° C. na na = inactive Example 10: Heat inactivation of insecticidal activity Heat stability of toxin Confirm. E. coli pCIB9369 strain (E. coli host DH5a, Xenorhabdu
overnight culture containing 3.0 kb DNA of S. nematophilus) with Luria Broth and Terrific
Grow in a 50:50 mixture of broth. Culture was performed in culture tubes on test tube rollers.
Grow at 7 ° C. Place 1 ml specimens of each culture in 1.5 ml Eppendorf tubes, 60 ° C, 8
Leave at 0 ° C. After 5 minutes the specimen is removed and allowed to cool to room temperature. The specimen, along with the untreated portion of the culture, is assayed for Conger. 50 μl of the specimen are sprayed on the feed and dried, and the new moth larvae of Plutella are applied to the surface. The assay is incubated at room temperature for 5 days.

Untreated specimens and specimens treated at 60 ° C. result in 100% mortality. Specimens treated at 80 ° C. and feed-only controls produce no observable mortality. Example 11: Protease treatment of insecticidal activity Xenorhabdus nematophilus cosmid clone pCIB9369 and S in E. coli host DH5
The tratagene's Blue Script vector is grown in medium containing 50% Terrific broth and 50% luria broth and supplemented with 50 μg / ml ampicillin. Shake flask cultures (300 ml in 1000 ml baffled flasks) are grown overnight at 37 ° C. at 250 RPM. Cultures of each strain are centrifuged at 7,000 RPM in a Sorvall GS-3 rotor at 4 ° C. Resuspend the pelleted cells in 30 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. Using a Branson Model 450 sonicator, the enriched cells are crushed by sonicating eight times for about 10 seconds while cooling on ice between cycles. The sonicates are centrifuged in a Sorvall SS34 rotor at 6,000 RPM for 10 minutes at 4 ° C. The resulting supernatant is filtered through a 0.2μ filter. The 1 ml samples of Josei by the addition of CaCl ₂ is adjusted to 5 MMCA ++. Protease K (Gibco BRL; Gaithersbur
g, MD) to make 500 μg / ml. The specimen is incubated at 37 ° C. for 2 and 24 hours. A control specimen is prepared, Ca ++ added and incubated without protease.

The pellet from the centrifuged sonicate is resuspended in 30 ml of 50 mM NaCl, 25 mM Tris base, pH 7.0. Sonicated filtrate from pCIB9369 and pC incubated in the presence of 5 mM Ca ++
The IB9369 specimen produces 100% mortality for the newborn larva Plutella xylostella. The pCIB9369 specimen incubated with Protease K in addition to Ca ++ shows a slightly lower mortality rate of about 90% for moths.

Example 12: Sequence comparison using cry3A protein sequence and juvenile hormone esterase protein sequence The nucleotide sequence of pCIB9369 (SEQ ID NO: 1) is shown in nucleotides 569-979 and 104.
At 5-2334, two open reading frames (ORFs) are specified. ORF # 1 is UWGCG Bla
After a search using the st and Gap program, there is no homology with any sequence in Genbank. Gap analysis of the protein encoded by ORF # 2 of pCIB9369 by the Blast program identifies a somewhat non-significant homology (21% identity) with the Bacillus Bacillus thuringensis cry3A protein. However, the protein encoded by ORF # 2 of pCIB9369 by the Blast program (SEQ ID NO: 3)
Gap analysis revealed homology with juvenile hormone esterase-related protein (30.6% AA)
Identity and 44.1% AA similarity) (GenBank accession number 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 in each of these clones. pCIB9381 and pCIB938
The nucleotide sequences of the two ORFs in each of 3-21 are over 90% identical in total to those of pCIB9369. Therefore, the ORF # 2 protein of pCIB9381 and pCIB9383-21 has essentially the same homology to the juvenile hormone esterase-related protein, as does the ORF # 2 protein of pCIB9369. ORF of pCIB9354
The nucleotide sequence of # 1 is 77% identical to the nucleotide sequence of ORF # 1 of pCIB9369, and the nucleotide sequence of ORF # 2 of pCIB9354 is 79% identical to the nucleotide sequence of ORF # 2 of pCIB9369. The ORF # 2 protein of pCIB9354 also has homology to juvenile hormone esterase-related proteins (29.2% AA identity and
42.2% AA similarity).

Example 13: Sequence comparison of sequences from pCIB9369 and WO98 / 08388 The nucleotide sequence of which is 6
22 sequences of 0 nucleotides (60-mers) are obtained and compared to the nucleotide sequence of CIB9362-3 including pCIB9369. The first 60-mer starts at base 1 of the 38.2 kb DNA fragment and the other 60-mers are located on the DNA fragment at approximately 2.0 kb intervals. Their positions on the 38.2 kb DNA fragment are shown below: 1-60; 2,041-2,100; 4,021-4,080; 6,001-6,060; 8,041-8,100; 10,021-10,08
0; 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. Compare sequences using the UWGCG Gap program and check each of the 22 60-mer sequences, as well as their complementary sequences. The results of these alignments indicated that the highest percent identity was 53%, which is not a significant homology in the art.

Example 14 Southern Blot Analysis Using Probes Obtained from WO98 / 08388 Sequences Oligonucleotide pairs were converted to 38.2 kb DNA fragment published in WO98 / 08388.
Design to amplify NA fragments. Oligonucleotides are Genosys Biotechnol
Order from ogies (The Woodlands, Texas). Their positions in the 38.2 kb DNA fragment are shown below. Also listed are the sizes of those and the amplified PCR fragments: Positions 13,360-13,380 Size of PCR fragment amplified using VK1048 and VK1049: 2,120 bp VK1050: Positions 26,581-26,601 VK1051: Positions 28,537-28,560 Size of PCR fragment amplified using VK1050 and VK1051: 1,979 bp VK1052: Positions 18,901-18,921 VK1053: Size of PCR fragment amplified using positions 20,321-20,340 VK1052 and VK1053: 1,439 bp VK1054: Positions 34,261-34,281 VK1055: Positions 35,320-35,340 of PCR fragments amplified using VK1054 and VK1055 Size: Using a 1,079 bp Perkin-Elmer 9600 thermocycler, complete the PCR reaction under the following conditions: 94 ° C, 2 minutes; then 30 times at 94 ° C for 30 seconds; 54 ° C for 30 seconds; 72 ° C. 4 minutes. The specimen is
800 ng of Xenorhabdus nematophilus DNA, 0.1-0.5 μM each pair of oligonucleotides, 250 μM dNTPs, 5 U Taq polymerase and 1 × in a final volume of 100 μl
Contains buffer (Perkin-Elmer). The completed reaction was precipitated in ethanol,
Resuspend in TE and load on 1% SeaPlaque (FMC, Rockland, Maine) TBE gel. After electrophoresis, the fragments are excised from the gel after ethidium bromide staining and visualization under UV light. Melt the gel slices at 65 ° C., mix a 10 μl aliquot with 10 μl distilled water, boil for 5 minutes and place on ice. Next, 15 μl of Random Priming labeling buffer (GIBCO-BRL, Gaithersburg, MD) and 6 μl of dNTP mix (dCT
P-free), 80 μCi of α-dCT ³² P and 1 μl of Klenow are mixed. The labeling reaction is performed at room temperature for 60 minutes. Nick columns (Ph
armacia Biotech). The probe is boiled for 5 minutes and placed on ice.

