RU2240003C2 - Isolated nucleic acid molecule, chimeric gene, recombinant vector, microorganism strain, toxin, insecticide composition, method for preparing toxin, method for preparing toxin-resistant plant and method for control of insects - Google Patents

Abstract

FIELD: molecular biology, biotechnology, agriculture, insecticides.

SUBSTANCE: invention relates to biotechnology of agricultural plants. Method involves isolating nucleotide sequences from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus lumintecens that produce insecticide toxins during their expression. Host-cell is transformed with chimeric gene comprising one of isolated nucleotide sequences and compositions comprising insecticide toxins useful for control of insect-pests are prepared. Invention provides enhancing the resistance of plants against insect-pests and to enhance the yield of agricultural production.

EFFECT: valuable biological properties of nucleic acid, improved method for plant preparing.

23 cl, 27 ex

Description

The invention relates to new toxins from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus lumintecens, to nucleotide sequences resulting in the expression of toxins, and to methods for producing and methods of using toxins and corresponding nucleotide sequences to control insects.

Pests are a major cause of crop loss. In the United States alone, annual crop losses due to damage by various types of insects are estimated at approximately $ 7.7 billion. In addition to causing crop losses, insect pests also complicate the work of growers and growers, producers of decorative flowers, and they are a source of trouble for gardeners and homeowners.

In general, insect pests are controlled by the intensive use of chemical insecticides that are active in inhibiting the growth of insects, preventing the nutrition or reproduction of insects, or leading to their death. This method allows you to effectively fight insects, but such chemical control agents sometimes also infect other beneficial species of insects. Another problem arising from the widespread use of chemical pesticides is the emergence of resistant insect strains. This problem is partially addressed through various resilience management strategies, but there is an increasing need for alternative pest control agents. Biological insect control agents, such as Bacillus thuringiensis strains expressing insecticidal toxins, such as δ-endotoxins, have also been used with satisfactory results, and they represent an alternative and addition to chemical insecticides. Recently, genes encoding some of these δ-endotoxins have been isolated, and it has been found that their expression in heterologous hosts is another means of combating economically important pests. In particular, the expression in transgenic plants of insecticidal toxins, such as δ-endotoxins of Bacillus thuringiensis, provides effective protection against certain insect pests, and transgenic plants expressing such toxins are commercially available, which allows farmers to reduce the amount of insect control chemicals used . However, even in this case, the possibility of developing sustainability remains and the number of only a small number of certain types of pests can be limited. Thus, there remains a long-recognized, but unsatisfied need for the creation of new and effective insect control agents that would bring economic benefits to farmers and be environmentally sound.

The present invention aims to solve the long-standing problem of creating new insect control agents. First of all, agents are needed that are aimed at combating economically important insect pests and which can effectively fight insect strains that are resistant to existing insect control agents. In addition, preferred are agents whose use minimally damages the environment.

In the search for new insect control agents, particular classes of nematodes from the genera Heterorhabdus and Steinernema are of particular interest, due to their insecticidal properties. They kill insect larvae and their offspring feed on dead larvae. In fact, insecticidal activity is due to symbiotic bacteria living in nematodes. Such symbiotic bacteria are Photorhabdus for Heterorhabdus and Xenorhabdus for Steinernema. The present invention relates to nucleotide sequences isolated from Xenorhabdus nematophilus, to nucleotide sequences practically similar to them, the expression of which leads to the production of insecticidal toxins that are highly toxic to economically important pests, especially plant pests. In addition, the invention relates to an insecticidal toxin resulting from the expression of a nucleotide sequence, and to compositions and formulations comprising an insectipid toxin that are able to inhibit (suppress) the survival of insect pests, their growth or reproductive ability, or limit insect damage or crop loss. In addition, the invention relates to a method for producing a toxin and to methods of using a nucleotide sequence, for example, in microorganisms, for controlling insects or in transgenic plants to confer resistance to insects, as well as to a method for using a toxin, and compositions and formulations comprising a toxin for example, by treating an area infected with insects with a toxin, composition or formulation, or for prophylactically treating insect-susceptible areas or plants that are potential targets, in order to provide protection or create resistance to harmful insects.

The new toxin has a high insecticidal activity against Plutella xylostella (cabbage moth), an economically important insect pest. The toxin can be used in numerous insect control strategies, leading to maximum efficiency combined with minimal environmental impact.

One of the objects of the present invention is an isolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or (b) the nucleotide sequence of the isocodonic nucleotide sequence specified in subparagraph (a); moreover, the expression of a nucleic acid molecule leads to the formation of at least one toxin with activity against insects. In one embodiment, the nucleotide sequence is an isocodone nucleotide sequence substantially similar to nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 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. Preferably, the nucleotide sequence is substantially similar to nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 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. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 7, 9, 11, 13 and 15. Most preferably, the nucleotide sequence includes nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045 -2334 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 a DNA fragment of approximately 3.0 kb in length contained in pCIB9369 (NRRL B-21883).

According to a preferred embodiment, the toxins resulting from the expression of the nucleic acid molecules of the invention have activity against Plutella xylostella.

Another object of the present invention is an isolated nucleic acid molecule comprising a part of a nucleotide sequence consisting of 20, 25, 30, 35, 40, 45 or 50 (preferably 20) base pairs that is identical to the corresponding consisting of 20, 25, 30, 35, 40, 45 or 50 (preferably 20) consecutive base pairs of a portion of a nucleotide sequence selected from the group consisting of: nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, wherein expression of the nucleic acid molecule is t to the formation of at least one toxin with activity against insects.

An object of the present invention is also a chimeric gene containing a heterologous promoter sequence operably linked to a nucleic acid molecule of the invention. In addition, an object of the present invention is a recombinant vector containing such a chimeric gene. Another object of the present invention is a host cell containing such a chimeric gene. A host cell according to this aspect of the invention may be a bacterial cell, a yeast cell or a plant cell, preferably a plant cell. And another object of the present invention is a plant containing such a plant cell. Preferably, the plant is corn.

Another object of the present invention are toxins resulting from the expression of DNA molecules of the present invention. In a preferred embodiment, the toxins of the present invention are active against Plutella xylostella.

In one embodiment, toxins are produced by an E. coli strain having registration number NRRL B-21883.

In another embodiment, the toxin of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, and 15.

Also an object of the invention is a composition comprising an insecticide effective amount of a toxin according to the invention. Another object of the present invention is a method for producing a toxin having activity against insects, comprising: (a) obtaining a host cell containing a chimeric gene that includes a heterologous promoter sequence operably linked to a nucleic acid molecule of the invention; and (b) expression of the nucleic acid molecule in the cell, which leads to the formation of at least one toxin having activity against insects.

Another object of the present invention is a method for producing an insect resistant plant, comprising introducing into the plant a nucleic acid molecule according to the invention, the nucleic acid molecule being able to be expressed in the plant in an amount effective against insects. In a preferred embodiment, the insect is Plutella xylostella.

And another object of the present invention is a method of controlling insects, comprising administering to the insect an effective amount of the toxin of the present invention. According to a preferred embodiment, the insect is Plutella xylostella. Preferably, the toxin is ingested orally.

Another object of the present invention is a method for mutating a nucleic acid molecule of the present invention, according to which the nucleic acid molecule is cleaved to obtain a population of random double-stranded fragments of the desired length and the following steps are carried out:

a) adding to the resulting population of random double-stranded fragments of one or more single or double-stranded oligonucleotides, each of the oligonucleotides containing an identity region and a region of heterology with respect to the double-stranded matrix polynucleotide;

b) denaturation of the resulting mixture of random double-stranded fragments and oligonucleotides to obtain single-stranded fragments;

c) incubation of the obtained population of single-stranded fragments with polymerase under conditions in which the renaturation of these single-stranded fragments takes place in the region of identity with the formation of pairs of renatured fragments, and these regions of identity are sufficient for one member of the pair to prime replication of the other, leading to the formation of double-stranded polynucleotide containing a mutation; and

d) repeating the second and third stages for at least two more cycles, and the resulting mixture in the second stage of the additional cycle contains a double-stranded polynucleotide with a mutation obtained in the third stage of the previous cycle, as a result of which further cycles lead to the formation of an additional amount of double-stranded polynucleotide, containing the mutation.

Other objects and advantages of the present invention will be apparent to those skilled in the art based on a study of the description of the invention and non-limiting examples.

Definitions

The term "activity" in relation to the toxins according to the invention means that the action of toxins as insect control agents with oral activity leads to toxic effects, or they have the ability to stop or disrupt the nutrition of the insect, which may or may not cause death insect. If the toxin of the invention enters the body of an insect, the result is usually the death of the insect, or the insect ceases to feed from the source from which the toxin enters the insect.

The term “coupled to / functionally linked to” refers to two nucleotide sequences that are physically or functionally linked. For example, a promoter or regulatory DNA sequence is “linked” to a DNA sequence encoding an RNA or protein if the two sequences are functionally linked or arranged so that the regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.

The term "chimeric gene" refers to a recombinant nucleotide sequence in which a promoter or regulatory nucleotide sequence is operably linked or connected to a nucleotide sequence that encodes an mRNA or that is expressed as a protein, so that the regulatory nucleotide sequence is capable of regulating the transcription or expression of its associated nucleotide sequence . The regulatory nucleotide sequence of a chimeric gene is normally not functionally linked to the nucleotide sequence connected to it, as is the case under natural conditions.

The term "coding sequence" means a nucleotide sequence that is transcribed with the formation of RNA, such as, for example, mRNA, rRNA, tRNA, snPHK (M.a. RNA), sense RNA or antisense RNA. Preferably, the RNA is then translated in the body to form a protein.

The term "control" of insects means to inhibit, as a result of a toxic effect, the ability of insect pests to survive, grow, eat and / or reproduce, or to limit insect damage or crop loss of cultivated plants. In the "fight" with insects, you can kill or not kill insects, although preferably this concept means to kill insects.

The term “introduction (ingestion)” of a toxin means that the toxin comes into contact with an insect, which leads to a toxic effect and control of the insect. The toxin can enter the body in many known ways, for example, orally when eaten by insects, or by contact with an insect as a result of the expression of a toxin by a transgenic plant, using a protein-based formulation (s), protein-based spray composition (s), baits or any other toxin administration systems known in the art.

The term “expression cassette” as used herein refers to a nucleotide sequence capable of expressing a particular nucleotide sequence in an appropriate host cell that includes a promoter operably linked to a nucleotide sequence of interest that is operably linked to termination signals. It normally also includes the sequences necessary for the correct translation of the nucleotide sequence. An expression cassette containing a nucleotide sequence of interest may be chimeric, which means that at least one of its components is heterologous to at least one of the other components. The expression cassette can also be a cassette that is found in vivo, but which was obtained in a recombinant form suitable for heterologous expression. However, as a rule, the expression cassette is heterologous with respect to the host, i.e. a specific DNA sequence of the expression cassette does not naturally occur in the host cell and must be introduced into the host cell or ancestor of the host cell by transformation. The expression of the nucleotide sequence in the expression cassette may be controlled by a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is treated with a specific external stimulus. In the case of a multicellular organism, such as a plant, the promoter may also be specific for a particular tissue or organ or developmental stage.

The term "gene" refers to a specific region within the genome that, in addition to the coding sequence indicated above, contains other, primarily regulatory, nucleotide sequences responsible for controlling expression, i.e. for transcription and translation of the coding region. A gene may also include other 5’- and 3’-untranslated sequences and termination sequences. Other elements that may also be present in the gene are, for example, introns.

The term “gene of interest” means any gene that, when transferred to a plant, gives the plant the desired properties, such as antibiotic resistance, virus resistance, insect resistance, disease resistance or resistance to other pests, herbicide resistance, improved nutritional value , improved characteristics in terms of industrial processing or modified ability to reproduce. A "gene of interest" may be a gene that is carried into plants to produce economically important enzymes or metabolites in a plant.

A “heterologous” nucleotide sequence refers to a nucleotide sequence that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of naturally occurring nucleotide sequences.

A “homologous” nucleotide sequence refers to a nucleotide sequence that is naturally associated with a host cell into which it is introduced.

The term "homologous recombination" refers to the mutual exchange of nucleic acid fragments between homologous nucleic acid molecules.

The term "insecticidal" refers to toxic biological activity by which it is possible to fight insects, preferably kill them.

The nucleotide sequence is the "isocodone" nucleotide sequence with which the comparison is made if the nucleotide sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the nucleotide sequence with which the comparison is made.

An “isolated” nucleic acid molecule or “isolated” enzyme refers to a nucleic acid molecule or enzyme that, thanks to humans, exists outside their natural environment and, therefore, are not natural products. An isolated nucleic acid molecule or enzyme may exist in purified form or may exist in an unnatural environment, such as, for example, a recombinant host cell.

"Nucleic acid molecule" or "nucleotide sequence" refers to 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 preferably denotes a DNA segment. The term "ORF" refers to an open reading frame.

The term "plant" means any plant at any stage of development, in particular the seed material of a plant.

The term "plant cell" refers to the structural and physiological unit of a plant, including protoplast and cell membrane. The plant cell may be in the form of an isolated single cell or cultured cell, or may be part of a highly organized unit, such as, for example, plant tissue, plant organ, or the whole plant.

The term "plant cell culture" refers to cultures of structural or physiological units of a plant, such as, for example, protoplasts, cells in a cell culture, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

The term "plant material" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, eggs, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

The term "plant organ" refers to a separate and structurally formed differentiated part of a plant, such as a root, stem, leaf, leaf bud or embryo.

The term "plant tissue" in the context of the present description refers to a group of plant cells organized in a structural or functional unit. Any plant tissue in planta or in culture is meant. This concept includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, and any groups of plant cells organized in structural and / or functional units. The use of this concept in combination with a specific type of plant tissue, as defined above, or by some other criteria, falls within this definition, or regardless of the type of tissue, does not imply that any other type of plant tissue is excluded.

The term "promoter" refers to an untranslated DNA sequence located upstream of the transcription of the coding region, which contains the binding site of RNA polymerase II and initiates DNA transcription. The promoter region may also include other elements that act as regulators of gene expression.

The term "protoplast" refers to an isolated plant cell without a cell membrane or only part of the cell membrane.

The term "regulatory elements" refers to sequences involved in the control of expression of a nucleotide sequence. Regulatory elements include a promoter operably linked to a nucleotide sequence of interest and termination signals. They also usually include the sequences necessary for proper translation of the nucleotide sequence.

In the broadest sense, the term "almost analogous", when used in the present description with reference to the nucleotide sequence, means a nucleotide sequence corresponding to the nucleotide sequence with which the comparison is made, and the sequence in question encodes a polypeptide having practically the same structure and functions as a polypeptide encoded by the nucleotide sequence with which the comparison is made, for example, a polypeptide having only such substitutions of amino acids that do not affect the function of the polypeptide. Preferably, a substantially similar nucleotide sequence encodes a polypeptide encoded by the comparison nucleotide sequence. It is desirable that the percent identity of a substantially similar nucleotide sequence and the nucleotide sequence to be compared is at least 80%, more preferably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%. The nucleotide sequence "almost analogous" to the comparison nucleotide sequence, as a rule, hybridizes with the nucleic acid molecule to be compared with under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C; washing with double SSC (2xSSC), 0.1% SDS at 50 ° C, more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C; washing with a single SSC (1xSSC), 0.1% SDS at 50 ° C, even more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C; washing with 0.5xSSC, 0.1% SDS at 50 ° C, preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C; washing with 0.1xSSC, 0.1% SDS at 50 ° C, more preferably 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C; washing with 0.1xSSC, 0.1% SDS at 65 ° C.

The term "synthetic" refers to a nucleotide sequence that includes structural features that are not present in a naturally occurring sequence. For example, a synthetic sequence is called an artificial sequence characterized by the closest G + C content and normal codon distribution characteristic of genes of dicotyledonous and / or monocotyledonous plants.

The term "transformation" means the introduction of a heterologous nucleotide sequence into a host cell or organism. In particular, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest.

