WO2015118207A1

WO2015118207A1 - New cry protein of bacillus thuringiensis with insecticide activity for controlling hemipterans

Info

Publication number: WO2015118207A1
Application number: PCT/ES2015/070083
Authority: WO
Inventors: Primitivo Caballero Murillo; Leopoldo PALMA DOVIS; Íñigo RUÍZ DE ESCUDERO FUENTEMILLA; Delia MUÑOZ LABIANO; Jesús MURILLO MARTÍNEZ; Colin Berry
Original assignee: Universidad Pública de Navarra; Consejo Superior De Investigaciones Científicas
Priority date: 2014-02-10
Filing date: 2015-02-10
Publication date: 2015-08-13
Also published as: ES2545683A1; ES2545683B1

Abstract

The invention relates to a novel gene encoding a Cry (or delta-endotoxin) protein, to the actual protein encoded by said gene, to the systems for the expression of same (vectors and host cells), and to the use of said gene and/or said protein for controlling hemipteran pests, preferably the green aphid, Myzus persicae, both by using a composition containing the novel protein and/or a system for the expression thereof, and by using transgenic plants wherein the gene is expressed. Said transgenic plants and compositions are especially interesting as the novel Cry protein has an especially good insecticide capacity against Myzus persicae compared to other known Cry proteins which exhibited a smaller insecticide capacity against aphids and none were known to be active against Myzus persicae.

Description

DESCRIPTION

New Cry protein from Bacillus thuringiensis with insecticidal activity to fight a hemiptera

Field of the Invention

The present invention relates to the field of biopesticides and transgenic plants capable of expressing them. More specifically, the invention relates to a new gene that encodes a Cry protein (or delta-endotoxin), to the protein itself encoded by said gene, to the systems for its expression (vectors and host cells), and to the use of said gene and / or said protein to fight pests of Myzus persicae (Sulzer), the aphid commonly known as a green aphid, both by using a composition containing the protein and by transgenic plants in which the gene is expressed.

Background of the invention

Bacillus thuringiensis (Bt) is a Gram positive, ubiquitous and sporulant bacterium that frequently synthesizes various proteins (Cry, Cyt and Vip) with insecticidal activity against a wide range of insects that cause pests such as lepidoptera (Dulmage, 1970; Estruch et al. , 1996), beetles (Estruch and Yu, 1998; Krieg et al., 1983), diptera (Federici et al., 2003; Goldberg, 1977) and hemiptera (Porcar et al., 2009; Sattar and Maiti, 201 1) . Bt produces insecticidal proteins during sporulation that appear as parasporal crystals, crystals that are predominantly composed of one or more Cry or Cyt proteins. Cry proteins are so named because they are the proteins of the parasporal inclusions (crystals, which give rise to the abbreviation Cry) of Bt that have an experimentally verifiable toxic effect against a target organism or that have a significant sequence similarity with a protein Known Cry, while Cyt proteins are the proteins of the Bes parasporal inclusions that exhibit hemolytic (cytolytic, which results in the abbreviation Cyt) or that have a significant sequence similarity with a known Cyt protein.

Cry proteins belong to a class of bacterial toxins known as pore-forming toxins. Bry Cry proteins in particular do not exhibit insecticidal activity until they have been ingested and solubilized in the gut of the insect, where the ingested form (protoxin) is hydrolyzed by proteases to give rise to the active toxic molecule (toxin). Together with some Cyt proteins, they are included in the delta-endotoxin group. Cry proteins generally have five conserved sequence blocks, and three conserved structural domains: domain I or domain N, domain II or domain M and domain III or domain C, which receive that denomination by the regions where they appear in the protein: zone of the amino end (N), middle zone (M) and carboxyl end zone (C). These three structural domains are responsible for their insecticidal activity against different insect species, as well as against human cancer cells (parasporins) (Ohba et al., 2009). Of the latter, it is considered that the M and C domains in particular are involved in recognition and receptor binding, and are therefore considered determinants of their specificity as toxins. Domain III (domain C) often has a structural homology with carbohydrate-binding proteins such as the cellulose binding domain of 1,4-glucanase C, galactose oxidase, sialidase, β-glucoronidase, the domain of binding to carbohydrates of xylanase U and β-galactosidase. The protoxin form of many Cry proteins has an extension at the carboxyl end that makes its length greater than that of other protoxins in the family, an extension that is considered to be dispensable for toxicity but is believed to play a role in the formation of the crystalline inclusion bodies within the bacteria.

Insecticides based on delta-endotoxins have been intensively used, which has even led to the emergence of resistance in some species.

Within the order of the hemiptera {Hemipterá), the family of aphids or aphids {Aphididae), better known as aphids, contains large numbers of parasites of angiosperm plants, which originate some of the main pests that affect different agricultural crops. The green aphid, Myzus persicae (Sulzer, 1776), is one of the main polyphagous pests in many regions of the world (Harrington, 2007) including Spain (Diaz et al., 2006). It also affects a large number of agricultural crops of great economic importance (Harrington, 2007), including different fruit trees (such as some citrus fruits, peach trees and other trees of the genus Prunus) and herbaceous crops such as Solanaceae, grasses and crucifixes In these crops, it not only produces direct food damage, but also because it is an efficient vector for the transmission of several phytopathogenic viruses that cause very harmful plant diseases (Nebreda et al., 2004). Moreover, it has recently been described that this species has become resistant to some of the most commonly used synthetic chemical insecticides for its control in Europe (Slater et al., 2012). Currently, several Bry Cry proteins with insecticidal activity against aphids are known. However, they have not been especially toxic against these insects, since high doses are necessary to kill between 50% and 100% of the treated insects (Chougule and Bonning, 2012; Porcar et al., 2009). Table 1 below summarizes the known toxicities of Cry proteins against aphid species that cause pests, according to data from Chougule and Bonning (Chougule and Boning, 2012).

Table 1

Known toxicities of Cry proteins against pest-causing aphids

Toxin Toxicity Specificity

Potato aphid

Cry2, Cry3, Cry4 Reduced

{Macrosiphum euphorbiae)

Cry4Aa 1X50: 70 - 100 μg / ml

Alfalfa green aphid

Cry1 1 Aa 100% mortality at 500 μg / ml

(Acyrthosiphon pisum)

Cry3A 60% mortality at 500 μg / ml

International patent application WO2010099365 (belonging to the same family as US patent US8318900B2), refers to nucleotide sequences encoding proteins that have homology to delta-endotoxins, compositions comprising said proteins and methods for conferring resistance against pests to bacteria, plants, plant cells, tissues and seeds by transforming said organisms with said nucleotide sequences. The application WO2010099365 discloses both natural sequences of genes identified in different strains of Bacillus thuríngiensis (SEQ ID NOs: 1 to 47 of said application) and synthetic sequences prepared from the above (SEQ ID Nos: 97 to 196 of said application WO2010099365) , which give rise to proteins that lack the C-terminal "crystalline domain" present in most delta-endotoxins. In the application WO2010099365 Myzus persicae is cited as one of the possible pests to combat. However, the application does not mention any test in which the efficiency to combat said aphid of the compositions and methods disclosed in WO2010099365 is tested, although general guidelines for possible aphid tests in Example 20 are defined, without showing preference for the use of any of the delta-endotoxins disclosed in the document. Example 19 of said application describes an assay in which efficacy against another aphid, Aphis glycines, of proteins is tested. Synthetic derivatives of the Axmi171 protein (a distant homologue of the Cry12Aa2 protein, encoded by the sequence inserted in the construction described in sequence number 204 of application WO2010099365 and patent US8318900B2, sequence encoding the amino acid sequence identified with the number 205); the maximum mortality value obtained, 2 (that is, 25-50% of the containers showed 100% mortality) is only achieved with a concentrated extract of E. coli that expresses the shape of the protein that lacks the N-terminal domain or with a form of the protein obtained after cleaving with the factor Xa a fusion protein that included the maltose binding protein and resuspending the precipitate formed in water, while the rest of the variants gave rise to 0-25% of containers with 100% mortality. However, in the previous example, Example 18, tests carried out also with derivatives of said Axmi171 protein are described, specifically with different concentrations of the form of the protein obtained after cleaving with the factor Xa a fusion protein that included the protein of union to the maltose and resuspend the precipitate formed in water, but where the activity against a hemiptera is determined that does not belong to the aphid family but to another family, the Miridae family, specifically Lygus hesperus, finding that a concentration of 50 ppm (50 μg / ml) is sufficient to give rise to 80% mortality in said hemiptera.

International patent application WO2009158470, belonging to the family of US Pat. US8461421 B2 and of the same applicant as the previous one, also describes new genes encoding pesticide proteins of the delta-endotoxin type. Proteins and their respective genes are used to prepare pesticide formulations and for the production of pest resistant transgenic plants. Myzus persicae is mentioned as one of the possible pests to combat by the invention described in said international patent application, without specifying the possible delta-endotoxin to be used or the specific conditions of application. In this application, there is also no mention of any example in which the usefulness of said formulations or of said transgenic plants against any aphid is demonstrated, but only against some lepidoptera, although some general guidelines for possible aphid tests are defined in the Example 8, again without showing preference for the use of any of the delta-endotoxins disclosed in the document to effectively carry them out.

U.S. patent applications published as US2012047607A1 and US201204760A1 (both with the same applicant, Pionner Hi-Bred International Inc.) also refer to new Bacillus thuríngiensis genes that encode Cry family pesticide proteins, compositions derived therefrom and methods of control of pests that use them, including the generation of transgenic plants that express them. Although several aphids are cited as possible pests of interest, including Myzus persicae in particular, the applications do not include trials demonstrating the effectiveness of the proteins of such inventions against any aphid. In the application US2012047605A1, for example, only trials with several lepidopterans, fed with an artificial diet including the Cry protein disclosed in said application, obtained by mutagenesis, are described. In the application US2012047607A1, on the other hand, a similar test is described with a beetle, Diabrotica virgifera, (Example 1), which also includes the calculation of LC ₅₀ and EC ₅₀ (average effective concentration) (Example 2).

International patent application W09516778 does specifically refer to a method to combat the damage that aphids cause in plants by means of a toxic Bacillus thuringiensis protein. Said international patent application mentions in the background of the invention the lack of activity against the aphids of the insecticides based on Bacillus thuringiensis when used independently, neither leading to an increase in effectiveness when combined with pyrethrins. The same lack of activity had been found with various transgenic plants capable of expressing Bacillus thuringiensis insecticidal proteins. Application W09516778 proposes to feed aphids with an artificial diet containing Cry proteins prepared in a suitable formulation (solubilized in an aqueous medium or in the form of a suspension of solids) by means of a feeder device, prepared on a laboratory scale, where aphids are separated from the diet by a thin plastic membrane. Alternatively, it is proposed to obtain transgenic plants in which the promoter to which the Cry protein gene is attached is chosen so that said Cry protein is expressed in the vascular tissue of the plant, preferably in the phloem, since that ensures that Aphids ingest protein when feeding on the plant. This request does not show any example in which the effectiveness of the Cry protein expression strategy in the phloem of transgenic plants is demonstrated.