DNA from Xenorhabdus nematophilus total DNA, cosmids pCIB9362 and pCIB9363 (these cosmids overlap over 25 kb and both contain a DNA fragment of CIB9369; pCIB9362 was used for subcloning), ClaI, S
Southern blots are performed by digesting DNA from subclones pCIB9362-3 (9 kb ScaII fragment) and pCIB9369 (2.96 kb ClaI fragment) digested with acII or HindIII. Run the digestion reaction on a 0.75% agarose TBE gel and run overnight. Take a picture and process the gel as described by Bio-Rad for blotting on a Zeta-Probe hybridization membrane. After blotting, bake out the membrane 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. After adding fresh solution and equilibrating to 67 ° C., add the radioactive probe described above and hybridize overnight. The membrane is placed in 2 × SSC, 0.5% SDS at 67 ° C. for 30 minutes, then 0.5 × S
Wash in SC, 0.5% SDS at 67 ° C. for 30 minutes. The membrane is exposed to the film for 1 hour and 3 hours. Develop the film. The results show that the PCR probe from the WO98 / 08388 sequence does not hybridize to the cosmid DNA or subclone DNA described in the present invention. However, using X. nematophilus, a strong hybridization signal is observed.

These results confirm the results of the sequence comparison and indicate that clone pCIB9369 differs from the nucleotide sequence described in WO 98/08388. B. Expression of a nucleic acid sequence of the invention in a heterologous microbial host Suitable microorganisms for heterologous expression of a nucleotide sequence of the invention are any microorganisms capable of colonizing a plant or rhizosphere. As such, they are directed into contact with pests. These include, for example, Gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, Gram-positive microorganisms,
Examples include the genus Bacillus, and the fungi Trichoderma, Gliocladium and the brewer's yeast Saccharomyces cerevisiae. Particularly preferred heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Ps
eudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, Bacillus cereus, Trichod
erma viride, Trichoderma harianum, Gliocladium virens and brewer's yeast S
accharomyces cerevisiae.

Example 19: Expression of nucleotide sequences in E. coli and other Gram-negative bacteria A number of genes have been expressed in Gram-negative bacteria in a heterologous manner. The expression vector pKK223-3 (Pharmacia catalog # 27-4935-01) allows for 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). A number of other expression systems have been developed for use in E. coli. The heat-inducible expression vector pPL (Pharmacea # 27-4946-01) uses a tightly regulated bacteriophage λ that allows for high level expression of the protein. The lac promoter provides another means of expression, but the promoter is not expressed at the same high levels as the tac promoter. The addition of a broad host range replicon to some of these expression system vectors allows expression of the nucleotide sequence in closely related Gram-negative bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia bacteria. For example, pLRKD211 (
Natl. Acad. Sci. USA 81: 5816-5820 (1984)) contains a broad host range replicon ori T that allows replication in a large number of Gram-negative bacteria.

In E. coli, induction by IPTG is required for expression of the tac (ie, trp-lac) promoter. This same promoter (eg, the broad host range plasmid pLRK)
When D211) is introduced into Pseudomonas, it is constitutively active without induction by IPTG. The trp-lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacteria for constitutive expression of such genes. Thus, the nucleotide sequence whose expression results in an insecticidal toxin is placed behind a strong constitutive promoter and is transferred to a plant or a bacterium with rhizosphere colonization properties, rendering the organism an insecticide. Other possible promoters can be used for constitutive expression of the nucleotide sequence in Gram-negative bacteria.
Examples of these include, for example, the pseudomonas regulatory genes gafA and lemA (WO94 / 0
1561), as well as the Pseudomonas savastanoi IAA 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 the best characterized. The promoter for the erythromycin resistance gene (ermR) from S. pneumoniae Streptococcus pneumoniae has been shown to be active in Gram-positive aerobic and anaerobic bacteria, as well as in E. coli (
Trieu-Cuot et al., Nucl. Acids Res. 18: 3660 (1990)). Yet another antibiotic resistance promoter from the thiostrepton gene has been used in Streptomyces cloning vectors (Bibb, Mol. Gen. Genet. 199: 26-36 (1985).
)). The shuttle vector pHT3101 is also suitable for expression in Bacillus (Lerec
lus, FEMS Microbiol Lett 60: 211-218 (1989)). A significant advantage of this approach is that many Gram-positive bacteria produce pollen that can be used in formulations that produce insecticides with a longer shelf life. Bacillus and Streptomyces are active migratory species of soil.

Example 21 Expression of Nucleotide Sequences in Fungi Trichoderma harianum and Gliocladium virens have been shown to provide varying levels of biocontrol in the field (US Pat. Nos. 5,165,928 and 4,996,157). (Cornell Research Foundation). Nucleotide sequences whose expression results in an insecticidal toxin can be expressed in such fungi. This can be accomplished by a number of methods well known in the art. One is protoplast-mediated transformation of fungi by PEG or electroporation-mediated techniques. Or,
Particle bombardment can be used to transform protoplasts or other fungal cells that have the ability to develop into rejuvenated mature structures. The vector pA, originally developed for the transformation of Aspergillus sp., Is now widely used for the transformation of fungi
N7-1 (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))
Is engineered to contain the nucleotide sequence. This plasmid is
Contains the E. coli hygromycin B resistance gene flanking the rgillus nidulans gpd promoter and trpC terminator (Punt et al., Gene 56: 117-124 (
1987)).

In a preferred embodiment, the nucleic acids of the invention are of the yeast brewer's yeast Saccharomyces.
cerevisiae. For example, pCIB9369, pCIB9381, pCIB9354 or pCI
Each of the two ORFs from B9383 is cloned in a separate vector with a GAL1 inducible promoter and a CYC1 terminator. Each vector has ampicillin resistance and a 2μ replicon. The vectors preferably differ in their yeast growth markers as well. The constructs are transformed separately and together in brewer's yeast. ORFs are expressed together and tested for protein expression and insecticidal activity.

C. The formulation isolation toxins insecticidal toxins, or it produces, and using encompasses active ingredient suspension or concentrate of cells described in the previous examples, producing insecticidal formulation I do. For example, pest insects can be controlled using E. coli cells expressing insecticidal toxins.
The formulations are manufactured in liquid or solid form. It is described below.

Embodiment 18 FIG. : Liquid formulation of insecticidal composition In the following examples, percentages of the composition are given by weight.

[0105]

[Table 2]

Emulsions of any required concentration can be produced from such concentrates by dilution with water.

[0107]

[Table 3]

[0108] These solutions are suitable for application in the form of microdroplets.

[0109]

[Table 4]

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

[0111]

[Table 5]

[0112] Easy-to-use dusts are obtained by intimately mixing the carrier with the active ingredient. Example 19: Solid formulation of an insecticidal composition In the following examples, percentages of the compositions are given by weight.

[0113]

[Table 6]

The active ingredient is thoroughly mixed with the adjuvant, and the mixture is triturated well with a suitable mill to produce a wettable powder that can be diluted with water to give a suspension of the desired concentration.

[0115]

[Table 7]

Dilution with water gives the emulsion of any required concentration from this concentrate.

[0117]

[Table 8]

Easy-to-use dusts are obtained by mixing the active ingredient with a carrier and comminuting the mixture with a suitable mill.

[0119]

[Table 9]

The active ingredient is mixed thoroughly with the adjuvant, ground and the mixture is then moistened with water. After extruding the mixture, it is dried in a stream of air.

[0121]

[Table 10]

The finely divided active ingredient is evenly applied in a mixer to kaolin moistened with polyethylene glycol. In this way, non-powder coated granules are obtained.