The term "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 can be stably integrated into the host genome, or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule may have the ability to self-replicate. It should be understood that the concepts of transformed cells, tissues or plants include not only the end product of the transformation process, but also its transgenic offspring. The terms "non-transformed", "non-transgenic" or "non-recombinant" refer to a wild-type organism, such as a bacterium or plant, which does not contain a heterologous nucleic acid molecule.

Nucleotides are labeled based on the following standard abbreviations: adenine (A), cytosine (C), thymine (T) and guanine (G). Similarly, amino acids are indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q) , glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met, M) phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). In addition, (Haa; X) denotes any amino acid.

A brief description of the sequences presented in the sequence listing

SEQ ID NO: 1 is a approximately 3.0 kbp DNA fragment sequence contained in the Xenorhabdus nematophilus clone pCIB9369 clone that includes the following ORFs at the indicated nucleotide positions:

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

SEQ ID NO: 3 is a sequence of a juvenile hormone similar to esterase protein with a molecular weight of ~ 47.7 kDa, encoded by orf2 of clone pCIB9369.

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

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

SEQ ID NO: 6 is the orf2 DNA sequence of clone Xenorhabdus nematophilus pCIB9381.

SEQ ID NO: 7 is a sequence similar to the protein esterase of juvenile hormone encoded by orf2 of the clone pCIB9381.

SEQ ID NO: 8 is the orf1 DNA sequence of clone Xenorhabdus poinarii pCIB9354.

SEQ ID NO: 9 is a protein sequence encoded by orf1 of clone pGIB9354.

SEQ ID NO: 10 is the orf2 DNA sequence of clone Xenorhabdus poinarii pCIB9354.

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

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

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

SEQ ID NO: 14 is the orf2 sequence of the Photorhabdus luminescens clone pCIB9383-21.

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

Escrow

The following products have been deposited with the Agricultural Research Service (Patent Culture Collection (NRRL), 1815 Northern University Street, Peoria, Illinois 61604, USA) under the Budapest Treaty on the International Recognition of Deposited Microorganisms for the Purpose of the Patent Procedure . All restrictions on the availability of deposited products must be irreversibly removed after the grant of a patent.

New nucleotide sequences whose expression leads to the formation of insecticidal toxins

The present invention relates to nucleotide sequences, the expression of which leads to the formation of new toxins, and to the production and use of toxins for controlling insect pests. Nucleotide sequences are isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii and Photorhabdus luminescens, which are members of the Enterobacteriaceae family. Bacteria of the genus Xenorhabdus are symbiotic bacteria of the nematodes of the genus Steinernema. Bacteria of the genus Photorhabdus are symbiotic bacteria of the nematodes of the genus Heterorhabditis. Nematodes colonize the insect larva, kill it and their offspring feeds on the dead larva. In fact, the insecticidal activity is due to the symbiotic bacteria of the river R. Xenorhabdus and Photorhabdus. Applicants were the first to isolate the nucleotide sequences of the present invention. The expression of the nucleotide sequences of the present invention leads to the formation of toxins that can be used to control lepidopteran insects, such as Plutella xylostella (cabbage moth).

The nucleotide sequence of the present invention, contained in clone pCIB9369, is characterized by the presence of a DNA fragment with a length of approximately 3.0 kb, which is deposited under the Budapest deposit agreement for patent purposes under registration number NRRL B-21883. The sequence of this DNA fragment is presented in SEQ ID NO: 1. SEQ ID NO: 1 contains two open reading frames (ORFs) (nucleotides 569-979 and nucleotides 1045-2334, respectively) encoding proteins with predicted molecular weights of 15 kDa and 47.7 kDa (SEQ ID NO: 2 and SEQ ID sequences NO: 3, respectively). Two ORFs are ordered as an operon-like structure. A search for known sequences that have homology with each individual ORF using the UWGCG Blast and Gap programs did not reveal a sequence that has any significant homology with ORF # 1, and revealed only 21% identity between ORF # 2 and the Bacillus cry3A protein sequence thuringiensis, which in this area is not considered significant. Gap analysis of the protein encoded by ORF No. 2 pCIB9369, performed using the Blast program, revealed a 30.6% amino acid sequence identity and 44.1% amino acid sequence similarity to the protein sequence associated with juvenile hormone esterase (GenBank, registration number 2921553, Henikoff et al., PNAS USA 89: 10915-10919 (1992)). The nucleotide sequence of the present invention was also compared with the known Xenorhabdus nematophilus sequences encoding the tox4 insecticidal toxin (WO 95/00647), but no significant homology was found. A 3.0 kb DNA fragment also compared with the nucleotide sequence published in WO 98/08388. Using the UWGCG Gap program, twenty-two sequences of 60 nucleotides each (60-mer) from a 38.2 kb DNA fragment described in WO 98/08388 were compared with a 3.0 DNA fragment. n according to the present invention. The nucleotide sequence of the first 60 mers begins at the base of 1 DNA fragment 38.2 kbp, and the remaining 60 mers are spaced at approximately 2.0 kbp. Each of 22 sequences was analyzed, as well as sequences complementary to them. The highest percentage of identity that was found between a 3.0 kb DNA fragment according to the present invention and one of these 60 measures, it was equal to 53%, which is not considered significant homology. In addition, five different DNA fragments of a 38.2 kbp DNA sequence were used by Southern blotting. tested for hybridization with a 3.0 kb DNA fragment according to the present invention. None of them revealed a positive hybridization signal.

Two open reading frames were also found in the nucleotide sequences of each of the clones pCIB9381, pCIB9354 and pCIB9383-21. The nucleotide sequences of two ORFs in each of pCIB9381 and pCIB9383-21 had a high degree of homology with the ORF sequences of pCIB9369. Consequently, ORF # 2 of the pCIB981 and pCIB9383-21 proteins have almost the same homology with the protein associated with juvenile hormone esterase as ORF # 2 of the pCIB9369 protein. The nucleotide sequence of ORF # 1 pCIB9354 is 77% identical to the nucleotide sequence of ORF # 1 pCIB9369, the nucleotide sequence of ORF # 2 pCIB9354 is 79% identical to the nucleotide sequence of ORF # 2 pCIB9369. ORF No. 2 of the pCIB9354 protein also has homology with a protein associated with juvenile hormone esterase (29.2% amino acid sequence identity, 42.2% amino acid sequence similarity).

In a preferred embodiment, the invention includes a nucleotide sequence substantially similar to nucleotides 569-979 of SEQ ID NO: 1, nucleotides 1045-2334 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, the expression of which leads to the formation of an insecticidal toxin. Recombinant vectors containing the nucleotide sequences of the present invention also fall within the scope of the present invention. In these vectors, nucleotide sequences are preferably included in expression cassettes containing regulatory elements necessary for the expression of nucleotide sequences in a host cell capable of expressing nucleotide sequences. Such regulatory elements typically include a promoter and termination signals, and they preferably also include elements enabling efficient translation of the polypeptides encoded by the nucleotide sequences of the present invention. Vectors including nucleotide sequences typically have the ability to replicate in specific host cells, preferably in the form of extrachromosomal molecules, and therefore can be used to amplify the nucleotide molecules of the present invention in host cells. In one embodiment, the host cells for such vectors are microorganisms, such as bacteria, especially E. coli. In another embodiment, the host cells for such recombinant vectors are endophytes or epiphytes. A preferred host cell for such vectors is a eukaryotic cell, such as a yeast cell, plant cell, or insect cell. Most preferred host cells are plant cells, such as corn cells. In another preferred embodiment, such vectors are viral vectors and are used to replicate nucleotide sequences in specific host cells, for example, insect cells or plant cells. Recombinant vectors are also used to transform host cells with nucleotide sequences of the present invention, and the nucleotide sequences are stably integrated into the DNA of such host cells. In an embodiment, such host cells are prokaryotic cells. 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 cells are plant cells, such as corn cells.

The nucleotide sequences of the invention can be isolated using the methods described in the examples below, or using PCR using the sequences listed in the sequence listing as the basis for constructing PCR primers. For example, oligonucleotides having sequences comprising the first and last 20-25 consecutive pairs of nucleotides of the sequence orf1 of SEQ ID NO: 1 (for example, nucleotides 569-588 and 957-976 of SEQ ID NO: 1) can be used as primers for PCR for amplification of the coding sequence of orf1 (nucleotides 569-976 of SEQ ID NO: 1) directly from the source strain (strain Xenorhabdus nematophilus ATCC 19061). Similarly, from the corresponding source strains using PCR using the ends of the coding sequences listed in the sequence listing, other gene sequences of the invention can be amplified as the basis for the PCR primers.

In another preferred embodiment, the insecticidal toxins comprise at least one polypeptide encoded by the nucleotide sequence of the invention. The molecular weight of the insecticidal toxin of the present invention according to experiments with size fractionation is more than 6000 Da. After proteinase K treatment in insect bioassay, only a minimal decrease in insecticidal activity is observed, which indicates that insecticidal toxins are practically resistant to proteinase K. Insecticidal toxins retain their insecticidal activity after storage for 2 weeks at 22 ° C or at 4 ° FROM. They also retain their insecticidal activity after freeze-drying and storage for 2 weeks at 22 ° C. Insecticidal toxins also still have activity after incubation for 5 min at 60 ° C, but they lose their insecticidal activity after incubation for 5 min at 100 ° C or at 80 ° C.

In other embodiments, the nucleotide sequences of the present invention can be modified by incorporating non-specific (random) mutations using a technique known as in-vitro recombination or DNA permutation. This method is described by Stemmer et al., Nature 370: 389-391 (1994) and in US Pat. No. 5,605,793, which are incorporated herein by reference. Millions of copies of mutated nucleotide sequences are obtained based on the original nucleotide sequence described herein and its variants with improved properties, such as increased insecticidal activity, increased stability, or different specificity or a wider range of target insect pests. The method involves the formation of a double-stranded polynucleotide with a mutation based on a matrix that is a double-stranded polynucleotide, which includes the nucleotide sequence of the present invention, wherein the double-stranded polynucleotide matrix is split into two double-stranded random fragments of the required size, and involves the steps of adding double-stranded random fragments of the same population to the resulting population or several single or double stranded oligonucleotides, and oligonucleotides include the region identity and the relative area of heterology matrix constituting double-stranded polynucleotide; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides to obtain single-stranded fragments; incubating the resulting population of single-stranded fragments with polymerase under conditions that lead to the renaturation of these single-stranded fragments in the indicated regions of identity to obtain pairs of renatured fragments, and these regions of identity are sufficient for priming replication of one of the members of the pair to the other, which results in a double-stranded polynucleotide with a mutation; and repeating the second and third stages for at least two more cycles, the mixture obtained in the second stage of the additional cycle comprising a double-stranded polynucleotide with a mutation obtained in the third stage of the previous cycle, and using an additional cycle, an additional amount of double-stranded polynucleotide with a mutation is obtained. According to a preferred embodiment, the concentration of the individual species of the double-stranded random fragment in the population of double-stranded random fragments is less than 1%, based on the weight of the total DNA. According to another preferred embodiment, the double stranded nucleotide template comprises at least about 100 kinds of polynucleotides. According to another preferred embodiment, the size of the double-stranded random fragments is from about 5 base pairs to about 5 kb. And according to another preferred embodiment, the fourth step of the method comprises repeating the second and third steps for at least 10 cycles.

Expression of nucleotide sequences in heterologous host microorganisms

Insecticidal toxins, which are biological insect control agents, are produced by expression of nucleotide sequences in heterologous host cells capable of expressing nucleotide sequences. In a first embodiment, Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens cells are described comprising modifications of at least one nucleotide sequence of the present invention at its location on the chromosome. Such modifications include mutations or deletions of existing regulatory elements, leading to a change in the expression of the nucleotide sequence, or the inclusion of new regulatory elements that control the expression of the nucleotide sequence. In another embodiment, additional copies of one or more nucleotide sequences are introduced into the cells of Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens either by embedding on the chromosome or by introducing molecules that replicate outside the chromosome that contain nucleotide sequences.

In yet another embodiment, at least one nucleotide sequence of the invention is inserted into a suitable expression cassette containing a promoter and termination signals. The expression of the nucleotide sequence may be constitutive or an inducible promoter that responds to various types of stimuli may be used to initiate translation. In a preferred embodiment, the cell in which the toxin is expressed is a microorganism, such as a virus, bacterium or fungus. In a preferred embodiment, a virus, such as a baculovirus, contains the nucleotide sequence of the invention in its genome and expresses large amounts of the corresponding insecticidal toxin after infection of the corresponding eukaryotic cells suitable for virus replication and expression of the nucleotide sequence. The insecticidal toxin thus produced is used as an insecticidal agent. Alternatively, baculoviruses are used that are designed to include a nucleotide sequence in order to infect and kill insects in vivo either by expression of an insecticidal toxin, or by a combination of viral infection and expression of an insecticidal toxin.

Bacterial cells can also serve as hosts for the expression of nucleotide sequences of the invention. In a preferred embodiment, non-pathogenic symbiotic bacteria that are able to live and multiply in plant tissues, the so-called endophytes, or non-pathogenic symbiotic bacteria that are able to colonize the phyllosphere or rhizosphere, the so-called epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flawobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium, are also possible hosts for the expression of nucleotide sequences of the invention for this purpose.

Methods for carrying out such genetic manipulations are known in the art and are specific for various suitable hosts. For example, pKK.223-3 and pKK.223-2 expression vectors inserted as transcriptional or translational fusion under the control of the tac or trc promoter can be used to express heterologous genes in E. coli. For the expression of operons encoding multiple ORFs, the simplest method is to embed the operon as a transcriptional fusion into a vector such as pKK.223-3, which allows the use of a related ribosome binding site for heterologous genes. Methods for overexpressing 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., Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz et al. (Eds.), American Society for Microbiology, Washington (1993)). Alternative overexpression systems are based, for example, on yeast vectors, and they include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekrishna, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman and Skatrud (eds.), American Society for Microbiology, Washington (1993)); Deguin and Barre, Biotechnology 12: 173-177 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990)).

In another preferred embodiment, at least one of the described nucleotide sequences is transferred and expressed in Pseudomonas fluorescens strain CGA267356 (described in published patent application EU 0472494 and in WO 94/01561), which has the characteristics necessary for biological control. In another preferred embodiment, the nucleotide sequence of the invention is transferred to the Pseudomonas auerofaciens strain 30-84, which also has the characteristics necessary for biological control. Expression in heterologous strains intended for biological control requires the selection of vectors suitable for replication in the selected host, and the appropriate choice of promoter. Methods for expressing in gram-negative and gram-positive bacteria and fungi are well known in the art.

Expression of nucleotide sequences in plant tissues

In a most preferred embodiment, at least one of the insecticidal toxins of the invention is expressed in a higher organism, for example a plant. In this case, transgenic plants expressing effective amounts of toxins protect themselves from pests. If an insect begins to eat such a transgenic plant, then it also absorbs expressed toxins. This can scare the insect away from further eating the plant tissue, or it can even have a negative effect on it or kill the insect. The nucleotide sequence of the present invention is inserted into an expression cassette, which is preferably stably integrated into the plant genome. In another preferred embodiment, the nucleotide sequence is included in a non-pathogenic, self-replicating virus. Plants transformed according to the present invention can be monocotyledonous or dicotyledonous plants, such as corn, wheat, barley, rye, sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus , onion, garlic, pepper, celery, large-fruited pumpkin, pepo pumpkin, hemp, zucchini, apple tree, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya , mango, banana, soy, tobacco, tomatoes, sorghum, sugarcane, with sugar beets, sunflowers, rape, clover, tobacco, carrots, cotton, alfalfa, rice, potatoes, eggplant, cucumber, Arabidopsis thalaiana and woody plants such as conifers and deciduous trees.

After certain plant species have been transformed with the required nucleotide sequence, it can be propagated in these species or transferred to other varieties of the same species, especially varieties of commercial importance, using traditional selection methods.