As regards artificial feeding, only W09516778 describes trials with the potato aphid, Macrosiphum euphorbiae (Thomas), which demonstrate that high concentrations of the CryIIIA protein are necessary to observe an effect (Example 1). In Example 2, meanwhile, try to compare the effectiveness of CrylA (c), CryIIA, CryIIIA, CrylllB2, CrylllB3, CryIVD and a mixture of Cry type I (CrylA (a), CrylA (b) proteins , CryIC and CryIF), all of them in aqueous suspension; Although the difference in the concentrations added in each case makes comparisons difficult, the result shows that only the CrylA (c) protein showed no activity insecticide against Macrosiphum euphorbiae, while the CryIIA protein (at 200 ng / μΙ) resulted in a rapid onset of mortality, which was close to 100% after two days of feeding, the other proteins being less effective. In the section dedicated to the detailed description of the preferred embodiments of the invention of application W09516778, it is considered that the most effective proteins for fighting aphids, together with the above, are the CryIIIA, CryIIIB and CryIVD proteins. This assessment, however, has not been performed in trials with Myzus persicae. The remaining proteins tested showed varying percentages of insecticidal activity, taking 4 to 5 days to observe a mortality close to 100% in the case of the Cryl protein mixture (where the total concentration was close to 400 μ9 / μΙ, equivalent to 400 mg / ml)), in the case of CryIIIA protein (at 400 ng / μΙ, equivalent to 400 μg / ml) and in that of CryIVD protein (at 350 ng / μΙ, equivalent to 350 μg / ml) and up to 7 days or more in the case of CrylllB2 proteins (at 150 ng / μΙ, equivalent to 150 μg ml) and CrylllB3 (132 μg / μ \, equivalent to 132 mg / ml).

According to the above, it can be said that no Cry protein that has insecticidal activity against Myzus persicae has been described so far.

Given the worldwide spread of Myzus persicae and the large number of agricultural crops it affects, it would be interesting to find a protein with insecticidal activity, for example of the endotoxin type, that would show significant insecticidal activity against aphids and, in particular, as Myzus persicae. The present invention provides a solution to said problem.

Brief Description of the Invention

The present invention relates, in a first aspect, to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a delta-endotoxin, characterized in that the nucleotide sequence encoding a delta-endotoxin is represented by SEQ ID NO : 1 or has at least 90% identity with it.

The nucleotide sequence encoding a delta-endotoxin and which is represented by SEQ ID NO: 1 will also be referred to hereinafter as "the gene of the present invention", "the cry gene of the present invention" or "the new cry gene ". The nucleic acid molecule defined in this first aspect of the invention will be referred to as the nucleic acid molecule of the present invention.

In further aspects of the invention, it refers to an expression system of the above nucleic acid molecule and / or of the coding sequence comprised therein, and to the expression vectors that may be part of said expression system. Therefore, the present invention also relates to a vector in which it is Insert the nucleic acid molecule of the present invention, a vector that will be considered hereafter a vector of the present invention. In addition, the invention also relates to a host cell comprising a vector of the invention, that is, a host cell of the present invention.

In a further aspect, the invention relates to a transgenic plant comprising a host cell of the present invention that is a plant cell, as well as a transgenic seed comprising a nucleic acid molecule of the present invention.

Another aspect of the invention is a polypeptide of the present invention, that is, a polypeptide comprising an amino acid sequence characterized in that said amino acid sequence:

a) is represented by SEQ ID NO: 2;

b) is the sequence of a delta-endotoxin that is encoded by the sequence represented by SEQ ID NO: 1 or by a sequence that has at least 90% identity with it, or

c) is the sequence of a delta-endotoxin that has at least 80% identity with the sequence represented by SEQ ID NO: 2.

The amino acid sequence represented by SEQ ID NO: 2, as well as the sequence that has at least 80% identity with it or the sequence of a delta-endotoxin that is encoded by the sequence represented by SEQ ID NO: 1 or by a sequence that has at least 90% identity with it, the delta-endotoxin of the present invention will be considered hereinafter.

An aspect of the invention is also a composition comprising a polypeptide of the present invention, a vector of the present invention and / or a host cell of the present invention. Such compositions will be considered the compositions of the present invention.

It is also an aspect of the invention to use a composition of the present invention to combat pests of at least one hemiptera, preferably because the hemiptera is an aphid, and especially preference for the aphid Myzus persicae.

A further aspect of the invention is a method for the production of a polypeptide of the present invention, comprising:

a) obtaining a recombinant organism that expresses the polypeptide of the present invention;

b) culturing said recombinant organism;

c) inducing, if necessary, the expression of the polypeptide in said recombinant organism; d) purify the polypeptide from the recombinant organism.

Finally, an aspect of the invention is also an antibody that specifically binds to a polypeptide of the present invention, especially an antibody that specifically binds to the fragment of said polypeptide that constitutes the delta-endotoxin of the present invention.

Brief Description of the Figures

Fig. 1: Multiple alignment of active Cry protein sequences against different aphid species. The Cry protein of the present invention (CryBMI) and the protein represented by the sequence with identification number 205 are included in US8318900B2 (Seq205-US8318900). Under each sequence, shaded rectangles appear, indicating, if any, the location of the N, M and C domains, characteristic of insecticidal delta-endotoxins, as well as that of other significant regions (Gal: galactose binding domain; CW: binding to the cell wall; TMR: transmembrane region; SP: signal peptide) Note the presence of a rectangle labeled "Ricin B" (indicating a domain similar to ricin B) under the right end of the corresponding protein representation CryBMI

Fig. 2: Representative dendrogram of the phylogenetic relationship between Cry proteins active against aphids. The Cry protein of the present invention (CryBMI) and the protein represented by the sequence with identification number 205 are included in US8318900B2 (Seq205-US8318900). The length of the horizontal bar located below the lower protein line, CryIAa, corresponds to a taxonomic distance of 0.2, calculated based on the percentage of identity of the alignments of the amino acid sequences, as indicated in the figure itself .

Fig. 3: Scheme of the recombinant expression vector pET-28b (+) used to insert the cryBMI gene and then used to transform Escherichia coli BL21 (DE3) cells and express the insecticidal protein of the present invention under control of the bacteriophage promoter T7 (Promoter T7) inducible by IPTG. The insertion position of the cryBMI gene and the size of the plasmid before insertion of said gene is indicated (5369 base pairs, abbreviated as bp).

Fig. 4: Photograph of the SDS-PAGE gel obtained by electrophoresis a sample of the CryBMI protein expressed in Escherichia coli and purified on a nickel column. The band corresponding to the sample (right lane, labeled "Cry") is indicated with an arrow and the estimated size (89 kDa); in the left lane, corresponding to the marker, the size of the two bands between which the CryBMI sample band is located is indicated. Fig. 5: Photograph of the elements used in the bioassay of insecticidal activity. From left to right: rear view of a fragment of the Parafilm® "M" plastic laboratory film strips; side view of a test box; top view of a test box, with 15 newborn nymphs of Myzus persicae; top view of a box covered with a layer of Parafilm® "M" with a drop of diet + CryBMI protein ("toxin" in the photo caption ") deposited on it; top view of a box with the same elements as the previous one, in which the diet drop + CryBMI protein has been covered with a second layer of "M" Parafilm®. Next to the boxes (at an upper level of the surface of the photograph) an end of the pipette is shown automatic used to deposit the drops of diet + CryBMI protein, in which the tip used to take and deposit the samples has been fitted.

Detailed description of the invention

The present invention is based on the identification and isolation of a Bacillus thuríngiensis (Bt) gene, as well as the identification, isolation, production and insecticidal evaluation of its product, a new Bt Cry protein that is toxic against the Myzus aphid Persicae or green aphid. This new gene and its product can be used for the development of new biopesticides and the construction of transgenic plants resistant to green aphids.

In the present application, the nucleotide sequence of the new isolated gene (represented by SEQ ID NO: 1) and the amino acid sequence of the protein encoded by said gene (represented by SEQ ID NO: 2) are detailed. Evidence of the insecticidal capacity of this protein is also provided, such as the calculation of the value of the mean lethal concentration (LC ₅₀ ), (which is the concentration of toxin that causes death in 50% of the insects that have been treated) , which has been found to be a lower concentration than those described for other Cry proteins applied to other pest hemiptera, particularly lower than those described so far for other aphids.

The present application provides a detailed description of the new Cry protein and the gene that encodes it, which are significantly different, in sequence and in some structural characteristics, from the sequences corresponding to other Cry proteins and, in particular, quite different from the proteins. Cry with proven insecticidal activity described so far.

The structural analysis schematized in Fig. 1 shows that the protein whose isolation and production is described later in the present application (indicated in said Fig. 1 as CryBMI) is a 89 kDa protein that has the characteristic structure of delta- endotoxins, including the N, M and C domains that generally They are present in Cry proteins. In the C domain in particular, an area similar to a carbohydrate binding domain is observed, specifically a galactose binding domain. Therefore, the protein whose isolation and production is disclosed in the present application, together with the identification and isolation of its coding sequence, is called in the present application the Cry protein of the present invention, the delta-endotoxin of the present application. invention or simply the protein of the present invention. The representative dendrogram of the phylogenetic relationship between active and aphid Cry proteins of Fig. 2 shows that the Cry protein of the present invention is not closely related to any of said proteins, indicating that it corresponds to a protein not described to date. Said protein has been provisionally assigned the abbreviated designation CryBMI in relation to the research group (Microbial Bioinsecticides).

The comparative analyzes performed, based on searches in databases of other known nucleotide sequences and their comparison with respect to SEQ ID NO: 1, on the one hand, and of known protein sequences and their comparison with respect to SEQ ID NO : 2, on the other hand, have allowed us to corroborate not only that the new Cry protein (or delta-endotoxin) of the present invention is new, as is its coding sequence, because its sequences do not coincide with any other amino acid sequence or Nucleotides, respectively, previously known, but also detect differences with other related sequences.