[0123]

[Table 11]

The finely divided active ingredient is thoroughly mixed with the adjuvant and diluted with water to produce a suspension concentrate that can produce a suspension of any desired concentration. D. Expression of nucleotide sequences in transgenic plants The nucleic acid sequences described in this application can be incorporated into plant cells using conventional recombinant DNA techniques. In general, this involves inserting the coding sequence of the invention into an expression system in which the coding sequence is heterogeneous (ie, not normally present) using standard cloning techniques known in the art. The vector contains the necessary elements for the transcription and translation of the inserted protein coding sequence. Many vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include viral vectors, such as the λ vector system,
λgtI1, λgtI0 and Sharon 4; plasmid vectors such as pBI121, pBR322
, PACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, p
LG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems. Components of the expression system may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be used.
The expression system described herein can be used to transform virtually any crop plant cell under appropriate conditions. Transformed cells can be regenerated in whole plants such that the nucleotide sequences of the present invention confer insect resistance on the transgenic plant.

Example 22 Modification of Coding Sequence and Flanking Sequences The nucleotide sequences described in this application may be modified for expression in a transgenic plant host. Host plants that express the nucleotide sequence and produce insecticidal toxins in their cells exhibit enhanced resistance to insect infestation and thus have good provisions to withstand crop losses associated with such infestation.

Transgenic expression in plants of genes obtained from microbial sources requires modification of those genes to achieve and optimize their expression in plants. In particular, bacterial ORFs that encode separate enzymes but are encoded by the same transcript in the original microorganism are best expressed in plants with respect to separate transcripts. To accomplish this, each microbial ORF is isolated separately and 5 ′ of the ORF.
The plant promoter sequence is cloned at the end and in a cassette providing a plant transcription terminator at the 3 'end of the ORF. The isolated ORF sequence is preferably
It includes a start ATG codon and a stop STOP codon, but may include additional sequences beyond the start ATG and stop STOP codon. In addition, the ORF is truncated, but still retains the required activity. Particularly for long ORFs, truncated versions that retain activity are preferred for expression in transgenic organisms. “Plant promoter” and “plant transcription terminator” shall mean promoters and transcription terminators that operate in plant cells. This includes promoters and transcription terminators obtained from non-plant sources, such as viruses (one example is cauliflower mosaic virus).

In some cases, no modifications to the ORF coding sequence and flanking sequences are required. It is sufficient to isolate the fragment containing the ORF and insert it downstream of the plant promoter. For example, Gaffney et al. (Science 261: 754-756)
(1993)), without modification of the coding sequence and with the Pseudomonas gene xbp upstream of the ATG and ybp downstream of the STOP codon, still with the nahGORF attached. The Pseudomonas nahG gene was successfully expressed in transgenic plants under the control of the terminator. Preferably, the small flanking microbial sequences are upstream of ATG and STOP.
It should remain linked downstream of the codon. Indeed, such a construction results from the availability of restriction sites.

In other cases, expression of a gene obtained from a microbial source may present a problem in expression. These problems have been well characterized in the art and are particularly common for genes obtained from certain sources, such as Bacillus. These issues also apply to the nucleotide sequences of the present invention, and modifications of these genes can be made using techniques well known in the art. The following problems can occur: Codon usage The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the use of codons in the cloned microbial ORF with the use of codons in plant genes (and, in particular, genes from the target plant) allows identification of codons in the ORF that are preferably to be changed. Typically, plant evolution has a strong propensity to select nucleotides C and G at the third base position of monocots, while dicots often use nucleotides A or T at this position. Modifying a gene to incorporate preferred codon usage for a particular target transgenic species overcomes many of the problems described below regarding GC / AT content and illegitimate splicing.

[0129] 2. GC / AT content Plant genes typically have a GC content of more than 35%. ORF sequences rich in A and T nucleotides can cause several problems in plants. First, the motif of ATTTA is thought to cause message destabilization and is found at the 3 'end of many short-lived mRNAs. Second, the occurrence of a polyadenylation signal, eg, AATAAA, at an inappropriate position in the message is likely to cause premature truncation of transcription. In addition, monocots may recognize AT-rich sequences as splice sites (see below).

[0130] 3. Sequences flanking the initiating methionine Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that the ribosome binds to the 5 'end of the message and probes for the first available ATG to initiate translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that microbial gene expression can be enhanced by inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210), which is incorporated herein by reference,
Suggests a sequence as a consensus translation initiator for expression of the E. coli uidA gene in plants. In addition, Joshi (NAR 15: 6643-6653 (1987))
The description of which is incorporated herein by reference) compares a number of plant sequences adjacent to the ATG and suggests another consensus sequence. In situations where difficulties are encountered in expressing the microbial ORF in plants, the inclusion of one of these sequences in the starting ATG may improve translation. In such cases, the last three nucleotides of the consensus sequence are not suitable for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences flanking the initiating methionine may vary between different plant species. A search for 14 corn genes located in the GenBank database provided the following results:

[0131]

[Table 12]

This analysis can be performed on the desired plant species into which the nucleotide sequence has been incorporated, as well as on the sequence adjacent to the ATG that is modified to incorporate the preferred nucleotide. 4. Removal of unorthodox splice sites Genes cloned from non-plant sources and not optimized for expression in plants are recognized in plants as 5 'or 3' splice sites and cleaved, Thus, it may also contain motifs that can generate truncated or deleted messages. These sites can be removed using techniques well known in the art.

Techniques for modifying coding sequences and flanking sequences are well known in the art. If the initial expression of the microbial ORF is low and it is deemed appropriate to alter the sequence as described above, construction of synthetic genes can be achieved by methods well known in the art. These are, for example, the published patent disclosures EP0385962 (Monsanto), EP035
9472 (Lubrizol) and WO 93/07278 (Ciba-Geigy), the contents of which are incorporated herein by reference. In most cases, short-term assay protocols (known in the art) are used prior to their transfer to transgenic plants.
Preferably, the expression of the gene construct is assayed using.

Example 23: Construction of a plant expression cassette A coding sequence intended for expression in a transgenic plant is first assembled into an expression cassette after a suitable promoter that can be expressed in the plant. The expression cassette may also include any additional sequences required or selected for expression of the transgene. Such sequences include transcription terminators, exogenous sequences to enhance expression, such as introns, life support sequences, and sequences intended to target gene products to particular organelles and cell compartments. However, it is not limited to these. These expression cassettes can then be easily transferred into the plant transformation vectors described below. The following is a description of the various components of a typical expression cassette.

[0135] 1. Promoter The selection of the promoter used in the expression cassette will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. The selection promoter expresses the transgene in a particular type of cell (eg, leaf epidermal cells, mesophyll cells, root cortical cells) or in a particular tissue or organ (eg, root, leaf or flower); And the choice reflects the desired location of accumulation of the gene product. Alternatively, a selection promoter can drive expression of the gene under various inducing conditions. Promoters differ in their strength, ie, their ability to promote transcription. Depending on the host cell system employed, any of a number of suitable promoters may be used, including the native promoter for the gene. The following are examples of, but not limited to, promoters that can be used in expression cassettes.

A. Constitutive expression, ubiquitin promoter: Ubiquitin is a gene product known to accumulate in many types of cells and its promoter has been cloned from several species for use in transgenic plants. (Eg, sunflower-Binet et al., Plant
Science 79: 87-94 (1991); Maize-Christensen et al., Plant Molec.
Biol. 12: 619-632 (1989); and Arabidopsis-Norris et al., Plant Mol.
Biol. 21: 895-906 (1993)). The corn ubiquitin promoter was developed in a transgenic monocot system, and its sequences and vectors constructed for monocot transformation are described in Patent Publication EP0342926 (Lubrizol), which is incorporated herein by reference. Included). Taylor et al. (Plant Cell Rep
12: 491-495 (1993)) is a vector containing the corn ubiquitin promoter and the first intron (pAHC25), and its presence in cell suspensions of multiple monocots when introduced by microinjection shock. Describe high activity. 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 both monocotyledonous and dicotyledonous transgenic plants. A suitable vector is pA
Either a derivative of HC25, or any of the transformation promoters described in this application, modified by the introduction of a suitable ubiquitin promoter and / or intron sequence.