The nucleotide sequence of the present invention is preferably expressed in transgenic plants, thereby leading to the biosynthesis of the corresponding toxin in the transgenic plant. In this way, transgenic plants having increased resistance to insects are obtained. For their expression in transgenic plants, DNA molecules may need modification and optimization. Although in many cases the genes of microorganisms can be expressed with a high level in plants without modification, low expression in transgenic plants may be due to the fact that the nucleotide sequences of microorganisms have codons that are not preferred for plants. It is known in the art that all organisms have specific preferences for the most common codon, and codons in the nucleotide sequence that includes the DNA molecules of the present invention can be replaced in order to meet the specific preferences of the plant, preserving the amino acid sequences that encoded by them. In addition, a high level of expression in plants is most easily achieved using coding sequences in which the GC content is at least about 35%, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60 % Nucleotide sequences of microorganisms having a low GC content may be poorly expressed due to the presence of ATTTA motifs that can destabilize transcripts and AATAAA motifs that can cause inadequate polyadenylation. Although preferred gene sequences can be appropriately expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to suit the specific preferences of monocotyledonous or dicotyledonous plants with respect to the codon and with respect to the GC content, since it has been found that these preferences are different (Murray et al., Nucl. Acid. Res. 17: 477-498 (1989)). In addition, nucleotide sequences are screened for illegal splicing sites that can shorten transcripts. All necessary changes in the nucleotide sequences, such as the changes described above, are carried out using well-known methods of site-directed mutagenesis, PCR and the construction of synthetic genes using the methods described in published patent applications EP 0385962 (in the name of Monsanto), EP 0359472 (in company name Lubrisol) and WO 93/07278 (company name Ciba-Geigy).

For efficient translation initiation, it may be necessary to modify sequences adjacent to the methionine initiation site. For example, they can be modified to include sequences for which it is known that they are effective in plants. Joshi proposed a consensus sequence suitable for plants (NAR 15: 6643-6653 (1987)), and Clontech proposed an additional consensus translation initiator (1993/1994 catalog, p. 210). These consensus sequences can be used with the nucleotide sequences of this invention. These sequences are included in constructs that contain a nucleotide sequence up to and including ATG (while leaving the second amino acid unmodified) or alternatively up to and including the GTC codon next to ATG (in this case, it is possible to modify the second amino acid of the transgene).

In transgenic plants, the expression of nucleotide sequences is controlled by a promoter for which the ability to function in plants has been established. The choice of promoter may vary depending on the temporal and spatial patterns of expression, as well as depending on the target species. So preferred is the expression of the nucleotide sequences of the present invention in leaves, on the cob, in inflorescences (for example, ears, panicles, corn cobs, etc.), in the roots and / or in the seedlings. However, in many cases, it is required to have protection against more than one type of insect pests, therefore, it is desirable that expression occurs in several types of tissue. Although it has been established that many promoters from dicotyledonous plants can function in monocotyledons and vice versa, ideally, dicotyledon promoters are chosen for expression in dicotyledons, and monocotyledon promoters are selected for expression in monocotyledons. However, there are no restrictions on the source of the selected promoters; it is sufficient that they are functionally active with respect to controlling the expression of DNA molecules in the desired cell.

Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and 35S and 19S CaMV promoters. The nucleotide sequences of the present invention can also be expressed under the control of chemically regulated promoters. This allows the synthesis of insecticidal toxins only when cultivated plants are treated with inducing chemical compounds. A preferred method for chemically inducing gene expression is described in detail in published application EP 0332104 (in the name of Ciba-Geigy) and in US Pat.

A preferred category of promoters are wound-induced promoters. Numerous promoters have been described that provide expression at the site of injury, as well as at the site of infection by the pathogen. Ideally, such a promoter should be active only locally at the site of infection, and in this case, insecticidal toxins accumulate only in those cells in which it is necessary to synthesize insecticidal toxins in order to kill the attacking insect pest. Preferred promoters of this type include those described by Stanford et al., Mol. Gen. Genet. 215: 200-208 (1989), in Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-161 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22: 783-792 (1993), from Firek et al. Plant Molec. Biol. 22: 129-142 (1993) and Warner et al. Plant J. 3: 191-201 (1993)).

Preferred tissue-specific expression patterns include expression specific for green tissue, specific for the root, specific for the stem and specific for the flower. Promoters suitable for expression in green tissue include many promoters that regulate the expression of genes involved in photosynthesis, and many of them originating from both monocotyledonous and dicotyledonous plants have been cloned. A preferred promoter is the PEPC promoter of the corn phosphoenolcarboxylase gene (Hudspeth and Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root-specific expression is the promoter described by de Framond (FEBS 290: 103-106 (1991); EP 0452269 in the name of Ciba-Geigy). A preferred stem-specific promoter is the promoter described in US Pat. No. 5,625,136 (to Ciba-Geigy), which controls the expression of the maize trpA gene.

In preferred embodiments, transgenic plants express the nucleotide sequences of the invention in a root-specific manner. In other preferred embodiments, transgenic plants express nucleotide sequences under the control of a promoter induced by injury or induced by infection by a pathogen.

In addition to choosing an acceptable promoter, constructs designed to express protein in plants require an appropriate transcription terminator that must be attached during transcription relative to the heterologous nucleotide sequence. Several such terminators are known and can be used in the art (e.g., tm1 CaMV, E9 rbcS). Any terminator that is known to function in plants can be used according to the present invention.

Numerous other sequences may be included in the expression cassettes provided herein. These include sequences that have been shown to be capable of enhancing expression, such as intron sequences (e.g., Adh1 and bronze1 genes) and viral leader sequences (e.g., tobacco mosaic virus (TMV), corn chlorose spotting virus (MCMV), and alfalfa mosaic virus (AMV) )).

It may be preferable to direct the expression of DNA molecules to various plant cell compartments. In some cases, localization in the cytosol may be required, while in other cases, localization in any subcellular organelle may be preferred. Subcellular localization of the enzyme encoded by the transgene can be carried out using methods well known in the art. Typically, DNA encoding a directing peptide from a known gene product that carries out targeted transfer into a specific organelle is processed and fused against the course of transcription relative to the nucleotide sequence. A number of such transfer-guiding sequences are known for chloroplast and it has been established that they function in heterologous constructs. The expression of the nucleotide sequences of the present invention is also directed to the endoplasmic reticulum or to the vacuoles of the host cells. Methods for achieving this are well known in the art.

Vectors suitable for transformation of plants are given below in the present description. For Adrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence can be used, however, any vector is suitable for direct gene transfer, and linear DNA containing only the construct of interest may be preferred. In the case of direct gene transfer, transformation with individual types of DNA or cotransformation can be used (Schocher et al., Biotechnology 4: 1093-1096 (1986)). For direct gene transfer as well as for Adrobacterium mediated transfer, transformation is usually (but not necessarily) used using a selectable marker that can confer resistance to an antibiotic (kanamycin, hygromycin or metatrexate) or herbicide (basta). Examples of such markers are neomitsintransferaza, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyl, dehalogenase 2,2-dichloropropionic acid, acetohydroxyacid synthase, 5-enolpyruvylshikimate-phosphate, galoarilnitrilaza, protoporphyrinogen oxidase, acetyl-CoA carboxylase, digidropteroatsintaza, chloramphenicol acetyl transferase and β-glucuronidase. However, the selection of a selectable marker is not critical to the invention.

The recombinant DNA molecules of the invention described above can be introduced into a plant cell by numerous methods known in the art. It will be apparent to those skilled in the art that the choice of method may depend on the type of plant to be transformed. Suitable methods for transforming plant cells include microinjection (Crossway et al., BioTechniques 4: 320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA, 83: 5602-5606 (1986); Agrobacterium mediated transformation (Hinchee et al., Biotechnology 6: 915-921 (1988); (for the transformation of maize plants, see Ishida et al., Nature Biotechnology 14: 745-750 (June 1996)), direct gene transfer ( Paszkowski et al., EMBO J. 3: 2717-2722 (1984)), ballistic particle injection using devices supplied by Agracetus, Inc., Madison, Wisconsin and Dupont, Inc. Wilmington, Delaware (see, e.g., Sanford et al., U.S. Patent 4,954,050; and McCabe et al., Biotechnolog y 6: 923-926 (1988)); see also Weissinger et al., Annual Rev. Genet. 22: 421-477 (1988); Sanford et al., Particulate Science and Technology 5: 27-37 (1987) ) (onion); Svab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al. Plant Physiol. 87: 671-674 (1988) (soybeans ); McCabe et al., Bio / Technology 6: 923-926 (1988) (soybeans); McCabe et al., Bio / Technology 6: 923-926 (1988) (soybeans); Klein et al., Proc. Natl. Acad. Sci. USA, 85: 4305-4309 (1988) (corn); Klein et al., Bio / Technology 6: 559-563 (1988) (corn); Klein et al. Plant Physiol. 91: 440-444 (1988) (corn); Fromm et al., Bio / Technology 8: 833-839 (1990); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) (corn); Koziel et al., Biotechnology 11: 194-200 (1993) (corn); Shimamoto et al., Nature 338: 274-277 (1989) (Figure); Christou et al., Biotechnology 9: 957-962 (Figure); Datta et al., Bio / Technology 8: 736-740 (1990) (Figure); European patent application EP 0332581 (hedgehog team and other species of Pooideae); Vasil et al., Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al. Plant Physiol. 102: 1077-1084 (1993) (wheat); Wan et al. Plant Physiol. 104; 37-48 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89: 525-533 (1994) (barley); Umbeck et al., Bio / Technology 5: 263-266 (1987) (cotton); Casas et al., Proc. Natl. Acad. Sci. USA 90: 11212-11216 (December 1993) (sorghum); Somers et al., Bio / Technology 10: 1589-1594 (December 1992) (oats); Torbert et al. Plant Cell Reports 14: 635-640 (1995) (oats); Weeks et al. Plant Physiol. 102: 1077-1084 (1993) (wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al., The Plant Journal 5: 285-297 (1994) (wheat). The most preferred range of embodiments of the introduction of recombinant DNA molecules into maize plants by micro-projectile bombardment can be found in Koziel et al., Biotechnology 11: 194-200 (1993), Hill et al., Euphytica 85: 119-123 (1995) and Koziel et al., Annals of the New York Academy of Sciences 792: 164-171 (1996). A further preferred embodiment is the maize protoplast transformation method described in EP 0292435. Transformation can be carried out using individual types of DNA or several types of DNA (ie cotransformation), and both methods are suitable for use using a peroxidase coding sequence.

According to another preferred embodiment, the nucleotide sequence of the present invention directly transforms the plastid genome. The main advantage of plastid transformation is that plastids are generally able to express bacterial genes without significant modification and that plastids are able to express multiple open reading frames under the control of a single promoter. The method of transformation of plastids is described in detail in patents US 5451513, 4545817 and 5545818; in international patent application WO 95/16783; and McBride et al., Proc. Natl. Acad. Sci. USA 91: 7301-7306 (1994). A basic chloroplast transformation technique involves introducing into a suitable target tissue portions of cloned plastid DNA flanking the selectable marker along with the gene of interest, for example, using a bio-ballistic transfer method or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). Flanking regions of 1-1.5 kbp length, called directed transfer sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastoma. Initially, point mutations in the chloroplast genes 16S-pPHK and rps12, which cause resistance to spectinomycin and / or streptomycin (Svab Z., Hajdukiewicz P. and Maliga P., Proc. Natl. Acad. Sci. USA), were used as selectable markers for transformation. 87: 8526-8530 (1990); Staub JM and Maliga P., Plant Cell 4: 39-45 (1992). This results in stable homoplasmic transformants with a frequency of about 1 transformant per 100 bombardment of target leaves. The presence of cloning sites between these markers allows you to create a vector for the introduction of foreign genes about supporting direct transfer to plastid (Staub JM and Maliga P., EMBO J. 12: 601-606 (1993)). A significant increase in the frequency of transformation is obtained by replacing the genes of recessive rRNA or p-protein, which determine antibiotic resistance by a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab Z. and Maliga P. Proc. Natl. Acad. Sci. USA 90: 913-917 (1993)). Previously, this marker was successfully used for the high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont M., Nucl. Acids Res. 19: 4083-4089 (1991)). Other selectable markers suitable for transforming plastids are known in the art and fall within the scope of the invention. As a rule, to achieve a homoplastid state after transformation, approximately 15-20 cycles of cell division are required. Expression in plastids, in which genes are inserted by homologous recombination into all several thousand copies of the circular plastid genome present in each plant cell, has the advantage that a huge number of copies exceeding the number of copies expressed by nuclear DNA genes allows for levels expression, which can easily exceed 10% of the total soluble protein of the plant. In a preferred embodiment, the nucleotide sequence of the present invention is inserted into a vector that provides targeted transfer to the plastid and transformed with it the plastid genome of the corresponding host plant. Plants are obtained homoplastic with respect to plastid genomes that contain the nucleotide sequence of the present invention and which are preferably capable of expressing the nucleotide sequence at a high level.

Preparation of formulations containing insecticidal compositions

Compositions comprising at least one of the insecticidal toxins of the present invention also fall within the scope of the invention. To effectively control pests, such compositions should preferably contain a sufficient amount of toxin. These amounts vary depending on the crop to be protected, on the particular target pest, and on environmental conditions such as moisture, temperature or soil type. In a preferred embodiment, the compositions comprising insecticidal toxins comprise host cells expressing toxins not further purified. In another preferred embodiment, cells expressing insecticidal toxins are lyophilized before use as an insecticidal agent. In another embodiment, such insecticidal toxins are created that are secreted by the host cells. In cases where it is desirable to purify the toxins from the host cells in which they are expressed, various degrees of purification of the insecticidal toxins can be achieved.

Also within the scope of the present invention is a method of preparing compositions comprising at least one insecticidal toxin of the present invention, mixed uniformly with one or more compounds or groups of compounds listed in the present description. The present invention also relates to methods for treating plants, comprising treating plants with insecticidal toxins or compositions containing insecticidal toxins. Insecticidal toxins can be applied in the form of compositions to the cultivated crop area or to the plant to be treated, simultaneously or sequentially with other biologically active compounds. These compounds can be either fertilizers or micronutrient donors, or other drugs that affect plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if necessary together with other carriers, surfactants or facilitating the application of adjuvants commonly used in the field of preparation forms. Suitable carriers and adjuvants may be solid or liquid, and they are substances commonly used in the field of formulation, i.e. suitable or regenerated minerals, solvents, dispersing agents, wetting agents, adhesives, binders or udobreniya.

A preferred method for using the insecticidal toxins of the present invention is to spray the habitat of a pest, for example, soil, water surface or plant leaves. The number of treatments and application rates depend on the type and intensity of infection by the pest. Insecticidal toxins can also penetrate the plant through roots from the soil (systemic effect) as a result of impregnation of the plant growth site with a liquid composition or as a result of adding compounds in solid form to the soil, for example in granular form (application to the soil). Insecticidal toxins can also be applied to the seed material (coating) either by impregnating the seeds with a liquid composition containing insecticidal toxins, or by coating them with a solid composition. In some cases, you can also apply other types of processing, for example selective processing of trunks or buds of a plant. Insecticidal toxins can also be used as baits placed on or below the surface of the soil.

Insecticidal toxins are used in unmodified form or preferably together with adjuvants, commonly used in the field of formulation technology, and therefore, they can be prepared in the usual way in the form of emulsion concentrates, coating pastes, ready to use sprayed or diluted solutions, diluted emulsions, wettable powders, soluble powders, dusts, granules, as well as in encapsulated form, for example, in polymeric substances. Depending on the nature of the compositions, treatment methods such as spraying, small-droplet spraying, dusting, scattering or irrigation are selected in accordance with the objectives and prevailing circumstances.

Formulations, compositions or preparations containing insecticidal toxins and, optionally, a solid or liquid adjuvant, are prepared in a known manner, for example, by homogeneous mixing and / or grinding of the active substances with excipients, for example with solvents, solid carriers and, if necessary, with surfactants (detergents).