Thus, for example, the comparison of the nucleotide sequence of the gene of the present invention (SEQ ID NO: 1) with other nucleotide sequences has allowed us to verify that no nucleotide sequence is found that has a percentage of identity that reaches the value of 90%, or higher, with respect to SEQ ID NO: 1. The maximum percentage of identity, 87%, corresponds to the nucleotide sequence with the number 38 in the family of the international patent application WO 2010099365 and US Pat. US8318900, to which Japanese application JP 2012519000-A also belongs, a sequence that encodes the protein that responds to sequence 87 of said family. Other nucleotide sequences disclosed in the same family of patent documents, such as sequence 169 of WO2010099365, also appear among the list of sequences with the highest percentage of homology with respect to SEQ ID NO: 1 (see Table 4 below). (sequence 122 of JP 2012519000-A), with which the gene of the present invention has an identity of 70%. A sequence disclosed in the family of patent documents of the international application WO 2009158470 and US Pat. US8461421 (which also includes the Japanese patent application JP201 1526150-A), the sequence with the number 24 of this second family of patents, with which SEQ ID NO: 1 of the present invention has a 71% identity (considering nucleotides 1 to 1345), which rises to 76% in the 215 nucleotide fragment between nucleotide 1372 and nucleotide 1586 of SEQ ID NO: 1; the fragment between nucleotides 1346 and 1371, however, does not appear among those of greater identity with the sequence with number 24 of the patent family of international application WO 2009158470 and US Pat. US8461421.

However, as previously mentioned herein, none of the documents cited shows tests carried out in which insecticidal activity of any of the proteins encoded by said nucleotide sequences is demonstrated, assuming that such activity exists by responding to all proteins. to the structure of delta-endotoxins. There is also no suggestion in these documents that any of those proteins encoded by sequences with a considerable percentage of identity with the gene of the present invention could be especially suitable for aphids. Moreover, sequence 169 of WO2010099365 belongs to the group of synthetic genes described in said patent application that lack the "crystalline" domain of the carboxyl end which is present in many delta-endotoxins, and which is believed to be involved in the formation of crystalline inclusion bodies within Bt.

The situation is similar in regard to other nucleotide sequences that also have an identity percentage equal to or greater than 70% with respect to the gene sequence of the present invention, represented by SEQ ID NO: 1. Thus, the CDS (coding sequence) with accession number KC156704, which is said to correspond to a pesticide protein of Bacillus thuríngiensis, strain ARP242, also has an identity percentage of 87% with respect to SEQ ID NO: 1, but not it appears to present among the information available from its accession number the mention of any publication or other document in which its pesticide activity is demonstrated, with no mention of any possible application to hemiptera, aphids in particular, or Myzus persists in particular. The same applies to the CDS corresponding to strains ARP256 (87% identity) or ARP277 (70% identity) of Bt. Finally, in addition to fragments of the complete genome sequencing of Bacillus subtilis, high percentages of identity also appear, but that do not reach 75% (74%) with reduced fragments (152 nucleotides) of coding sequences (CDS) that, hypothetically, could encode Cry proteins that are being assigned a non-pesticide application, but to kill cancer cells.

The comparison made directly using the sequence of the protein itself of the present invention (SEQ ID NO: 2) results in the identity percentages being reduce. Among the sequences extracted from patent documents (Table 3), the highest percentage of identity does not reach 80% (76.20%) and corresponds to sequence 87 of the family of US8318900 and international patent application WO2010099365, which is precisely the amino acid sequence encoded by the sequence 38 of said applications, that is, the nucleotide sequence with which SEQ ID NO: 1 has the highest percentage of identity. Next, only sequence 84 of the family of US Pat. US 8461421 and the international patent application WO2009158470 has an identity percentage greater than 50% (57.91%). The rest of the sequences extracted from patent applications, all of which belong to the latter family cited, have identity percentages below 40% with respect to SEQ ID NO: 2.

The comparison with other non-redundant proteins does not allow to locate proteins with a higher degree of identity with the delta-endotoxin sequence of the present invention. Only Cry41 Aa1-like protein (Genbank accession number AEH76822.1) has an identity percentage close to 60%, 57.91%. In addition to this protein, only one sequence, which corresponds to a hypothetical protein (Genbank accession number EJR94916.1) has an identity percentage greater than 50% with SEQ ID NO: 2.

Thus, neither the comparative search by the gene sequence nor the search by the protein sequence allows to locate sequences that correspond to Cry proteins with known activity against hemiptera (especially against aphids) and that have a high percentage of identity (at least 50%) with the gene and / or protein of the present invention. In particular, the sequence corresponding to the Cry protein or derivatives thereof with which insecticidal activity tests are carried out in the patent family of the international application WO2010099365 and the patent are not shown in any of Tables 2, 3 or 4 from the USA US8318900B2.

The delta-endotoxin of the present invention is quite different from other Cry proteins active against aphids described so far. As shown in Table 5 below, the Cry protein with known activity against aphids that has a higher percentage of identity with the Cry protein of the present invention is the Cry3Aa protein, with which it only shares a 26.3% identity With respect to other proteins for which its possible activity against aphids has been tested (Cry2Aa, Cry4Aa, Cry1 1 Aa and, even, the protein represented by the sequence with identification number 205 in US8318900B2 and the international patent application WO2010099365), the identity does not reach 20%. And yet, surprisingly, the experimental assays described below in Examples of the present application demonstrate that the new Cry protein of the present invention is the first Ct protein of Bt with significant insecticidal activity against aphids in general and, in particular, the Bry's first Cry protein for which it is reported to possess insecticidal activity against the green aphid, Myzus persicae, so, consequently, it is an object of the present invention, together with the gene that encodes it, as well as the use as insecticide of the compositions containing the gene and / or the protein, including the generation of transgenic plants that express said gene.

Moreover, the experimental results show that this protein has a high insecticidal activity, its CL ₅₀ being only 32.7 μg / ml. This value is much lower than the values obtained with other Cry proteins previously evaluated in other aphid species related to M. persicae (Table 1). The difference in order of magnitude is also significant with the amounts of other Cry proteins used in tests such as those described in international patent application W09516778 where some analogous proteins, such as the Cryl protein mixture, become used at a close concentration. at 400 mg / ml, and where other proteins, such as CrylllB2, used at a concentration almost an order of magnitude greater (150 μg ml), need up to 7 days to result in a mortality close to 100% in aphid-directed assays, in which Myzus persicae is not mentioned. Even extending the scope of comparison to known activities on other hemiptera, the value can be considered high, as can be seen from Example 18 of the application WO2010099365 and US8318900B2, made with unnatural derivatives of the protein represented by the sequence 205 of these documents, where the concentration of 50 μg / ml results in an 80% mortality of the hemiptera Lygus hesperus, from the Miridae family, a concentrated extract obtained from E. coli being necessary for one of the derivatives thereof Protein results in 100% mortality in 25-50% of the containers when the test is performed with an aphid, Aphis glycines. In general, as previously mentioned in the present application, aphids are not very susceptible to Bt Cry toxins (Chougule and Bonning, 2012). This fact, together with the ability of aphids to generate resistance to synthetic chemical insecticides, increases the importance of discovering new, more toxic proteins to implement alternative control methods based on Bt toxins. The new Cry protein of the present invention is It presents as such an alternative, and can be adapted to the development of control methods based on new formulated bioinsecticides and the construction of transgenic plants resistant to green aphids and other susceptible aphid species. The coding sequence of this new protein can be used to transform bacterial heterologous hosts or plant cells by well known and routine techniques in genetic engineering, either to produce new copies of the protein or as an intermediate step for the generation of transgenic plants that express it. For this, a previous step is usually its insertion into a "vector", a DNA molecule that allows the objective to be achieved to be achieved, either the expression of the gene (expression vectors) or the transfer of molecules or DNA constructs between different host cells, such as the transformation of plant cells, often including the subsequent obtaining of a transgenic plant (the so-called transformation vectors, which are often also expression vectors, or include what is they call "expression cassettes"). In both cases, it is common that a previous step is the generation of "expression cassettes", in which the gene to be expressed is operatively linked to a promoter that allows its expression in a system of interest and that often they also contain some additional regulatory sequence or useful for expression, such as a 3 'untranslated region or, especially when expression in eukaryotic cells, a signal sequence or a leader sequence that facilitates the transport of the peptide being formed is desired , or once translated, to a particular cellular organelle, such as chloroplasts, endoplasmic reticulum, Golgi apparatus ...; It is also common for the "expression cassette" to be formed by inserting the coding sequence to be expressed in the vector in a precise place, so that it is attached to the desired elements.

The choice of promoter depends on the system in which it is desired that the gene to which it will be operatively linked be expressed. Thus, for example, when the protein is to be produced, for example, in a microorganism, such as a bacterium, it will be necessary to choose a promoter that allows expression in said bacterium. One of the most commonly used bacteria for protein expression is Escherichia coli, for which there are many promoters and expression vectors that contain them well known to those skilled in the art. In the case of Escherichia coli, it is usual to insert the genes to be expressed under the control of the lac operon, as in Example 2 of the present invention, which allows to control the expression of the protein, inducing it by the addition of IPTG (isopropyl- β-Dl-thiogalactopyranoside). There are many known and commercially available expression vectors that allow expression in E. coli, of which plasmids are widely used, but some recombinant viruses prepared from the natural forms that infect such bacteria are also common and known, such as the bacteriophage lambda. Plasmid-like expression vectors are easy to locate and choose not only from commercial catalogs, but from databases dedicated to these types of vectors such as Addgene's (http://www.addgene.org), where, for example, the structure of plasmid pET-28b (+) used in the examples of the present invention can be consulted.

Other microbial hosts available for transformation with expression vectors, of particular interest in the present case, could be the Bt 4Q7 crystalline strain (not producing other endotoxins) available at the Bacillus Genetic Stock Center, Bacillus megaterium (Mobitec, Germany), Pseudomona spp. or yeasts In these microorganisms, the insecticidal protein produced remains encapsulated in the bacterial soma so that the same microorganism would become the active material of the biopesticide, easily recoverable by centrifugation. This microencapsulation would also protect the protein from environmental factors (eg UV), extending its useful life in the environment. The option of coating said proteins with substances (nanoparticles) that similarly protect said toxin is also an attractive alternative.

This new cry gene can also be optimized for expression in plant cells in the construction of transgenic insect-resistant plants, through the use of techniques already described and well known in genetic engineering.

The plant transformation methods of the invention involve the introduction of a nucleotide construct, including in gene to be transferred, into a plant. By "introduction" is meant to provide the plant with the construction of nucleotides in such a way that the construction accesses the interior of a plant cell. The methods of the present invention do not require that a particular method be used for the introduction of a nucleotide construct into a plant. Methods for introducing nucleotide constructs into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus mediated methods.

"Plant" means whole plants, plant organs (for example, leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and their progeny. Plant cells (or plant cells) can be differentiated or undifferentiated (for example, calluses, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen).