B. Constitutive expression, CaMV 35S promoter: The construction of plasmid pCGN1761 is described in published patent application EP0392225 (Example 23), the contents of which are incorporated herein by reference. pCGN1761
Is a "double" C with a unique EcoRI site between the promoter and the terminator
Contains the aMV 35S promoter and tml transcription terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed that has a modified polylinker containing NotI and XhoI sites in addition to the existing EcoRI site. This derivative is called pCGN1761ENX. pCGN1761ENX is useful for cloning cDNA or coding sequences (including microbial ORF sequences) within its polylinker for their expression under the control of the 35S promoter in transgenic plants. All 35 of such constructs
The S promoter coding sequence-tml terminator cassette contains HindIII, Sph 5 ′ to the promoter, for example, for transfer into a transformation vector as described below.
It can be excised with I, SaII and XbaI sites, and XbaI, BamHI and BgII sites 3 ′ to the terminator. In addition, the double 35S promoter fragment was excised 5 ′ with HindIII, SphI, SaII, XbaI or PstI for replacement by another promoter, and a polylinker restriction site (EcoRI, NotI or XhoI).
By 3 'excision. If desired, modifications around the cloning site can be made by introducing sequences that can enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761ENX can be modified by optimization of the translation initiation site as described in Example 37 of US Pat. No. 5,639,949, which is incorporated herein by reference.

C. Constitutive expression, actin promoter: Several isoforms of actin are known to be expressed in most types of cells, and thus the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice ActI gene has 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 all the regulatory elements required for expression in rice protoplasts. In addition, a number of 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)). These are Act
Incorporate I-intron 1, AdhI 5 'flanking sequence and AdhI-intron 1 (from corn alcohol dehydrogenase gene), and the CaMV 35S promoter. Vectors showing the highest expression are 35S and ActI-intron or Ad
It is a fusion of the hI 5 'flanking sequence and AdhI-intron. Start ATG (GU
Optimization of surrounding sequences (of the S reporter gene) also enhanced expression. McElroy et al. (M
ol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for gene expression and is particularly suitable for use in monocot hosts. For example, the promoter-containing fragment is removed from the McElroy construct and used to replace the double 35S promoter in pCGN1761ENX, which is then available for insertion of specific gene sequences. The fusion gene thus constructed can then be transferred to a suitable transformation vector. In another report,
The rice ActI promoter, together with its first intron, has been shown to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12:
506-509 (1993)).

D. Inducible expression, PR-1 promoter: The dual 35S promoter in pCGN1761ENX can be replaced with any other promoter of choice that produces appropriately high expression levels. By way of example, U.S. Pat.No. 5,614,395
One of the chemically regulatable promoters described in the above paragraph can replace the dual 35S promoter. The promoter of choice will be cleaved from its source, preferably by restriction enzymes, but can alternatively be PCR amplified using primers bearing appropriate terminal restriction sites. PCR amplification should be done, in which case the promoter should be resequenced after cloning of the amplification promoter in the target vector to check for amplification errors. The chemical / pathogen regulatable tobacco PR-1a promoter was cut from plasmid pCIB1004 (for construction, see Example 21 of EP0332104, which is incorporated herein by reference) and the plasmid pCGN1761ENX Populated (Uknes e
t al., 1992). pCIB1004 is cleaved by NcoI and the resulting 3 ′ overhang of the linearized fragment is made blunt by treatment with T4 DNA polymerase.
The fragment is then cleaved by HindIII, and the resulting PR-1a promoter-containing fragment is gel purified and cloned into pCGN1761ENX with the double 35S promoter removed. This is accomplished by cleavage with XhoI and blunting with T4 polymerase, followed by HindIII cleavage and isolation of the large vector terminator containing the fragment into which the pCIB1004 promoter fragment is cloned. This is a PR
This results in a pCGN1761ENX derivative with the -1a promoter and tmI terminator, and an intervening polylinker with unique EcoRI and NotI sites. The selection coding sequence is inserted into this vector, and the fusion product (ie, promoter-gene-terminator) can then be transferred to any selection transformation vector, such as those described below. Various chemical modulators such as U.S. Pat.Nos. 5,523,311 and 5
The benzothiadiazole, isonicotinate and salicylic acid compounds disclosed in U.S. Pat. No. 6,614,395 can be used to induce expression of a selection coding sequence in plants transformed according to the present invention.

E. Inducible Expression, Ethanol-Inducible Promoter: Promoters inducible by certain alcohols or ketones, such as ethanol, can also be used to confer inducible expression of a coding sequence of the present invention. Such a promoter is, for example, the alcA gene promoter from Aspergillus nidulans of the genus Aspergillus (Caddick et al. (1998) Nat. Biotechnol.
16: 177-180). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factor in the presence of a chemical inducer. For purposes of the present invention, the CAT coding sequence (Cadd in plasmid palA: CAT containing the alcA gene promoter sequence fused to a minimal 35S promoter
ick et al. (1998) Nat. Biotechnol. 16: 177-180) are replaced by a coding sequence of the invention to produce an expression cassette having the coding sequence under the control of the alcA gene promoter. This is performed 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 a steroid hormone-based system is also contemplated. For example, a glucocorticoid-mediated induction system has been used (Aoyama and
Chua (1997) The Plant Journal 11: 605-612), a glucocorticoid such as a synthetic glucocorticoid, preferably dexamethasone, preferably 0.1 mM to 1 mM.
More preferably, application at a concentration in the range of 10 mM to 100 mM induces gene expression. For the purposes of the present invention, a luciferase gene sequence is replaced by a nucleic acid sequence of the invention, and the expression comprising the nucleic acid sequence of the invention under the control of six copies of the GAL4 upstream activation sequence fused to a 35S minimal promoter. Generate a cassette. This is performed using methods well known in the art. The trans-acting factor is a hormone-binding domain of the rat glucocorticoid receptor (Picard et al., (1988))
Transactivation domain of the herpesvirus protein VP16 (Trie
zenberg et al. (1988) Genes Devel. 2: 718-729) fused to the GAL4 DNA binding domain (Keegan et al. (1986)). Expression of the fusion protein is controlled by any promoter suitable for expression in plants known in the art or described herein. This expression cassette is also included in plants that contain a nucleic acid sequence of the invention fused to the 6XGAL4 / minimal promoter. Thus, tissue or organ specificity of the fusion protein is achieved, resulting in inducible tissue or organ specificity of the insecticidal toxin.

G. Root-specific expression: Another pattern of gene expression is root expression. A suitable root promoter is de F
ramond (FEBS 290: 103-106 (1991)) and published patent application EP0452269.
(This description is also included herein by reference). This promoter is transferred to a suitable vector, such as pCGN1761ENX, for insertion of the selection gene and subsequent transfer of the entire promoter-gene-terminator cassette into the transformation vector.

H. Wound-inducible promoter: Wound-inducible promoters are suitable for gene expression. A number of such promoters have been described (eg, Xu et al. Plant Molec. Biol. 22: 573-58).
8 (1993); Logemann et al. Plant Cell 1: 151-158 (1989); Rohrmeier & Leh
le, Plant Molec. Biol. 22: 783-792 (1993); Firek et al. Plant Molec. Bio
l. 22: 129-142 (1993); Warner et al. Plant J. 3: 191-201 (1993)), and all are suitable for use with the present invention. Logemann et al describe the 5 'upstream sequence of the dicotyledon potato wunI gene. Xu etc. are dicotyledon potatoes (pin2)
2 shows that the wound-inducible promoter from is active in monocotyledonous rice. In addition, Rohrmeier & Lehle describes the cloning of a maize Wipl cDNA that is wound inducible and can be used to isolate a cognate promoter using standard techniques. Similarly, Firek et al. And Warner et al. Describe a wound-inducing gene from the monocotyledon Asparagus officinalis that is expressed at local wounds and sites of pathogen infestation. Using cloning techniques well known in the art, these promoters can be transferred into a suitable vector, fused to the genes belonging to the present invention, and used to express these genes at plant wound sites.