Suitable solvents include aromatic hydrocarbons, preferably fractions having 8-12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters such as ethanol, ethylene glycol monomethyl or monoethyl ethers, ketones such as cyclohexanone, highly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethylformamide, and also cooking oils or epoxidized vegetable oils, such as epoxidized coconut oil or soybean oil, or water.

Solid carriers used, for example, for dusts and dispersible powders, are typically natural mineral fillers such as calcite, talc, kaolin, montmorillonite or attapulgite. In order to improve physical properties, it is also possible to add finely divided silicic acid or highly dispersed adsorbent polymers. Suitable granular adsorption carriers are those of a porous type, for example pumice, broken brick, sepiolite or bentonite; and suitable non-absorbent carriers are materials such as calcite or sand. In addition, a large number of pre-granulated materials of inorganic or organic origin can be used, for example, dolomite or powdered plant residues are particularly preferred.

Suitable surfactants are nonionic, cationic and / or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" also includes mixtures of surfactants. Suitable anionic surfactants can be both water soluble soaps and water soluble synthetic surfactants.

Suitable soaps are alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and higher fatty acids (having chains containing 10-22 carbon atoms), for example, sodium or potassium salts of oleic or stearic acid, or mixtures of natural fatty acids, which may be obtained, for example, from coconut or tall oil. In addition, methyl taurine fatty acid salts may also be used.

More often, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylaryl sulfonates.

Fatty sulfonates or sulfates are usually used in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts, and they usually have an alkyl radical containing 8-22 carbon atoms, which also includes an alkyl moiety of acyl radicals, for example, in the form of sodium or calcium salt of lignosulfonic acid, dodecyl sulfate or a mixture of sulfates of fatty alcohols derived from natural fatty acids. These compounds also include salts of sulfuric acid esters and sulfonic acid adducts of a fatty alcohol / ethylene oxide. Sulfonated derivatives of benzimidazole preferably contain 2 sulfonic acid groups and one fatty acid radical containing from 8 to 22 carbon atoms. Examples of alkylaryl sulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid or a naphthalenesulfonic acid / formaldehyde condensation product. Suitable phosphates are also suitable, for example salts of a phosphoric acid ester and an adduct of paranylphenol with 4-14 moles of ethylene oxide.

Nonionic surfactants are preferably derivatives of polyglycol ether and aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkyl phenols, these derivatives contain 3-30 glycol ether groups and 8-20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6- 18 carbon atoms in the alkyl fragment of alkyl phenols.

Further suitable nonionic surfactants are water-soluble adducts of polyethylene oxide and polypropylene glycol, ethylene diamine propylene glycol and alkyl propylene glycol containing 1-10 carbon atoms in the alkyl chain, the adducts containing 20-250 groups of ethylene glycol ether and 10-100 groups of propylene glycol ether. These compounds typically contain 1-5 ethylene glycol moieties per propylene glycol moiety.

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

Cationic surfactants are preferably quaternary ammonium salts which have at least one C ₈ -C ₂₂ alkyl radical as an N substituent and, in addition, unsubstituted or halogenated lower alkyl, benzyl or hydroxy ( lower) alkyl radicals. Salts in the form of halides, methyl sulfates or ethyl sulfates are preferred, for example, stearyl trimethyl ammonium chloride or benzyldi (2-chloroethyl) ethyl ammonium bromide.

Surfactants commonly used in the field of formulation are described, for example, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp. Ridgwood, New Jersey, 1979; and Sisley and Wood, Encyclopedia of Surface Active Agents, Chemical Publoshing Co., Inc. New York, 1980.

Examples

The invention is described below with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention, unless otherwise indicated. The standard recombinant DNA and molecular cloning techniques used are well known in the art and are described by Ausubel (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); at T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory, Cold Spring Harbor, NY (1989); and T.J. Silhavy, M. L. Berman and L. W. Enquist, Ezperiments with Gene Fusions, Cold Spring Laboratory, Cold Spring Harbor, NY (1984).

A. Isolation of nucleotide sequences, the expression of which leads to the formation of toxins with activity against insects from the order Lepidoptera

Example 1. Growing strains of Xenorhabdus and Photorhabdus

For analyzes of biological activity against insects, the following strains are grown in a nutrient medium recommended by ATCC at 25 ° C for 3 days. For DNA isolation, cultures were grown under the same conditions for 24 hours.

Strain Xenorhabdus nematophilus ATCC 19061.

Strain Xenorhabdus nematophilus Pst1, isolate USDA.

Strain Xenorhabdus poinarii ATCC 49122.

Strain Photorhabdus luminescens Ps5, isolate USDA.

Example 2

Biological activity assays for Plutella xylostella (Px) are performed by aliquoting 50 μl of each of the E. coli cultures onto a solid artificial nutrient medium for P. xylostella (Biever and Boldt, Annals of Entomological Society of America, 1971; Shelton et al. , J. Ent. Sci. 26:17). Five newly hatched P. xylostella caterpillars from a laboratory colony adapted to the feed are placed in each dish containing feed, and then closed with a white paper cover (Bioserve product No. 9049). The activity for at each concentration is analyzed using 10 tracks. Trays with cups are placed in a thermostat at 72 ° F for 3 days with a light cycle of light: darkness 14:10 (in hours). Record the number of live caterpillars in each cup.

Example 3. The results of the analysis of biological activity

In the analysis of biological activity according to the method described in example 2, the use of a broth containing a strain of Xenorhabdus nematophilus ATCC 19061, leads to 100% mortality of Plutella xylostella (Px). Similarly, in the analysis of biological activity according to the method described in example 2, the use of each of the broths containing Xenorhabdus nematophilus Pst1 strain, Xenorhabdus poinarii strain ATCC 49122 and Photorhabdus luminescens Ps5 strain leads to 100% mortality of Plutella xylostella (Px).

Example 4. Construction of a cosmid library

Complete DNA was isolated from Xenorhabdus nematophilus ATCC 19061 strain by treating freshly grown cells resuspended in 100 mM Tris, pH 8, 10 mM EDTA for 30 min at 37 ° C with lysozyme taken at a concentration of 2 mg / ml. Proteinase K was added at a final concentration of 100 μg / ml in 0.5% SDS and incubated at 45 ° C. The solution becomes clear and very viscous. The concentration of SDS was increased to 1% and 300 mM NaCl and an equal amount of a phenol-chloroform-isoamyl alcohol mixture were added. The sample was gently mixed for 5 min and centrifuged at 3K (3 × lysine). The process is repeated twice. Then the aqueous phase is mixed with 0.7 volumes of isopropanol and centrifuged. Debris DNA is washed three times with 70% ethanol and carefully resuspended in 0.5xTU (mixing element). 6 μg of DNA is treated with 0.3 units Sau3A / μg DNA at 37 ° C for 3.5 min in a volume of 100 μl. Then, the sample was incubated for 30 min at 65 ° C to inactivate the enzyme, and then incubated for 30 min at 37 ° C with 2 units. alkaline phosphatase from the calf intestine. The sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed and mixed with 0.7 volumes of isopropanol and centrifuged. Debris is resuspended in 0.5xTE at a concentration of 100 ng / ml.

The SuperCos cosmid vector (Stratagene, La Jolla, Calif.) Is prepared according to the supplier’s instructions using the BamHI cloning site. The resulting SuperCos vector at a concentration of 100 ng / ml was ligated overnight at 6 ° C in a volume of 5 μl in a 2: 1 ratio with X. nematophilus DNA, previously digested with Sau3A. The resulting ligation mixture is packaged using a Gigapack XL III kit (Stratagene) according to the supplier's instructions. Packed phages infect cells of the E. coli strain XL-1MR (Stratagene) according to the instructions of the supplier. The cosmid library was plated on L-agar containing 50 μg / ml kanamycin and incubated for 16 hours at 37 ° C. 500 colonies are plaque-plated on freshly processed L-kan plates with a density of 50 colonies / plate. Cells are washed with L-broth and mixed with 20% glycerol and frozen at -80 ° C.

For the genome of X. nematophilus, the size of 4.2 bp 450 clones with an average size of 40 kb correspond to a 4-fold overlap of the genome. Therefore, when screening for 450 clones, there is a 99% chance of detecting any gene.

Cosmid libraries of strain Xenorhabdus nematophilus Pst1, strain Xenorhabdus poinarii ATCC 49122 and strain Photorhabdus luminescens Ps5 are constructed in a similar manner.

Example 5. The results of biological analysis of cosmids and the identification of clones with insecticidal activity

For analysis of biological activity against insects, 400 E. coli clones from each cosmid library were screened to identify clones with activity against Plutella xylostella. The cloned cosmid of the strain Xenorhabdus nematophilus ATCC 19061, having insecticidal activity, is designated as pCIB9362. A cloned cosmid with insecticidal activity, 42 kb in length from strain Xenorhabdus nematophilus Pst1 is designated as pCIB9379. A cloned cosmid with insecticidal activity, 42 kb in length from strain Xenorhabdus poinarii ATCC 49122 is designated as pCIB9354. An insecticidal cloned cosmid with a length of 7 kb from strain Photorhabdus luminescens Ps5 are designated as pCIB9383-21.

Example 6. Isolation of Subclones with Insecticidal Activity Clone pCIB9362 was digested with SacII and a 9 kb DNA fragment was isolated. This fragment is inserted into a Bluescript vector digested with SacII. The resulting ligation mixture transform cells of E. coli strain DH5α according to the method described in Molecular Cloning, 2nd edition (Sambrook et al.). The resulting transformation mixture was plated on L-agar containing 100 μg / ml ampicillin and incubated overnight at 37 ° C. Isolated colonies were grown in L-broth containing 100 μg / ml ampicillin, and plasmid DNA was isolated using alkaline miniprep according to the method described in Molecular Cloning, 2nd edition. A 9 kbp clone isolated using SacII designated as pCIB9362-3, and it has insecticidal activity causing 100% mortality of Plutella xylostella. 3 μg of pCIB9362-3 is isolated, digested with 0.3 units. Sau3A per 1 μg of DNA for 4, 6 and 8 minutes at 37 ° C and kept at 75 ° C for 15 minutes. Samples are grouped and inserted into the pUC19 plasmid, previously digested with BamHI, and treated with calf intestinal alkaline phosphatase. The resulting ligation product transforms the cells of the E. coli strain DH5α, seeded on L-agar containing XgaI / Amp, according to the method described in Molecular Cloning, and grown overnight at 37 ° C. White colonies were selected and grown in L-broth containing 100 μg / ml ampicillin, and plasmid DNA was isolated according to the method described above. DNA was digested with EcoRI / HindIII and the new restriction fragments were sequenced. Sequencing primers are obtained from Genosys Biotechnologies (Woodland, Texas). Sequencing is carried out according to the dideoxy method based on chain breaking and is performed using an automatic DNA sequencer model 377 from Applied Biosystems Inc. (Forster City, California). Sequences are combined using Sequencher 3.0 software from Gene Code Corporation (Ann Arbor, MI).

After the identification of restriction sites and possible ORFs, plasmid pCIB9362-3 was cleaved with ClaI and a 3.0 kbp fragment. isolate, clone in the Bluescript vector and transform cells of E. coli DH5α strain with it. Isolated colonies were grown according to the method described above, and plasmid DNA was isolated by alkaline lysis. The subclone is identified as pCIB9369 and it causes 100% death of Plutella xylostella in experiments on the study of biological activity. Strain pCIB9369 was deposited in the Culture Collection for patenting purposes by the USDA ARS on November 12, 1997 under registration number NRRL B-21883.

A 20 kbp fragment designated pCIB9381 is subcloned from the cosmid pCIB9379 using NotI digestion.

Example 7. The results of the analysis of biological activity

PCIB9369 biological activity assays are carried out for various species of insects.

on the. - no activity detected

+ - significant growth inhibition,

+ + - death> 40%, but less than 100%,

+ + + - 100% death.

These results indicate that an insectic toxin resulting from the expression of a 3.0 kb nucleotide sequence in pCIB9369 has a high activity against Plutella xylostella.

Analyzes of the biological activity of strains pCIB9381, pCIB9354 and pCIB9383-21 show that these strains also have high insecticidal activity against Plutella xylostella.

Example 8. The determination of the size of the fraction, causing insecticidal activity

The cosmid clone Xenorhabdus nematophilus pCIB9369 and the BlueScript vector from Stratagene in E. coli strain DH5α were grown in a medium consisting of 50% Terrific broth and 50% Luria broth supplemented with 50 μg / ml ampicillin. Cultures are grown in shake flasks (300 ml in flasks with 1000 ml septa) at 250 rpm overnight at 37 ° C. The cultures of each strain are centrifuged at 7000 rpm in a Sorvall GS type centrifuge at 4 ° C. Pelletized cells are resuspended in 30 ml of 50 mM NaCI, 25 mM Tris buffer, pH 7.0. Concentrated cells were destroyed by ultrasonic irradiation using a Branson Model 450 Sonicator type device for approximately eight 10 second cycles using ice cooling between cycles. The product obtained as a result of ultrasonic irradiation is centrifuged in a Sorvall SS34 type centrifuge at 6000 rpm for 10 min at 4 ° C. The resulting supernatants are filtered through a 0.2 μm filter. The debris of centrifuged products obtained by ultrasonic irradiation is resuspended in 30 ml of 50 mM NaCl, 25 mM Tris buffer, pH 7.0.

3 ml filtrate fractions were added to Bio-Rad Econo-Pac 10DG columns, pre-equilibrated with 10 ml of 50 mM NaCl, 25 mM Tris buffer, pH 7.0. Discard the stream collected during sample loading. Samples are fractionated by the sequential addition of two 4 ml portions of each NaCl-Tris equilibration buffer. The first three fractions are selected for analysis. The first fraction should contain all products having a molecular weight above about 6000 Da. The following fractions should contain products with a molecular weight of less than 6000 Da.

A sample of the ultrasonic irradiated filtrate and resuspended debris after ultrasonic irradiation were tested along with three fractions obtained using a 10DG column for their activity against freshly hatched P. xylostella caterpillars by contacting with an infected surface. The filtered supernatant of the product obtained by irradiation and the first fraction of sample 9369 obtained from the column were highly effective against P. xylostella. The second and third fractions of sample 9369 were inactive. None of the samples obtained from the culture of strain DH5α in the BlueScript plasmid had activity. These results indicate that the insecticidal activity inherent in clone X. nematophilus pCIB9369, as well as its homologs pCIB9381, pCIB9354 and pCIB9383-21, is due to a fragment having a molecular weight exceeding 6000 Da.

Example 9. The stability of insecticidal activity

300 ml of Luria broth supplemented with 100 μg / ml of ampicillin are inoculated with plasmid pCIB9369 and grown overnight at 37 ° C.

Samples are placed in sterile 15 ml screw caps and stored at 22 ° C and 4 ° C. One sample is centrifuged, the supernatant is removed, freeze dried and stored at 22 ° C. These samples are stored under the indicated conditions for 2 weeks and then their biological activity against P. xylostella is analyzed. The product obtained by freeze-drying is resuspended in an equal volume as previously described for a freeze-dried sample. All samples are resuspended by shaking. Another sample was kept at 100 ° C for 5 minutes.

on the. - no activity detected

Example 10. Inactivation of insecticidal activity when heated. Determine the stability of the toxin when heated. Cultures of E. coli strain pCIB9369 (E. coli DH5α host strain carrying a 3.0 kb Xenorhabdus nematophilus DNA fragment) were grown in a 50:50 mixture of Luria broth and Terrific broth. Cultures are grown at 37 ° C in test tubes on a rotary shaker. Samples of each culture with a volume of 1 ml are placed in an Eppendorf tube with a volume of 1.5 ml and maintained at 60 ° C and 80 ° C. After 5 minutes, the samples were removed from the thermostat and allowed to cool to room temperature. The sample along with the untreated portion of the culture is analyzed in relation to activity against P. xylostella. 50 μl of the sample is sprayed onto the feed, allowed to dry and freshly hatched P. xylostella caterpillars are placed on the surface. Samples are incubated for 5 days at room temperature.