The terms "transgenic plants" or "transformed plants" or "stably transformed" cells or tissues plants refer to plants that have incorporated or integrated nucleic acid sequences or exogenous DNA fragments into one or more plant cells. These nucleic acid sequences include those that are exogenous, or not present in the cell of the non-transformed plant, as well as those that may be endogenous, or that are present in the cell of the non-transformed plant. The term "heterologous" generally refers to nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and that have been added to the cell by infection, transfection, microinjection, electroporation , microprojection, or similar.

The transgenic plants of the present invention will be those that express the gene of the present invention or a sequence coding for a delta-endotoxin that has at least 90% identity with the sequence of the gene of the present invention (SEQ ID NO: 1 ). The transgenic plant may further comprise one or more additional genes for insect resistance, and / or any additional gene that imparts an agronomic trait of interest.

The transformation of plant cells can be achieved by various techniques well known to those skilled in the art. Normally, the gene to be expressed (in this case, the gene of the present invention, or coding sequences that have at least 90% identity with it), will be part of a construct (an expression cassette, such as described above) in which the gene will be operatively linked to a promoter that will control its transcription, as well as to a non-translated 3 'zone that will allow termination of transcription and polyadenylation of the transcript. Among the possible termination regions, for example, those present in the Ti plasmid of Agrobacterium tumefaciens, such as the termination regions of octopine synthase and nopaline synthase may be suitable. If desired, a signal peptide may also be included that facilitates the transfer of the peptide that is being formed to the endopiásmic reticulum. It may also be interesting to design the plant expression cassette so that it contains at least one intron, such that the processing of the mRNA intron is required for its expression.

Normally, this "plant expression cassette" is inserted or inserted into a "plant transformation vector". This plant transformation vector may be composed of one or more DNA vectors necessary to achieve plant transformation. For example, it is a common practice in the art to use plant transformation vectors that are composed of more than one contiguous DNA segment. These vectors are often referred to as "binary vectors." Binary vectors, as well as vectors with auxiliary plasmids, are the most commonly used for Agrobacterium-mediated transformation, when the size and complexity of the DNA segments necessary to achieve efficient transformation is quite large. Binary vectors typically contain a plasmid vector containing the cis-acting sequences necessary for the transfer of T-DNA (such as the left border and the right border), a selection marker that is designed to be able to express itself in a plant cell, and a "gene of interest" (a gene designed to be able to express itself in a cell of a plant from which generation is desired of transgenic plants which, in the present case, would be the gene of the present invention or a coding sequence that has at least 90% identity with it). Also present in this plasmid vector are sequences required for replication in bacteria. The cis-acting sequences are arranged in such a way as to allow efficient transfer in plant cells and expression therein. For example, the selection marker gene and the gene of the present invention lie between the left and right edges. Often a second plasmid vector contains the trans-acting factors that mediate the transfer of Agrobacterium T-DNA to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow the infection of plant cells by Agrobacterium, and the transfer of DNA by cleavage of the sequences of the borders and the transfer of DNA mediated by vir, as understood in the technique (Heliens and uilineaux, 2000). Various types of Agrobacterium strains can be used for plant transformation (for example, LBA4404, GV3101, EHAIOI, EHA105, etc.). The second plasmid vector is not necessary for the transformation of plants by other methods such as microprojection, microinjection, electroporation, polyethiengiicol, etc.

In general, plant transformation methods involve the transfer of heterogeneous DNA to the plant's target cells (for example, immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of a level maximum threshold for appropriate selection (depending on the marker gene will be selected) to recover cells from transformed plants from a group of an unprocessed cell mass. The expianies are normally transferred to a fresh supply of the same medium and are routinely grown. Subsequently, the transformed cells differentiate into outbreaks after placing them in regeneration medium supplemented with a maximum threshold level of selection agent. The shoots are then transferred to a selective rooting medium for the recovery of shoots with roots or rooted seedlings. The transgenic seedling then grows into a mature plant and produces fertile seeds. The explants are normally transferred to a fresh supply of the same medium and are routinely grown. A general description of the techniques and methods to generate transgenic plants can be found in publications such as Ayres and Park (Ayres and Park, 1994) and Bommineni and Jauhar (1997). Since the transformed material contains many cells, Any sample of said transformed material contains transformed and non-transformed cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in the cultivation of transformed plants. Often, the ability to eliminate non-transformed cells is a limitation for the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

The transformation protocols as well as the protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell that is to be transformed, that is, monocot or dicot. As previously mentioned, the generation of transgenic plants can be carried out by various methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA through Agrobacterium into plant cells (transformation mediated by Agrobacterium), bombardment of plant cells with DNA adhered to particles, acceleration of ballistic particles, transformation by means of an aerosol beam, transformation with Lec1, and various other methods not mediated by particles.

After the integration of the heterologous foreign DNA into plant cells, an appropriate maximum threshold level of selection is then applied in the medium to eliminate the non-transformed cells and separate and proliferate the hypothetically transformed cells that survive this selection treatment by periodically transferring them to a fresh medium, through continuous passes and maintaining the appropriate selection conditions. Numerous markers have been developed for use with plant cells, such as chloramphenicol resistance, G418 aminogycoside, hygromycin resistance, or the like.

Molecular and biochemical methods can be used to confirm the presence of the heterologous gene of interest integrated in the genome of the transgenic plant, a gene of interest that, in the case of the transgenic plants of the present invention, is the gene of the present invention or a coding sequence with an identity percentage of at least 90% with it.

Cells that have been transformed can be cultured to give rise to plants in a conventional manner. These plants can then be cultured, and either be pollinated with the same transformed strain or with different strains, and the resulting hybrid has constitutive expression of the desired phenotypic characteristic, in this case, the expression of the Cry protein of the present invention Two or more generations can be cultivated to ensure that the expression of the desired phenotypic characteristic is maintained and inherited stably and then harvest the seeds to ensure that the expression of the desired phenotypic characteristic has been achieved. Thus, the present invention provides transformed seeds (also referred to as "transgenic seeds") having a nucieotidic construction of the present invention, (eg, an expression cassette in which the gene of the invention it is linked, stably, to a promoter that allows its expression in plants and to a suitable sequence of termination, stably incorporated into its genome).

After the introduction of heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome can be confirmed by various methods such as the analysis of nucleic acids, proteins and metabolites associated with the integrated gene. In this particular case, the analysis of the presence of the integrated gene, the generation of its mRNA or the presence of the protein to be expressed, the protein of the present invention or a protein that has a high degree can be performed. of identity with it.

PCR analysis is a rapid method to detect in cells, tissues or transformed outbreaks, the presence of the gene incorporated at an earlier stage before transplanting it into the soil, by routine methods for those skilled in the art (see, for example, Sambrook and Russeli, 2001). PCR can be carried out using specific oligonucleotide primers that allow amplifying the gene of interest from the start codon (ATG) to the translation stop codon (TTA) (direct primer oligonucieotide 5 ^' -ATGAACCAAAATTATAAGAACAATG-3', (SEQ ID NO: 3) Reverse primer oligonucieotide 5 ^: CAAATCAAAAATTCAAACTGAATAAGTTA3 '(SEQ ID NO: 4)). By amplification by PCR, the amplicon could also be obtained, purified and labeled either by radioactive ( ³² P) or non-radioactive (digoxigenin) methods and used as a specific probe.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russeli, 2001). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated on an agarose gei and transferred to a nitroceiuous or nylon membrane. Next, a membrane assay is performed with the DNA transferred with, for example, a fragment of the target DNA (a fragment of the gene of the present invention, in this case, or a sequence that has at least 90% identity with it) to confirm the integration of the gene introduced into the genome of the plant.

In Northern blot analysis, RNA is isolated from specific tissues of the transformant, fractionated on an agarose-formaldehyde gel, and transferred to a nyion filter according to standard procedures that are routinely used by Those skilled in the art (Sambrook and Russeli, 2001). The expression of the RNA encoded by the gene of the present invention or by a coding sequence that has at least 90% identity with it is tested by hybridization of the filter with a radioactive probe derived from the sequence of the α to endotoxin gene of the invention, by methods known in the art (Sambrook and Russel !, 2001).

The presence of the protein encoded by the delta-endotoxin gene of the present invention can also be confirmed by Western blotting, biochemical tests or the like carried out in transgenic plants by standard procedures (Sambrook and Russeil, 2001, supra). ), using antibodies that bind to one or more epitopes present in the delta-endotoxin protein of the present invention. For this purpose, for example, antibodies directed to other Cry proteins with which the Cry protein of the present invention share epitopes can be used. Another alternative is the preparation of specific antibodies against the new Cry protein of the present invention and, similarly, using Anti-6xHis_Tag (Sigma-Aldrich) antibodies against the tail of 6 histidines fused to the carboxyl end of the recombinant CyBM1 protein and which are encoded in the vector pET-28b (+). The generation of antibodies against any protein is a common technique, known to any person skilled in the art, which can be considered routine.

Preference is given to monoclonal antibodies, which are homogeneous antibodies produced by a hybrid cell resulting from the fusion of a clone of B lymphocytes descended from a single and single stem cell and a tumor plasma cell, which guarantees its perpetuity (Kohier & Milstein, 1975). With this fusion of two cells, one programmed to produce a specific antibody but that does not multiply indefinitely (lymphocyte) and another immortal with great growth capacity but that does not produce immunoglobulin (myeloma cell), combines the genetic information necessary for synthesis of the desired antibody and a capacity for protein synthesis, allowing its indefinite multiplication both in vitro and in vivo. For the production of monoclonal antibodies against the Cry protein of the present invention, the present authors of the present invention have a preference for the methodology described by Iglesias et al. (1 997). Briefly: splenocytes obtained from female BALB / c mice, previously injected intraperitoneally with the complete form of the Cry protein of the present invention, are fused with murine myeloma cells X63-Ag8.653, in the presence of 50% polyethylene glycol (PEG) 4000 (Boehringer Mannheim, Barcelona, Spain). Hybridoma cultures can be maintained as described by Estévez et al. (1994). Subsequently, anti-Cry monoclonal antibodies are obtained from ascites fluid injected mice, intraperitoneally, with the cells of the Selected hybridoma (2x10 ⁶ cells / mouse, for example). Partial purification of the antibodies can be performed by precipitation of ascites with saturated ammonium sulfate (SAS). In any case, currently in Spain there are many biomedical companies specializing in the production of monoclonal antibodies on demand, which can be ordered to produce.

In the present case, the presence of the delta-endotoxin transgene of the invention can also be detected by tests of its pesticidal activity in fertile plants. Plants that show optimal activity are selected for later reproduction. There are methods available in the art for testing pest activity, such as mixing the protein and using it in feeding assays, as in the Examples shown later in the present application.