I. Medullary-preferred expression: Patent application WO 93/07278, which is incorporated herein by reference, describes the isolation of a maize trpA gene that is selectively expressed in marrow cells. Gene sequences and promoters extending from the start of transcription to 11726 bp are shown.
Using standard molecular biology techniques, this promoter or a portion thereof
It can be transferred into a vector capable of replacing the S promoter, for example pCGN1761, and used to drive expression of the foreign gene in a pith-preferred manner. Indeed, the fragment containing the pith-preferred promoter or a portion thereof can be transferred to any vector and modified for use in transgenic plants.

J. Leaf-specific expression: The maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec. Biol. 12: 579-589 (1989)). Using standard molecular biology techniques, the promoter for this gene can be used to drive expression of any gene in a leaf-specific manner in transgenic plants.

K. Pollen-specific expression: WO 93/07278 describes the isolation of a maize calcium-dependent protein kinase (CDPK) gene expressed in pollen cells. The gene sequence and promoter extend up to 1400 bp from the start of transcription. Using standard molecular biology techniques, this promoter, or a portion thereof, is a vector in which it can replace the 35S promoter,
For example, it can be transferred to pCGN1761 and used to drive expression of a nucleic acid sequence of the invention in a pollen-specific manner.

[0147] 2. Transcription terminators A variety of transcription terminators are available for use in expression cassettes. These are involved in the termination of transcription across the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants, examples of which include the CaMV 35S terminator, the tmI terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocots and dicots. In addition, the native transcription terminator of the gene may be used.

[0148] 3. Sequences for Enhancing or Regulating Expression A number of sequences have been found to enhance gene expression from within transcription units, and these sequences may be used to enhance their expression in transgenic plants. It can be used with the gene of the invention. Various intron sequences have been shown to enhance expression, especially in monocot cells. For example, it has been found that the intron of the maize AdhI gene, when introduced into maize cells, significantly enhances the expression of the wild-type gene under its cognate promoter. Intron 1 has been found to be particularly effective and has enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987).
)). In the same experimental system, the intron from the maize bronze1 gene was
A similar effect was exhibited upon enhancing expression. Intron sequences are routinely incorporated into plant transformation vectors, typically within a non-translated leader.

A number of non-translated leader sequences derived from viruses are also known to enhance expression, and are particularly effective in monocot cells. In particular, leader sequences from tobacco mosaic virus (TMV, "W sequence"), maize yellow spot virus (MCMV) and alfalfa mosaic virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al. Nucl Acids Res. 15: 8693-
8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).

[0150] 4. Targeting gene products in cells Various mechanisms for targeting gene products are known to exist in plants, and the sequences that control the function of these mechanisms have been characterized in some detail. ing. For example, the targeting of gene products to chloroplasts is controlled by signal sequences found at the amino terminus of various proteins that are cleaved during chloroplast transfer to produce mature proteins (eg, Comai).
et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused with a heterologous gene product to perform the transfer of the heterologous product into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). The DNA encoding the appropriate signal sequence is 5 'of the cDNA encoding the RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein, and many other proteins known to be localized chloroplasts. It can be isolated from the termini (see also "Expression by Chloroplast Targeting" in Example 37 of US Patent No. 5,639,949).

Other gene products are localized to other organelles, such as mitochondria and peroxisomes (eg, Unger et al. Plant Molec. Biol. 13: 4
11-418 (1989)). The cDNAs encoding these products can also be manipulated to perform targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPase and mitochondrial specific aspartate aminotransferase isoforms. The targeting cell protein body is described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82:65
12-6516 (1985)).

In addition, sequences that cause targeting of the gene product to other cellular compartments have been characterized. Amino-terminal sequences are involved in targeting ER, apoplasts, and extracellular secretion from aleurone cells (Koehler
& Ho, Plant Cell 2: 769-783 (1990)). In addition, the amino terminal sequence, together with the carboxy terminal sequence, is involved in vacuolar targeting to gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

The transgene product can be directed to any organelle or cell compartment by fusing the appropriate targeting sequence with the transgene sequence. For example, for chloroplast targeting, a chloroplast signal sequence from the RUBISCO gene, CAB gene, EPSP synthase gene or GS2 gene is fused in frame to the amino-terminal ATG of the transgene. The signal sequence chosen should include a known cleavage site, and the constructed fusion should take into account any amino acids after the cleavage site required for cleavage. In some cases, this requirement is due to the addition of a few amino acids between the cleavage site and the transgene ATG.
Alternatively, it may be satisfied by substitution of some amino acids in the transgene sequence. Fusions constructed for chloroplast transfer were obtained by chloroplast uptake by in vitro translation of in vitro transcribed constructs, followed by Bartlett et al. (Edelmann et al. (Eds.) Metho.
ds in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982)) and the efficacy of in vitro chloroplast uptake using the technique described by Wasmann et al. (Mol. Gen. Genet. 205: 446-453 (1986)). Can be inspected. These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The mechanism described above for cell targeting, not only with their cognate promoters, but also with heterologous promoters, under the transcriptional control of promoters that have a different expression pattern than the promoters driven by the targeting signal It can be used to perform specific cell targeting goals.

Example 24: Construction of Plant Transformation Vectors A large number of transformation vectors available for plant transformation are known to those skilled in the plant transformation art, and the genes belonging to the invention It can be used with a vector. The choice of vector will depend on the preferred transformation technique and target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be selected. Selectable markers routinely used for transformation include nptII, which confers resistance to kanamycin and related antibiotics.
Gene (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature
304: 184-187 (1983)), a bar that confers resistance to the herbicide phosphinothricin
Gene (White et al., Nucl. Acids Res. 18: 1062 (1990); Spencer et al.,
Theor. Appl. Genet. 79: 625-631 (1990)), the hph gene that confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell Biol. 4:29).
29-2931), and the dhfr gene (Bouro, which confers resistance to metatrexate)
uis et al., EMBO J. 2 (7): 1099-1104 (1983)), and the EPSPS gene that confers resistance to glyphosate (US Pat. Nos. 4,940,935 and 5,188,642).
No.).

[0156] 1. Vectors Suitable for Agrobacterium Transformation Numerous vectors are available for transformation of Agrobacterium with Agrobacterium tumefaciens. These typically have at least one TD
Possesses the NA border sequence, for example, pBIN19 (Bevan, Nucl.
ds Res. (1984)) and pXYZ. The following describes the construction of two exemplary vectors suitable for Agrobacterium transformation.

A. pCIB200 and pCIB2001: The binary vectors pCIB200 and pCIB2001 are used to construct recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS
75kan was made by NarI digestion of pTJS75 (Schmidhauser & Helinski, J. Bac
teriol. 164: 446-455 (1985)), excision of the tetracycline resistance gene,
The subsequent insertion of the AccI fragment from pUC4K carrying NPTII is possible (Messing
& Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (19)
83); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). XhoI
The linker was ligated to the EcoRV fragment of PCIB7 containing the left and right T-DNA borders, the plant-selective nos / nptII chimeric gene and the pUC polylinker (Rothstein et al.
t al., Gene 53: 153-161 (1987)), and the XhoI digested fragment was SaII digested pTJS75ka.
to create pCIB200 (see also Example 19 of EP0332104)
. pCIB200 contains the following unique polylinker restriction sites: EcoRI, SstI, K
pnI, BglII, XbaI and SaII. pCIB2001 is a derivative of pCIB200 created by inserting an additional restriction site into the polylinker. The unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI, BglII, XbaI, SaII, MluI, BclI, Avr
II, ApaI, HpaI and StuI. In addition to these unique restriction sites, pCIB2001
Are selected for plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, for mobility between E. coli and other hosts.
It has a trfA function from RK2- and an OriT and OriV function from RK2. pCIB2001
Polylinkers are suitable for cloning plant expression cassettes that contain their own regulatory signals.