The untreated sample and the sample treated by heating to 60 ° C caused 100% death. A sample treated by heating to 80 ° C and a control sample, which is only feed, did not cause noticeable death.

Example 11. The effect of protease treatment on insecticidal activity

The cosmid clone Xenorhabdus nematophilus pCIB9369 and the Stratagene BlueScript vector in E. coli DH5α host strain were grown in a medium consisting of 50% Terrific broth and 50% Luria broth supplemented with 50 μg / ml ampicillin. Cultures are grown in shake flasks (300 ml in flasks with 1000 ml septa) at 250 rpm overnight at 37 ° C. The cultures of each strain are centrifuged at 7000 rpm in a Sorvall GS type centrifuge at 4 ° C. Pelletized cells are resuspended in 30 ml of 50 mM NaCl, 25 mM Tris buffer, pH 7.0. Concentrated cells are destroyed by ultrasonic irradiation using a device such as a Branson Model 450 Sonicator for approximately eight 10-second cycles using ice cooling in between cycles. The product obtained as a result of ultrasonic irradiation is centrifuged in a Sorvall SS34 type centrifuge at 6000 rpm for 10 min at 4 ° C. The resulting supernatants are filtered through a 0.2 μm filter. The concentration of Ca ⁺⁺ in 1 ml supernatant samples was adjusted to 5 mM by adding CaCl ₂ . Protease K (Gibco BRL; Gaithersburg, MD) was added to a concentration of 500 μg / ml. Samples were incubated at 37 ° C for 2 and 24 hours. Control samples were prepared and incubated, to which Ca ⁺⁺ was added but no protease was added.

Ultrasound-irradiated filtrate obtained from samples containing pCIB9369 and samples containing pCIB9369 incubated in the presence of 5 mM Ca ⁺⁺ caused 100% death of freshly hatched Plutella xylostella caterpillars. Samples containing pCIB9369 incubated in the presence of protease K (with the addition of Ca ⁺⁺ ) caused a slightly smaller death of Plutella caterpillars, amounting to approximately 90%).

Example 12. Comparative analysis of the sequence and sequence of protein cry3A and the protein sequence of the juvenile hormone esterase

The nucleotide sequence of pCIB9369 (SEQ ID NO: 1) has two open reading frames (ORFs) at nucleotide positions 569-976 and 1045-2334. According to the search results using the UWGCG Blast and Gap programs, ORF No. 1 has no homology with any of the sequences from GenBank. Gap analysis of the protein encoded by ORF No. 2 pCIB9369, performed using the Blast program, revealed a slight homology (21% identity) with the protein Bacillus thuringensis cry3A. Moreover, the Gap analysis of the protein encoded by ORF No. 2 pCIB9369, carried out using the Blast program, revealed a certain degree of homology (30.6% amino acid sequence identity and 44.1% amino acid sequence similarity) with a protein similar to esterase juvenile hormone (GenBank registration number 2921553; Henikoff et al., PNAS USA 89: 10915-10919 (1992).

The nucleotide sequences pCIB9381, pCIB9354 and pCIB9383-21 also have two open reading frames each. The nucleotide sequences of two ORFs in each of pCIB9381 and pCIB9383-21 are more than 90% identical to ORF pCIB9369) Therefore, ORF No. 2 of the pCIB9381 and pCIB9383-21 proteins has almost the same homology with the protein associated with juvenile hormone esterase as ORF No. 2 protein pCIB9369. The nucleotide sequence of ORF No. 1 of the protein pCIB9354 is identical to 77% of the nucleotide sequence of ORF No. 1 of the protein pCIB9369, and the nucleotide sequence of ORF No. 1 of the protein pCIB9354 is identical to 79% of the nucleotide sequence ORF No. 2 of the protein pCIB9369. ORF No. 2 of the protein pCIB9354 also has homology with a protein associated with esterase of juvenile hormone (amino acid sequence identity of 29.2%, amino acid sequence similarity of 42.2%).

Example 13. Comparison of the sequence of pCIB9369 with sequences described in WO 98/08388

From a 38.2 kb DNA fragment whose nucleotide sequence is described in WO 98/08388, twenty-two sequences of 60 nucleotides each (60-mer) are isolated and compared with the pCIB9362-3 nucleotide sequence including pCIB9369. The first 60-mer begins with the base of 1 DNA fragment 38.2 kbp in length, and the rest are located on the DNA fragment at approximately 2 kbp intervals Their positions on the 38.2 kb DNA fragment are listed below:

1-60; 2041-2100; 4021-4080; 6001-6060; 8041-8100; 10021-10080; 12001-12060; 14041-14100; 16021-16080; 18001-18060; 20041-20100; 22021-22080; 24001-24060; 26041-26100; 28021-28080; 30001-30060; 32041-32100; 34021-34080; 36001-36060; 38041-38100; 38161-38220.

Sequences were compared using the UWGCG Gap program, and both each of the 22 60-dimensional sequences and their complementary sequences were analyzed. The results of this comparative analysis indicate that the largest percentage of identity is 53%, which in this area is not considered a significant degree of homology.

Example 14. Southern blot analysis using probes based on sequences from WO 98/08388

To amplify DNA fragments from a 38.2 kb DNA fragment described in WO 98/08388, pairs of oligonucleotides are constructed. Oligonucleotides are obtained from Genosys Biotechnologies (The Woodlands, Texas) and their positions on a 38.2 kb DNA fragment. are listed below. Their sizes and sizes of fragments amplified by PCR are also indicated:

VK1046: positions 20-40,

VK1047: Provisions 2078-2100.

The size of the fragment amplified by PCR using primers VK1046 and VK1047: 2080 base pairs,

VK1048: provisions 11221-11241,

VK1049: Provisions 13360-13380.

The size of the fragment amplified by PCR using primers VK1048 and VK1049: 2120 base pairs,

VK1050: provisions 26581-26601,

VK1051: Provisions 28537-28560.

The size of the fragment amplified by PCR using primers VK1050 and VK1051: 1979 base pairs,

VK1052: provisions 18901-18921,

VK1053: provisions 20321-20340.

The size of the fragment amplified by PCR using primers VK1052 and VK1053: 1439 base pairs.

VK1054: provisions 34261-34281,

VK1055: positions 35320-35340 base pairs,

The size of the fragment amplified by PCR using primers VK1054 and VK1055: 1079 base pairs.

PCR is carried out in a Perkin-Elmer 9600 type cell using the following conditions: 94 ° C, 2 min; then 30 cycles at 94 ° C, 30 s; 54 ° C, 30 s; 72 ° C, 4 min. Samples contain 800 ng of Xenorhabdus nematophilus DNA, 0.1-0.5 μM of each oligonucleotide pair, 250 μM dNTP, 5 units. Taq polymerase and a single (1x) buffer (Perkin-Elmer) in a final volume of 100 μl. The resulting reaction mixtures were precipitated with ethanol, resuspended in TE and added to SeaPlaque 1% TBE gel (FMC, Rocklend, Maine). After electrophoresis and ethidium bromide staining, the fragments are cut out from the gel and visualized in UV light. Slices of the gel are melted at 65 ° C and 10 μl aliquots are mixed with 10 μl of distilled water, boiled for 5 minutes and placed on ice. Then mixed with 15 μl of labeling buffer by random priming (GIBCO-BRL, Gaitersburg, MD), 6 μl of a mixture of dNTP (not containing dTCP), 80 μCi α-dTTP ³² P and 1 μl of Klenov polymerase. The labeling reaction is carried out for 60 minutes at room temperature. Samples were purified on Nick type columns (Pharmacia Biotech) as recommended by the supplier. The probes are boiled for 5 minutes and placed on ice.

Southern blot analysis is carried out by digesting the total DNA of Xenorhabdus nematophilus, DNA isolated from cosmids pCIB9362 and pCIB9363 (these cosmids have an overlapping length of 25 kb and they both contain a pCIB9369 DNA fragment; pCIB9362 is used for subcloning), DNA isolated from pClIB9362-3 subclones (SacII fragment 9 kb in length) and pCIB9369 (ClaI fragment 2.96 kb in length) cleaved with ClaI, SacII or HindIII. The products obtained as a result of cleavage are placed on a 0.75% agarose TBE gel and dispersed overnight. Pictures are taken and the gel is processed according to Bio-Rad instructions regarding blotting using a membrane for hybridization with a Zeta probe. After blotting, the membrane is kept at 80 ° C for 30 minutes. The membrane is then placed in 7% SDS containing 250 mM sodium phosphate, pH 7.2, and incubated at 67 ° C for 30 minutes. A fresh solution was added and, after equilibration at 67 ° C., the above radioactive probes were added and hybridization was carried out overnight. The membrane is washed with 2xSSC, 0.5% SDS for 30 minutes at 67 ° C, and then 0.5xSSC, 0.5% SDS for 30 minutes at 67 ° C. The membrane is exposed to the film for 1 h and 3 h. The film is developed, and the results indicate that the PCR probes based on the sequence described in WO 98/08388 do not hybridize with cosmid DNA or with the subclone DNA described in this application. However, a strong hybridization signal is observed for X. nematophilus DNA.

These results support the results of a comparative sequence analysis and indicate that the nucleotide sequence of clone pCIB9369 is different from the nucleotide sequence described in WO 98/08388.

B. Expression of the nucleotide sequences of the invention in heterologous host microorganisms

Microorganisms suitable for heterologous expression of the nucleotide sequences of the invention can be any microorganisms that are capable of colonizing plants or the rhizosphere. To do this, they must be brought into contact with pests. These include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, the gram-positive microorganism Bacillus and the fungi Trichoderma and Gliocladium, and the yeast Saccharomyces cerevisiae. The most preferred heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesiummeria cereus cereus cereus cereus cereus cereus cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereum cereus cereum

Example 15. The expression of nucleotide sequences in E. coil and other gram-negative bacteria

Many genes were expressed in gram-negative bacteria in a heterologous manner. Expression vector pKK223-3 (catalog number of the company Pharmacia No. 27-4935-01) allows expression in E. coli. This vector has a strong tac promoter (Brosius J. et al., Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG (isopropyl-β-D-thiogalactoside). A large number of other expression systems have been developed for use in E. coli. The heat-induced expression vector pP _L (catalog number Pharmacia No. 27-4946-01) contains a highly regulated bacteriophage λ promoter, allowing expression of high-level proteins. The lac promoter is another expression promoter, however, it does not achieve such high levels of expression as the tac promoter. When replicons with a wide spectrum of hosts are added to these expression vector systems, expression of nucleotide sequences in closely related gram-negative bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia becomes possible. For example, plasmid pLRKD211 (Kaiser and Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820 (1984)) contains an ori T replicon with a broad host spectrum that allows replication in many types of gram-negative bacteria.

Expression in E. coli under the control of the tac promoter (i.e., trp-lac) requires induction by IPTG. If this promoter (for example, as part of a plasmid with a wide spectrum of hosts pLRKD211) is inserted into Pseudomonas, then it will have constitutive activity that does not require induction using IPTG. This trp-lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacteria in order to effect constitutive expression of such a gene. Thus, the nucleotide sequence, the expression of which leads to the formation of an insecticidal toxin, can be controlled by a strong constitutive promoter, and transferred to a bacterium with the ability to colonize a plant or rhizosphere, which turns this organism into an insecticidal agent. Other suitable promoters may be used to effect constitutive expression of the nucleotide sequence in gram-negative bacteria. These include, for example, the gafA and lemA regulatory gene promoter (WO 94/01561) from Pseudomonas and the IAA operon promoter Pseudomonas savastanoi (Gaffney et al., J. Bacteriol. 172: 5593-5601 (1990)).

Example 16. The expression of nucleotide sequences in gram-positive bacteria

Heterologous expression of the nucleotide sequence in gram-positive bacteria is another means of producing insecticidal toxins. The expression systems for Bacillus and Streptomyces are most fully characterized. The promoter of the erythromycin resistance gene (ermR) from Streptococcus pneumoniae has been found to be active in gram-positive aerobic and anaerobic microorganisms, as well as in E. coli (Trieu-Cuot et al., Nucl. Acids Res. 18: 3660 (1990) ) Another promoter of the antibiotic thiostrepton resistance gene was used in Streptomyces cloning vectors (Bibb, Mol. Gen. Genet. 199: 26-36 (1985)). Shuttle vector pHT3101 (Lereclus, FEMS Microbiol. Lett. 60: 211-218 (1989)) is also suitable for expression in Bacillus. The great advantage of this approach is that many gram-positive bacteria form spores that can be used in compositions, which allows the creation of insecticidal agents with a long shelf life. Species Bacillus and Streptomyces are active soil colonizers.

Example 17. Expression of nucleotide sequences in fungi

It has been found that Trichoderma harzianum and Gliocladium virens can achieve different levels of biological control in the field (US 5165928 and US 4996157, both patents in the name of the Cornell Research Foundation). The nucleotide sequence, the expression of which leads to the formation of an insecticidal toxin, can also be expressed in fungi. This can be accomplished by various methods well known in the art. One of them is the transformation of fungal protoplasts, mediated by PEG or carried out by electroporation. Alternatively, particle bombardment can be used to transform protoplasts or other fungal cells capable of developing into regenerated mature structures. Vector pAN7-1, originally created for the transformation of Aspergillus and currently widely used for the transformation of fungi (Curragh et al., Mycol. Res. 97 (3): 313-317 (1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 56: 117-124 (1987)), are designed to contain a nucleotide sequence. This plasmid contains a gene for hygromycin B resistance from E. coli flanked by the gpd promoter and the trpC terminator Aspergillus nidulans (Punt et al., Gene 56: 117-124 (1987)).

In a preferred embodiment, the nucleotide sequences of the invention are expressed in Saccharomyces cerevisiae yeast. For example, each of the two ORFs, pCIB9369, pCIB9381, pCIB9354, or pCIB9383, are cloned in separate vectors together with the inducible GAL1 promoter and CYC1 terminator. Each vector has a gene for resistance to ampicillin and a replicon of 2 microns in size. Vectors are preferably distinguished by their growth markers in yeast. S. cerevisiae is transformed with constructs individually and in combination with each other. ORFs are expressed together and tested for protein expression and insecticidal activity.

B. Formulations containing an insecticidal toxin

 Insecticidal formulations are prepared using active ingredients that include either an isolated toxin or, alternatively, suspensions or concentrates of cells producing it, and which are described above in the examples. For example, E. coli cells expressing an insecticidal toxin can be used to control pests. Formulations that can be prepared in liquid or solid form are described below.

Example 18. Liquid formulations of insecticidal compositions

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

By thoroughly mixing the carriers with the active substance, ready-to-use dusts are obtained.

Example 19. Solid formulations of insecticidal compositions

In the examples below, the percentage of ingredients in the composition is given in wt.%.

The active substance is thoroughly mixed with adjuvants and the mixture is thoroughly ground in a suitable mill to obtain wettable powders that can be diluted with water to obtain suspensions of the desired concentration.

Emulsions of the desired concentration are obtained from this concentrate by dilution with water.

Ready-to-use dusts are prepared by mixing the active substance with carriers and grinding the mixture in a suitable mill.

The active substance is mixed and ground with adjuvants and then the mixture is moistened with water. The mixture is extruded and then dried in a stream of air.

The finely divided active substance is uniformly fed into the mixer to kaolin moistened with polyethylene glycol. In this way, dust-free coated granules are obtained.

The finely divided active substance is thoroughly mixed with adjuvants to obtain a suspension concentrate from which suspensions of the desired concentration can be obtained by dilution with water.

The plants described above are treated with insecticidal formulations according to methods well known in the art in amounts sufficient to control insect pests with an insecticidal toxin.