The use of any of the methodologies described above gives rise to transgenic plants that express the delta-endotoxin of the present invention, whereby said transgenic plants will have insecticidal activity against at least one aphid, preferably Myzus persicae, so that the The use of such transgenic plants to combat aphid pests and / or confer resistance to the plants, in particular Myzus persicae, is also an object of the present invention. In principle, the way in which the cells of transgenic plants are generated is not critical for the purposes of the present invention. However, since the main objective of the present invention is to combat hemiptera pests, preferably aphids, including Myzus persicae in particular, it is a particularly interesting embodiment of the present invention that in which selectively transformed (transgenic) plants express the delta-endotoxin of the present invention in the vascular tissue of the plant, in particular in phloem tissue, as disclosed in application W095 / 16778, to facilitate ingestion of delta-endotoxin by aphids to fight. This can be achieved by choosing as a promoter operably linked to the gene of the present invention, or to a coding sequence with a percentage of identity of at least 90% therewith, a promoter that facilitates expression in said tissues. For this, suitable promoters are those mentioned in said international patent application, such as the 4xB2 + A CaMV35S cauliflower mosaic virus promoter.

The present invention can be used for the transformation of any type of plant. Plants for which special preference is given are those that suffer damage caused by aphids, in particular by Myzus persicae, or in which it has been found that said aphid facilitates the transmission of viruses. Thus, among others, there is a preference for transgenic peach (Prunus pérsica), apricot, plum and other plants of the genus Prunus, citrus, crucifixes, nightshade, grasses and even ornamental plants.

Alternatively, the plants can be protected by applying a composition containing the delta-endotoxin of the invention or a system capable of expressing it. Compositions comprising the Bacillus strain containing in its genome the nucleotide sequence from which the gene of the present invention, Bacillus thuringiensis Bt H1 .5, or other recombinant microorganisms in which the delta is expressed can also be used -endotoxin of the invention.

The active ingredients (the delta-endotoxin of the present invention or a microorganism that expresses it) of the present invention are normally applied in the form of compositions (the pesticidal compositions of the present invention) and can be applied to the crop or plant area to be treated. , simultaneously or consecutively, with other compounds. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, soaps, oils, latent pesticides, polymers, or biodegradable vehicle formulations that allow long-term dosing in a target area after a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucidal, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, administered, if desired, together with other agriculturally acceptable vehicles, surfactants or adjuvants of the application commonly used in the technique of pesticide formulation. Suitable carriers and adjuvants may be solid or liquid and correspond to the substances normally employed in the formulation technology, for example, natural mineral substances, solvents, dispersants, wetting agents, binders or fertilizers. Similarly, the formulations can be prepared in the form of "edible baits" or "pest traps" be formed to allow feeding or ingestion by a target pest of the pesticide formulation; An example of such devices can be found in application W09516778.

The methods of applying a composition of the present invention, comprising at least the delta-endotoxin of the present invention or an expression system thereof, such as Bacillus thuringiensis Bt H1.5 cells or a recombinant microorganism comprising The gene of the invention operatively linked to a promoter that allows expression in said microorganism, includes application on the leaves, seed coating and application in the soil. The composition may be formulated in the form of powder, granule, aerosol, emulsion, colloid, solution, or the like, and may be Prepare by conventional means such as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a cell culture comprising the polypeptide corresponding to the delta-endotoxin of the present invention.

The formulations may also vary with respect to weather conditions, environmental considerations, the desired frequency of application and / or the severity of the infestation.

The compositions described can be carried out by formulating any bacterial cell of the aforementioned, (which, in the case of Bacillus thuringiensis, can be, for example, a suspension of spores or crystalline forms), or the formulation of the protein of the present invention, previously produced and isolated, with the desired agriculturally acceptable vehicle.

An example of a composition comprising the delta-endotoxin of the present invention can be found in Example 3 of the present application, where the soluble protein was diluted in an aqueous solution containing sucrose, specifically 20% w / v sucrose, which was the artificial diet prepared for aphids. Other formulation examples can be found, for example, in the international application W09516778, where delta-endotoxins are in crystalline form and are prepared in the form of an aqueous suspension.

In general, the compositions may be formulated prior to administration in an appropriate medium, and may be prepared in the desired manner by known means such as lyophilization, subsequent freeze drying, or drying, or in a suitable aqueous vehicle, medium or diluent. , such as saline or other buffer. The formulated compositions may be in the form of a powder or granular material, or a suspension in oil (vegetable or mineral), or water or oil / water emulsions, or as a wettable powder, or in combination with any other suitable vehicle for agricultural application The term "agriculturally acceptable vehicle" covers all adjuvants, inert components, dispersants, surfactants, adhesives, binders, etc., which are commonly used in pesticide formulation technology, which are well known to those skilled in pesticide formulation. The formulations can be mixed with one or more solid or liquid adjuvants and prepared by various means, for example, mixing until homogenization, mixing and / or milling of the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described, for example, in US Pat. N ^and 6468523, incorporated herein by reference. Another possible alternative is that the gene of the present invention is cloned for expression in strain Bt 4Q7, or in microorganisms such as Bacillus megateríum, Pseudomonas spp., Or yeasts, in which the produced delta-endotoxin produced remains encapsulated in the soma bacterial, so that the same microorganism would become the active biopesticide, easily recoverable by centrifugation. This microencapsulation would also protect the protein from environmental factors (eg UV), extending its useful life in the environment. The option of coating said proteins with substances (nanoparticles) that protect similarly to delta-endotoxin is also a possible alternative.

Microencapsulated delta-endotoxin can be used in spray applications, either independently, or in rotations with Bt-based insecticides that express other possible delta-endotoxins of interest.

Plants can also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides.

Therefore, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a delta-endotoxin, characterized in that the nucleotide sequence encoding a delta-endotoxin is represented by SEQ ID NO: 1 or has at least 90% identity with it. In possible embodiments, the percentage of identity can be selected, for example, from the group of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 %, 99.6%, 99.7%, 99.8%, 99.9%, percentages of identity that are considered to give rise to a delta-endotoxin sufficiently identical to delta-endotoxin whose isolation and production is described more later in Examples of the present invention. Logically, another possible embodiment is one in which the identity is 100%, an embodiment that would correspond to the alternative in which the nucleotide sequence encoding a delta-endotoxin is exactly that represented by SEQ ID NO: 1, thus being a possible embodiment of this aspect of the invention that the nucleotide sequence of the nucleic acid molecule be represented exactly by SEQ ID NO: 1.

The determination of the percentage of identity between two sequences can be carried out with the usual computer resources, based on mathematical algorithms, such as the BLASTN and BLASTP programs used in the later Examples of the present application, using, for example, the parameters which appear by default, as well as with other alternative applications known to those skilled in the art, such as Gapped Blast and PSI-Blast (Altschul et al., 1997), or others.

As discussed below, the Cry protein of the present invention has a domain similar to ricin b, which is not present in other Cry proteins with activity against aphids. Therefore, it is preferred that, whatever the percent identity with SEQ ID NO: 1, it is maintained that the nucleotide sequence encoding a delta-endotoxin comprises a sequence fragment encoding a domain similar to ricin b.

Preferred embodiments of the present invention, compatible with the foregoing, are those in which the nucleotide sequence encoding a delta-endotoxin has activity against at least one species of hemiptera insect. It is especially preferred that it has activity against at least one species of aphid and, especially, that it has activity against aphid Myzus persicae.

Another aspect of the invention is the "constructs" or "expression cassettes" that enable the gene to be expressed in an expression system or host of interest, as well as the vectors containing said constructs and, which facilitate the introduction of the gene in said systems and / or their expression in them.

Therefore, the invention also relates to an isolated nucleic acid molecule as any of those defined above as part of the present invention, in which the nucleotide sequence encoding the delta-endotoxin of the invention is operably linked to a promoter. . Depending on where it is desired to express the gene (the nucleotide sequence encoding the delta-endotoxin of the invention), preference will be given to different possible promoters, which may be promoters capable of directing the expression of the sequence in a plant cell, in a bacterium (preferably selected from the group Escheríchia coli, Bacillus thuringiensis, Pseudomonas spp.) or in a yeast such as Saccharomyces cerevisiae. Embodiments of this aspect of the invention are those in which the delta-endotoxin coding sequence is operably linked to some additional control sequence, such as a 3 'untranslated region.

Another aspect of the invention are the vectors comprising the delta-endotoxin coding sequence of the present invention, preferably linked to other elements so as to form the "constructs" or "expression cassettes" previously defined. Thus, another aspect of the invention is a vector comprising a nucleotide sequence encoding a delta-endotoxin, characterized in that the nucleotide sequence encoding a delta-endotoxin is represented by SEQ ID NO: 1 or has at least 90 % identity with it, or a nucleic acid molecule in which said nucleotide sequence is comprised, that is, a vector into which the nucleic acid molecule that constitutes the first aspect of the present invention is inserted. The vector is chosen according to the system where the expression is desired or which You want to transform with him. In a preferred embodiment, the vector is a plasmid, which allows obtaining a high number of copies in suitable bacteria transformed with it and, if the promoter is suitable, also allows the expression of the delta-endotoxin of the present invention. in the bacterium, being possible the production and purification of delta-endotoxin from bacterial cultures. Also within the scope of the invention is the embodiment in which the plasmid is a plasmid that can be replicated (and expressed) in yeasts, as is the case of the so-called 2 micron plasmid (2 μ), from Saccharomyces cerívisiae. A preferred embodiment is that in which the plasmid allows the expression of the delta-endotoxin of the present invention in a bacterium chosen from Escherichia coli, Pseudomonas spp., Bacillus megaterium or Bacillus thuríngiensis; In the latter case, there is a special preference for strain Bt 4Q7.

The vector can also be a plant transformation vector, an embodiment compatible, among others, with the alternative that the vector is a plasmid. It is especially preferred that said transformation vector is constructed in such a way that the nucleotide sequence encoding the delta-endotoxin represented by SEQ ID NO: 1 or the coding sequence that has at least 90% identity thereto is operably linked to a promoter capable of directing the expression of said nucleotide sequence in a plant cell, such as the cauliflower mosaic virus promoter (CaMV promoter) or the 4xB2 + A CaMV35S variant; Within this embodiment, it is particularly preferred that the coding sequence is also operatively linked to other control or regulatory sequences that facilitate or enable expression in plants, such as termination sequences or even leader sequences, although the presence of regulatory sequences is equally compatible with any vector, including those of expression in bacteria. With particular preference, the structure of the transformation vector should be such that the entire construction or "expression cassette" (promoter, coding sequence and regulatory or control sequences) can be transferred as such a structural assembly to the plant genome. It is also a preferred embodiment, compatible with all of the above, that in which the delta-endotoxin coding sequence of the present invention is linked to the coding sequence of a second polypeptide, in phase with it, and the entire assembly under the control of the same promoter, so that the expression of the sequence of coding sequences results in a fusion protein. Another aspect of the invention is the host cells comprising at least one vector of the present invention. As previously discussed, among the possible preferred hosts are bacteria cells, especially those of the Escheríchia coli group, Pseudomonas spp., Bacillus megateríum or Bacillus thuringiensis, the latter species in which there is a special preference for the strain Bt 4Q7. The cell can also be a plant cell. It is also possible, among other embodiments, that the cell is a yeast cell, particularly Saccharomyces cerívisiae.