B. pCIB10 and its Hygromycin-Selective Derivatives The binary vector pCIB10 contains the gene encoding kanamycin resistance for selection in plants, as well as T-DNA right and left border sequences, and transforms it in both E. coli and Agrobacterium. Broad host range plasmids to replicate
Incorporate the sequence from pRK252. Its construction is described by Rothstein et al. (Gene 53: 153-161 (
1987)). Various derivatives are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives are hygromycin only (pC
IB743) or transgenic plant cells for hygromycin and kanamycin (pCIB715, pCIB717).

[0159] 2. Vectors Suitable for Non-Agrobacterium Transformation Transformation without Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the selected transformation vectors, and therefore, vectors lacking these sequences will require T-DNA In addition to the vectors containing the sequences described above, they can be utilized. Transformation techniques that do not rely on Agrobacterium include particle bombardment, protoplast uptake (eg, PEG and electroporation), and microinjection. The choice of vector is largely dependent on the preferred selection for the species being transformed. The following describes the construction of a typical vector suitable for non-Agrobacterium transformation.

A. pCIB3064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer technology in combination with selection with the herbicide vasta (or phosphinothricin). Plasmid pCIB246 contains
CaMV 35S by operable fusion of Escherichia coli GUS gene with CaMV 35S transcription terminator
It includes a promoter and is described in PCT published application WO93 / 07278. The 35S promoter of this vector contains the two ATG sequences 5 'of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATG and generate the restriction sites SspI and PvuII. The new restriction site is unique Sa
96 and 37 bp away from the II site and 101 and 42 bp away from the actual start site.
The resulting derivative of pCIB246 is called pCIB3025. Next, SalI and Sac
The GUS gene is excised from pCIB3025 by digestion with I, the ends are blunted,
Religated to yield plasmid pCIB3060. Plasmid pJIT82 is from John Innes C
entre, Norwich, Streptomyces viridochromog
A small 400 bp fragment containing the bar gene from enes is excised and inserted into the HpaI site of pCIB3060 (Thompson et al., EMBO J. 6: 2519-2523 (1987)). this is,
Bar under control of CaMV 35S promoter and terminator for herbicide selection
Includes gene, gene for ampicillin resistance (for selection in E. coli), and polylinker with unique sites SphI, PstI, HindIII and BamHI
This produces pCIB3064. This vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

B. pSOG19 and pSOG35: pSOG35 is a transformation vector that utilizes the E. coli gene dihydrophore reductase (DFR) as a selectable marker that confers resistance to methotrexate. PCR is used to amplify the 35S promoter (〜800 bp), intron 6 (〜550 bp) from the maize Adh1 gene and the 18 bp GUS untranslated leader sequence from pSOG10. Escherichia coli dihydrofolate reductase II
A 250 bp fragment encoding the type gene was also amplified by PCR, and these two PCR
The fragment is assembled with the SacI-PstI fragment from pB1221 (Clontech), which includes the pUC19 vector backbone and the nopaline synthase terminator. The assembly of these fragments contains a pS containing the 35S promoter fused to the intron 6 sequence, GUS leader, DHFR gene and nopaline synthase terminator.
Generate SOG19. Replacement of the GUS leader in pSOG19 with the leader sequence from maize yellow spot virus (MCMV) yields the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI and EcoRI sites available for cloning foreign materials.

[0162] 3. Vectors Suitable for Chloroplast Transformation For the expression of the nucleotide sequence of the invention in plant plastids, the plastid transformation vector pPH143 (WO97 / 32011, Example 36) is used. The nucleotide sequence is inserted into pPH143, thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, a nucleotide is inserted into pPH143 such that it replaces the aadH gene. In this case, transformants are selected for resistance to the PROTOX inhibitor.

Example 25: Transformation Once a nucleic acid sequence of the invention has been cloned into an expression system, it is transformed into plant cells. Methods for plant transformation and regeneration are well known in the art.
For example, Ti plasmid vectors can be used to deliver foreign DNA, as well as direct DN
It has been used for A uptake, liposomes, electroporation, microinjection and microprojection. In addition, bacteria from the genus Agrobacterium can be used to transform plant cells. The following is a description of representative techniques for transforming both dicotyledonous and monocotyledonous plants.

1. Transformation of dicots Transformation techniques for dicots 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 protoplasts or cells. This can be achieved by PEG or electroporation mediated uptake, particle impact mediated delivery, or microinjection. Examples of these techniques are described in the following literature: Paszkowski e
t al., EMBO J. 3: 2717-2722 (1984); Potrykus et al., Mol. Gen. Genet. 19
9: 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 dicotyledon transformation due to its high transformation efficacy and its widespread utility with many different species. Agrobacterium transformation is typically performed on a suitable Agrobacterium strain (e.g., with respect to pCIB200 or pCIB2001) on the co-resident Ti plasmid or chromosomally, obtained by complementing the vir gene carried by the host Agrobacterium strain. Is a binary vector (for example, pCIB200 or pCIB2001) carrying the foreign DNA into the CIB542 strain (Uknes et al. Plant Cell 5: 159-169 (1993)).
The transfer of E. coli harboring a recombinant binary vector, recombination into Agrobacterium by a triparental mating method using a helper E. coli strain that harbors a plasmid such as pRK2013 and can mobilize the recombinant binary vector into a target Agrobacterium strain The transfer of the binary vector is accomplished. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of a target plant species with a recombinant Agrobacterium usually involves co-culturing Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissues are regenerated on a selectable medium that carries an antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.

Another approach to transforming plant cells with the gene involves forcing plant tissues and cells into inert or biologically active particles. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006 and 5,100,792 (all of Sanford
Etc.). In general, this technique involves forcing the cells to pass inert or biologically active particles under conditions effective to penetrate the outer surface of the cell and effect its incorporation therein. When using inert particles, the vector can be introduced into cells by coating the particles with a vector containing the desired gene.
Alternatively, the target cells may be transported such that the vector is carried into the cells by the wake of the particles,
It can be surrounded by vectors. Biologically active particles, such as dried yeast cells, dried bacteria or bacteriophage, each containing the DNA to be introduced, can also be propelled into the plant cell tissue.

[0168] 2. Transformation of monocotyledons Transformation of most monocotyledons is now becoming routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, as well as particle bombardment of callus tissue. Transformation is single DNA
Species or multi-DNA species (ie, co-transformation), but both of these techniques are suitable for use with the present invention. Co-transformation avoids complete vector construction and transgenic plants that have unlinked loci for the gene of interest and a selectable marker, allowing the removal of the selectable marker in subsequent generations to be considered desirable. It may have the advantage of regeneration. However, a disadvantage of the use of co-transformation is that the frequency of distinct DNA species being integrated into the genome is less than 100% (Schocher et al. Biotechnology 4: 1093-10).
96 (1986)).

Patent applications EP0292435, EP0392225 and WO93 / 07278 disclose the preparation of callus and protoplasts from inbred maize lines, transformation of protoplasts using PEG or electroporation, and regeneration of maize plants from transformed protoplasts. Describe the techniques for: Gordon-Kamm et al. (Plant Cell 2: 603-61
8 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have described techniques for the transformation of A188-derived corn lines using particle bombardment. Further, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of a selected maize inbred line by particle bombardment. This technique is 1.5 to 2 lengths cut from corn ears 14 to 15 days after pollination
Utilizes a .5 mm immature corn embryo and a PDS-1000He Biolistics instrument for impact.