D. Expression of nucleotide sequences in transgenic plants

The nucleotide sequences described herein can be introduced into plant cells using conventional recombinant DNA methods. Typically, they utilize embedding the coding sequence of the invention in an expression system with respect to which the coding sequence is heterologous (i.e., is not present in it in a normal state) using standard cloning techniques known in the art. The vector contains the elements necessary for transcription and translation of the embedded protein-coding sequences. Numerous vector systems known in the art can be used, such as plasmids, bacteriophages and other modified viruses. Suitable vectors include viral vectors, such as lambda vector systems λgtl1, λgt10 and Charon 4; plasmid vectors such as pBl121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similar systems, but not limited to. The components of the expression system can also be modified to increase the level of expression. For example, shortened sequences, nucleotide substitutions, or other modifications may be used. The expression systems described herein can be used to transform almost any cell of a cultivated plant under appropriate conditions. Transformed cells can be regenerated to produce whole plants, and the nucleotide sequence of the invention confers insect resistance to transgenic plants.

Example 22. Modification of coding sequences and adjacent sequences

The DNA molecules described herein can be modified for expression in transgenic host plants. A host plant that expresses nucleotide sequences and which produces insecticidal toxins in its cells is highly resistant to insect attack and is thus better able to withstand crop losses associated with such an attack.

Transgenic expression in plants of genes derived from microorganisms may require modification of these genes in order to achieve and optimize their expression in plants. In particular, bacterial ORFs that encode various enzymes, but which are encoded by the same transcript in the native microorganism, are better expressed in the plant if they are encoded by different transcripts. To achieve this, each ORF originating from a microorganism is isolated separately and cloned into a cassette that has a plant promoter sequence at the 5 ′ end of ORF and a plant transcription terminator at the 3 ′ end of ORF. The selected ORF sequence preferably includes an ATG initiation codon and a stop stop codon, but it may contain an additional sequence besides the ATG initiation codon and a stop codon. In addition, ORF can be shortened, but still maintain the required activity; in the case of particularly long ORFs, truncated activity-retaining versions may be preferred for expression in transgenic organisms. It is understood that the terms “plant promoter” and “plant transcription terminator” mean promoters and transcription terminators that function in a plant. They also include promoters and transcription terminators that can be isolated from non-plant sources, such as viruses (for example, cauliflower mosaic virus (CaMV)).

In some cases, modification of the ORF coding sequences and adjacent sequences may not be required. It is enough to isolate the fragment containing the ORF of interest and insert it along the transcription relative to the plant promoter. For example, Gaffney et al., (Science 261: 754-756 (1993)) successfully expressed the nahG Pseudomonas gene in transgenic plants under the control of the 35S CaMV promoter and tml CaMV terminator without modifying the coding sequence, while x base pairs of the Pseudomonas gene still remained attached to ORF nahG against the course of transcription relative to the ATG codon and at base pairs still remained attached to it during transcription relative to the stop codon. Preferably, small contiguous portions of the sequence from the microorganism remain attached upstream of the transcription relative to the ATG codon and along the transcription relative to the stop codon. In practice, the possibility of creating such a design may depend on the availability of restriction sites.

In other cases, the expression of genes isolated from sources representing microorganisms may cause problems. These problems are well known in the art and are common to genes isolated from certain sources, such as Bacillus. These problems can arise for the nucleotide sequences of the present invention, and to overcome them, modifications of these genes can be carried out using methods well known in the art. The following problems may occur:

1. The most common codons

In plants, the most common codons differ from the most common codons in some types of microorganisms. Comparison of the most common codons in a cloned ORF of a microorganism with the most common codons of plant genes (and especially genes of a target plant) makes it possible to identify codons in ORF that should preferably be modified. As a rule, as a result of plant evolution, monocotyledonous plants have a pronounced preference for nucleotides C and G in the third position of the nucleotide sequence, while in dicotyledonous plants in this position, nucleotides A or T are most common. of the occurring codon for a particular transgenic target species, many of the problems described below regarding the content of GC / AT and illegal splicing sites can be overcome.

2. GC / AT Content

Typically, plants have a GC content of more than 35%. The use of ORF sequences having a high content of nucleotides A and T can pose serious problems for plants. First, ATTTA motifs apparently destabilize transcripts and are found at the 3’-end of many short-lived mRNAs. Secondly, the presence of polyadenylation signals, such as AATAAA, at inappropriate positions in the transcript, apparently causes a premature termination of transcription. Furthermore, in monocotyledonous plants, sequences with a high AT content can be recognized as splicing sites (see below).

3. Sequences adjacent to the initiating methionine

Plants differ from microorganisms in that their transcripts do not have a specific ribosome binding site. Apparently, the ribosomes attach to the 5’-end of the transcript and look for the first available ATG codon, in which translation can start. However, there is an assumption that there is a preference for certain nucleotides adjacent to the ATG codon, and that expression of the DNA molecules of the present invention can be enhanced by including a eukaryotic consensus translation initiator adjacent to the ATG. Clontech (1993/1994 catalog, p. 210) proposed a sequence as a consensus translation initiator for the expression of the E. coli uidA gene in plants. In addition, Joshi (NAR 15: 6643-6653 (1987)) conducted a comparative analysis of a large number of plant sequences adjacent to the ATG codon and proposed a different consensus sequence. In situations where there are difficulties associated with the expression of DNA molecules in plants, the inclusion of one of these sequences next to the ATG initiation codon can improve translation. In such cases, the last three nucleotides of the consensus sequence may not be suitable for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may be different in different plant species. Sequence analysis of 14 maize genes deposited in the GenBank database yielded the following results.

Position in front of the initiating ATG codon in the sequences of 14 maize genes:

Such an analysis can be carried out for the target species of the plant into which the nucleotide sequence is inserted, and the sequence adjacent to the ATG can be modified to include preferred nucleotides.

4. Removal of illegal splicing sites

Genes cloned from non-plant sources and not optimized for expression in plants can also contain motifs that can be recognized in plants as 5'- or 3'-splicing sites and cleavage can occur in them, resulting in shortened or deletion transcripts. Such sites can be deleted using methods known in the art.

Modification techniques for coding sequences and adjacent sequences are well known in the art. In cases where the initial level of expression of the DNA molecule of the present invention is low and it seems desirable to implement the above sequence modifications, the construction of synthetic genes can be carried out according to methods well known in this field. These methods are described, for example, in published patent applications EP 0385962 (in the name of Monsanto), EP 0359472 (in the name of Lubrizol) and WO 93/07278 (in the name of Ciba-Geygi), all of which are included in the present description in the form links. In many cases, it is preferable to analyze the level of expression of gene constructs using short-term assay protocols (which are well known in the art) before being transferred to transgenic plants.

Example 23. Construction of plant expression cassettes

Coding sequences intended for expression in transgenic plants are first combined into expression cassettes behind a suitable promoter capable of being expressed in a plant. Expression cassettes may also include any other sequences necessary or selected for transgene expression. Such sequences include, but are not limited to, transcription terminators, foreign sequences designed to enhance expression, such as introns, vital sequences, and sequences intended for targeted transfer of a gene product to specific organelles and cell compartments. After that, such expression cassettes can easily be transferred to plant transforming vectors according to the method described below. The following is a description of the various components of typical expression cassettes.

1. Promoters

The choice of promoter used in expression cassettes determines the spatial and temporal pattern of transgene expression in a transgenic plant. The selected promoters allow the expression of transgenes in certain types of cells (such as epidermal leaf cells, mesophilic cells, root bark cells) or in certain tissues or organs (e.g. roots, leaves or flowers), and this choice depends on the desired accumulation site gene product. Alternatively, the selected promoter may control gene expression under various induction conditions. Promoters can vary in strength, i.e. ability to stimulate transcription. Depending on the host cell system used, any of numerous suitable promoters, including a native gene promoter, can be used. The following are examples of promoters that can be used in expression cassettes, not limiting the scope of the invention.

a. Constitutive expression: ubiquitin promoter

Ubiquitin is another gene product known for its ability to accumulate in many types of cells, and its promoter has been cloned from several species for use in transgenic plants (for example, in sunflower - Binet et al., Plant Science 79: 87-94 (1991), corn, Christensen et al., Plant Molec. Biol. 12: 619-632 (1989); and in Arabidopsis, Norris et al. Plant Mol. Biol. 21: 895-906 (1993). The maize ubiquitin gene promoter was investigated in transgenic systems of monocotyledonous plants and its sequence and vectors designed for transformation of monocotyledonous plants are described in published patent EP 0342926 (addressed to Lubrizol), incorporated herein by reference. In addition, Taylor et al. (Plant Cell Rep. 12: 491-495 (1993) described a vector (pANC25) that contains the maize ubiquitin promoter and the first intron and noted its high activity in cell suspensions of numerous monocotyledonous plants when introduced with using microprojectile bombardment The Arabidopsis ubiquitin promoter is ideal for use with the nucleotide sequences of the present invention. The ubiquitin promoter is suitable for gene expression in transgenic plants, both in monocotyledonous and dicotyledonous. Suitable vectors are derivatives in RANS25 vector or any transforming vectors described herein, modified by embedding the corresponding ubiquitin promoter and / or intron sequences.

b. Constitutive expression, 35S CaMV promoter

The construction of plasmid pCGN1761 is described in published patent application EP 0392225 (Example 23), which is incorporated herein by reference. Plasmid pCGN1761 contains a “double” 35S promoter and tml transcription terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type framework. A plasmid derived from pCGN1761 is constructed which carries a modified polylinker comprising NotI and XhoI sites in addition to the existing EcoRI site. This derivative plasmid is designated as pCGN1761ENX. pCGN1761ENX can be used to clone cDNA or gene sequences (including microbial ORF sequences) within its polylinker for expression in transgenic plants under the control of the 35S promoter. A complete cassette of this design, including the tml 35S coding terminator sequence promoter, can be cleaved at the HindIII, SphI, SalI and XbaI sites at the 5'-end of the promoter and the XbaI, BamHI and BglI sites at the 3'-end of the terminator for transfer to transforming vectors, such as described above. In addition, a 35S double promoter fragment can be removed by 5’ cleavage with HindIII, SphI, SalI, XbaI, or PstI, at the 3’ end at any polylinker restriction sites (EcoRI, NotI, or XhoI) to be replaced with another promoter. If necessary, modifications can be made around cloning sites by embedding sequences that can enhance translation. This is particularly preferred in cases where overexpression is required. For example, pCGN1761ENX can be modified by optimizing a translation initiation site according to the method described in Example 37 of US Pat. No. 5,639,949, which is incorporated herein by reference.

in. Constitutive expression: actin promoter

Several actin isoforms are known to be expressed in most cell types, and therefore, the actin promoter is a good choice as a constitutive promoter. In particular, the Act1 rice gene promoter was cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). It was found that the promoter fragment is 1.3 kb in length. contains all regulatory elements necessary for expression in rice protoplasts. In addition, numerous expression vectors have been constructed, including the Act1 promoter, especially for use in monocotyledonous plants (McElroy et al., Mol. Gen. Genet. 231: 150-160 (1991)). These include Act1 intron 1, the Adh1 5 ′ flanking sequence and Adh1 intron 1 (from maize alcohol dehydrogenase gene) and a sequence from the 35S CaMV promoter. The most expressed vectors were fusions of the 35S and the Act1 intron or the 5 ′ flanking sequence of Adh1 and the Act1 intron. Optimization of sequences adjacent to the ATG initiation codon (GUS reporter gene) also enhances expression. The expression cassette promoter described in (McElroy et al., Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified to express the gene of the present invention and is particularly suitable for use in monocotyledonous host plants. For example, fragments containing the promoter can be removed from McElroy constructs and used to replace the double 35S promoter in pCGN1761ENX, which then becomes suitable for insertion of specific gene sequences. The fusion genes constructed in this way can then be transferred to the corresponding transforming vectors. Another scientific report reported that the Act1 rice promoter, with its first intron, also causes high levels of expression in cultured barley cells (Chibbar et al., Plant Cell Rep. 12: 506-509 (1993)).

Inducible expression, PR-1a promoter

The 35S double promoter in pCGN1761ENX can be replaced with any other selected promoter that provides a high level of expression. For example, the 35S double promoter can be replaced by one of the chemically regulated promoters described in US Pat. No. 5,614,395. The selected promoter is preferably cleaved from its source by restriction enzymes, but in the alternative, it can be amplified by PCR using primers carrying the appropriate termination restriction sites. After PCR amplification, the promoter must be re-sequenced to verify amplification errors after cloning the amplified promoter in the target vector. A chemically / pathogen-regulated tobacco PR-1a promoter is isolated from plasmid pCIB1004 (for construction see Example 21 of patent specification EP 0332104, incorporated herein by reference) and transferred to plasmid pCGN1761ENX (Ukness et al., 1992). Plasmid pC1B1004 was digested with NcoI, and the resulting 3 ′ protruding end of the linearized fragment was blunted by treatment with T4 phage DNA polymerase. The fragment was then digested with HindIII and the resulting fragment containing the PR-la promoter was gel purified and cloned into pCGN1761ENX from which the 35S double promoter was removed. This is accomplished by cleavage with XhoI and "blunting" the end with T4 polymerase, followed by cleavage with HindIII and isolating a larger fragment containing a terminator vector in which the fragment of the pCIB1004 promoter is cloned. This makes it possible to obtain a vector derived from pCGN1761ENX, which contains the PR-1a promoter and tml terminator and an integrated polylinker with unique EcoRI and NotI sites. The selected coding sequence can be inserted into this vector, and the fusion products (i.e., the terminator promoter gene) can then be transferred to any selected transformation vector, including the vectors below. Various chemical regulators can be used to induce expression of the coding sequence in plants transformed according to the present invention, including derivatives of benzothiadiazole, isonicotinic acid and salicylic acid, as described in US patents 5523311 and 5614395.

D. Inducible expression, promoter. ethanol induced

A promoter induced by certain alcohols or ketones, for example ethanol, can also be used to provide inducible expression of the coding sequence of the present invention. Such a promoter is, for example, the promoter of the alcA gene of Aspergillus nidulans (Caddick et al., Nat. Biotechnol. 16: 177-180 (1998)). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by AlcR transcription factors in the presence of a chemical inducer. For the purposes of the present invention, the coding sequences of CAT in the plasmid pa1CA: CAT, including the alCA gene promoter sequence, fused to the minimum 35S promoter (Caddick et al., Nat. Biotechnol. 16: 177-180 (1998)) are replaced by the coding sequence of the present invention obtaining an expression cassette containing a coding sequence controlled by an alcA gene promoter. This can be done using methods well known in the art.

e. Inducible expression, glucocorticoid inducible promoter

Can also be considered the induction of expression of the nucleotide sequence of the present invention using systems based on steroid hormones. For example, you can use an induction system mediated by a glucocorticoid (Aoyama and Chua, The Plant Journal, 11: 605-612 (1997)) and induce gene expression by treatment with a glucocorticoid, for example, a synthetic glucocorticoid, preferably dexamethasone, preferably in a concentration in the range from 0.1 to 1 mm, more preferably in the range from 10 to 100 mm. For the purposes of the present invention, the luciferase gene sequences are replaced by the nucleotide sequence of the invention, constructing an expression cassette containing the nucleotide sequence of the invention under the control of six copies of GAL4, located opposite the transcription relative to the activating sequences fused with the minimum 35S promoter. This is accomplished using methods well known in the art. The transactivating factor includes the GAL4 DNA binding domain (Keegan et al., Science 231: 699-704 (1986)) fused to the transactivating domain of the herpes virus protein VP16 (Triezenberg et al., Genes Devel. 2: 718-729 (1988) ) fused to the hormone binding domain of the rat glucocorticoid receptor (Picard et al., Cell 54: 1073-1080 (1988)). The expression of the fusion protein is controlled by any promoter known in the art suitable for expression in plants. This expression cassette containing the nucleotide sequence of the invention fused to the 6GAL4 / minimal promoter construct is inserted into the plant. In this way, the fusion protein is specific for the tissue or organ, resulting in inducible expression of the insecticidal toxin in a tissue or organ specific manner.

g. Root-specific expression

Another pattern of gene expression is root expression. A suitable promoter for root expression is the promoter described by de Framound (FEBS 290: 103-106 (1991), as well as in published patent application EP 0452269, which is incorporated herein by reference. This promoter is transferred to a suitable vector, such as pCGN1761ENX, for embedding the selected gene and subsequent transfer of the complete promoter-gene-terminator cassette into a transforming vector of interest.

h. Wound Induced Promoters

Wound-induced promoters can also be used to express a gene. Numerous such promoters have been described (see, for example, Hu et al., Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-151 (1989), Rohrmeier Lehle, Plant Molec Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993), and they are all suitable for use in accordance with the present invention. Logemann et al. described sequences opposite the transcription with respect to the 5'-end of the wun1 gene of a dicotyledonous plant - potato. Hu et al. activity in a monocotyledonous plant, rice. In addition, Rohrmeier and Lehle described the cloning of Wip1 maize cDNA, which is induced by injury and can be used to isolate a related promoter using standard methods. Similarly, Firek et al. and Warmer et al. described a wound-induced gene from a monocot Asparagus officinalis plants, which are locally expressed at the site of injury and at the pathogen introduction site. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused with genes, and used to express these genes at injured plant sites.

and. Core preferred expression

Patent application WO 93/07278, incorporated herein by reference, describes the isolation of a maize trpA gene, which is preferred for expression in core cells. The sequence of the gene and the promoter located from the start of transcription to nucleotide 1726 are shown. Using standard molecular biology methods, this promoter or parts thereof can be transferred to a vector such as pCGN1761, in which it can replace the 35S promoter and be used to ensure the expression of a foreign gene preferred for the core. In essence, fragments containing the preferred core promoter or parts thereof can be transferred to any vector and modified for use in transgenic plants.