A further aspect of the present invention is transgenic plants comprising cells transformed with vectors of the present invention. Therefore, an aspect of the present invention is a transgenic plant comprising at least one host cell comprising at least one vector of the present invention. By plant, as already mentioned above, a complete plant or any part thereof can be understood, as well as the previous forms in its development that give rise to the completely developed plant, including seedlings and seeds. Because of its importance, a further aspect of the invention may be considered to be a seed comprising a nucleic acid molecule of the present invention, that is, an isolated nucleic acid molecule comprising a nucleotide sequence encoding a delta-endotoxin, characterized in that the nucleotide sequence encoding a delta-endotoxin is represented by SEQ ID NO: 1 or has at least 90% identity with it. Both in the case of plants and in the case of seeds, plants that suffer damage caused by aphids {Myzus persicae in particular) are preferred, or where it has been found that these insects facilitate virus transmission. Thus, among others, there is preference for plants, parts of plants or seeds of peach tree {Prunus pérsica), apricot tree {Prunus armeniaca), plum {Prunus domestica), almond tree {Prunus dulcís), other species of the genus Prunus, citrus, potato , tomato and other species of the Solanaceae family, tobacco, beet, sugar beet, sugar cane, sunflower, celery, mustard, pepper, squash, artichoke, asparagus, beans, broccoli, Brussels sprouts, cabbage, cauliflower, cabbage, cabbage curly, carrot, cucumber, turnip, eggplant, lettuce, parsley, parsnip, pea, alfalfa, pepper, radish, spinach, watercress, watermelon, melon, corn, sorghum, millet, wheat, barley, rye, oats, rice, soybeans, rapeseed, safflower, peanut, sweet potato, yucca, coffee, coconut, pineapple, cocoa, tea, banana, avocado, fig, guava, mango, papaya, olive, cotton, coniferous, azalea, hydrangea, hibiscus, roses, tulips, daffodils, Petunias, carnations, poinsettia, and chrysanthemums.

It is also an aspect of the present invention any polypeptide comprising the sequence of the new delta-endotoxin of the present invention or sequences with a high degree of identity therewith that can be considered delta-endotoxins. Thus, the present invention also relates to a polypeptide comprising an amino acid sequence characterized in that said amino acid sequence: a) is represented by SEQ ID NO: 2;

Any of the sequences that meet the definition of a), b) or c) is considered a protein (delta-endotoxin) of the present invention. It is especially preferred that the protein has three structural domains that correspond to the N, M and C domains of the Cry proteins. Very especially, it is preferred that the amino acid sequence of delta-endotoxin comprises a domain similar to ricin b.

In the case of alternative c), the percentage of identity may be, among others, 100% being option a). It is a possible embodiment that the polypeptide corresponds exactly to that represented by SEQ ID NO: 2.

In any of the options, it is very especially preferred that the polypeptide has insecticidal activity against at least one hemipter, preferably at least one species of aphid, having special preference for having insecticidal activity against the aphid Myzus persicae.

In a possible embodiment, compatible with the foregoing, the delta-endotoxin sequence of the present invention is linked to a second polypeptide, forming a fusion protein. Among the possible examples of interest as a second polypeptide can be mentioned the maltose binding protein, as in example 18 of the application WO2010 / 099365. The delta-endotoxin of the present invention may also be linked to other amino acid sequences of possible interest, such as a 5 'leader sequence or, for example, a histidine tail that facilitates its subsequent isolation.

Additionally, an aspect of the present invention is also a composition comprising a polypeptide of the present invention as defined in the preceding paragraphs, a vector of the present invention or a host cell of the present invention, a composition that is considered a composition. of the present invention. Preferably, the composition also comprises an excipient and / or an agronomically acceptable carrier. The composition may be in various forms, preferably suitable forms so that the composition can be administered as pesticidal compositions, such as powder, granulate, emulsion, colloid, suspension or solution. The suspension or solution may be aqueous or it may be that the liquid vehicle is, for example, an oil or an emulsion. It is preferred that the composition comprises a polypeptide of the present invention or a host cell of the present invention. In this second case, as previously mentioned, preference is given to bacterial or yeast host cells, particularly cells of a species selected from Pseudomonas spp., Bacillus megateríum, Bacillus thuríngiensis Bt 4Q7 or Saccharomyces cerivisae.

In the possible embodiment in which the composition comprises a polypeptide of the present invention, it may be part of a solid, liquid or prepared composition to be administered as an aerosol. The polypeptide may be in crystalline form, so that it will form a suspension in the event that the composition is prepared in an aqueous vehicle, or it may be solubilized in a liquid vehicle, which may be aqueous.

The compositions of the present invention, as previously discussed, may contain other different additional components, including various other pesticides or compounds that increase their action, including Bacillus thuringiensis spores, which may belong to strain Bt 1.5H, to strain Bt 4Q7, to any other natural strain, or to recombinant strains, including recombinant strains comprising the vectors of the present invention.

Since one of the main objectives of the present invention is to combat the Myzus persicae aphid pests, an additional aspect of the present invention is the use of the compositions of the present invention to combat hemiptera pests, especially aphids, and very especially pests. of the aphid Myzus persicae.

Preferably, the use of the present invention is to combat pests that cause damage to plants, plant crops, or to the products of their harvest. It is especially preferred that the plants to be treated are plants that are damaged by Myzus persicae infestations or plants in which said aphid can act as a virus transmitter. As already mentioned, these plants are mainly the peach tree and other fruit trees of the genus Prunus, as well as citrus, crucifixes, solnaceae and even conifers and ornamental plants. Among the species to be treated can be mentioned peach tree, apricot tree, plum, almond tree, other species of the genus Prunus, citrus, potato, tomato and other species of the Solanaceae family, tobacco, beet, sugar beet, sugar cane, sunflower, celery, mustard, pepper, pumpkin, artichoke, asparagus, beans, broccoli, Brussels sprouts, cabbage, cauliflower, cabbage, kale, carrot, cucumber, turnip, eggplant, lettuce, parsley, parsnip, pea, alfalfa, pepper, radish, spinach , watercress, watermelon, melon, corn, sorghum, millet, wheat, barley, rye, oats, rice, soybeans, rape, safflower, peanuts, sweet potatoes, cassava, coffee, coconut, pineapple, cocoa, Tea, banana, avocado, fig, guava, mango, papaya, olive, cotton, conifers, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettia, and chrysanthemums. Special preference is given for fruit trees of the genus Prunus, on the one hand, and for tobacco and potatoes, for the special importance it has in the latter for virus transmission.

The use comprises the administration of the compositions of the invention by any known method, as previously mentioned, appropriate according to the formulation of the composition, such as spraying, administration in irrigation water, direct deposition on plant organs or together at the base of it ...

Another aspect of the invention is a method for the production of a polypeptide of the present invention. The method comprises the steps of:

b) culturing said recombinant organism;

c) inducing, if necessary, the expression of the polypeptide in said recombinant organism;

d) purify the polypeptide from the recombinant organism.

The recombinant organism may be, for example, a transgenic plant of the present invention or a host cell of the present invention or another recombinant microorganism, preferably a bacterial or yeast cell that either comprises an expression vector of the present invention, or either it has integrated into its genome a nucleic acid molecule of the present invention, especially if in said nucleic acid molecule the nucleotide sequence encoding a delta-endotoxin was already operably linked to a promoter that allows its expression in said recombinant microorganism. An example of purification of the polypeptide of the present invention, in this case the delta-endotoxin of SEQ ID NO: 2, is presented in Example 2 of the present invention, in which recombinant Escherichia coli cells are obtained and cultured. with a plasmid expressing said delta-endotoxin, inducing the expression of delta-endotoxin with IPTG and proceeding to purification from the culture, purification that can be carried out by any known means, such as affinity chromatography as in the Example 2 cited. In the event that production and purification occurred from transgenic plants, the polypeptide forms of the present invention comprising fusion proteins or amino acid helper sequences such as histidine tails may be of particular utility for the isolation of the polypeptide from the present invention, an additional step may be necessary in which the auxiliary sequence or the second polypeptide of the fusion protein is cleaved, for example by the use of a suitable endoprotease.

Finally, a further aspect of the present invention is an antibody that specifically binds to a polypeptide of the present invention. Preferably, the antibody specifically binds to the fragment of the polypeptide constituting the delta-endotoxin of the present invention, with particular preference for the embodiment in which the delta-endotoxin sequence is that represented by SEQ ID NO: 2. In a preferred embodiment, the antibody is a monoclonal antibody.

The invention will now be explained in more detail with the help of the Examples and Figures below.

Examples

- Example 1: identification and isolation of the new cry gene

eleven . Identification and isolation of the new gene

The new cry gene was identified in the strain of Bacillus thuringiensis Bt H1 .5, a strain belonging to the Bt collection of the Public University of Navarra, Laboratory of Microbial Bioinsecticides (Iriarte et al., 1998; Iriarte et al., 2000: articles that describe how the sampling was done and the collection was built). The strain Bt H1 .5 specifically was isolated on the island of El Hierro, Canary Islands, Spain.

This strain was grown in LB medium (Luria-Bertani) at 28 ^e C for 16 hours at 200 rpm and its total DNA (genomic plus plasmid) was purified with the Wizard® DNA purification kit (Promega), following the manufacturer's instructions for the isolation of DNA from Gram positive bacteria, which implies the pretreatment of bacterial cells with lysozyme at a final concentration of 10 mg / ml and a 30 minute incubation at 37 ^e C.

The total DNA was used to generate a combined "pooled" library according to the instructions of lllumina, Inc. for the preparation of samples with the TruSeq ™ lepreps truseq / truseqsampleprep / truseq sample prep poolinq quide 15042173 a.pdf) system. It was sequenced using HiSeq ™ 2000 Sequencing System technology (lllumina® Sequencing) in single reading mode, with a reading size of 50 nucleotides (GATC Biotech, Germany).

The sequences obtained were analyzed using Blast (Altschul et al., 1990) with databases constructed with sequences of Bt toxins such as Cry (crystalline), Cyt (cytolytic), Vip1, Vip2 and Vip3 (vegetative insecticidal proteins), Sip ( protein secretable insecticides) already described as well as those produced by the bacterium Bacillus sphaericus such as Mtx 1, Mtx2 and Mtx3 (mosquito toxins) and Bina BinB binaries (Berry, 2012), which were obtained from public databases such as Genbank (Benson et al., 2005). Alternatively, the RAST (Rapid Annotation using Subsystem Technology) automated annotation server (Aziz et al., 2008) was also used, which is a fully automated system to annotate complete or almost complete bacterial and archaea genomes, providing genome annotations. high quality of the entire phylogenetic tree (http://rast.nmpdr.org). In addition to identifying the coding regions or CDSs, it identifies the rRNA (ribosomal RNA) and tRNA (transfer RNA) genes, assigns functions to the genes and uses this information to reconstruct the metabolic network. This server also allows the download of the results in different formats (GenBank, Fasta, GFF, etc.) and navigation in the annotated genome thus facilitating comparative analysis with other annotated genomes.

The new cry gene was identified by using Blast (Blastx) and the ORF (Open Reading Frame) or manually defined coding sequence from the start codon (ATG) to the stop codon (TAA) and with regarding the homologous sequence of reference with greater coverage and percentage of identity. The typical ribosome binding sequence or Shine-Dalgarno sequence was also taken into account, which consists of the following 5'- AAGGAGG-3 'nucleotides, prior to the ATG start codon and separated by 7 nucleotides in sequence 5 '-TTTATGC-3'. This ribosome binding sequence or Shine-Dalgarno sequence has already been identified in Bacillus anthracis, a species that is closely related to Bacillus thuríngiensis (Steichen et al., 2003). The prediction of start codons, ATG in our case, based on the presence of already known Shine-Dalgarno sequences; significantly minimizes the probability of making a mistake in predicting said codon (Starmer et al., 2006), especially in genes such as the present invention, where there are two ATG codons close and separated by nucleotide C (TTTATG CATG).

This ORF, whose sequence is represented by SEQ ID NO: 1, had a total length of 2,397 bp (base pairs), encodes a protein of 799 amino acids, with an approximate molecular weight of 89 kDa, amino acid sequence that is represented by SEQ ID NO: 2.

1 .2. Bioinformatic and comparative analysis of the protein sequence and its coding sequence

The amino acid sequence of the new protein had a maximum Blast (BlastP) identity of 76.56% and 76.20% when compared against other proteins previously described in public databases, both comparing with "nr" sequences (non-redundant protein sequences), and comparing with "pat" sequences (proprietary protein sequences), respectively. Since the proteins with the highest percentage of identity were all Cry proteins or delta-endotoxins, the protein of the present invention is a new Cry protein.

Tables 2 and 3 show the results obtained when performing the BlastP through public use resources accessible for this purpose on the website of the National Center for Biotechnology Information (NCBI) in the USA.

(http: / blast.ncbi.nlm.nih.gov/Blast.ca¡?PROGRAM=blastp&PAGE TYPE = BlastSearch & LI NK LOC ^ blasthome), both by selecting the non-redundant protein database (nr) (Table2), and by selecting the patented protein database (pat) (Table 3). Table 4, for its part, shows the results obtained with the analogous tools available on the website of the EBI (European Bioinformatics Institute: European Bioinformatics Institute) for the ENA (European Nucleotide Archive) consultation (http : // www. eb i. ac. uk / en a '), by querying the nucleotide sequence of SEQ ID NO: 1.

Table 2

List of non-redundant proteins with a higher percentage of identity with respect to the delta-endotoxin of the present invention

a Start point of the comparison in the search sequence (delta-endotoxin of the invention)

b End point of the comparison in the search sequence (delta-endotoxin of the invention)

c Start point of the comparison in the localized sequence

d End point of the comparison in the localized sequence

Table 3

Protein list of patent documents with a higher percentage of identity with respect to the delta-endotoxin of the present invention

c Start point of the comparison in the localized sequence

d End point of the comparison in the localized sequence

Table 4

List of nucleotide sequences with the highest percentage of identity with respect to the delta-endotoxin coding sequence of the present invention

a Start point of the comparison in the search sequence (delta-endotoxin gene of the invention) b End point of the comparison in the search sequence (delta-endotoxin gene of the invention) 0 Start point of comparison in the localized sequence

d End point of the comparison in the localized sequence

The Geneious Pro v6.1 .7 software (Drummond et al., 2014) and its Geneious Alignment tool were used to perform multiple alignment of the CryBMI protein sequence against other known Cry proteins, for which it has been described that they present activity against aphids, including the protein represented by the sequence with identification number 205 in US8318900B2 and in international application WO2010099365 (referred to hereinafter as Seq205- US8318900). The results of said alignment are shown in Fig. 1. This alignment was subsequently used to generate the representative dendrogram of the phylogenetic relationship between the various Cry active proteins against aphids that is represented in Fig. 2 with the Geneious Tree Builder tool and obtain a matrix of% identity of each of them ( Table 5).

Table 5

Similarity percentage between active Cry proteins against aphids, including the Cry protein of the invention (CryBMI)

As can be seen in Fig. 1, the new Cry protein, like the others (except Cry2Aa, Cry1 1Aa and Seq205-US8318900), turned out to be a Cry protein with three domains N, M and C, the domains that are considered responsible for insecticidal activity against different insect species.

The new Cry protein of the present invention (CryBMI), however, has a unique feature represented by a sequence fragment homologous to a conserved domain of ricin (InterPro access number IPR000772) consisting of 148 amino acids located at the carboxyl end between positions 652 to 799 and represented by the sequence TPTPEPVVDGIYQIVTALNNSSVAENGGPTTRGGPDQVKLSPFYNSTDQKWEFIYDSNE DVYTIRNLAGGFLTYFMLNPGYPVLAIRPQWATENQKWIVEPAGNGYYYLRSKSVPTEAA FAPNAADGSVVRSTMSAGSVVRNMSKGDV more specifically, a domain similar to that of the B chain of natural ricin, which is the chain that has activity as lectin (i.e., a protein that binds carbohydrates), as it binds to terminal galactose residues present in cell surfaces The natural ricin that is produced in the seeds of castor plants (Ricinus communis) is a heterodimeric protein, composed of a chain A and a chain B, which turns out to be a highly toxic lectin for a wide variety of organisms (Chougule and Bonning , 2012). Without wishing to limit itself to any specific theory, it is proposed that this ricin domain could be responsible for the high toxicity of the Cry protein of the present invention against M. persicae which is demonstrated in later examples of the present invention. - Example 2: Cloning of the gene and production of the recombinant protein

Once the new cry gene was identified, it was amplified by PCR {Polymerase Chain Reactiorí) using an error-proof Taq polymerase (PrimeSTAR HS DNA polymerase, Takara) with a direct primer that allowed to add a restriction target Λ / col (5 ' -CC / 47GGATGAACCAAAATTATAACAACAATG ~ 3} (SEQ ID NO: 5) immediately before the start codon (ATG) and a reverse primer that allowed to add a Saft restriction target (5'-

G7UG IGTAACTTATTCAGTTTGAATTTTTGATTTG -3 ') (SEQ ID NO: 6), detailed in italics in the sequence. The reverse primer also lacked the stop codon (TAA) allowing the fusion of the recombinant protein to the 6 histidine tail encoded in the vector. The reactions were carried out in a final volume of 25 μΙ with 5 μΙ of 5X reaction buffer, 10 mM dNTPs, 6.3 pmol of each primer, 0.5 U of the PrimeSTAR HS DNA error-proof DNA polymerase polymerase (Takara) and 100 ng of total DNA. Each reaction was carried out under the following conditions: 4 min. of initial denaturation at 94 ^e C, 35 cycles of amplification (denaturation of 1 min. denaturation at 94 ^e C, 1 min. of annealing (mating) at 52 ^e C and 2.5 min. of extension at 72 ^e C) and with a final extension step at 72 ^e C for 10 min.

The amplified fragment was ligated to the pJET vector (Thermo Scientific) following the instructions provided by the manufacturer, separated from the cloning vector by digestion with the restriction enzymes A / col and Sa / I and purified from the agarose gel with the Nucleospin Gel kit and PCR Cleanup (Macherey-Nagel). The purified fragment was subcloned into the expression vector pET-28b (+) (Novagen) (see Fig. 3) predigested with the Restriction restriction enzymes col / col and Sa / I and following routine molecular biology techniques described by Sambrook and Russell (Sambrook and Russell, 2001). This vector has a strong promoter for viral RNA polymerase of Phage T7 and an antibiotic resistance gene Kanamycin to facilitate its selection.

Once the recombinant expression vector, represented in Fig. 3, was obtained, it was used to transform Escheríchia coli BL21 (DE3) cells (Life Technologies), following routine molecular biology techniques described by Sambrook and Russell (Sambrook and Russell , 2001). These cells carry in their genome the gene encoding the viral polymerase RNA of phage T7 inducible by IPTG (isopropyl ^ -D-1-thiogalactopyranoside), which recognizes the promoter encoded in the expression vector pET-28b (+) (Novagen ) and transcribes the cloned gene leading to the production of the protein encoded by such a gene.

The production of the CryBMI recombinant protein was carried out in the obtained Escheríchia coli cells, inducing its expression with 1 mM (final concentration) of IPTG at 37 ^e C and a 200 rpm of agitation for 16 hours.

The purification of the recombinant carrier protein of a 6 histidine tail was carried out by means of affinity chromatography on nickel columns with TED (triscarboxymethyl-ethylene diamine) as chelating ligand, with the Protino Ni-Ted 1000 kit (Macherey -Nagel), following the manufacturer's instructions. The concentration of the purified soluble protein was determined by the Bradford method (Bradford, 1976). The new Cry protein produced in E. coli was obtained in the soluble phase at an approximate concentration of 1 mg / ml from one liter of culture medium. Upon electrophoresis in SDS-PAGE gel an aliquot of the purified sample obtained, a band at a height corresponding to a size of 89 kDa was observed, comparable to the theoretical molecular weight of the recombinant protein sequence and shown in the Fig. 4.

- Example 3: Analysis of insecticidal activity

The analysis of the insecticidal activity was carried out by means of a simplification of the bioassay methodology described by Sadeghi et al. (Sadeghi et al., 2009) using nymphs of M. persicae of first stage (N1) supplied by the insectarium of the research center of the present inventors. Briefly, the soluble protein was diluted in an artificial aphid diet which consisted of 20% P / V sucrose in sterile distilled water. Each dilution was incorporated between two sheets of Parafilm® "M" (Bemis Flexible Packaging, Nena, Wiscosin, USA) and covering small circular boxes 3 cm in diameter (1.5 cm high) without lid. Each box contained 15 nymphs N1. In this way, the nymphs were able to feed through the first membrane of Parafilm® "M" and directly on the mixture of artificial diet + toxin in the treatments or simply of the artificial diet in the case of negative control. Fig. 5 shows photographs of the boxes used, covered with Parafilm® "M" and with the artificial diet drop between two layers of said material.