Transformation of rice can also be done by direct gene transfer techniques utilizing protoplasts or by particle bombardment. Protoplast-mediated transformation has been described for the Japonica and Indica types (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 types can be routinely transformed using particle bombardment (Christou et al., Biotechnology 9: 957-962 (1991)).
. Further, WO 93/21335 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 allow for the transformation of Dactylis and wheat. In addition, wheat transformation was performed using Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment of cells of type C long-term renewable calli, and using calli derived from immature embryos and immature embryos, Vasil et al. (Biotechnology 11: 1553-
1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)). However, preferred techniques for wheat transformation include transformation of wheat by particle bombardment of immature embryos, including a high sucrose or high maltose step prior to gene delivery. Before impact, any number of embryos (0.75-1 mm in length)
3% sucrose (Murashiga & Skoog, Physiologia Planta
rum 15: 473-497 (1962)) and 3 mg / l of 2,4-D on MS medium, which proceeds in the dark. On the day of bombardment, the embryos are removed from the induction medium and plated on osmochicum (ie, an induction medium containing sucrose or maltose added at the desired concentration, typically at 15%). Embryos are subjected to protoplasmic separation for 2-3 hours and then subjected to shock treatment. Twenty embryos per target plate are typical, but this is not critical. A suitable gene-bearing plasmid (eg, pCIB3064
Alternatively, pSG35) is precipitated on μm-sized gold particles using standard techniques. Standard 80
Each plate of embryos is shot with a DuPont Biolistics® Helium instrument using a mesh screen and a burst pressure of 〜1000 psi. After bombardment, the embryos are returned to the dark and allowed to recover for approximately 24 hours (still on Osmochikum). After 24 hours, the embryos are removed from the osmoticum and returned to the induction medium, where they stay for about a month before regenerating. After about one month, the embryo explants, together with the developing embryogenic callus, are regenerated into regeneration medium (
MS + 1 mg / l NAA, 5 mg / l GA) and contain the appropriate selective agent (10 mg / l vasta for pCIB3064, 2 mg / l methoxylate for pSOG35). Approximately one month later, GA7 containing half strength MS, 2% sucrose and the same concentration of selective agent
Transfer the generated shoots to a large sterile container known as "".

Transformation of monocots using Agrobacterium has also been described (WO94 / 009).
77 and U.S. Pat. No. 5,591,616) (both of which are incorporated herein by reference). 3. Transformation of plastids Tobacco Nicotiana tabacum at 7 / plate in 1 inch circular array on T agar medium
cv 'Xanthi nc' seeds are germinated, essentially as described above (Svab, Z. and Maliga, P.
(1993) PNAS 90, 913-917) and impacted 12-14 days after seeding with 1 μm tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145. give. The impacted seedlings are incubated on T medium for 2 days, after which the leaves are cut off and RMOP medium (Svab, Z., Hajdukiewic) containing 500 μg / ml spectinomycin dihydrochloride (Stigma, St. Louis, MO).
z, P. and Maliga, P. (1990) PNAS 87, 8526-8530)
500500 μmol photons / m ² / s) with the abaxial side up. Resistant shoots that appear just below the bleached leaves 3-8 weeks after bombardment are subcloned on the same selection medium to form callus, and secondary shoots are isolated and subcloned. Complete isolation of homologous transforming plastid genome copies in individual subclones
asmicity)) is assessed by standard techniques of Southern blotting (Sambrook e
t al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor). BamHI / EcoRI-digested whole cell DNA (Mettle
r, IJ (1987) Plant Mol Biol Reporter 5, 346-349) was separated on a 1% tris-borate (TBE) agarose gel, transferred to a nylon membrane (Amersham) and rp
0.7 kb BamHI / HindIII DN from pC8 containing part of the s7 / 12 plastid target sequence
Probe using the ³² P-labeled random primed DNA sequence corresponding to the A fragment. The homoplasmic shoots were transferred to an MS / IBA medium containing spectinomycin (McBride, KE et.
al. (1994) PNAS 91, 7301-7305) and aseptically rooted and transferred to a greenhouse.

E. Crosses and Seed Production Example 26: Crosses Plants obtained by transformation with the nucleic acid sequences of the present invention can be of any of a wide variety of plant species, for example, monocots and dicots. The plants used in the method of the invention are preferably selected from the list of agriculturally important target crops mentioned above. The expression of the gene of the present invention in combination with other features important for the production and quality of the present invention is incorporated into the plant line by crossing. Hybridization approaches and techniques are known in the art (eg, Welsh JR, Fundamenta
ls of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop B
reeding, Wood DR (Ed.) American Society of Agronomy Madison, Wisconsin
(1983); Mayo O., The Theory of Plant Breeding, Second Edition, Clarend
on Press, Oxford (1987); Singh, DP, Breeding for Resistance to Disea
ses 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 characteristics engineered in the transgenic seeds and plants described above are transferred by reproductive or plant growth, and thus can be retained and propagated in progeny plants. In general, the maintenance and propagation employ known agricultural methods that have been developed for specific purposes, such as cultivation, sowing or harvesting. Special methods such as hydroponics or greenhouse methods may also be applied. Growing crops are vulnerable to assaults or damage caused by insects or infections and to competition by weed plants, so weeds, plant diseases, insects, nematodes and other adverse effects to improve yield Conditions are controlled. These include mechanical measurements, such as soil tillage operations, or removal of weeds and infected plants, and agrochemicals, such as herbicides (
herbicides), fungicides, gametocides, nematicides, growth regulants, ripening agen
ts), and the application of insecticides.

The use of the beneficial genetic properties of the transgenic plants and seeds of the present invention may further improve the forcing such as tolerance of pests, herbicides or stress, improve nutritional values, increase yields, or lose by collapse or fruit drop. This can be done in plant crosses aimed at developing plants that exhibit reduced structural improvements. The various mating steps are characterized by well-defined human intervention, such as selection of the line to be crossed, instructions for pollination of the parent line, or selection of appropriate progeny plants. Depending on the properties desired, different hybridization measurements are employed. Related techniques are well known in the art, and include, but are not limited to, hybridization, inbreeding, backcrossing, multiline breeding, variant fusion, interspecies hybridization, aneuploid techniques, and the like. . Hybridization techniques further include sterilizing the plants to produce female or male sterile plants by mechanical, chemical or biochemical means. Cross-pollination of male-sterile plants with pollen from different lines ensures that the genomes of the male-sterile, but female-fertile plants obtain the homogeneity of the characteristics of both parental lines. Thus, the transgenic seeds and plants of the present invention are improved plants that increase the effectiveness of conventional methods, such as, for example, herbicide or pesticide treatment, or that are distributed using the methods described above due to their modified genetic properties. It can be used for crossing of lines. Alternatively, the optimized genetic "
Due to the "equipment" new crops are obtained which show improved stress tolerance, which produce better quality harvested products than those which cannot tolerate comparable adverse conditions.

Example 27: Seed production In seed production, seed germination quality and uniformity are essential product characteristics, while seed germination quality and uniformity are harvested and sold by farmers. Is not important. Because it is difficult to keep the crops from being mixed with other crops and weed seeds, the growth, conditioning and control of genuine seeds to control seed-bearing diseases and produce seeds that show good germination A fairly extensive and well-defined means of seed production has been developed by seed producers with experience in the marketing industry. Thus, instead of using seeds from his own crop, it is common for farmers to purchase guaranteed seeds that meet certain quality standards. Reproductive substances used as seeds are usually treated with a prophylactic protectant coating, including herbicides, insecticides, fungicides, bactericides, nematicides, molluscides or mixtures thereof. You. Conventionally used prophylactic protective coatings include compounds such as captan, carboxin, thiram (TMTD ™), metalaxyl (Apron ™) and pirimiphos-methyl (Acteric ™). If desired, these compounds may be further formulated with carriers, surfactants, or application-enhancing adjuvants that are commonly used in the formulation art to provide protection against damage caused by bacteria, fungi or animal pests. The prophylactic protectant coating may be applied by impregnating the liquid formulation with the propagation material, or by coating with a combined wet or dry formulation. Other methods of application, such as treatment directed at buds or fruits, are also possible.