K. Leaf-specific expression

The corn gene encoding phosphoenol carboxylase (PEPC) is described by Hudspeth and Grula (Plant Molec. Biol. 12: 579-589 (1989)). Using standard molecular biology methods, the promoter of this gene can be used to ensure expression of any gene in transgenic plants in a leaf-specific manner.

l Pollen-specific expression

Patent Application WO 93/07278 describes the isolation of a maize calcium dependent protein kinase (CDPK) gene that is expressed in pollen cells. The sequence of the gene and promoter extends to the position of 1400 base pairs from the site of transcription initiation. Using standard molecular biology techniques, this promoter or parts thereof can be transferred to a vector, such as pCGN1761, where it can replace the 35S promoter, and it can be used to provide expression of the nucleotide sequence of the present invention in a pollen-specific manner.

2. Transcription terminators

A wide variety of transcription terminators are suitable for use in expression cassettes. They are responsible for terminating transcription for the transgene and for its correct polyadenylation. Suitable transcription terminators are terminators for which they are known to have the ability to function in plants and they include the 35S CaMV terminator, tml terminator, nopaline synthase terminator, and pea r9 terminator E9. They can be used both in monocotyledonous and dicotyledonous plants. In addition, native gene transcription terminators can be used.

3. Sequences for enhancing or regulating expression

Numerous sequences are known that enhance the expression of a gene transcription unit, and these sequences can be used in combination with the genes of the present invention to enhance their expression in transgenic plants.

It was found that various intron sequences enhance expression, primarily in the cells of monocotyledonous plants. For example, it was found that the introns of the maize Adhl gene when introduced into maize cells significantly enhance the expression of the wild-type gene under the control of a related promoter. Intron 1 has been found to be particularly effective and enhances expression in hybrid constructs when fused to the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the maize bronzel intron gene exhibits a similar effect with respect to increased expression (Callis et al., Supra). Intron sequences are incorporated in the usual manner into plant transforming vectors, usually as part of an untranslated leader sequence.

A large number of untranslated leader sequences originating from viruses that enhance expression and which are especially effective in dicotyledonous cells are also known. In particular, it was found that leader sequences from the tobacco mosaic virus (TMV, "Ω sequence"), from the chlorine spotting virus of the corn (MCMV), and from the alfalfa mosaic virus (AMV) effectively enhance expression (see, for example, Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al., Plant Molec. Biol. 15: 65-79 (1990)).

4. Directional transfer of a gene product in a cell

In plants, various mechanisms of targeted transfer of gene products are known, and the sequences that control the functioning of these mechanisms are described in great detail. For example, the targeted transfer of gene products into the chloroplast is controlled by a signal sequence found at the N-terminus of various proteins that cleaves during import to the chloroplast, resulting in a mature protein (see, for example, Comai et al., J. Biol. Chem. 263: 15104-15109 (1988)). These signal sequences can be fused to heterologous gene products to efficiently import heterologous products into chloroplast (van den Broeck et al., Nature 313: 358-363 1985)). DNA encoding the corresponding signal sequences can be isolated from the 5 ′ end of the cDNA encoding the RUBISCO protein, CAB protein, EPSP synthase enzyme, GS2 protein and a number of other proteins for which it is known that they are localized in the chloroplast. See also the section entitled "Expression with directional transfer to chloroplast" in example 37 of US patent 5639949).

Other gene products are localized in other organelles, such as mitochondria and peroxisome (see, for example, Unger et al., Plant Molec. Biol. 13: 411-418 (1989)). cDNAs encoding these products may also be exposed to effect the targeted transfer of heterologous gene products to these organelles. Examples of such sequences are ATPase encoded nuclear DNA and mitochondria-specific aspartate aminotransferase isoforms. Targeted transfer to cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad Sci. USA 82: 6512-6516 (1985)).

In addition, sequences have been characterized that result in targeted transfer of gene products to other cell compartments. N-terminal sequences are responsible for targeted transfer to the endoplasmic reticulum (ER), apoplast, and extracellular secretion from aleuron cells (Koehler and Ho, Plant Cell 2: 769-783 (1990)). In addition, N-terminal sequences in combination with C-terminal sequences are responsible for the directed transfer of gene products into vacuoles (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

By fusing the corresponding guide sequences described above with the transgenic sequences of interest, the transgenic product can be directed to any organelle or any cell compartment. For directional transfer, for example, into the chloroplast, the signal sequence of the chloroplast of the RUBISCO gene, CAB gene, EPSP synthase gene or GS2 gene is fused to the reading frame with the N-terminal codon of the ATG transgene. The selected signal sequence should include a known cleavage site, and any amino acids located behind the cleavage site that are necessary for cleavage should be considered when constructing hybrids. In some cases, this requirement can be met by adding a small amount of amino acids between the cleavage site and the ATG of the transgene, or alternatively by replacing several amino acids within the transgene sequence. Hybrids designed to be imported into chloroplast can be evaluated for chloroplast uptake efficiency by translating in vitro transcribed in vitro constructs followed by in vitro uptake with chloroplast using the methods described by Bartlett et al. In: Edelmann et al. (Ed.) Methods in Chloroplast Molecular Biology, Elsevier. p. 1081-1091 (1982)); in Wasmann et al. (Mol. Gen. Genet. 205: 446-453 (1986)). These construction methods are well known in the art and are equally suitable for both mitochondria and peroxisomes.

The above-described mechanisms of directional transfer in a cell can be used not only in combination with their related promoters, but also with heterologous promoters in order to carry out specific target directed transfer in the regulation of transcription by a promoter that has an expression scheme different from the expression scheme of the promoter which emits a directional transfer signal.

Example 24. The construction of plant transforming vectors

Numerous transforming vectors suitable for transforming plants are known to those skilled in the art, and the genes of the present invention can be used in combination with any of these vectors. The choice of vector depends on the preferred transformation method and on the target species intended for transformation. For certain target species, various markers for selection based on antibiotic or herbicide resistance may be preferred. Selectable markers commonly used for transformation include the nptII gene, which causes resistance to kanamycin and related antibiotics (Messing and Vierra, Gene, 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)), the bar gene for the herbicide resistance of phosphinotricin (White et al., Nucl. Acids Res. 18: 1062 (1990), Spencer et al., Theor. Appl. Genet. 79: 625-631 (1990)), the hpH gene, causing antibiotic resistance to hygromycin (Blochinger and Diggelmann, Mol. Cell Biol, 4: 2929-2931 (1984)) and the dhfr gene, which determines resistance to methotrexate (Bourouis et al., EMBO J., 2: 1099-1104 (1983 )) and the EPSPS gene, which determines Resistant to glyphosate (US 4,940,935 and 5,188,642 patent).

1. Construction of vectors suitable for transformation using Agrobacterium

Numerous vectors are suitable for transformation using Agrobacterium tumefaciens. They usually carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res., (1984)) and pXYZ. The construction of two typical vectors suitable for transformation using Agrobacterium is described below.

a. pCIB200 and pCIB2001:

The binary vectors pCIB200 and pCIB2001 are used to construct recombinant vectors intended for use with Agrobacterium, and they are constructed as follows. Create pTJS75kan by cleaving pTJS75 (Schmidhauser and Helinski, J. Bacteriol, 164: 446-455 (1985)) using NarI, which allows to cut the tetracycline resistance gene, followed by the insertion of the AccI fragment from the pUC4K plasmid carrying NPTII (Messing and V Gene, 19: 259-268 (1982)); Bevan et al., Nature, 304: 184-187 (1983); McBridge et al., Plant Molecular Biology, 14: 266-276, (1990)). XhoI linkers are ligated with the pC1B7 EcoRV fragment containing the left and right T-DNA border sequences, the nos / nptII chimeric gene and pUC polylinker selected in the plant (Rothstein et al., Gene, 53: 153-161 (1987)), and cloned XhoI cleaved fragment in plasmid pTJS75 cleaved with SalI to generate pCIB200 (see also EP 0332104, Example 19 [1338]). The pCIB200 vector contains the following unique sites recognized by restriction enzymes (restriction sites) in polylinker: EcoRI, SstI, KpnI, BglII, XbaI and SalI. The vector pCIB2001 is a derivative of pC1B200 and is created by inserting additional restriction sites into the polylinker. The unique restriction sites in the polylinker of the pCIB2001 vector are EcoRI, SstI, KpnI, BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI and StuI. In addition to the fact that pCIB2001 contains these unique restriction sites, it also contains a gene for selecting kanamycin-transformed plants and bacteria, the left and right T-DNA border sequences for Agrobacterium mediated transformation, the trfA function, which is a derivative of RK2, for mobilization between E. coli and other hosts, and the functions of OriT and OriV, also derived from RK2. The polylinker pCIB2001 is suitable for cloning plant expression cassettes containing their own regulatory signals.

b. Vector pCIB10 and its derivatives selected for hygromycin resistance

The binary vector pCIB10 contains the gene responsible for resistance to kanamycin in plants, the right and left border sequences of T-DNA and includes sequences from the plasmid pRK252 with a wide range of hosts, which allows its replication both in E. coli and in Agrobacterium. Its construction is described by Rothstein et al., Gene, 53: 153-161, (1987)). Gritz et al. (Gene, 25: 179-188, (1983)) describe various engineered derivatives of pCIB10, which include the phosphotransferase gene, which determines resistance to hygromycin B. These derivatives make it possible to select cells of transgenic plants or only on the basis of hygromycin resistance (pCIB743), or based on hygromycin and kanamycin resistance (pCIB715, pCIB717).

2. Vectors suitable for transformation without the use of Agrobacterium

Transformation without the use of Agrobacterium tumefaciens makes it possible to circumvent the requirement for the presence of T-DNA sequences in a selected transforming vector and, therefore, vectors that lack these sequences can be used in addition to vectors, such as those described above, which contain T-DNA sequences. Transformation methods that are not based on Agrobacterium include particle bombardment transformation, protoplast uptake (e.g., PEG and electroporation) and microinjection. The choice of vector is largely dependent on the choice of preferred species to be transformed. The construction of some typical vectors is described below.

a. pCIB3064:

pCIB3064 is a pUC derived vector suitable for direct gene transfer methods in combination with selection for a basta herbicide (or phosphinotricin). Plasmid pCIB246 includes the 35S CaMV promoter operably linked to the E. coli GUS gene and the 35S CaMV transcription terminator, and is described in PCT published application WO 93/07278. The 35S promoter of this vector contains two ATG sequences at the 5 ′ end of the initiation site. These sites are mutated using standard PCR methods so as to remove both ATGs and form restriction sites SspI and PvuII. New restriction sites are located at a distance of 96 and 37 base pairs from the unique Sall site and at a distance of 101 and 42 base pairs from the actual initiation site. The resulting derivative of plasmid pCIB246 was designated as pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sall and SacI, the ends are blunted and re-ligated to obtain plasmid pCIB3060. The plasmid pJIT82 was obtained from John Innes Center, Norwich, and a 400 bp SmaI fragment containing the bar gene from Streptomyces viridochromogenes was excised and inserted into the HpaI site of the plasmid pCIB3060 (Thompson et al., EMBO J., 6: 2519-2523, ( 1987)). This makes it possible to obtain plasmid pCIB3064 containing the bar gene under the control of the 35S CaMV promoter and terminator for selection based on herbicide resistance, the gene determining ampicillin resistance (for selection in E. coli) and the polylinker with unique sites SphI, PstI, HindIII and Bamhi. This vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

b. pSOG19 and pSOG35:

pSOG35 is a transforming vector in which E. coli dihydrofolate reductase (DHFR) gene, which determines resistance to methotrexate, is present as a selectable marker. PCR is used to amplify the 35S promoter (~ 800 base pairs), intron 6 of the maize Adh1 gene (~ 550 base pairs), and a fragment of 18 base pairs long of the untranslated GUS leader sequence from plasmid pSOG10. A 250 bp fragment encoding the E. coli type II dihydrofolate reductase gene is also amplified by PCR and the two PCR fragments are combined with the SacI-PstI fragment from pBI221 (Clontech), which contains the vector base pUC19 and the nopalin synthase terminator. The assembly of these fragments makes it possible to obtain the plasmid pSOG19 containing the 35S promoter in fusion with the sequence of intron 6, the GUS leader, the DHFR gene, and the nopalin synthase terminator. Replacing the GUS leader in pSOG19 with the leader sequence of the maize chlorose mosaic virus yields the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have HindIII, SphI, PstI, and EcoRI sites suitable for cloning foreign sequences.

3. A vector suitable for the transformation of chloroplast

To express the nucleotide sequence of the present invention in plant plastids, the pRH143 plastid transforming vector is used (WO 97/32011, example 36). The nucleotide sequence is inserted into pPH143, replacing it with the PROTOX (protoporphyrinogen oxidase) coding sequence. This vector is then used to transform plastids and select transformants based on spectinomycin resistance. Alternatively, the nucleotide sequence is inserted into pRH143 so that it replaces the aadH gene. In this case, transformants are selected based on their resistance to PROTOX inhibitors.

Example 25. Transformation

After the nucleotide sequence is cloned in the expression system, the plant cell is transformed. Methods for transforming and regenerating plants are well known in the art. For example, Ti plasmids are used for introducing foreign DNA, as well as for direct DNA uptake, administration by liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria of the genus Agrobacterium can be used to transform plant cells. Representative methods for transforming both dicotyledonous and monocotyledonous plants, as well as a representative method for transforming plastids, are described below.

1. Transformation of dicotyledonous plants

Methods for transforming dicotyledonous plants are well known in the art and include methods based on the use of Agrobacterium and methods for which Agrobacterium is not required. Methods for which Agrobacterium is not required include the uptake of exogenous genetic material directly by protoplasts or cells. This may be accomplished by PEG mediated absorption or electroporation, particle bombardment, or microinjection. Examples of these methods are described by Paszkowski et al., EMBO J. 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al. Biotechnology 4: 1001-1004 (1986) and Klein et al. Nature 327: 70-73 (1987). In each case, the transformed cells regenerate to whole plants using standard methods known in the art.

и Willmitzer, Nucl. Acids Res. 16:9877 (1988)).Agrobacterium mediated transformation is the preferred method for dicotyledonous transformation due to the high transformation efficiency when using this method and its wide applicability to a number of different species. Agrobacterium transformation typically involves transferring a binary vector carrying foreign DNA of interest (e.g., pCIB200 or pCIB2001) to a suitable Agrobacterium strain, which may depend on the complement of the vir genes of the Agrobacterium host strain, which can either be located on the coreresident Ti- plasmid, or on the chromosome (for example, strain CIB542 for pC1B200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Transfer of the recombinant binary vector to Agrobacterium is carried out by three parents using E. coli carrying the recombinant bin an ary vector of an E. coli helper strain carrying a plasmid such as pRK2031 and which is capable of mobilizing a recombinant binary vector for targeted transfer to the Agrobacterium target strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium using DNA transformation (

and Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of target plant species using recombinant Agrobacterium typically involves co-cultivation of Agrobacterium with plant explants and is performed in accordance with protocols well known in the art. The transformed tissue is regenerated on a breeding medium containing a marker for selection based on antibiotic or herbicide resistance, located between the boundary sequences of the T-DNA of the binary plasmid.

Another method for transforming plant cells to the genome is based on the introduction of inert or bioactive particles into the tissue or cells of the plant. This method is described in US patents 4945050, 5036006 and 5100792 (all in the name of Sanford and others). Typically, this method consists of introducing particles that are inert or having biological activity into the cells under conditions that allow them to penetrate the outer membrane of the cell and integrate into it. If inert particles are used, then the vector can be introduced into the cell by applying to the particles a vector containing the target gene. Alternatively, the target cell may be surrounded by a vector such that the vector is transferred into the cell following the particle. Biologically active particles (for example, dried yeast cells, dried bacteria or bacteriophages, each of which contains DNA intended for introduction) can also be used to introduce plant cells into the tissue.

2. Transformation of monocotyledonous plants

The transformation of most monocotyledonous plant species has now also become traditional. Preferred methods include direct gene transfer to protoplasts using PEG-based techniques or electroporation and particle bombardment of callus tissue. Transformations can be performed with single types of DNA or with multiple types of DNA (i.e. cotransformation), and both of these methods are suitable for use in the present invention. The advantage of cotransformation may be that it does not require a complete vector construction and makes it possible to obtain transgenic plants with unlinked loci for the gene of interest and a selectable marker, which allows you to remove the selectable marker in subsequent generations, if necessary. However, the disadvantage of using the cotransformation method is that the frequency with which different types of DNA integrate into the genome is less than 100% (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent applications EP 0292435, EP 0392225 and WO 93/072778 describe methods for producing callus and protoplasts of an elite inbred line of maize, transformation of protoplasts using PEG, or electroporation and regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al., In Plant Cell 2: 603-618 (1990) and Fromm et al. In Biotechnology 8: 833-839 (1990)) described methods for transforming a maize line obtained from the A188 line using particle bombardment . Methods for transforming elite inbred maize lines by particle bombardment are also described in WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)). This method involves the use of immature corn kernels 1.5-2.5 mm long, cut from a corn cob 14-15 days after pollination, and a bombardment device such as PDS-l000 He Biolistics.

Rice transformation can also be carried out using direct gene transfer methods using protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japanese and Indian rice 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 of rice are also transformed using standard particle bombardment techniques (Christou et al., Biotechnology 9: 957-962 (1991)). In addition, WP 93/21335 describes methods for transforming rice using electroporation.

In patent application EP 0332581 describes methods for producing, transforming and regenerating plant protoplasts of the family. bluegrass (Pooideae). These methods make it possible to transform the hedgehog (Dactylis spp.) And wheat. In addition, Vasil et al. (Biotechnology 10: 667-674 (1992)) described wheat transformation using particle bombardment of callus type C cells with long regeneration times, and also Vasil et al. (Biotechnology 11: 1553-1558 (1993) ) and Weekes et al. (Plant Physiol. 102: 1077-1084 (1993)) described transformation using particle bombardment of immature embryos and immature callus of embryonic origin. However, a preferred method for transforming wheat involves transforming wheat by particle bombardment of immature embryos and requires either a high sucrose or high maltose stage prior to gene introduction. Before the bombardment, any number of embryos (0.75-1 mm long) are plated in MS medium with the addition of 3% sucrose (Murashige and Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg / L 2,4-D (dichlorophenoxyacetic acid) for the induction of somatic embryos that are allowed to develop in the dark. On the designated bombardment day, the embryos are removed from the induction medium and placed in the osmoticum (i.e., in the induction medium with the addition of sucrose or maltose in the desired concentration, usually 15%). The embryos are given the opportunity to undergo plasmolysis within 2-3 hours and then subjected to bombardment. In one target cup, 20 embryos are usually used, although this amount is not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is deposited onto gold particles of the order of micron size using standard methods. Each embryo cup is bombarded with a DuPont Biolistics ^® helium-based device at a volley pressure of ~ 1000 psi and a standard screen with a pore size of 80 mesh. After the bombardment, the embryos are placed back in the dark for recovery for approximately 24 hours (still in the osmoticum). After 24 hours, the embryos are removed from the osmoticum and placed back into the medium for induction, where they remain for about a month before regeneration. After approximately one month, the explants of embryos with developing embryogenic callus are transferred to a regeneration medium (MS + 1 mg / L NIK, 5 mg / L HA (hibernellic acid)), which also contains an appropriate selection agent (10 mg / L bast in the case of pCIB3064 and 2 mg / L methotrexate in the case of pSOG35). After approximately one month, the developed shoots are transferred to larger sterile containers known as “GA7s” containing half-strength MS, 2% sucrose and the same concentration of selective agent.

The transformation of monocotyledonous plants using Agrobacterium is also described (see WP 94/00977 and US Pat. No. 5,591,616, both of which are incorporated herein by reference).

3. Plastid transformation

Seeds of Nicotiana tabacum cultivar "Xanthi nc" are planted at the rate of 7 pieces per cup on a circle with a diameter of 1 inch on T-agar medium and 12-14 days after sowing, they are bombarded with 1 μm tungsten particles (M10, Biorad, Hercules, CA) coated with plasmid DNA rRH143 and rRH145, mainly according to the method described in Svab Z. and Maliga P., PNAS 90, 913-917 (1993). The bombarded seedlings are incubated on a T-medium for two days, after which the leaves are cut off and placed upside down under bright illumination (350-500 μmol photons / m ² / s) on plates with RMOP medium (Svab Z., Hajdukiewicz P, and Maliga P. PNAS 87, 8526-8530 (1990)) containing 500 μg / ml spectinomycin dihydrochloride (Sigma, St. Louis, MO). Resistant seedlings, in which leaves decolored from below appear 3–8 weeks after the bombardment, are subcloned on the same selective medium, they can callus and secondary seedlings are secreted and subcloned. Complete separation of copies of the transformed plastid genome (homoplasmic state) in independent subclones is determined using standard Southern blotting methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989)). BamHI / EcoRI digested total cellular DNA (Mettler IJ Plant Mol. Biol. Reporter 5, 346-349 (1987)) was separated on 1% Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with ³² P labeled randomly primed DNA sequences corresponding to a 0.7 kb BamHI / HindIII DNA fragment from pC8, which contains part of the rps7 / 12 sequence, providing directional transfer to plastid. Homoplasmic shoots are planted under aseptic conditions on spectinomycin-containing MS / IBA medium (McBride K.E. et al. PNAS 91, 7301-7305 (1994)) and transferred to a greenhouse.

D. Selection and seed production

Plants obtained by transformation with the nucleotide sequence of the present invention may relate to any of a wide variety of plant species, including monocotyledonous and dicotyledonous plants; however, preferably, the plants used in the method of the invention are selected from the above list of agriculturally important target crops. The ability to express a gene of the present invention in combination with other characteristics important in terms of productivity and quality can be imparted to plant lines by selection. Selection principles and methods are known in the art (see, e.g., Welsh JR, Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood DR (ed.) American Society of Agronomy, Madison, Wisconsin (1983); Mayo O. The Theory of Plant Breeding, 2nd ed., Clarendon Press, Oxford (1987); Singh DP, Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986) ; and Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).

Genetic traits constructed in the transgenic seeds and plants described above are transmitted through sexual reproduction or vegetative growth, and thus can be preserved and inherited by the progeny of plants. Typically, said preservation and transmission to offspring is carried out using known agricultural methods designed for specific purposes, such as tillage, sowing or harvesting. Specialized methods such as hydroponics or indoor cultivation can also be used. Since cultivated crops are susceptible to attack and damage caused by insects or infections, and are subject to competition from weeds, measures are taken to control weeds, plant diseases, insects, nematodes and other harmful factors to improve crops. They include mechanical methods, such as tillage or removal of weeds and infected plants, as well as the use of plant protection products, such as herbicides, fungicides, gametocides, nematicides, growth regulators, ripening agents, and insecticides.

In addition, the advantageous genetic features of the transgenic plants and seeds of the invention can be used in plant breeding with the aim of breeding plants with improved properties, such as tolerance to pests, herbicides or stress, with improved nutritional value, increased yield or improved structure, which helps to reduce loss of lodging or shedding. The various stages of selection are characterized by targeted human intervention, such as selection of lines to be crossed, direct pollination of parental lines or selection of the corresponding offspring of plants. Depending on the desired properties, various selection methods are selected. Appropriate methods are well known in the art and include, but are not limited to, hybridization, inbreeding, backcrossing, multi-linecrossing, cultivar mixing, interspecific hybridization, aneuplodization techniques, etc. Hybridization methods also include sterilizing plants to obtain male or female sterile plants using mechanical, chemical or biochemical methods. Cross-pollination of a male sterile plant with pollen from various lines ensures that the genome of a male sterile but fertile female plant will equally acquire the properties of both parent lines. Thus, the transgenic seeds and plants of the present invention can be used for breeding improved plant lines, which, for example, can increase the efficiency of conventional methods, such as treatment with a herbicide or pesticide, or makes it possible to do without these methods due to the modified genetic characteristics of these plants. In addition, new cultivated plants with improved stress tolerance can be obtained by optimizing the genetic “apparatus”, which allows to obtain a crop of better quality products compared to products obtained from plants that do not have the ability to resist similar harmful conditions in the process of their development.

Example 27. Seed production

In seed production, germination quality and seed uniformity are important characteristics of the product, while germination quality and uniformity of seeds collected and sold by the farmer are not important. Since it is difficult to grow a cultivated plant out of contact with the seeds of another cultivated and weed plant, quite extensive and targeted methods for producing seeds have been developed to control seed-borne diseases and to obtain seeds with good germination by manufacturers involved in growing, conditioning and selling pure seeds. So, usually a farmer practices the purchase of certified seed material that meets certain quality standards, instead of using seed material obtained from his own crop. Propagating material, which is used as seed, is usually treated with a protective coating, including herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. Commonly used protective coatings include compounds such as captan, carboxin, thiram (TMTD ^® ), metalaxyl (Apron ^® ) and pyrimifosmethyl (Actelinc ^® ). If necessary, these compounds are formulated with additional carriers, surfactants, or adjuvants commonly used in the field of formulations to protect against damage caused by bacteria, fungi, or pests. Protective coatings can be applied by impregnating the propagation material with a liquid composition or by coating with a combined wet or dry composition. Other application methods are also possible, such as treatment of the kidneys or fruits.

Another subject of the invention is new agricultural methods, such as the above methods, which are characterized by the use of transgenic plants, transgenic plant material or transgenic seeds of the present invention.

Seeds can be placed in a bag, container or vessel made of suitable packaging material, the bag or container must be able to be closed to preserve the seeds. A bag, container or vessel can be designed either for short-term or long-term storage of seeds, or for both at the same time. Examples of suitable packaging material include paper such as kraft paper, hard or flexible plastic or other polymeric material, glass or metal. Preferably, the bag, container or vessel consisted of several layers of packaging materials of the same type or different types. In one embodiment, the implementation of the bag, container or vessel is made in such a way as to exclude or limit the contact of seeds with water or moisture. In one example, a bag, container or vessel is sealed, for example, sealed by heating, in order to prevent the ingress of water or moisture. In another embodiment, water absorbent materials are placed between or on layers of packaging material. In yet another embodiment, a bag, container or vessel, or the packaging material of which it is composed, is processed to limit, suppress, or prevent infection, contamination, or other harmful factors that occur during storage or transportation of seeds.

An example of such a treatment is sterilization, for example, by chemical means or by irradiation. A commercial bag containing seeds of a transgenic plant carrying the gene of the present invention, which is expressed in a transformed plant at a higher level than a wild-type plant, together with a suitable carrier and with instructions for use to provide plant resistance to a wide range of diseases.

The embodiments described above are provided for purposes of illustration. This description of the invention enables a person skilled in the art to make numerous variations of the invention. It should be understood that all such obvious and predictable variations fall within the scope of the attached claims.

Claims (23)

1. The selected nucleic acid molecule comprising a nucleotide sequence that encodes at least one toxin active against Plutella xylostella, where this nucleotide sequence (a) hybridizes with a nucleotide sequence selected from the group comprising nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 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 under the following conditions : 7% sodium dodecyl sulfate (SDS), 0.5M NaPO _4, 1mM EDTA at 50 ° C with 0.1 x SSC rinsing, 0.01 × SDS at 65 ° C, or (b) the nucleotide sequence st, izokodonnuyu nucleotide sequence specified in (a). 2. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence is an isocodonic nucleotide sequence that hybridizes with nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 SEQ ID NO: 1, SEQ ID NO: 4 , SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 12 or SEQ ID NO: 14 under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C with flushing 0.1 x SSC, 0.01 x SDS at 65 ° C. 3. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence hybridizes with nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 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 under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C with flushing 0.1 x SSC, 0.01 x SDS at 65 ° C. 4. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence encodes an amino acid sequence selected from the group comprising SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, and 15. 5. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence includes nucleotides 569-979 SEQ ID NO: 1, nucleotides 1045-2334 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. 6. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence hybridizes with nucleotides 569-979 SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12 under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C with washing with 0.1 x SSC, 0.01 x SDS at 65 ° C. 7. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 13. 8. The selected nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence hybridizes with nucleotides 1045-2334 SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 14 under the following conditions: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA at 50 ° C with washing with 0.1 x SSC, 0.01 x SDS at 65 ° C. 9. The isolated nucleic acid molecule according to claim 1, characterized in that the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11 or SEQ ID NO: 15. 10. The isolated nucleic acid molecule according to claim 1, characterized in that said nucleotide sequence is a DNA fragment of approximately 3.0 kbp, which is included in the plasmid pCIB9369 (NRRL B-21883). 11. A chimeric gene comprising a heterologous promoter sequence operably linked to a nucleic acid molecule according to claim 1, wherein the chimeric gene is capable of expression in a microbial or plant host cell. 12. Recombinant plasmid comprising the chimeric gene according to claim 11. 13. The bacterial strain E. coli NRRL B-21883 is a producer of the toxin active against Plutella xylostella. 14. The toxin resulting from the expression of a DNA molecule according to claim 1, wherein the toxin is active against Plutella xylostella. 15. The toxin of claim 14, wherein the toxin is produced by an E. coli strain having registration number NRRL B-21883. 16. The toxin of claim 14, wherein the toxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 5, 7, 9, 11, 13, and 15. 17. The toxin according to clause 16, where the toxin includes an amino acid sequence selected from the group comprising SEQ ID NO: 2, 5, 9 and 13. 18. The toxin according to clause 16, where the toxin includes an amino acid sequence selected from the group comprising SEQ ID NO: 3, 7, 11 and 15. 19. An insecticidal composition comprising an insecticidal amount of toxin according to claim 14 and an agriculturally acceptable carrier. 20. A method of producing a toxin having activity against Plutella xylostella, comprising (a) transforming a host cell strain with a nucleic acid molecule according to any one of claims 1 to 9; and (b) expressing a nucleic acid molecule in these host cells, which leads to the formation of at least one toxin with activity against insects. 21. A method for producing an insect resistant plant, comprising introducing into the plant a nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is able to be expressed in the plant in an amount effective against Plutella xylostella. 22. A method of controlling Plutella xylostella, comprising administering to an insect an effective amount of a toxin according to claim 14. 23. The method of claim 22, wherein the toxin is administered orally to the insect.