The bioassays were carried out in a chamber under the following controlled conditions: temperature 25 ° C, 70% rdative humidity and under a 16: 8 hour light / dark photoperiod, respectively. The mortality produced was recorded at 72 hours.

Initially, preliminary tests were performed in two repetitions and two concentrations of 100 and 1000 μg / ml of the Cry protein of the present invention, CryBMI. These trials allowed to determine the approximate concentrations that would produce a mortality of between 5 and 95%. The mean lethal concentration (LC ₅₀ ) was then determined at four concentrations of 20, 25, 32 and 40 μg / ml and at three repetitions for each concentration and the negative control.

The results obtained are shown below in Table 6:

Table 6

Mortality percentages produced by different concentrations of CryBMI when ingested by neonatal nymphs of Myzus persicae

Concentration (μςΛτιΙ) Insects treated Dead insects% mortality

0 120 15 12.5

20 126 20 15.9

25 125 29 23.2

32 1 19 65 54.6

40 120 100 83.3 The CL ₅₀ was estimated from the previous data, using the statistical program Polo PC (LeOra-Software, 1987) based on Finney's probit analysis (1971). The value obtained and the details of the probit analysis on which it is based are shown below in Table 7. Table 7

Average lethal concentration (LC ₅₀ ) estimated for the new CryBMI protein against neonatal nymphs of Myzus persicae

LC ₅₀ Linear regression Goodness of fit

Treatment (Mfl / ml) Pending ± EE ^* to ^* ± EE ^* χ ² gi. ^*

Cry protein 32.7 10.3 + 1, 4 -10.6 + 2.1 0.55 2

* a: intersection with the regression line

EE: standard error

g.l .: degrees of freedom

The LC ₅₀ determined was only 32.7 μg / ml. This value can be considered the lowest LC ₅₀ described to date for a Cry protein of Bt applied to a species of hemiptera plague.

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Claims

one . An isolated nucleic acid molecule comprising a nucleotide sequence encoding a delta-endotoxin, characterized in that the nucleotide sequence encoding a delta-endotoxin is represented by SEQ ID NO: 1 or has at least 90% identity with the same.

2. The nucleic acid molecule according to claim 1, wherein the nucleotide sequence encoding a delta-endotoxin has at least a percentage of identity with the sequence represented by SEQ ID NO: 1 selected from the group of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9 %.

3. The nucleic acid molecule according to claim 1 or 2, wherein the nucleotide sequence encoding a delta-endotoxin comprising a sequence fragment encoding a domain similar to ricin b.

4. Nucleic acid molecule according to any one of the preceding claims, comprising a nucleotide sequence encoding a delta-endotoxin that has activity against at least one hemiptera insect.

5. Nucleic acid molecule according to claim 4, comprising a nucleotide sequence encoding a delta-endotoxin having activity against at least one species of aphid.

6. Nucleic acid molecule according to claim 5, comprising a nucleotide sequence encoding a delta-endotoxin having activity against the aphid Myzus persicae.

7. Nucleic acid molecule according to claim 1, whose nucleotide sequence is represented by SEQ ID NO: 1.

8. The nucleic acid molecule according to any one of claims 1 to 6, wherein the nucleotide sequence encoding a delta-endotoxin is operably linked to a promoter.

9. The nucleic acid molecule according to claim 8, wherein the promoter is selected from the group of a promoter capable of directing the expression of said nucleotide sequence in a plant cell, in a bacterium that is selected from the Escherichia coli group , Bacillus thuringiensis, Pseudomonas spp., Or in a cell of Saccharomyces cerevisiae.

10. Nucleic acid molecule according to claim 8 or 9, which is additionally operably linked to an additional control sequence to the promoter.

1 1. A vector in which a nucleic acid molecule of any one of claims 1 to 10 is inserted.

12. Vector according to claim 1, which is a plasmid.

13. Vector according to claim 12, which is an expression vector that allows expression in a bacterium of the nucleotide sequence encoding a delta-endotoxin represented by SEQ ID NO: 1 or of a coding sequence having at least 90 % identity with it.

14. Vector according to claim 12, wherein the bacterium is selected from the group of Escherichia coli, Pseudomonas spp., Bacillus megaterium or Bacillus thuringiensis.

15. Vector according to claim 1 1 or 12, which is a plant transformation vector.

16. Vector according to claim 15, wherein the nucleotide sequence encoding the delta-endotoxin represented by SEQ ID NO: 1 or the coding sequence having at least 90% identity thereto is operably linked to a promoter capable of directing the expression of said nucleotide sequence in a plant cell.

17. Vector according to any one of the preceding claims, wherein the delta-endotoxin coding sequence represented by SEQ ID NO: 1 or the coding sequence that has at least 90% identity with it is linked in reading phase to the coding sequence of a second polypeptide.

18. A host cell comprising a vector according to any one of claims 1 to 17.

19. Host cell according to claim 18, which is a yeast cell.

20. Host cell according to claim 18, which is a bacterial cell.

21. Host cell according to claim 20, which is a cell of a bacterium that is selected from the group Escherichia coli, Pseudomonas spp., Bacillus megaterium or Bacillus thuringiensis.

22. Host cell according to claim 18, which is a plant cell.

23. A transgenic plant comprising the host cell of claim 22.

24. A transgenic seed comprising a nucleic acid molecule of any one of claims 1 to 9.

25. Transgenic plant according to claim 23 or transgenic seed according to claim 24, which is selected from the group of peach, apricot, plum, almond tree, other species of the genus Prunus, citrus, potato, tomato and other species of the Solanaceae family, tobacco , beet, sugar beet, sugar cane, sunflower, celery, mustard, pepper, squash, artichoke, asparagus, beans, broccoli, Brussels sprouts, cabbage, cauliflower, cabbage, kale, carrot, cucumber, turnip, eggplant, lettuce , parsley, parsnip, pea, alfalfa, pepper, radish, spinach, watercress, watermelon, melon, corn, sorghum, millet, wheat, barley, rye, oatmeal, rice, soybeans, rape, safflower, peanut, sweet potato, cassava, coffee , coconut, pineapple, cocoa, tea, banana, avocado, fig, guava, mango, papaya, olive, cotton, conifers, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettia, and chrysanthemums.

26. A polypeptide comprising an amino acid sequence characterized in that said amino acid sequence:

a) is represented by SEQ ID NO: 2;

b) is the sequence of a delta-endotoxin that is encoded by the sequence represented by SEQ ID NO: 1 or by a sequence that has at least one 90% identity with it, or

27. Polypeptide according to claim 26, wherein the amino acid sequence represented by SEQ ID NO: 2 or the delta-endotoxin defined in sections b) or c) of claim 26 has three structural domains that correspond to the domains N, M and C of Cry proteins.

28. Polypeptide according to claim 26 or 27, comprising an amino acid sequence that is the sequence of a delta-endotoxin that has at least 80% identity with the sequence represented by SEQ ID NO: 2, wherein the percentage Identity is selected from the group of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%.

29. Polypeptide according to claim 26, the sequence of which is represented by SEQ ID NO: 2.

30. Polypeptide according to any one of claims 26 to 29, which exhibits insecticidal activity against at least one hemiptera.

31. Polypeptide according to claim 30, which exhibits insecticidal activity against at least one aphid.

32. Polypeptide according to claim 31, which exhibits insecticidal activity against Myzus persicae.

33. Polypeptide according to any one of claims 26 to 32, wherein the amino acid sequence represented by SEQ ID NO: 2 or corresponding to a delta-endotoxin encoded by a sequence that has at least 90% identity with SEQ ID NO: 1 or corresponding to a delta-endotoxin that has at least 80% identity with the sequence represented by SEQ ID NO: 2, is linked to a second polypeptide, forming a fusion protein.

34. A composition comprising a polypeptide of any one of claims 26 to 33, a vector of any one of claims 1 to 17 and / or a host cell of any one of claims 18 to 22.

35. Composition according to claim 34, further comprising an agronomically acceptable excipient and / or an agronomically acceptable carrier.

36. Composition according to claim 34 or 35, comprising a host cell of any one of claims 19 to 21.

37. Composition according to claim 36, wherein the host cells are selected from the cells of the species Pseudomonas spp., Bacillus megaterium, Bacillus thuringiensis Bt 4Q7 or Saccharomyces cerivisae.

38. Composition according to claim 34 or 35, comprising a polypeptide of any one of claims 26 to 33, in crystalline form or solubilized in a liquid carrier.

39. Composition according to any one of claims 34 to 38, which is in the form of powder, granulate, emulsion, colloid, suspension or solution.

Composition according to any one of claims 34 to 39, which comprises an additional pesticidal compound, a compound that increases the pesticidal action or spores of Bacillus thuringiensis.

41. Use of a composition of any one of claims 34 to 40 to combat pests of at least one hemiptera.

42. Use according to claim 41, wherein the hemiptera is an aphid.

43. Use according to claim 42, wherein the aphid is Myzus persicae.

44. Use according to any one of claims 41 to 43, wherein the pest to be affected affects a plant, a crop of plants, or the products of their harvest.

45. Use according to claim 44, wherein the plant is selected from the group of peach, apricot tree, plum, almond, other species of the genus Prunus, citrus, potato, tomato and other species of the Solanaceae family, tobacco, beet, beet sugar bowl, sugar cane, sunflower, celery, mustard, pepper, squash, artichoke, asparagus, beans, broccoli, Brussels sprouts, cabbage, cauliflower, cabbage, kale, carrot, cucumber, turnip, eggplant, lettuce, parsley, parsnip , pea, alfalfa, pepper, radish, spinach, watercress, watermelon, melon, corn, sorghum, millet, wheat, barley, rye, oats, rice, soybeans, rapeseed, safflower, peanut, sweet potato, yucca, coffee, coconut, pineapple , cocoa, tea, banana, avocado, fig, guava, mango, papaya, olive, cotton, coniferous, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettia, and chrysanthemums.

46. A method for the production of a polypeptide of any one of claims 26 to 33, comprising:

b) culturing said recombinant organism;

d) purify the polypeptide from the recombinant organism.

47. Method according to claim 46, wherein the recombinant organism is a transgenic plant.

48. The method of claim 46, wherein the recombinant organism is a host cell of the present invention, a recombinant bacterium or a recombinant yeast

49. An antibody that specifically binds to a polypeptide of any one of claims 26 to 33.

50. Antibody according to claim 49, which specifically binds to the polypeptide fragment whose sequence:

a) is represented by SEQ ID NO: 2;

51. Antibody according to claim 50, which specifically binds to the sequence represented by SEQ ID NO: 2.

52. Antibody according to any one of claims 49 to 51, which is a monoclonal antibody.