It is yet another aspect of the present invention to provide a novel agricultural method, characterized by the use of the transgenic plant, transgenic plant material or transgenic seed of the present invention as exemplified above. . Seeds are provided in bags, containers or containers made of suitable packaging material,
The bag or container may be closed to contain the seed. Bags, containers or containers may be designed for short or long term storage of seeds, or both. Examples of suitable packaging materials include paper, such as kraft paper, rigid or flexible plastic or other polymeric material, glass or metal. Desirably, the bag, container or container consists of multiple layers of the same or different types of packaging material. In one embodiment, a bag, container or container is provided to eliminate or limit water or moisture from contacting the seed. In one embodiment, the bag, container or container is sealed, for example, sealed, to prevent water or moisture from entering. In another embodiment, the water-absorbing material is located between or adjacent to the package material layers. In yet another embodiment, the bag, container or container, or the contained packaging material, is treated to limit, inhibit, or prevent disease, contamination, or other adverse effects of seed storage or transport. An example of such a treatment is, for example, sterilization by chemical means or by exposure to radiation. Transgenic plant seeds comprising the genes of the present invention expressed in said transformed plants at higher levels than wild-type plants, together with suitable carriers, are used to confer a wide range of disease resistance to the plants. Commercially available pouches containing labels with instructions for the use of are included in the present invention.

The above disclosed embodiments are illustrative. This disclosure of the invention allows those skilled in the art to recognize that the invention has many variations. All such obvious and foreseeable changes are intended to be covered by the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int. Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 15/00 ZNAA C12P 21/02 5/00 C (81) Designated countries EP (AT, BE) , CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Anderson, Arn Robert United States of America, North Carolina 27597, Zebulon, Green-Payce Road 1005 F-term (reference) 2B030 AA02 AB03 AD20 CA06 CA17 CA19 CB02 CD03 CD07 CD10 4B024 AA07 AA08 BA80 CA05 DA01 DA05 DA11 DA12 EA04 EA06 GA11 GA17 GA19 HA12 4B064 AG01 AG30 CA02 CA05 CA06 CA11 CA19 CC24 DA12 4B065 AA01 BAA14A24A14A24A18A24 CA11 DA83 EA06 FA74

Claims (34)

[Claims] 1. The following: (a) nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045- of SEQ ID NO: 1
2334, a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or (b) (a) An isolated nucleic acid molecule comprising a nucleotide sequence that is isocoding to the nucleotide sequence of claim 1, wherein expression of the isolated nucleic acid molecule results in at least one toxin that is active against insects. 2. The method of claim 1 wherein said nucleotide sequence is nucleotides 569-979 of SEQ ID NO: 1.
SEQ ID NO: 1, nucleotides 1045-2334, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
The isolated nucleic acid molecule of claim 1, which isocodes with a nucleotide sequence substantially similar to SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. 3. The method according to claim 2, wherein said nucleotide sequence is nucleotides 569-979 of SEQ ID NO: 1.
SEQ ID NO: 1, nucleotides 1045-2334, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is substantially similar to SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. 4. The method according to claim 1, wherein the nucleotide sequence is SEQ ID NO: 2, 3, 5, 7, 9, 1, or 2.
2. The isolated nucleic acid molecule of claim 1, which encodes an amino acid sequence selected from the group consisting of 1, 13, and 15. 5. The method according to claim 5, wherein the nucleotide sequence is nucleotides 569-97 of SEQ ID NO: 1.
9, nucleotides 1045-2334 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8
The isolated nucleic acid molecule of claim 1, comprising SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. 6. The method of claim 1, wherein said nucleotide sequence is nucleotides 569-97 of SEQ ID NO: 1.
9, substantially similar to SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12.
Isolated nucleic acid molecule. 7. The isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 13. 8. The nucleotide sequence of claim 1 wherein the nucleotide sequence is nucleotides 1045-233 of SEQ ID NO: 1.
4. The isolated nucleic acid molecule of claim 1, which is substantially similar to SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 14. 9. The isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 15. 10. The isolated nucleic acid molecule of claim 1, wherein said nucleotide sequence comprises an approximately 3.0 kb DNA fragment contained in pCIB9369 (NRRL B-21883). 11. The isolated nucleic acid molecule of claim 1, wherein the toxin is active against Plutella xylostella. 12. A member consisting of nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. Sequence of selected nucleotide sequences
An isolated nucleic acid molecule comprising a 20 base pair nucleotide portion that is identical in sequence to the 20 base pair nucleotide portion, the expression of which produces at least one toxin that is active against insects. 13. A chimeric gene comprising a heterologous promoter sequence operably linked to the nucleic acid molecule of claim 1 or 12. 14. A recombinant vector comprising the chimeric gene according to claim 13. 15. A host cell comprising the chimeric gene of claim 13. 16. The host cell of claim 15, which is a bacterial cell. 17. The host cell of claim 15, which is a yeast cell. 18. The host cell according to claim 15, which is a plant cell. 19. A plant comprising the plant cell of claim 18. 20. The plant of claim 19, which is maize. 21. A toxin produced by expression of the DNA molecule of claim 1 or 12. 22. The toxin of claim 21, which is active against Plutella xylostella. 23. The toxin of claim 21 produced by an E. coli strain designated as NRRL Accession No. B-21883. 24. The toxin of claim 21, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 7, 9, 11, 13, and 15. 25. The toxin of claim 24, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9, and 13. 26. The toxin of claim 24, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 11, and 15. 27. A composition comprising an insecticidal effective amount of the toxin of claim 21. 28. A method for producing a toxin that is active against insects, comprising: (a) obtaining the host cell of claim 15, and (b) expressing a nucleic acid molecule in said cell; A method which comprises the step of producing at least one toxin that is active against insects. 29. A method for producing an insect-tolerant plant comprising introducing the nucleic acid molecule of claim 1 or 12 into the plant, wherein the nucleic acid molecule is contained in an amount effective to control insects. A method that can be expressed in the plant. 30. The insect of claim 29, wherein the insect is Plutella xylostella.
the method of. 31. A method for controlling insects comprising delivering an effective amount of the toxin of claim 21 to the insects. 32. The insect of claim 31 wherein the insect is Plutella xylostella.
the method of. 33. The method of claim 32, wherein the toxin is delivered orally to the insect. 34. The method of mutating a nucleic acid molecule of claim 1 or 12 cleaved into a population of double-stranded random fragments of a desired size, comprising: (a) double-stranded random fragments; Adding to the population of engineered fragments one or more single or double-stranded oligonucleotides, each comprising a region of identical structure and a region of heterologous structure to the double-stranded template polynucleotide; (b) Denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments, and (c) priming the population of single-stranded fragments with respect to one member of the pair for the other copy. Incubation with a polymerase under conditions that cause annealing of the single-stranded fragment in a region of sufficient identity to produce a pair of annealed fragments, thereby producing a mutated double-stranded polynucleotide. And (d) repeating the second and third steps at least two more times, wherein
The mixture resulting from performing the second step one more time contains the mutated double-stranded polynucleotide from the previous third step, and performing one more time yields more mutated double-stranded polynucleotides; A method comprising the steps of: