MX2012012370A - Combinations including cry34ab/35ab and cry6aaproteins to prevent development of resistance corn rootworms(diabrotica spp.). - Google Patents

Abstract

The subject invention relates in part to Cry34Ab/35Ab in combination with Cry6Aa. The subject invention relates in part to the surprising discovery that combinations of Cry34Ab/Cry35Ab and Cry6Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm (Diabrotica spp.) population. Included within the subject invention are plants producing these insecticidal Cry proteins, which are useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone. Plants (and acreage planted with such plants) that produce these two insecticidal protein systems are included within the scope of the subject invention. The subject invention also relates in part to combinations of Cry34Ab/35Ab and Cry3Aa proteins "triple stacked" with a Cry6Aa protein. Transgenic plants, including corn, comprising a cry6Aa gene, cry34Ab/35Ab genes, and a cry3Aa gene are included within the scope of the subject invention. Thus, such embodiments target rootworms with three modes of action.

Description

COMBINATIONS THAT INCLUDE PROTEINS CRY34AB / 35AB AND CRY6AA TO PREVENT THE DEVELOPMENT OF RESISTANCE IN CORN ROOT WORMS (DIABROTICA SPP.) BACKGROUND OF THE INVENTION Men grow corn for food and energy applications. Corn is an important crop. It is an important source of food, food products and animal feed in many parts of the world. Insects eat and damage plants and thus undermine the efforts made by man. Billions of dollars are spent every year to control insect pests and additional billions are lost because of the damage they cause.

The damage caused by insect pests is one of the main factors in the loss of global corn crops, despite the use of preventive measures, such as chemical pesticides. Based on this, insect resistance has been genetically engineered in crops such as maize in order to control the damage caused by insects and reduce the need for traditional chemical pesticides.

More than 4046856.42 hectares of corn from the United States are infected with the complex of corn rootworm. The complex of the corn rootworm species includes the rootworm of northern corn (Diabrotica barben), the rootworm of southern corn (D. undecimpunctata howardi), and the rootworm of the West corn (D. virgifera virgifera). (Other species include Diabrotica virgifera zeae (Mexican corn rootworm), Diabrotica balteata (Brazilian corn rootworm), and the Brazilian corn rootworm complex (Diabrotica viridula and Diabrotica speciosa). ) The larvae that inhabit the soil of these Diabrotica species feed on the root of the corn plant, which causes them to remain there. Their permanence finally reduces the yield of corn and generally results in the death of the plant. When feeding on corn beards, adult beetles reduce pollination and, therefore, affect the yield of corn per plant to the detriment. Also, adults and larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetables and field crops in commercial production as well as those grown in the gardens of households.

Synthetic organic chemical insecticides have been primary tools used to control insect pests, but biological insecticides, such as insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect-resistant plants through transformation with Bt insecticide protein genes has revolutionized modern agriculture and increased the importance and value of insecticidal proteins and their genes.

The insecticidal crystal proteins of some strains of Bacillus thuringiensis (B.t.) are well known in the art. See, for example, Hofte et al., Microbial Reviews, Vol. 53, No. 0 2, p. 242-255 (1989). These proteins are usually produced by bacteria such as approximately 130 kDa protoxins that are then cleaved by proteases in the insect's midgut, after ingestion by the insect, to provide a scarce 60 kDa core toxin. These proteins are known as crystal proteins because distinctive crystalline inclusions with spores can be observed in some strains of B.t. These crystalline inclusions are usually composed of several distinctive proteins.

A group of genes that have been used for the production of insect-resistant transgenic crops are the delta-endotoxins of Bacillus thuringiensis (B.t.). Delta endotoxins have been successfully expressed in crop plants such as cotton, potatoes, rice, sunflower, as well as corn, and have been shown to provide excellent control over insect pests. (Perlak, F. J et al. (1990) Bio / Technology 8, 939-943; Perlak, FJ et al. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al. (1993) Bio / Technology 1 1: 1151-1 155; Tu et al. (2000) Nature Biotechnology 18: 1 101-1 104; PCT publication number WO 01/13731; and Bing JW et al. (2000) Efficacy of CryI F Transgenic Maize, 14. Biennial International Plant Resistance to Insects Workshop, Fort Collins, Coló.) Several Bt proteins have been used to create transgenic insect resistant plants that have been successfully registered and marketed to date. These include CryIAb, CryIAc, CryI F, Cry1A.105, Cry2Ab, Cry3Aa, Cry3Bb, and Cry34 / 35Ab in corn, CryIAc and Cry2Ab in cotton, and Cry3A in potato.

Commercial products that express these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins (for example, CryIAb and Cry3Bb in combined corn to provide resistance to lepidoptera and rootworm pests, respectively) is desired. or where the independent action of proteins makes them useful as a tool to delay the development of resistance in susceptible insect populations (for example, CryIAc and Cry2Ab in combined cotton to provide resistance management to the tobacco worm).

Some qualities of transgenic insect-resistant plants that have led to a rapid and widespread adoption of this technology have also caused concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested to preserve the usefulness of Bt-based insect resistance characteristics that include high-dose protein shuffling in combination with shelter, and alternation with, or joint deployment of, different toxins (McGaughey et al. (1998), "Bt Resistance Management," Nature Biotechnol 16: 144-146).

The proteins selected for use in the insect resistance management (MRI) stack must be active so that the resistance developed to a protein does not confer resistance to the second protein (ie, there is no cross-resistance to the proteins). If, for example, a pest population selected for "Protein A" resistance is sensitive to "Protein B", one would conclude that there is no cross-resistance and that a combination of Protein A and Protein B would be effective in delaying the Resistance to Protein A alone.

In the absence of resistant insect populations, assessments can be made based on other characteristics presumed to be related to cross-resistance potential. The usefulness of receptor-mediated binding in the identification of insecticidal proteins that probably do not present cross-resistance has been suggested (van Mellaert et al., 1999). The key predictor is the lack of cross-resistance inherent in this approach is that insecticidal proteins do not compete for receptors in a sensitive insect species.

In the case that two Bt toxins compete for the same receptor, then if that receptor mutates in that insect so that one of the toxins does not bind to that receptor and, therefore, is not more insecticidal to the insect, it can it is also the case that the insect is also resistant to the second toxin (which binds competitively to the same receptor). That is, it is said that the insect is cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.

A relatively new insecticidal protein system was discovered in Bacillus thuringiensis as described in WO 97/40162. This system comprises two proteins - one of approximately 14-15 kDa and the other two of approximately 44-45 kDa. See also United States Patent No. 6,083,499 and 6,127,180. These proteins have now been assigned to their own classes, and therefore they received the Cry designations of Cry34 and Cry35, respectively. See Crickmore et al. website (biols.susx.ac.uk/home/Neil_Crickmore/Bt/). Many other related proteins of this type of system have not been described. See, for example, U.S. Patent No. 6,372,480; WO 01/14417; and WO 00/66742. Optimized plant genes encoding such proteins, where genes are designed to use codons for optimized expression in plants, have also been described. See, for example, U.S. Patent No. 6,218,188.

The exact mode of action of the Cry34 / 35 system has yet to be determined, but it seems to form pores in the membranes of the insect's gut cells. See Moellenbeck et al., Nature Biotechnology, vol. 19, p. 668 Gulio 2001); Masson et al., Biochemistry, 43 (12349-12357) (2004). The exact mechanisms of action are not yet clear despite the 3D atomic coordinates and crystal structures that are known for a protein of Cry34 and a Cry35. See U.S. Patent No. 7,524,810 and 7,309,785. For example, it is not clear if one or both of these proteins bind a typical type of receptor, such as an alkaline phosphatase or an aminopeptidase.

Also, because there are different mechanisms by which an insect can develop resistance to the Cry protein (such as by altered glycosylation of the receptor [see Jurat-Fuentes et al. (2002) 68 AEM 5711-5717], by elimination of the receptor protein [see Lee et al. (1995) 61 AEM 3836-3842], by mutation of the receptor, or other mechanisms [see Heckel et al., J. Inv. Pathol. 95 (2007) 192-197]), it was impossible to predict a priori if there would be cross-resistance between Cry34 / 35 and other Cry proteins. Lefko et al. explains a phenomenon of complex resistance in the rootworm. J. Appl. Entomol 132 (2008) 189-204.

Predicting competitive binding for the Cry34 / 35 system is further complicated by the fact that two proteins are involved in the Cry34 / 35 binary system. Again, it is not clear if these proteins effectively bind the insect / intestine gut and how they do it, and if they interact or bind with each other and how they do it.

Other options for controlling coleoptera include toxins Cry3Bb, Cry3C, Cry6B, ET29, ET33 with ET34, TIC407, TIC435, TIC417, TIC901, TIC1201, ET29 with TIC810, ET70, ET76 with ET80, TIC851, and others. RNAi approaches have also been proposed. See, for example, Baum et al., Nature Biotechnology, vol. 25, No. 11 (Nov. 2007) p. 1322-1326.

Meihls et al. suggests the use of shelters for the management of resistance in the corn rootworm. PNAS (2008) vol. 105, No. 49, 19177-19182.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates in part to Cry34Ab / 35Ab in combination with Cry6Aa. The present invention is related in part to the surprising discovery that Cry34Ab / 35Ab and Cry6Aa are useful for preventing the development of resistance (to any insecticidal protein system alone) by a population of corn rootworm. { Diabrotica spp.). One skilled in the art will recognize with the benefit of the present disclosure, plants that prodthese proteins. Cry insecticides will be useful in mitigating concern that a population of corn rootworm could develop which would be resistant to any of these protein systems alone. .

The present invention is based in part on the discovery that the components of these Cry protein systems do not compete with each other for binding of corn rootworm gut receptors.

The present invention also relates in part to triple piles or "pyramids" of three (or more) toxin systems, with Cry34Ab / 35Ab and Cry6Aa as the base pair. Therefore, the plants (and surface in hectares cultivated with said plants) that prodthese two insecticidal protein systems are included within the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1 B. Binding of 125l-Cry35Ab1 (figure 1A) and 125l-Cry6Aa1 (figure 1B) as a function of input of radiolabeled Cry toxins to BBMV prepared from larvae of the western corn rootworm. Specific union = total union - non-specific union, error bar = SEM (standard error of average).

Figure 2. Union of 125l-Cry35Ab1 to BBMV prepared from worm larvae of western corn root at different concentrations of unlabeled competitor (log 0.1-1.0 log 1 = 0, log 10 = 1.0, log100 = 2.0, log1000 = 3.0).

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1: Cry35Ab1 full-length native protein sequence.

SEQ ID NO: 2: Core protein sequence of Cry35Ab1, truncated chymotrypsin.

SEQ ID NO: 3: Cry34Ab1 full-length native protein sequence.

SEQ ID NO: 4: Sequence of full-length native Cry6Aa1 protein.

DETAILED DESCRIPTION OF THE INVENTION Sequences for the Cry34Ab / 35Ab protein can be obtained from Bacillus thruingiensis isolate PS149B1, for example. For other genes, protein sequences and source isolates for use in accordance with the present invention, see, for example, the Crickmore et al. (lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html).

The present invention includes the use of Cry34Ab / 35Ab insecticidal proteins in combination with a Cry6Aa toxin to protect maize from damage and loss of yield caused by feeding the corn rootworm by populations of corn rootworm that can develop resistance to any of these Cry protein systems alone (without the other).

The present invention also explains a stack of Insect Resistance Management (MRI) to prevent the development of resistance by a corn rootworm to Cry6Aa and / or Cry34Ab / 35Ab.

The present invention provides compositions for controlling corn rootworm pests comprising cells that produce a Cry6Aa toxin protein and a Cry34Ab / 35Ab toxin system.

The invention further comprises a host transformed to produce a Cry6Aa protein and a Cry34Ab / 35Ab binary toxin, wherein said host is a microorganism or plant cell.

It is further sought that the invention provides a method of control of rootworm pests comprising contacting said pests or the environment of said pests with an effective amount of a composition containing a Cry6Aa protein and also containing a binary Cry34Ab / 35Ab toxin.

One embodiment of the invention comprises a corn plant comprising a gene that can be expressed in plants encoding a Cry34Ab / 35Ab binary toxin and a gene that can be expressed in plant that encodes a Cry6Aa protein, and seed of said plant.

Another embodiment of the invention comprises a maize plant wherein the gene that can be expressed in plants encoding a Cry34Ab / 35Ab binary toxin and a gene that can be expressed in plant that encodes a Cry6Aa protein has been introgressed into said corn plant , and seed of said plant.

As described in the Examples, competitive receptor binding studies using radiolabeled Cry35Ab core toxin protein show that the Cry6Aa core toxin protein does not compete for binding in the CRW insect tissue samples to which Cry35Ab binds. See Figure 2. The results indicate that the combination of Cry6Aa and Cry34Ab / 35Ab is an effective means to mitigate the development of resistance in the CRW populations to any single protein system.

Thus, in part based on the data described above and elsewhere herein, the Cry34Ab / 35Ab and Cry6Aa proteins can be used to produce IRM combinations for the prevention or mitigation of resistance development by CRW. For example, other proteins can be added to this combination to expand the insect control spectrum. The present pair / combination can also be used in some preferred "triple piles" or "pyramids" in combination with even another protein to control rootworms, such as Cry3Ba and / or Cry3Aa. Even another option is RNAi against rootworms. See, for example, Baum et al., Nature Biotechnology, vol. 25, No. 1 1 (Nov. 2007) p. 1322-1326. Therefore the present combination provides multiple forms of action against a root worm.

In light of the description of USSN 61 / 327,240 (filed April 23, 2010) relating to the combinations of the Cry34Ab / 35Ab and Cry3Aa proteins, USSN 61 / 476,005 (filed April 15, 201 1) relating to combinations of the Cry34Ab / 35Ab and Cry3Ba proteins, and USSN 61 / 477,447 (filed on April 20, 201 1) relating to combinations of Cry3Aa and Cry6Aa, some preferred "triple piles" of the present invention include a Cry6Aa protein combined with Cry34Ab / 35Ab and Cry3Aa and / or Cry3Ba proteins. Transgenic plants, including corn, comprising a cry6Aa gene, cry34Ab / 35Ab genes and a cry3Aa and / or Cry3Ba gene are included within the scope of the present invention. Therefore, these modalities are directed to the insect with at least three modes of action. Also, based on these data and explanations, one could substitute Cry3Ba or Cry3Aa instead of Cy6Aa exemplified herein with the base combination pair with Cry34A / 35A.

The deployment options of the present invention include the use of Cry6Aa and Cry34Ab / 35Ab proteins in corn growing regions where Diabrotica spp. They are problematic. Another deployment option would be to use one or both of the Cry6Aa and Cry34Ab / 35Ab proteins in combination with other characteristics.

A person skilled in the art will appreciate that Bt toxins, even within a certain class, such as Cry6Aa and Cry34Ab / 35Ab may vary to some extent.

Genes and toxins The term "isolated" refers to a polynucleotide in an unnatural construct, or to a protein in a purified or at least unnatural state. The genes and toxins useful according to the present invention include not only the full-length sequences described but also fragments of the sequences, variants, mutants, and fusion proteins that retain the pesticidal activity characteristic of the toxins specifically exemplified herein. . As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences that encode the same toxins or that encode equivalent toxins that have pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins that have the same or essentially the same biological activity against the target pests as the toxins claimed. The same applies to Cry3's if it is used in triple piles according to the present invention. The domains / subdomains of these proteins can be exchanged to form chimeric proteins. See, for example,. U.S. Patent No. 7,309,785 and 7,524,810 in relation to Cry34 / 35 proteins. The 785 patent also explains truncated Cry35 proteins. Truncated toxins are also exemplified herein.

As used herein, the limits represent approximately 95% (Cry6Aa's and Cry34Ab's and Cry35Ab's), 78% (Cry6A's and Cry34A's and Cry35A's), and 45% (Cry6's and Cry 34's and Cry 35's) of sequence identity, according to " Review of the Nomenciature for the Bacillus thunngiensis Pesticidal Crystal Proteins, "N. Crickmore, DR Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. The same applies to Cry3's if it is used in triple piles, for example, in accordance with the present invention.

It should be apparent to a person skilled in the art that the genes encoding the active toxins can be identified and obtained by various means. The specific genes or gene portions exemplified herein can be obtained from isolates deposited in a culture reservoir. These genes, or portions or variants thereof, can also be constructed synthetically, for example, by the use of a gene synthesizer. Variations of genes can easily be constructed using standard techniques to make point mutations.

Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut nucleotides from the ends of these genes. Genes encoding active fragments can also be obtained using a variety of restriction enzymes. The proteases can be used to directly obtain active fragments of these protein toxins.

Fragments and equivalents that retain the pesticidal activity of the exemplified toxins would be within the scope of the present invention. Also, due to the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences described herein. A person skilled in the art may well create these alternative DNA sequences that encode the same or essentially the same toxins. Variant DNA sequences are within the scope of the present invention. As used herein, reference to "essentially the same" sequence refers to sequences that have insertions, additions, deletions or amino acid substitutions that significantly affect pesticidal activity. The fragments of genes that encode proteins that retain pesticidal activity are also included in this definition.

Another method for identifying the genes encoding the gene and toxin portions useful in accordance with the present invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences can be detected by an appropriate label or can be made inherently fluorescent as described in International Application No. WO93 / 16094. As is well known in the art, if the sounding molecule and nucleic acid sample are hybridized by the formation of a strong bond between two molecules, it can be reasonably assumed that the probe and the sample have substantial homology. Preferably, the hybridization is carried out under stringent conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., p. 169-170. Some examples of salt concentrations and temperature combinations are as follows (in order of greater stringency): 2X SSPE or SSC at room temperature; 1X SSPE or SSC at 42 ° C; 0.1X SSPE or SSC at 42 ° C; 0.1X SSPE or SSC at 65 ° C. Probe detection provides a means to determine in a known manner whether hybridization has occurred. Said probe analysis provides a rapid method for identifying toxin-encoding genes of the present invention. The nucleotide segments that are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the present invention.

Variant toxins.

Certain toxins of the present invention have been specifically exemplified herein. Since these toxins are simply exemplary of the toxins of the present invention, it should be readily apparent that the present invention comprises equivalent variant or toxins (and nucleotide sequences encoding equivalent toxins) that have the same pesticidal or similar activity of the exemplified toxin. . The equivalent toxins will have amino acid homology with an exemplified toxin. The amino acid identity will generally be greater than 75%, or preferably greater than 85%, preferably greater than 90%, preferably greater than 95%, preferably greater than 96%, preferably greater than 97%, preferably greater than 98%, or preferably greater than 99% in some modalities. The amino acid identity will generally be the highest in the critical regions of the toxin that represent the biological activity or are involved in the determination of the three-dimensional configuration that is ultimately responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions that are not critical for activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be classified into the following classes: non-polar, polar non-charged, basic, and acidic. Conservative substitutions by which an amino acid of one kind is replaced with another amino acid of the same type are within the scope of the present invention so long as the substitution does not significantly affect the biological activity of the compound. Table 1 provides a list of examples of amino acids that belong to each class.

TABLE 1 In some cases, non-conservative substitutions can also be made. The critical factor is that these substitutions should not detract significantly from the biological activity of the toxin.

Recombinant guests.

The genes encoding the toxins of the present invention can be introduced into a wide variety of plant or microbial hosts. The expression of the toxin genes results, directly and indirectly, in intracellular production and pesticide maintenance. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the present invention. Other host organisms can also be transformed with one or both of the toxin genes and then used to achieve the synergistic effect. With suitable microbial hosts, for example, Pseudomonas, the microbes they can be applied at the site of the pest, where they will proliferate and will be ingested. The result is the control of the plague. Alternatively, the microbe that inhabits the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the environment of the white plague. The non-regenerable / non-totipotent plant cells of a plant of the present invention (comprising at least one of the present IRM genes) are included in the present invention.

Plant transformation.

A preferred embodiment of the present invention is the transformation of the plants with genes encoding the subject insecticidal protein or its variants. The transformed plants are resistant to attack by a white plague of insects by means of the presence of control amounts of the target insecticidal protein or its variants in the cells of the transformed plant. By incorporating genetic material that encodes the insecticidal properties of the insecticidal toxins of B.t. in a genome of a plant eaten by a particular insect pest, the adult or larvae would die after consuming the food plant. Several members of the monocotyledonous or dicotyledonous classifications have been transformed. Transgenic agronomic crops as well as fruits and vegetables are of commercial interest. Said crops include, by way of illustration, corn, rice, soybean, cañola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. There are several techniques for introducing foreign genetic material into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of microparticulate-coated genetic material directly in the cells (U.S. Patent No. 4945050 and U.S. Patent No. 5,141,131). Plants can be transformed using Agrobacterium technology, see U.S. Patent No. 5177010, U.S. Patent No. 5104310, European Patent Application No. 0131624B1, European Patent Application No. 120516, Patent Application. European Patent No. 159418B1, European Patent Application No. 1761 12, United States Patent No. 5149645, United States Patent No. 5469976, United States Patent No. 5464763, Patent of the United States of America. U.S. Patent No. 4940838, U.S. Patent No. 4693976, European Patent Application No. 116718, European Patent Application No. 290799, European Patent Application No. 320500, European Patent Application No. 604662, European Patent Application No. 627752, European Patent Application No. 0267159, European Patent Application No. 0292435, United States Patent No. 5231019, United States Patent No. 5463174, Patent. of the United States No. 4762785, Patent of United States No. 5004863; and United States Patent No. 5159135. Another transformation technology includes WHISKERS ™ technology, see United States Patent No. 5302523 and United States Patent No. 5464765. Electroporation technology has also been used to transform plants, see WO 87/06614, U.S. Patent No. 5472869, U.S. Patent No. 5384253, WO 9209696, and WO 9321335. All of these publications and patents of transformation are incorporated herein by way of reference. In addition to the numerous technologies for transforming plants, the type of tissue that comes into contact with foreign genes can also vary. Such tissue would include but not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem and the like. Almost all plant tissues can be transformed during dedifferentiation using suitable techniques within the skill in the art.

The genes encoding any of the target toxins can be inserted into plant cells using a variety of techniques that are well known in the art as described above. For example, a large number of cloning vectors comprising a marker that allows the selection of transformed microbial cells and a functional replication system in Escherichia coli are available for the preparation and modification of foreign genes for insertion into higher plants. Such manipulations may include, for example, the insertion of mutations, truncations, additions, or substitutions as desired for the proposed use. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Cry protein or variants can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation of E. coli cells, the cells of which are cultured in a suitable nutrient medium, then extracted and lysed so that workable amounts of the plasmid are recovered. Sequence analysis, restriction fragment analysis, electrophoresis, and other biological biochemical-molecular methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be excised and bound to the next DNA sequence. Each manipulated DNA sequence can be cloned into the same or other plasmids. The use of vectors containing T-DNA for the transformation of plant cells has been investigated intensively and has been described in EP 120516; Lee and Gelvin (2008), Fraley et al. (1986), and An et al. (1985), and is well established in the field.

Once the inserted DNA has been integrated into the genome of the plant, it is relatively stable over subsequent generations. The vector used to transform the plant cell normally contains a selectable marker that encodes a protein that confers on transformed plant cells resistance to a herbicide or an antibiotic, such as bialaphos, kanamycin, G418, bleomycin, or hygromycin, inter alia. The selectable marker gene used individually should therefore allow the selection of transformed cells while the growth of cells not containing the inserted DNA is suppressed by the selective compound.

A large number of techniques are available for insert DNA into a host plant cell. Such techniques include the transformation with T-DNA provided by Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent. Likewise, the fusion of protoplasts of plants with liposomes containing the DNA to be administered, direct injection of DNA, biolistic transformation (bombardment of microparticles), or electroporation, as well as other possible methods, can be used.

In a preferred embodiment of the present invention, the plants will be transformed with genes where the codon usage of the protein encoding the region has been optimized for the plants. See, for example, U.S. Patent No. 5380831, which is incorporated herein by reference. In addition, advantageously, the plates encoding a truncated toxin will be used. The truncated toxin will generally encode approximately 55% to approximately 80% of the full-length toxin. Methods for creating synthetic B.t genes for use in plants are known in the art (Stewart, 2007).

In spite of the transformation technique, the gene is preferably incorporated into a gene transfer vector adapted to express B.t. insecticidal toxin genes and variants in the plant cell by inclusion in the vector of a plant promoter. In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, one can use promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, and the manpina synthase promoter. In some preferred embodiments, promoters other than Bacillus-thuringiensis may be used. Promoters of plant virus origin can be used, for example the 35S and 19S promoters of the Cauliflower Mosaic Virus, a promoter of the Cassava Nerve Mosaic Virus, and the like. Plant promoters include, by way of illustration, ribulose-1, 6-bisphosphate (RUBP) small carboxylase subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat shock promoters, promoter ADF (actin depolymerization factor), ubiquitin promoter, actin promoter, and tissue specific promoters. The promoters may also contain certain enhancer sequence elements that can improve transcription efficiency. Typical enhancers include, by way of illustration, ADH1-intron 1 and ADH1-intron 6. Constitutive promoters can be used. Constitutive promoters direct the expression of continuous gene in almost all types of cells and at almost every moment (for example, actin, ubiquitin, CaMV 35S). Tissue-specific promoters are responsible for the expression of genes in specific cells or tissue types, such as leaves or seeds (eg, zein promoters)., oleosin, napin, ACP (Acyl Carrier Protein)), and these promoters can also be used. Promoters that are active during a certain stage of plant development as well as active in specific plant tissues and organs can also be used. Examples of such promoters include, by way of illustration, promoters that are not specific to the root, specific to the pollen, specific to the embryo, specific to the cotton barbs, specific to the cotton fiber, specific to the endosperm of the seed, specific of phloem, and the like.

In certain circumstances it may be convenient to use an inducible promoter. An inducible promoter is responsible for the expression of genes in response to a specific signal, such as: physical stimulus (for example, heat shock genes); light (eg, RUBP carboxylase); hormone (for example, glucocorticoid); antibiotic (for example, tetracycline); metabolites; and stress (for example, drought). Other convenient translation and transcription elements that function in plants can be used, such as 5 'untranslated leader sequences, RNA transcription termination sequences and poly adenylate addition signal sequences. Various plant-specific gene transfer vectors are known in the art.

Transgenic crops that contain insect resistance (IR) characteristics prevail in corn and cotton plants throughout the United States, and the use of these characteristics expands globally. Commercial transgenic crops that combine characteristics of IR and herbicide tolerance (HT) have been developed by several cereal companies. These include combinations of IR characteristics conferred by B.t. insecticidal proteins. and characteristics of HT, such as tolerance to Acetolactate Syntase (ALS) inhibitors, such as sulfonylureas, imidazolinones, triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase (GS) inhibitors, such as bialaphos, glufosinate, and the like, 4-HydroxyPenylPiruvate inhibitors Dioxygenase (HPPD), such as mesotrione, isoxaflutole, and the like, inhibitors of 5-EnolPiruvilShikimate-3-Phosphate Synthase (EPSPS), such as glyphosate and the like, and acetyl-Coenzyme A Carboxylase (ACCase) inhibitors, such as haloxifop, quizalofop, diclofop , and similar. Other examples are known in which the proteins provided transgenically provide tolerance of the plant to chemical classes of herbicide, such as phenoxy acid gerbicides and auxin pyridyloxyacetate herbicides (see WO 2007/053482 A2), or phenoxy acid herbicides and aryloxyphenoxypropionate herbicides (see WO 2005107437 A2, A3). The ability to control various pest problems through the characteristics of IR is a valuable commercial product concept, and the convenience of this product concept increases its insect control characteristics and weed control characteristics are combined in the same plant. In addition, improved value can be obtained through combinations of solar plants of IR characteristics provided by the Bt insecticidal protein, such as that of the present invention, with one or more additional HT characteristics, such as those mentioned above, plus one or more characteristics additional aggregates (eg, other insect resistance conferred by Bt-derived from other insecticidal proteins, insect resistance conferred by mechanisms, such as RNAi and the like, nematode resistance, disease resistance, stress tolerance, improved nitrogen utilization, and similar), or performance characteristic (eg, high oil content, healthy oil composition, nutritional improvement, and the like). Such combinations can be obtained either through conventional breeding (breeding stack) or collectively as a novel transformation event involving the simultaneous introduction of multiple genes (molecular stack). Benefits include the ability to manage insect pests and improved weed control in a crop plant that provides secondary benefits for the producer and / or consumer. Therefore, the present invention can be used in combination with other features to provide a complete agronomic package of improved culture quality with the ability to control a variety of agronomic issues in a flexible and cost-effective manner.

The transformed cells grow inside the plants in the usual way. They can form germ cells that transmit the transformed characteristics to the progeny plants.

These plants can be grown in the normal way and crossed with plants that have the same hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.

In a preferred embodiment of the present invention, plants will be transformed with genes where codon usage has been optimized for plants. See, for example, U.S. Patent No. 5,380,831.

In addition, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). A non-limiting example of a preferred transformed plant is a fertile maize plant comprising a gene that can be expressed in a plant that encodes a Cry6Aa protein, and further comprises a second set of genes that can be expressed in plants that encode Cry34Ab / proteins. 35Ab.

The transfer (or introgression) of the characteristic (s) determined by Cry6Aa and Cry34Ab / 35Ab in inbred corn lines can be achieved by improving recurrent selection, eg, backcrossing. In this case, a desired recurrent parent is first crossed with an inbred donor (the non-recurrent parent) carrying the appropriate gene or genes for the characteristics determined by Cry. The progeny of this crossover are then backcrossed with the recurrent father after selection in the resulting progeny for the desired characteristics or to be transferred from the nonrecurring parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent father with the selection of the desired characteristics or characteristics, the progeny will be heterozygous for the locus that controls the characteristic (s) to be transferred, but will be like the parent recurrent for most or almost all other genes (see, for example, Poehlman &Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivation Development, Vol. 1: Theory and Technique, 360-376).

Insect Resistance Management Strategies (MRI) Roush et al., For example, describes two toxin strategies, also called "pyramids" or "piles," for the management of transgenic insecticidal crops. (The Royal Society, Phil, Trans, R. Soc., Lond. B. (1998) 353, 1777-1786).

On its website, the United States Environmental Protection Agency (epa.gov/oppbppd1/biopesticides/pips/bt_corn_refuge_2006.htm) publishes the following requirements to provide non-transgenic shelters (that is, not -Bt) (a block of non-Bt / maize crops) for use with transgenic crops that produce a single Bt protein active against white pests.

"The specific structured requirements for the corn products of Bt (CryIAb or CryI F) protected from the corn borer are the following: Structured shelters: 20% shelter of Bt non-Lepidoptera Bt corn in the corn producing area; 50% shelter of Bt no-Lepidoptera Bt in the corn producing area; Blocks Internal (that is, within the Bt field) External (ie, separate fields within ½ mile (1 km) (1/4 mile if possible) of the Bt field to maximize random crossing Sashes within the field The strips must have at least 4 rows wide (preferably 6 rows) to reduce the effects of the movement of larvae " In addition, the National Maize Cultivators Association, on its website: (ncga.com/insect-resistance-management-fact-sheet-bt-corn) also provides similar guidelines regarding shelter requirements. For example: "IRM Requirements to the Corn Borer: - Plant at least 20% of your hectares of corn to shelter hybrids -In cotton producing regions, the refuge must be at least 50%.

-It must be planted within ½ mile (1 km) of shelter hybrids.

- The refuge can be planted as strips inside the Bt field, the refuge strips must be at least 4 rows wide.

- The refuge can be treated with conventional pesticides only if the economic thresholds for the white insect are reached.

- Insecticides that can be sprayed based on Bt can not be used in refuge corn.

-The appropriate shelter should be planted on each farm with corn Bt. " As established by Roush et al. (on pages 1780 and 1784 right column, for example), the piles or pyramids of two different proteins each effective against white pests and with little or no cross-resistance may allow the use of a smaller shelter. Roush suggests that for a successful structure, a refuge size of less than 10% refuge, can provide resistance management comparable to approximately 50% refuge for a single (non-pyramidal) feature. For currently available Bt corn-pyramid products, the Environmental Protection Agency requires that significantly less (usually 5%) of structured non-Bt corn be planted than for single-commodity products (usually 20%).

There are several ways to provide IRM effects for a refuge, including several geometric planting patterns in the fields (as mentioned above) and mixtures of seeds in sacks, as explained in more detail by Roush et al. (supra), and U.S. Patent No. 6,551, 962.

The above percentages, or similar refuge relationships, can be used for the present pyramids or double or triple piles.

All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety insofar as they are not inconsistent with the explicit teachings of the present specification.

The following are examples that illustrate procedures for practicing the invention. These examples should not be interpreted as limiting. All percentages are by weight and all proportions of solvent mixture are by volume unless otherwise indicated. All temperatures are in degrees Celsius.

Unless specifically implied or indicated, the terms "a", "an", "he" mean "at least one" in this.

EXAMPLES EXAMPLE 1 Construction of expression plasmids that encode full length toxins of Crv34Ab1. Crv35Ab1 v Crv6Aa1 Standard cloning methods were used in the construction of Pseudomonas fluorescens expression plasmids. { Pf) designed to produce full-length proteins Cry34Ab1, Cry35Ab1 and Cry6Aa1, respectively. Restriction endonucleases from New England BioLabs (NEB; Ipswich, MA) were used for DNA digestion and Invitrogen T4 DNA ligase was used for DNA ligation. The plasmid preparations were made by the Plasmid Mini kit (Qiagen, Valencia, CA), according to the supplier's instructions. DNA fragments were purified by Millipore Ultrafree®-DA cartridge (Billerica, MA) after electrophoresis in Tris-acetate agarose gel. The basic cloning strategy involved the subcloning of the coding sequences (CDS) of the full length Cry proteins in pMYC1803 for Cry34Ab1 and Cry35Ab1 in pDOW1169 for Cry6Aa1 in the restriction sites Spel and Xhol (or Sali which is compatible with Xhol) respectively , by which they were placed under the control of expression of the Ptac promoter and the terminator of rrnBT1T2 or rrnBT2 of the plasmid pKK223-3 (PL Pharmacia, Milwaukee, Wl), respectively. pMYC1803 is a plasmid of average copy number with the origin of replication RSF1010, a gene resistant to tetracycline, and a ribosome binding site in a manner preceding the recognition sites of enzymes in which fragments of DNA containing Protein coding regions (U.S. Patent Application No. 20080193974).

The expression plasmids for Cry34Ab1 and Cry35Ab1 were transformed by electroporation into a P. fluorescens MB214 strain, recovered in SOC-Soy hydrolyzate medium, and plated in lysogenic broth (LB) medium with 20 pg / ml tetracycline. . The expression vector pDOW1 169 is similar to pMYC1803 but pDOW1169 carries the pyrF gene encoding uracil, which is used as a marker to identify transformants when an auxotrophic strain of P. fluorescens uracil (as DPf10) is used for transformation into a middle plate M9 minimum that did not contain uracil (Schneider et al., 2005). Details about microbiological manipulations are available in U.S. Patent Application No. 20060008877, U.S. Patent Application No. 20080193974, and U.S. Patent Application No. 20080058262, which they are included here as a reference. The colonies were further checked by restriction digestion of the miniprep plasmid DNA. The plasmid DNA of the selected clones containing grafts was sequenced by contracting with a commercial sequencing vendor such as eurofins MWG Operon (Huntsville, AL). Sequence data was collected and analyzed with the Sequencher ™ software (Gene Codes Corp., Ann Arbor, MI).

EXAMPLE 2 Growth and expression Analyzes of growth and expression in shake flask production of Cry34Ab1, Cry35Ab1 and Cry6Aa1 toxins for characterization with the inclusion of Bt receptor binding and insect bioassay were obtained by P. fluorescens strains grown in shake flasks harboring expression constructs (e.g., clone pMYC2593 for Cry34Ab1, pMYC3122 for Cry35Ab1, and pDAB102018 for Cry6Aa1). Seed culture for Cry34Ab1 and Cry35Ab1 grown in P. fluorescens medium overnight, which was supplemented with 20 pg / ml tetracycline was used to inoculate 200 mL of the same medium with 20 Mg / ml tetracycline. However, seed culture for Cry6Aa1 was cultured in minimal M9 broth overnight and used to inoculate 200 mL of P. fluorescens medium without antibiotic. Expressions of the Cry34Ab1, Cry35Ab1 and Cry6Aa1 toxins by the Rae promoter were included by the addition of isopropyl-D-1-thiogalactopyranoside (IPTG) after an initial 24-hour incubation at 28-30 ° C with 300 shaking. rpm. Samples of the cultures were taken at the time of induction and at various times after the induction. The density of the cells was measured by optical density at 600 nm (OD600) - EXAMPLE 3 Fractionation of cells and SDS-PAGE analysis of the samples of the shake flasks At each time a sample was taken, the cell density was adjusted to OD600 = 20 and aliquots of 1-mL were centrifuged at 14,000 x g for five minutes. The cell pellets were frozen at -80 ° C. Soluble and insoluble fractions were generated from the cell pellet samples by using EasyLyse ™ Bacterial Protein Extraction Solution (EPICENTRE® Biotechnologies, Madison, Wl). Each cell pellet was resuspended in 1 mL of EasyLyse ™ solution and further diluted 1: 4 in lysis pH buffer and incubated with shaking room temperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at 4 ° C and the supernatant was recovered as the soluble fraction. The pellet (the insoluble fraction) was then resuspended in an equal volume of phosphate pH regulator saline solution (PBS, 1.9 mM Na2HP04, 137 mM NaCl, 2.7 mM KCl, pH7.4). Samples were mixed at 3: 1 with 4X Laemmli of sample pH regulator with β-mercaptoethanol and boiled for 5 minutes prior to loading on NuxAGE Novex 4-20% Bis-Tris gels (Invitrogen, Carlsbad, CA ). Electrophoresis was performed in the recommended NuPAGE MOPS pH regulator. The gels were stained with the SirnplyBlue ™ Safe Stain according to the manufacturer's protocol (Invitrogen) and visualized by the Typhoon imaging system (GE Healthcare Life Sciences, Pittsburgh, PA).

EXAMPLE 4 Preparation of the inclusion body The inclusion body preparations of the Cry protein (IB) were made from the fermentations of P. fluorescens that produced the insecticidal protein of B.t. not soluble, as demonstrated by SDS-PAGE and MALDI-MS (Desorption / Laser-assisted Ionization of Matrix / Mass Spectrometry). The P. fluorescens cell pellets created 48 hours after induction were thawed in a 37 ° C water bath. Cells were resuspended at 25% w / v in lysis pH buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mM Dithiothreitol (DTT), 5 mL / L of bacterial protease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis, MO) was added just before use only for Cry34Ab1 and Cry35Ab1 .The cells were resuspended by a homogenizer in the lower setting (Tissue Tearor, BioSpec Products, Inc., Bartlesville, OK) Twenty-five mg of lysozyme (Sigma L7651, white chicken egg) was added to the cell suspension by mixing with a metal spatula, and the suspension was incubated at room temperature for one hour, the suspension was chilled on ice for 15 minutes, then subjected to sonication by a Branson Sonifier 250 (two-minute sessions, 50% duty cycle, 30% production). Cell lysis was controlled by micro Another 25 mg of lysozyme was added if necessary, and incubation and sonication were repeated. When cell lysis was confirmed by microscopy, the lysate was centrifuged at 11,500 x g for 25 minutes (4 ° C) to form the IB pellet, and the supernatant discarded. The IB pellet was resuspended with 100 mL of lysis pH regulator, homogenized with the hand mixer and centrifuged as previously performed. The IB pellet was washed repeatedly by resuspension (in 50 mL of pH regulator for lysis), homogenization, sonication, and centrifugation until the supernatant became colorless and the IB pellet became firm and whitish in color. For final washing, the IB pellet was resuspended in filtered sterile distilled water (0.22 pm) with 2 mM EDTA, and centrifuged. The final pellet was resuspended in sterile distilled water filtered with 2 mM EDTA, and stored in 1 mL of aliquots at -80 ° C.

EXAMPLE 5 SDS-PAGE analysis and quantification The SDS-PAGE analysis and the protein quantification in the IB preparations were carried out by thawing 1 mL of the aliquot of the IB pellet and the 1:20 dilution with filtered sterile distilled water. The diluted sample was then boiled with 4X of reducing pH regulator [250 mM Tris, pH6.8, 40% glycerol (v / v), 0.4% Bromophenol Blue (w / v), 8% SDS (p / v) v) and 8% β-Mercapto-ethanol (v / v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12 + 2 well gel (Invitrogen) processed with 1X Tris / Glycine / pH regulator SDS (Invitrogen). The gel was processed for approximately 60 min at 200 volts, then stained and discolored following the SirnplyBlue ™ Safe Stain procedures (Invitrogen). The quantification of the target bands was performed by comparing the densitometric values for the bands with the Bovine Serum Albumin (BSA) samples processed in the same gel to produce a standard curve by using the Bio-Rad Quantity One software.

EXAMPLE 6 Solubilization of inclusion bodies Ten mL of inclusion bodies suspensions of P. fluorescens clones R1253, MR1636 and PDF 13032 (with 50-70 mg / mL of Cry34Ab1, Cry35Ab1 and Cry6Aa1 proteins respectively) were centrifuged in the highest configuration of an Eppendorf centrifuge model 5415C (approximately 14,000 xg) to granulate the inclusions. The supernatant of the storage pH regulator was removed and replaced with 25 mL of 100 mM sodium acetate pH buffer, pH 3.0, for Cry34Ab1 and Cry35Ab1, and 50 mM CAPS pH regulator [3- (cyclohexamino) 1-propanesulfonic acid], pH10.5, for Cry6Aa1, in a 50 mL conical tube respectively. The inclusions were resuspended by a pipette and shaken to mix thoroughly. The tubes were placed on a platform that gently rocked at 4 ° C overnight to extract the full-length Cry34Ab1, Cry35Ab1 and Cry6Aa1 proteins. The extracts were centrifuged at 30,000 x g for 30 min at 4 ° C, and the resulting supernatants containing the full-length solubilized Cry proteins were stored.

EXAMPLE 7 Truncation of full-length protoxin The full-length Cry35Ab1 was truncated or digested with chymotrypsin to obtain its chymotrypsin nucleus which is an active form of the Cry protein. Specifically, the full length solubilized Cry35Ab1 was incubated with chymotrypsin (bovine pancreas) (Sigma, St. MO) at (50: 1 = Cry protein: enzyme, w / w) in 100 mM sodium acetate pH buffer, pH3 .0, at 4 ° C with gentle shaking for 2-3 days. Full activation or truncation was confirmed by SDS-PAGE analysis. The molecular mass of the full-length Cry35Ab1 was "44 kDa, and the chymotrypsin nucleus was" 40 kDa, respectively. The full length and chymotrypsin nucleic acid sequences are provided as SEQ ID NO: 1 and SEQ ID NO: 2. Neither the chymotrypsin nor the trypsin nuclei are available for Cry34Ab1, and also CryAal is significantly more active for Attack worms from the root of insects that the chymotrypsin or trypsin nucleus. Therefore, the full-length Cy34Ab1 and Cry6Aa were used for the binding assays. The amino acid sequences of full length Cy34Ab1 and Cry6Aa are provided as SEQ ID NO: 3 and SEQ ID NO: 4.

EXAMPLE 8 Purification of Crv toxins The chymotrypsinised Cry35Ab1 and full length Cry6Aa1 were purified using an ion exchange chromatography system. Specifically, the digestion reaction of Cry35Ab1 was centrifuged at 30,000 x g for 30 min at 4 ° C and then continued with the pass through a 0.22 μ? T filter. to remove lipids and all other particles, and the resulting solution was concentrated 5 times using an Amicon Ultra-15 regenerated cellulose centrifugation device (10,000 Molecular Weight Limit; Millipore). The sample pH regulators were then changed to 20 mM sodium acetate pH buffer, pH 3.5, for Cry34Ab1 and Cry35Ab1 using disposable PD-10 columns (GE Healthcare, Piscataway, NJ) or dialysis. Then they were further purified using ATKA Explorer (Amersham Biosciences). For Cry35Ab1, the pH A regulator was 20 mM sodium acetate pH regulator, pH 3.5, and the pH B regulator was pH A + 1 M NaCl regulator, pH 3.5, while passing Cry6Aa1 the pH regulator A was 50mM CAPS, pH 10.5 and buffer B was 50 mM CAPS, pH 10.5, 1 M NaCl. A HiTrap SP column (5 ml) (GE) was used for truncated Cry35Ab1. After the column was completely equilibrated using the pH A regulator, the Cry35Ab1 solution was injected into the column at a flow rate of 5 ml / min. The elution was carried out using the gradient 0-100% of the pH regulator B at 5 ml / min with 1 ml / fraction.

For full-length Cry6Aa1, a Capto Q column, 5 ml (5 ml) (GE) was used and all other procedures were similar to those applied for Cry35Ab1. After the SDS-PAGE analysis of the fractions selected for selection plus fractions containing the best quality white protein, said fractions were grouped. The pH regulator was changed to the purified chymotrypsin nucleus of Cry35Ab1 with 20 mM Bist-Tris, pH 6.0, as described above. For purified Cry6Aa1, the salt was removed through dialysis against 10mM CAPS, pH 10.5. Samples were removed at 4 ° C for a subsequent binding assay after quantification using SDS-PAGE analysis and Typhoon imaging system (GE) with BSA as a standard. Cry34Ab1 was already pure after being solubilized in the acidic pH regulator as determined by the SDS-PAGE analysis, and therefore without further purification.

EXAMPLE 9 Preparations of BBMV Brush border membrane vesicle (BBMV) preparations of insect midgut have been widely used for Cry toxin receptor binding assays. The BBMV preparations used in the present invention were prepared from isolated medium intestines of third instar insects of western maize root (Diabrotica virgifera virgifera LeConte) by the method described by Wolferberger et al. (1987). Leucine aminopeptidase was used as a marker of membrane proteins in the preparation. , and the leucine aminopeptidase activities of the crude homogenate and the preparation of BBMV were determined as previously described. (Li et al., 2004a). The protein concentration of the BBMV preparation was measured by the Bradford method (1976).

EXAMPLE 10 Marked 125l Purified full length Cry34Ab1, chymotryped Cry35Ab1 chimeric and full length Cry6Aa1 were labeled by 125l for saturation and competitive binding assays. In order to ensure that the radio-labeling does not cancel the biological activity of the Cry toxins, cold iodination was carried out by Nal and according to the instructions of Pierce® lodination Beads (Pierce Biotechnology, Thermo Scientific, Rockford IL) . The bioassay results indicated that both the full-length Cry35Ab1 and Cry6Aa1 nuclei remained active against the western corn root larva, but the inactive iodination Cry34Ab1. As expected, 125l-Cry34Ab1 did not specifically bind to insect BBMV, and therefore Cry34Ab1 requires another method of labeling to assess the binding of the membrane receptor. 25l-Cry35Ab1 and 25l-Cry6Aa1 were obtained with Pierce® lodination Beads (Pierce) and Na125l. Zeba ™ Desalt Spin Columns (Pierce) were used to extract unbound or free Na125l from the iodinated protein. The specific radioactivities of the iodinated Cry proteins were from 1-5 uCi / ug. Multiple batches of labeling and binding assays were performed.

EXAMPLE 11 Saturation binding assays Saturation binding assays were performed using 125-well labeled Cry toxins as previously described (Li et al., 2004b). In order to determine the specific binding and estimate the binding affinity (dissociation constant, Kd) and the concentration of the binding site (the maximum amount of toxin specifically bound to a certain amount of BBMV, Bmax) of Cry35Ab1 and Cry6Aa1 to the BB V of the insect, a series of increasing concentrations of 125l-Cry35Ab1 or 125l-Cry35Ab1 were incubated with a determined concentration (0.1 mg / ml) of the BBMV of the insect respectively, in 150 ul of 20 mM Bis-Tris, pH 6.0, 150 mM KCI, supplemented with 0.1% BSA at room temperature for 60 min with gentle shaking. The toxin bound to BBMV was separated from the free toxins in the suspension by centrifugation at 20,000 x g at room temperature for 8 min. The pellet was washed twice with 900 ul of the same pH regulator frozen with 0.1% BSA. The radioactivity remaining in the pellet was measured with a COBRAII Auto-Gamma counter (Packard, a Canberra) and considered the total union.

Another series of binding reactions was established side-by-side, and a 500-1,000-fold excess of the corresponding unlabeled toxin was included in each of the binding reactions to fully occupy all specific binding sites in the BBMV, which was used to determine the non-specific binding. The specific binding was estimated by subtracting the non-specific binding of the total binding. The Kd and Bmax values of these toxins were estimated by using the number of molecules of the toxin (pmol) specifically bound to the BBMV protein per microgram against the concentrations of the labeled toxin used by the operation of GraphPad Prism 5.01 (GraphPad Software , San Diego, CA). The graphs were made with the Microsoft Excel or GraphPad Prism programs. The experiments were reproduced at least three times. These binding experiments demonstrated that both 125l-Cry35Ab1 and 125l-Cry6Aa1 were able to bind in a specific manner to BBMV (Figures 1A and 1B). 125l-Cry35Ab1 and 125l-Cry6Aa1 had a binding affinity of Kd = 11.66 ± 11.35, 7.99 ± 4.89 (nM), respectively and a binding site concentration of Bmax = 5.19 ± 3.02 pmol / mg, 2.71 ± 0.90 (pmol, / mg BBMV), respectively.

EXAMPLE 12 Competitiveness union tests Additional competitive binding assays were performed to determine whether Cry35Ab1 and Cry6Aa1 share the same group of receptors. For the competitive binding assays homologues of Cry35Ab1, increasing amounts were first mixed (0-5, 000 nM) of unlabeled Cry35Ab1 with Cry35Ab1 labeled with 5 nM, and then incubated with a certain concentration (0.1 mg / ml) of BBMV at room temperature for 60 min, respectively. The percentages of 125l-Cry35Ab1 bound with BBMV were determined for each of the reactions compared to the initial specific binding in the absence of an unlabeled competitor. Heterologous competitive binding assays were performed between 125l-Cry35Ab1 and unlabeled Cry6Aa1 to identify whether they share the same receptor group (s). This was obtained by increasing unlabeled Cry6Aa1 as a competitor introduced in the reactions to compete for the putative receptor (s) in the BBMV with the labeled Cry35Ab1. The experiment was reproduced at least three times.

The results of the experiment showed that Cry35Ab1 was able to displace approximately 50% when the molar concentration increased to approximately 100 nM (a 20-fold excess compared to 5 nM 125l-Cry35Ab1). The remaining 50% was considered as a non-specific binding that was not able to move based on the saturation binding result described elsewhere and the definition of non-specific binding, and therefore, the non-specific binding was subtracted and not showed here This suggests that the specific binding was completely displaced by Cry35Ab1 unlabeled with a 20-fold excess (Figure 2). However, Cry6Aa1 does not was able to displace 125l-Cry35Ab1. These data indicate that Cry35Ab1 does not share a receptor with Cry6Aa1. Whether Cry34Ab1 and Cry6Aa1 share a receptor or not is pending testing using Cry34Ab1 to compete for binding with radiolabelled Cry6Aa or unlabeled Cry6Aa1 to compete for binding with labeled Cry34Ab1 with another tagging method.

References Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein uting the principle of protein-dye binding, Anal. Biochem. 72, 248-254.

Li, H., Oppert, B., Higgins, R.A., Huang, F., Zhu, K.Y., Buschman, L.L., 2004a. Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and-susceptible Ostrínia nubilalis (Lepidoptera: Crambidae). Insect Biochem. Mol. Biol. 34, 753-762.

Li, H., Oppert, B., Gonzalez-Cabrera, J., Ferre, J., Higgins, R.A., Buschman, L.L. and Zhu, K.Y. and Huang, F. 2004b. Binding analysis of CryIAb and CryIAc with membrane vesicles from Bacillus thuringiensis-res stan \ and-susceptible Ostrínia nubilalis (Lepidoptera: Crambidae). Biochem. Biophys. Res. Commun. 323, 52-57.

Schneider, J.C. Jenings AF, Mun DM, McGovern PM, Chew LC. 2005. Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation. Biotechnology Progress 21, 343-348.

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Claims (29)

NOVELTY OF THE INVENTION CLAIMS

1. - A transgenic plant that produces a Cry34 protein, a Cry35 protein, and a Cry6A insecticidal protein.

2. - The transgenic plant according to claim 1, further characterized in that said plant also produces a fourth insecticidal protein selected from the group consisting of Cry3B, and Cry3A.

3. - The seed of a plant according to any of claims 1-2, wherein said seed comprises said DNA.

4. - A field of plants comprising a plurality of plants according to any of claims 1-2.

5. - The field of plants according to claim 4, further characterized in that said field also comprises non-Bt refuge plants, wherein said refuge plants comprise less than 40% of all the crop plants in said field.

6. - The field of plants according to claim 5, further characterized in that said refuge plants comprise less than 30% of all the crop plants in said field.

7. - The field of plants according to claim 5, further characterized in that said refuge plants comprise less than 20% of all the crop plants in said field.

8. - The field of plants according to claim 5, further characterized in that said refuge plants comprise less than 10% of all the crop plants in said field.

9. - The field of plants according to claim 5, further characterized in that said refuge plants comprise less than 5% of all the crop plants in said field.

10. - The field of plants according to claim 4, further characterized in that said field lacks refuge plants.

11. - The plant field according to claim 5, further characterized in that said refuge plants are in blocks or strips.

12. A seed mixture comprising non-Bt refuge plant refuge seeds, and a plurality of seeds of claim 3, wherein said refuge seeds comprise less than 40% of all seeds in the mixture.

13. - The seed mixture according to claim 12, further characterized in that said refuge seeds comprise less than 30% of all the seeds in the mixture.

14. - The seed mixture according to claim 12, further characterized in that said refuge seeds comprise less than 20% of all the seeds in the mixture.

15. - The seed mixture according to claim 12, further characterized in that said refuge seeds comprise less than 10% of all the seeds in the mixture.

16. - The seed mixture according to claim 12, further characterized in that said refuge seeds comprise less than 5% of all the seeds in the mixture.

17. - A container or bag of seeds comprising a plurality of seeds of claim 3, said container or bag has zero seeds of shelter.

18. - A method of managing the development of resistance to a Cry protein by an insect, said method comprising planting seeds to produce a field of plants of claim 5 or 10.

19. - The field according to any of claims 5-11, further characterized because said plants occupy more than 10 acres (4.04 hectares).

20. The plant according to any of claims 1-2, further characterized in that said plant is a corn plant.

21. - A plant cell of any one of claims 1-2, wherein said Cry35 protein is at least 95% identical with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, said protein Cry6A insecticide is at least 95% identical with SEQ ID NO: 4, and said Cry34 protein is at least 95% identical with SEQ ID NO: 3.

22. - The plant in accordance with any of the claims 1-2, further characterized in that said Cry35 protein comprises a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, said insecticidal protein Cry6A comprises SEQ ID NO: 4, and said Cry34 protein comprises SEQ ID NO: 3

23. - A method of production of the plant cell of claim 21.

24. A method of controlling the rootworm insect by contacting said insect with a Cry34 protein, a Cry35 protein, and a Cry6A insecticidal protein.

25. - The plant according to claim 1, further characterized in that said Cry34 protein is a Cry34A protein, said Cry35 protein is a Cry35A protein, and said Cry6A protein is a Cry6Aa protein.

26. - The plant according to claim 1, further characterized in that said Cry34 protein is a Cry34Aa protein and said Cry35 protein is a Cry35Aa protein.

27. - The plant according to claim 2, further characterized in that said Cry3A protein is a Cry3Aa protein and said Cry3B protein is a Cry3Ba protein.

28. - The method according to claim 24, further characterized in that said Cry34 protein is a Cry34A protein, said Cry35 protein is a Cry35A protein, and said Cry6A protein is a Cry6Aa protein.

29. The method according to claim 24, further characterized in that said Cry34 protein is a Cry34Aa protein and said Cry35 protein is a Cry35Aa protein.