WO2011009182A2

WO2011009182A2 - Isolated nucleic acid molecule, genetic construct, vector, transgenic cell, method for producing a transgenic cell and plant, isolated and purified polypeptide, biodegradable pesticide composition, pest control method, method for producing transgenic strains resistant to insect pests

Info

Publication number: WO2011009182A2
Application number: PCT/BR2010/000242
Authority: WO
Inventors: Maria Fátima GROSSI DE SA; Gustavo Ramos De Oliveira; Maria Cristina Mattar Da Silva; Thales Lima Rocha; QUEZADO DE MAGALHÃES Mariana TORQUATO
Original assignee: Embrapa - Empresa Brasileira De Pesquisa Agropecuária
Priority date: 2009-07-24
Filing date: 2010-07-26
Publication date: 2011-01-27
Also published as: WO2011009182A3

Abstract

The present invention pertains to an insecticide compound derived from a strain of Bacillus thuringiensis and relates to the field of plant pest control, particularly to the control of the boll weevil - Anthonomus grandis. More specifically, the subject matter of the invention relates to a new gene for a new delta-endotoxin designated Cry8Ha and to the cloning and expression of the gene that encodes for the protein Cry8Ha in Escherichia coli. The nucleotide and protein sequence that encodes the new delta-endotoxin, recombinant vectors and host cells are disclosed. Also disclosed are processes and means for the recombinant production and use of the new delta-endotoxin for controlling the boll weevil. Additionally, the invention also provides an optimized synthetic gene for expression in cotton plants. Using the gene described herein, it is possible to transform plants using techniques known by specialists in the field for the expression of the endotoxin active against the boll weevil.

Description

Report of the Invention Patent for "INSULATED NUCLEIC ACID MOLECULE, GENETIC CONSTRUCTION, VECTOR, TRANSGENIC CELL, METHOD FOR OBTAINING A TRANSGENIC CELL AND PLANT-FLEXED POLYDIOID PULUS, SULIFIDATED POLYPEID, A PRAGUE, METHOD FOR OBTAINING TRANSGENIC LINES RESISTANT TO A PRAGUE INSECT ".

FIELD OF INVENTION

The following patent specification report relates to the field of control of insect pests that attack agricultural crops using methods and compositions comprising δ-endotoxins derived from the Bacillus thuringiensis microorganism.

BACKGROUND OF THE INVENTION

Among all domestic and cultivated plants, cotton is one of the most attacked by diseases and insect pests, besides having a high sensitivity to the occurrence imposed by weeds (Beltrão, EM, Souza, JG. Cotton agribusiness in Brazil. Embrapa: Brasilia, v.01, 1999). Among the main pest insects, we highlight the cotton boll weevil, Anthonomus grandis (Boheman, CH Description of new species. In Schoenherr, Genera and species Curculionidum cum synonymia hujus Familiae, vol. 7, pt. 2. Paris: Roret 461 pp. 1843), considered one of the most serious pests of cotton cultivation, found in Mexico, Cuba, Haiti, Venezuela, Colombia, Paraguay and Brazil. This insect uses the flower buds and fruits of its host as food source and development site, causing direct damages to the commercialization of cotton fiber. Infestation levels increase rapidly and losses can reach up to 100% of production if control measures are not adequate. This insect represents a great potential for damage, being considered a key pest in the planning and control of insects harmful to the crop, mainly due to the difficulty of control by chemical insecticides.

Cotton and its pests coexist for a long time and Volutive. Plant and insect form an interdependent and competitive morphological and biochemical system, most often resulting in the insect's use of part of the plant. This part used represents the damage caused by the insect to the plant, and depends on the size of the pest population, and the ability of the plant to resist the attack and to recover from the damage suffered (Beltrão, EM, Souza, JG. Embrapa: Brasilia, v.01, 1999).

The plant versus insect interaction can be visualized in at least two ways: from the insect's point of view, with the plant varying from adequate to completely unsuitable as a host and, on the other hand, from the plant's point of view where the smaller the number of species and abundance of insects associated with it, and the lower the effect these insects have on them, the greater their resistance to these insects (Santos, WJ Identification, biology, sampling and control of cotton pests. In: Embrapa Agropecuária Oeste Embrapa Cotton, Cotton: Production Technology, pp. 296p.

Between the extremes of plant resistance to insect pests, there is a complete and extensive arsenal of insect attack and counterattack mechanisms, ranging from simple morphological impediment to complex phytochemical components, which directly affect the metabolic process involved in the use of the plant as host of the insect. In practical terms, cotton resistance to pest insects represents the ability of certain cultivars to produce better quality cotton in greater quantities than other cultivars under the same pest insect population (Freire, EC Cultivars and seed production). improving cotton quality in northeastern and central-western Brazil (Embrapa / CNPA Newsletter 1997).

In most countries where cotton is grown, pest vulnerability represents the main problem of this crop. Without more effective control alternatives, farmers routinely continue to believe that chemical insecticides are the only form of crop protection. Although efficient, they are costly, potentially harmful to man, the environment and, in the long term, cause the triggering of resistance processes, pest resurgence and reduction in the incidence of natural enemies (Panizzi, AR Effect of insecticides in the population of the main soybean pests. An. Soe. Entomol. Brazil, v.6, p.264-275. 1977). Therefore, the present invention aims to increase plant resistance by generating transgenic plants capable of expressing genes of high entomotoxic activity, thus solving the problem of chemical insecticide abuse.

The stable introduction of exogenous genes in cotton plants to induce resistance to pest insects is an excellent alternative for reducing most of the problems associated with chemical methods. This technology has several advantages, mainly because it does not pollute the environment. General data show that transformed cotton plants did not have negative effects on the environment, being the heritable characteristics normally expressed in the plant (Adamczyk, JJ, LC Adams L. C, Hardee, DD Field efficacy and seasonal expression profiles for terminal Ieaves of singie and doubly Bacillus thuringiensis toxin cotton genotypes Journal of Economic Entomology, v.94, no.6, DEC, p.1589-1593 (2001).

The availability of microorganisms and organic compounds in nature for biological use is very large, and they provide a wide variety of raw materials for the development of new products, with greater pathogenicity against the insect and broad spectrum of action. Among these microorganisms, a major discovery was the soil bacterium Bacillus thuringiensis, which is widely used as a biological control agent and as a source of potential molecules for biotechnological programs to obtain transgenic insect-resistant plants. With this strategy it is possible to reduce tolerable levels of agricultural pests of economic interest (Perlak, FJ, RW Deaton, TA Armstrong, RL Fuchs, SR Sims, JT Greenplate and DA Fischhoff. Insect resistant cotton plants. Biotechnology (NY), v.8, no.10, p.939-943, 1990). Although some δ-endotoxins with activity on cotton boll weevil have already been identified and described, the endophytic habit of this pest makes it difficult or even impossible to use these toxins by conventional means, which are marketed as bioinsecticides, such as protein formulations containing Cry toxins. They present instability in the environment, low yield in purification from natural sources, and the easy loss of activity of these toxins due to weather conditions such as rain and sun. Given this problem, the most efficient strategy is to use Cry toxin-encoding genes in the generation of genetically modified plants.

The use of coding genes for this type of entomotoxic proteins and their expression in heterologous systems (bacteria or transgenic plants) circumvents the difficulties generated by the use of bioinsecticides. This strategy has been highlighted in recent years in the field of transgenics, due to the specificity of these toxins in relation to pest insects, efficiency, targeted expression and their innocuity in animals and humans. In this way, genetically modified plants with specific insect pest resistance can be generated in high efficiency systems.

There are some Bt genes and transgenes with activity for cholesters, such as plants expressing a cry8 gene from DU PONT DE MENOURS with toxicity to Leptinotarsa decemlinea (US20030177528), transgenic maize with a cry8-like gene. of PIONEER & DU PONT with toxicity to Diabrotica virgifera, Diabrotica un-decimpunctata howardi, Leptinotarsa decemlineata and Anthonomus grandis (US20060021096, as also mentioned in US7105332 and US2005166284), Feng, S et al., 2005 also describe. modified cry8, cry8Ca, with activity specific to beetle insects (CN1609220-A) and, more recently, PIONEER & DU PONT describe a synthetic cry8 gene with toxicity to Diabrotica virgifera virgifera in monocot plants as an example in maize plants (as mentioned in US20060288448 patent application). Currently, plants expressing cry8-type Bt genes are all monocotyledons (eg maize). Thus, to date, no invention has described a gene of this nature, with potential application to dicotyledonous plants, such as a cotton plant.

Modern molecular biology techniques, such as the construction of combinatorial libraries, are used to develop and identify mutant analog genes for specific purposes.

The construction of variant analog gene libraries using in vitro molecular evolution techniques has been employed for the past three decades. This is due to the emergence of biotechnological tools, which serve as a genetic engineering platform in the development of new molecules with improved activity, mainly targeting agriculture and the pharmaceutical industry (Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution, Microbiology and Molecular Biology Review, Vol. 69, No. 3, pp. 373-392, 2005). There are several techniques which can be applied to generate mutations in a gene sequence, in this invention the DNA shuffling technique (Rosic, NN, Huang, W., Johnston, WA, James J. Devoss, JJ, Gillam, EMJ Extending the diversity of cytochrome P450 enzymes by DNA family shuffling Gene, Vol. 35762, No of Pages 9, 2007; Ling Yuan, L. Kurek, I., English, J. and Keenan, R. Laboratory-directed protein evolution Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, pp 373-392, 2005; Abecassis, V, Pompon, D. and Truan, G. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Research, Vol. 28, No. 20: E 88, 2000; Zhao, H. and Arnold, FH Functional and nonfunctional mutations dis - tinguished by random recombination of homologous genes, Proc Natl Acad Sci USA, Vol 94, pp 7997-8000, 1997; mer, WPC Rapid evolution of a protein in vitro By DNA shufflina. Nature London, Vol. 370, p. 389-391, 1994). The DNA shuffling technique consists of directed molecular evolution, which generates point changes in the primary structure of DNA molecules through random mutations (Ling Yuan, L. Kurek, I., J., and Keenan, R. Laboratory-directed protein evolution Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, pp 373-392, 2005; Stemmer, WPC Rapid evolution of a protein in vitro By DNA shuffling, Nature, London, Vol. 370, pp. 389-391, 1994, US5605793, US5811238, US5830721). The gene of interest is first randomly fragmented into small sequences of 30-50 bp, and this product is recombined in a PCR (Polymerase Chain Reaction) reaction, which is conducted without the addition of oligonucleotides. In a second consecutive reaction, first reaction products and specific oligonucleotides are added. This allows the amplification of a population of mutant / variant analog genes (Stemmer, WPC Rapid evolution of a protein in vitro by DNA Shuffling. Nature. London, Vol. 370, pp. 389-391, 1994; Zhao, H. and Arnold, FH Functional and nonfunctional mutations distinguished by random recombination of homologous genes (Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 7997-8000, 1997).

The efficiency of the technique to produce analogous molecules with higher biological activity could be proven in several works, such as, for example, Jager et al. (Jager, SAW, Jekel, PA and Janssen, DB Hybrid penicillin acylases with improved properties for synthesis of β -lactam antibiotics Enzyme And Microbial Technology, Vol. 40, pp. 1335 - 1344, 2007), where the enzymatic activity of penicillin acyclase increased by 90%. The technique may utilize one or more homologous genes and their success depends on a delicate arrangement between library size, originated biological diversity and a methodology for selecting variants with the desired trait (Ling Yuan, L. Kurek, I. , English, J. and Keenan, R. Laboratory-directed protein evolution Microbiology and Molecular Biology Reviews, Vol. 69, No. 3, pp. 373-392, 2005).

The combination of DNA shuffling techniques libraries (Phage Display) and the presentation of proteins on the surface of bacteriophages - make the selection and expression of new molecules much more efficient (Stoop, AA, Jespers, L, Lasters, I., Ellington, E. And Pannekoek , H. High density mutagenesis by combined DNA shuffling and Phage display to assign essential amino acid residues in protein-protein interactions: application to study structure-function of plasminogen activation inhibitor 1 (PAN). J. Mol. Biol., Vol 301, pp. 1135-1147, 2000).

SUMMARY OF THE INVENTION

The present invention provides molecules encoding new natural δ-endotoxins, mutant analogs and synthetic analogs for pest insect control, particularly cotton boll weevil (Anthonomus grandis), which is susceptible to new toxins.

Also aspects of the invention are gene constructs containing δ-endotoxin-encoding nucleic acid molecules, transformation and expression vectors, cells and transgenic organisms, methods for heterologous expression of novel δ-endotoxins in transgenic organisms, as well as the use of them in pest control. The invention also comprises a method for obtaining a transgenic plant comprising the following steps: a) transforming a plant cell with a gene construct according to claim 4; b) culturing the transformed cell containing the gene construct of interest stably inserted into its genome under ideal cell culture growth conditions; and c) regenerate a transgenic plant expressing the product of the inserted construct from the transformed cell and obtaining transgenic plants.

The invention also provides synthetic analog genes which are optimized for transformation and expression thereof in plants, particularly cotton plants.

Another embodiment of the invention relates to synthetic δ-endotoxin peptides used for the treatment of infected plants, the control of pest insects and their use in the preparation of biodegradable pesticide compositions. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Cry8 gene amplification. PCR amplification reaction using specific oligonucleotides described by Bravo et al (1998). Ethidium bromide stained 1.0% agarose gel (A) First round with oligonucleotides described by Bravo et al (1998). Line 1. Molecular weight marker 1 Kb ladder plus. Line 2. Amplified bands of approximately 400 bp with cry8b oligonucleotides, 2a. round. The arrow indicates the desired probable product. Lane 3. cry8a, 2a oligonucleotides. round. Lane 4. cry8general oligonucleotides. Lane 5. Cry8a oligonucleotides, 1a. Round. Lane 6. cry8general oligonucleotides, 1a. Round. (B) Second round with cr oligonucleotide Only Line 1. Molecular weight marker 1 Kb ladder plus. Lines 2 and 3. Amplification using 1 pL from reaction 1 (Figure A) and sample from line 2 with approximately 450 bp band.

Figure 2: TAIL-PCR. Schematic representation of the TAIL-PCR technique (Liu ef a /., 1995). 1. First amplification with specific 1 and arbitrary oligonucleotides. 2. Result of the first amplification generating nonspecific products (a, b) and the specific product (c). 3. Second amplification with the same arbitrary oligonucleotide and innermost specific oligonucleotide generating the second specific product (d). 4. Third amplification with the same arbitrary oligonucleotide and specific oligonucleotide 3 generating the final product (e). 5. Specific end product.

Figure 3: Cloning of strain S811 cry8 gene by TAIL-PCR. 1.0% agarose gels stained with ethidium bromide and 1 Kb ladder plus molecular weight marker. (A) First TAIL-PCR using arbitrary oligonucleotides AD1, AD2, AD3, AD4, showing successive rounds of amplifications with each specific oligonucleotide. (B) First TAIL-PCR using arbitrary oligonucleotides AD5, AD10, AD11, W4, showing successive rounds of amplifications with each specific oligonucleotide. (C) According to TAIL-PCR using arbitrary oligonucleotides AD3, AD4, AD2 and AD1, showing successive rounds of amplifications with each specific oligonucleotide. The arrows indicate potentially positive products that were subsequently cloned and sequenced.

Figure 4: Dendrogram of the alignment of the new Cry8Ka1 toxin obtained after the two rounds of TAIL-PCR. Analysis with the other Cry8 toxins deposited in the database so far, showing the high identity between them and that the cloned gene encodes a distinct protein from the others. The scale indicates that in the represented space there is an exchange of 0.1 aa. The dendrogram was made using the MEGA4 program (Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599).

Figure 5: Commercial vector map pET101 / D-TOPO for heterologous expression in Escherichia coli. Schematic representation of the vector, including the pT7 promoter. Pomotor T7: Induced by IPTG allows large-scale expression in some Escherichia coli strains; Operon lac (/ acO): Lac repressor binding site, important for reducing basal expression of recombinant proteins (their function may be regulated by the presence or absence of glucose in the culture medium); RBS: Ribosome binding site, located above the 5 'region of the gene to be cloned in the ideal position for the initiation of the translation process; Cloning Site TOP: Region comprising the exact location from which the insert will be cloned; V5 Epitope (Gly-Lys-Pro-lle-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr): Used for western blot detection of recombinant proteins using anti-V5 antibodies; C-terminal 6His: Important for protein purification using resins which have metal coupled; T7 Terminator: T7 bacteriophage sequence that allows for the completion of gene transcription; Bla promoter: ampicillin resistance gene promoter; Ampicillin resistance gene (β-lactamase): Selects E.co// ^' resistant plasmids; Origin of replication pBR322 (ori): Element of replication and maintenance of the E.coli plasmid.

Figure 6: Wiring product wiring system diagram PCR in the pET101 / D-TOP vector. (A) The cohesive end of the vector where the PCR product will be cloned is demonstrated together with the presence of the topoisomerase enzyme. (B) The PCR product is directly cloned by the addition of the 4 base pairs of the direct orientation oligonucleotide. The cohesive end of the cloning vector (GTGG) invades the 5 ^' end of the PCR product, annealing with the four added bases (CACC) and stabilizing the PCR product in the correct orientation. The topoisomerase then cleaves the spare part of the PCR product for binding to be effective.

Figure 7: 12% SDS-PAGE analysis of affinity chromatography purified Cry8Ka1 recombinant protein (Ni-NTA). Line 1. Molecular weight marker. Lane 2. Total extract of E. coli expressing recombinant protein Cry8Ka1. Line 3. Fraction not retained in Ni-NTA resin. Lines 4, 5 and 6. Cry8Ka1 protein eluted from Ni-NTA resin at different concentrations to confirm its purity. 5 pg (lines 2, 3 and 4), 10 pg (line 5) and 15 pg (line 6) of sample were added to sample buffer, subjected to 12% SDS-PAGE, 25 mA and stained with silver nitrate. .

Figure 8: Bioassays against A. grandis and S. frugiperda with recombinant Cry8Ka1 and Cry8Ka1 toxins. (A) Bioassay against S. frugiperda using Cry8Ka1. Surface diet showing 50% mortality at 5 pg / mL concentration. (B) Bioassay against A. grandis using Cry8Ka1. Incorporated diet demonstrating 50% mortality at 230 pg / mL concentration. (C) Bioassay against A. grandis using Cry8Ka1. Incorporated diet demonstrating 50% mortality at 160pg / mL concentration. As control was used the dialysis water in which the proteins were submitted. All experiments were performed in triplicates with 30 A. grandis and 90 S. frugiperda insects.

Figure 9. Schematic of DNA shuffling technique using a single gene as substrate. Scheme of cry8Ka1 gene amplification with specific oligonucleotides for insertion of the restriction site to the enzyme Sf / Ί. Fragmentation, amplification and reconstruction of mutant analog genes.

Figure 10. Interaction between A. grandis BBMVs and fusion phages. For absorbance reading the wavelength of 405nm was used. R-1 to R-6 - Selection cycles and the number of washes per cycle. The greater the absorbance, ie greater number of fusion phage with specificity insect BBMVs A. grandis occurred from ^the fifth selection cycle.

Figure 11. PCR of colony BI variants using specific primer oligonucleotides. 1% agarose gel photo showing amplified DNA of approximately 2000bp expected size. In this gel five colonies, besides the positive control, presented the expected size (4, 6, 10, 17 and 18). 1 - Marker 1 Kb plus® (INVITROGEN). 2 to 18 - Cry8Ka1 gene variants. 19 - Negative control (PCR without DNA). 20 - Positive control, cry8Ka1 gene with specific primer oligonucleotides.

Figure 12. Bioassay with neonate larvae of A. grandis for determination of insecticidal activity of proteins Cry8Ka1 and Cry8Ka5 (mutant). Control - Negative control, diet without the addition of proteins under study. A - Mortality of Cry8Ka1-fed larvae; B - Mortality of Cry8Ka5-fed larvae. At a concentration of 6 pg / mL diet, a two-fold increase in insecticidal activity of the new molecule was observed.

Figure 13. Representation of modeled structure of native Cry8Ka1 toxin using Modeller program and visualized by PyMOL (Delano, WL The PyMOL Molecular Graphics System (2002) on World Web http://www.pymol.org). In the Cry8Ka5 analog the framework backbone remains the same with only the side chains of the substituted amino acid residues. Shown in the Figure are the amino acid residues of Cry8Ka1 that have been substituted in the sequence of the Cry8Ka5 analog. In A, representation of the molecule with the three domains I, II and III. In B, highlight for domain I, formed by seven α-propellers. Glutamine-substituted arginine 132 is located on helix 3. In C, highlight for the three domain II anti-parallel β leaves, indicating native Cry 8 residues substituted on the analog molecule: tyrosine 311 substituted on Cry8Ka5 by cysteine and proline 372 by alanine. In D, the Domain III β sandwich, and indication of the three residues substituted on the analog molecule (arginine 559 for glycine, lysine 589 for glutamic acid and glutamic acid 645 for asparagine.

Figure 14. Graph of entomotoxic activity of cry8 genes analogous to native cry8Ka1 gene. The bioassay was conducted with the fusion phages. Caption: C- - Negative control using HELPER phages. Cry8Ka1 - Original protein expressed in the phage system. Cry8Ka2, Cr 8Ka3, Cry8Ka4 and Cry8Ka5 - Cry8 toxin variants expressed in the phage system.

Figure 15. Alignment of Cry8Ka1 nucleotide sequence with Cry8AB00.1, 50C (b) and Cry8Bb1 sequences. The first line represents the sequence of Cry8Ka1; the second line, Cry8AB00.1, sequence 3 of US patent 73297361; the third line, Cry8AB00.1, sequence 5 of US patent 7339092; the fourth line is the 50C (b) sequence of US 5554534; the fifth line, Cry8Bb1, sequence 15 of WO2005083095; the sixth line, Cry8Bb1, sequence 17 of patent WO2005083095. The numbers above the alignments refer to the position of each nucleotide in the sequence. The sequences were aligned using the CLUSTALW2 program. (http://www.ebi.ac.uk/Tools/clustalw2/) (Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; Mc-William, H; Valentin, F; Wallace , IM; Wilm, A; Lopez, R; Thompson, JD; Gibson, TJ; Higgins, DG. ClustalW and ClustalX version 2. Bioinformatics. 2007; 23: 2947-2948. Doi: 10.1093 / bioinformatics / btm404). Full sequence alignment can be seen in the corresponding Brazilian Patent Application filed under number 012090001018.

Figure 16. Alignment of Cry8Ka1 nucleotide sequence to Cry8Bb1. The first line represents the sequence of Cry8Ka1; the second line to the 32 line, Cry8Bb1, sequences 1, 3, 5, 7, 11, 13, 17, 18, 21, 25, 29, 33, 39, 41, 43, 45, 47, 49, 51, 59 , 61, 67, 69, 71, 73, 75, 77, 79, 81, 83, 91 and 93 of WO2005063996. The numbers above the alignments refer to the position of each nucleotide in the sequence. The sequences were aligned using the CLUSTALW2 program. (http://www.ebi.ac.uk/Tools/clustalw2/) (Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; McWilliam, H; Valentin, F; Wallace, IM ; Wilm, A; Lopez, R; Thompson, JD; Gibson, TJ; Higgins, DG. ClustalW and ClustalX version 2. Bioinformatics. 2007; 23: 2947-2948. Doi: 10.1093 / bioinformatics / btm404). The complete alignment of the sequences can be seen in the corresponding Brazilian Patent Application under number 012090001018.

Figure 17. Alignment of Cry8Ka1 nucleotide sequence to Cry8Bb1. The first line represents the sequence of Cry8Ka1; the second line to the 31 line, Cry8Bb1, sequences 1, 3, 7, 11, 13, 17, 18, 21, 25, 29, 33, 39, 41, 43, 45, 47, 49, 51, 59, 61 , 67, 69, 71, 73, 75, 77, 79, 81, 83, 91 and 93 of US 7105332. The numbers above the alignments refer to the position of each nucleotide in the sequence. The sequences were aligned using the CLUSTALW2 program. (http: //www.ebi. ac.uk/Tools/clustalw2/) (Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; McWilliam, H; Valentin, F; Wallace, IM ; Wilm, A; Lopez, R; Thompson, JD; Gibson, TJ; Higgins, DG. ClustalW and ClustalX version 2. Bioinformatics. 2007; 23: 2947-2948. Doi: 10.1093 / bioinformatics / btm404). Full sequence alignment can be seen in the corresponding Brazilian Patent Application filed under number 012090001018.

Figure 18. Alignment of the nucleotide sequence of

Cry8Ka1 with Cry8Bb1. The first line represents the sequence of Cry8Ka1; the second line to the 31 line, Cry8Bb1, sequences 1, 3, 7, 11, 13, 17, 18, 21, 25, 29, 33, 39, 41, 43, 45, 47, 49, 51, 59, 61 , 67, 69, 71, 73, 75, 77, 79, 81, 83, 91 and 93 of US 7378499. The numbers above the alignments refer to the position of each nucleotide in the sequence. The sequences were aligned using the CLUSTALW2 program. (http://www.ebi.ac.uk/Tools/clustalw2/) (Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; McWilliam, H; Valentin, F; Wallace, IM; Wilm, A; Lopez, R; Thompson, JD; Gibson, TJ; Higgins, DG. ClustalW and ClustalX version 2. Bioinformatics. 2007; 23: 2947-2948. doi: 10.1093 / bioinformatics / btm404). Full sequence alignment can be seen in the corresponding Brazilian Patent Application filed under number 012090001018.

Figure 19. Alignment of amino acid sequences of the new Cry8Ka1 δ-endotoxin with Cry8 sequences. The sequences were aligned using the CLUSTALW2 program (http://www.ebi.ac.uk/Tools/clustalw2/) (Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; McWilliam, H ; Valentin, F; Wallace, IM; Wilm, A; Lopez, R; Thompson, JD; Gibson, TJ; Higgins, DG. ClustalW and ClustalX version 2. Bioinformatics 2007; 23: 2947-2948 doi: 10.1093 / bioinformatics / btm404). Residues containing * are conserved amino acids; :, conservative substitutions; . , semi-conservative substitutions. Full sequence alignment can be seen in the corresponding Brazilian Patent Application filed under number 012090001018.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a new gene belonging to the cry8 family was identified and cloned with high toxicity to Coleoptera insects, specifically to cotton boll weevil. The codons of this sequence have been optimized for their expression in plants, specifically for cotton plants. In addition, a combinatorial library was constructed using the DNA shuffling technique to develop mutant analog genes, which also encode the Cry8 family protein. The generated mutant analog genes, as well as the original gene, have potential effect on the control of cotton boll weevil.

In order to reach the desired objective, that is, δ-endotoxin genes with activity on cotton weevil, an initial scan of the B. thuringiensis germplasm bank of Embrapa Genetic Resources and Biotechnology was performed, in order to identify strains with activity on cotton boll weevil. The effective strains had their genetic material extracted and submitted to molecular biology techniques for identification, characterization and subsequent cloning of cry genes. From this scan, we identified a strain, called S811, highly effective against cotton boll weevil.

For the cloning of cry genes of strain S811 (Germplasm Bank, Embrapa Genetic Resources and Biotechnology) an initial PCR amplification was performed with oligonucleotides specific for several δ-endotoxin families. Amplification with specific Cry8 family oligonucleotides resulted in a fragment of about 500 bp corresponding to the 5 'end of a new cry8 family gene. To obtain the complete gene sequence, the TAIL-PCR (Asymmetric Thermal Interlacing Chain Polymerase Reaction) technique was used, with specific oligonucleotides derived from the previously amplified sequences and eight arbitrary primer oligonucleotides. TAIL-PCR is an application of the PCR technique that allows the isolation of DNA segments adjacent to known sequences (Liu & Whittier, Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. 8: 457-463. 1995).

Briefly, three reactions of

PCR using three sequence-specific oligonucleotides on one side and one arbitrary sequence oligonucleotide on the other side. An initial low stringency loop is made to allow annealing of the arbitrary oligonucleotide to the unknown sequence target segment, followed by a few rounds at high stringency to favor specific oligonucleotide annealing and linear amplification of the target sequence. By changing high and low stringency cycles, double-stranded molecules are formed and amplification of the target sequence becomes logarithmic. In a second and third round of amplification non-specific products are no longer amplified and are eliminated.

Potentially positive, amplified TAIL-PCR fragments were cloned and sequenced in both directions in one sequence. Automatic copier. In total, two sequential TAIL-PCR reactions were performed and 2688 bp (SEQ ID NO: 1) equivalent to 896 amino acids (SEQ ID NO: 2) amplified from a new B. thuringgiensis gene belonging to the Cry8 δ-endotoxin family . The predicted protein sequence of the new gene, called cry8Ka1 (nomenclature following official rules http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/), presents the three structural domains characteristic of the activated N-terminal end of the δ-endotoxins and 240 amino acids of the C-terminal extension. This original sequence served as a template for the generation of analogues with improved enthoxy activity.

To obtain analogous cry genes with high entomotoxicity for cotton weevil, the cry8Ka1 gene isolated from the B. thuríngiensis strain S811 was used. This gene was used as a substrate in the process of generating variant genes by DNA shuffling technique. The variants were selected for their ability to bind to receptors present on the cotton boll weevil midgut membrane (BBMVs) by the protein presentation technique on the surface of bacteriophages - Phage display (Barbas III, CF; Burton , DR; Scott, JK; Silverman, GJ Selection from antibody libraries In: Phage display-laboratory manual (USA: Cold Spring Laboratory, pp. 0.1-20, 2001).

For the selection of cry8 gene variants of the present invention we used the bacteriophage surface protein presentation technique - Phage Display (Zhang, Q., Bai, G., Cheng, J., Yu, Y., Tian, W. and Yang, W. Use of an enhanced green fluorescence protein linked to a single chain fragment variable antibody to localize Bursaphelenchus xylophilus cellulose Biosci Biotechnol Biochem, Vol. 71, No 6, 1514 - 1520, 2007 ; Andris-Widhopf, J., Rader, C., Steinberger, P., Fuller, R., Barbas III, CF Methods for the generation of monoclonal chicken antibody fragments by Phage display.Journal of Immunological Methods, Vol. 159 - 181, 2000; Stoop, AA, Jespers, L, Lasters, I., Eldering, E. and Pannekoek, H. High density mutagenesis by combined DNA shuffling and Phage display to assign essential amino acid residues in protein-protein interactions : applica- tion to study structure-function of plasminogen activation inhibitor 1 (PAI-I). J. Mol. Biol., Vol. 301, p. 1135-1147, 2000; Barbas III, CF, Bain, JD, Hoekstra, MD, And Lerner, RA Proc. Nati. Acad. Sci. USA, Vol. 89, p. 4457-446, 1992).

In the end, the native Cry8Ka1 toxin, its mutant and synthetic analogues had their entomotoxic effects evaluated in vitro by selective bioassays. For this purpose, the selected analogous genes were cloned into heterologous expression vectors (Escherichia colí) and the generated recombinant toxins used in bioassays against cotton pest insects (SEQ ID N ° 5 to 12).

The invention describes novel entomotoxins and methods which enable the generation of technologies capable of controlling pest insects of great economic interest. More specifically, the nucleic acids (genes) of the present invention, including fragments and variants thereof, comprise nucleotide sequences which encode entomotoxic proteins (polypeptides). The described entomotoxic proteins are biologically active against some pest insects belonging to the Coleoptera order, such as the cotton boll weevil, Anthonomus grandis; the western corn root worm, Diabrotica virgifera virgifera; the cow, Diabrotica longicornis barberi; the cucumber beetle, Diabrotica undecimpunctata howardi. Additional pests include: larvae of electic beetles such as Melanotus, Eleodes, Conoderus, and Aeolus spp; Japanese beetle, Polyponia japonica; whiteworm, Phyllophaga crinita; maize and rice flea, Chaetocnema pulicaria; sunflower stem beetle, Cylindrocupturus adspersus; gray sunflower seed beetle, Smicronyx sordidus; sunflower beetle, Zygogramma exclamationis; alfalfa beetle, Hypera nigrirostris; 'wingless' beetle of crucifers, Phyllotreta cruciferae; Colorado potato beetle, Leptinotarsa decemlineata; striped wingless beetle, Phyllotreta striolata; Striped 'wingless' beetle of mustard root, Phyllotreta nemorum and Brassica beetle, Meligethes aeneus.

In addition to nucleotide sequences, the present invention also It also describes an expression vector comprising gene sequences encoding proteins with high entomotoxic activity.

The nucleotide sequences of the invention have direct use in pest insect control methods, particularly of the Coleoptera order. The present invention provides new techniques which do not depend on the use of traditional synthetic chemical pesticides. The invention relates to naturally occurring biodegradable pesticides and genes encoding them.

In some embodiments the invention provides a gene coding for Cry8 family δ-endotoxins, obtained from natural sources, called cry8Ka1 (following official nomenclature of these genes; wvvw.http: //epunix.biols.susx.ac.uk/Home / Neil_Crickmore / Bt.html). Mutant analog genes and synthetic genes to the native gene were also created by in vitro mutation, also coding for δ-endotoxins. In other embodiments, the invention provides genetically modified microorganisms and plants capable of expressing (producing) the new δ-endotoxins, as well as methods involving the use of nucleic acids in pesticide compositions and / or products to act against pest insects in question. The invention also relates to possible coding sequences or variant fragments encoding δ-endotoxins.

In the following description, various terms are used extensively. The following definitions are provided to facilitate understanding of the invention.

As described herein, the term "analog" describes nucleotide or protein sequences other than the specifically identified parent sequences, where one or more nucleotides or amino acid residues have been deleted, substituted and / or added. These sequences may be characterized by the percent identity of their sequences, by common algorithms employed in the state of the art, with the nucleotide (SEQ ID NO: 1-2) or protein sequences (SEQ ID NO: 3-4) described herein. Percentage identity is determined by aligning two sequences to be compared by determining the number of identical residues in the proportion. by dividing this number by the total number of residues in the searched sequence and multiplying the result by 100. This alignment can be done through public domain tools such as BLASTN and BLASTP, available on the National Center for Biotechnological Information / NCBI (http://www.ncbi.nlm.nih.gov). Sequence alignment and percent identity calculation of the present invention were performed as described with sequences deposited in the Gene Bank. Figures 15-19 show the alignment of the sequence of the present invention (Cry8Ka1) with the sequences described in the prior art.

As used herein, the terms "nucleic acid" and "nucleotide sequences" refer to a double-stranded deoxyribonucleotide (DNA) polymer, encompassing known analogs that possess the natural essence of natural nucleotides and specifically hybridize to single-stranded nucleic acids. similar to naturally occurring nucleotides.

The term "oligonucleotide" is referred to herein as primers and probes of the present invention, and is defined as a nucleic acid molecule comprising from ten to thirty deoxyribonucleotides, preferably more than eight. The exact size of oligonucleotides is dependent on the particular experimental factors of each process step.

As used herein, the terms "encoding", "encoding" or "encoded" mean that a nucleotide sequence has information, which will be biologically translated from the nucleotide sequence into a specific protein sequence. Coded information for a protein is specified by codons expressed in the nucleotide sequence. These codons are explored by each living organism differently, and parts of distinct nucleotide sequences can be translated biologically into identical peptides.

As used herein, the term "antisense", used in the context of orientation of a nucleotide sequence, refers to a complementary sequence of a polynucleotide coding sequence, which is operably linked in the 3'-5 'direction therefore from the 5' end of a gene. The antisense strand is complementary to the sense orientation strand by generating a final mRNA capable of hybridizing to the mRNA produced from the original sequence transcription.

The term "gene" corresponds to a specific nucleotide sequence located in a particular region of the chromosome and is responsible for encoding a specific end product. The gene also carries in its primary structure all the information necessary for transcription and biological translation processes, such as transcription promoter and regulatory regions. In the case of the present invention, gene comprises a coding nucleotide sequence corresponding to Cry toxins from Bacillus thuringiensis.

The term "vector" refers to a replicon, such as plasmid, phage or virus, to which other gene sequences or elements (whether DNA or RNA) may be linked. In this way the genes can be replicated along with the vector. Preferably one of the vectors of interest of the present invention concerns the phagemid. The term "phagemid" refers to a vector containing sequence for phage and bacterial replication, this vector has characteristics that meet host cell specifications as well as selection agents and promoters. An example is the pComb3X phagemid (Andris-Widhopf, J.; Rader, C.; Steinberger, P.; Fuller, R., Barbas III, CF Methods for Generation of Chicken Monoclonal Antibody Fragments by Phage display. Journal of Immunological Methods, 242: 159-181, 2000), which has the characteristic of fusing the sequence of interest to the protein III gene of the filamentous bacteriophage M13, located in the viral capsid. The term "recombinant vector" results from the combination of a commercial vector with genes of the present invention operably linked to an endogenous and / or heterologous polynucleotide of interest which is in turn operably linked to a termination signal. Such vectors may be obtained commercially, including those provided by Clontech Laboratories, Inc. (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New York, California. gland Bioiabs (Beverly, Mass.) and Promega (Madison, Wis.). Some but not limited examples of vectors used in the present invention are pGEM-T easy (Promega Corporation), pET101 / D-TOPO (Invitrogen), pComb3X (Andris-Widhopf, J .; Rader, C; Steinberger) vectors. Fuller, R., Barbas III, CF Methods for the generation of monoclonal chicken antibody fragment by Phage display.Journal of Immunological Methods, 242: 59-181, 2000). Obtaining recombinant vectors comprising nucleic acid-linked promoters is known in the art and can be found in Sambrook et al. (Sambrook, J., Russell, DW, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. 1989).

An "expression vector" is a specialized vector that contains a gene with regulatory regions required for expression in a host cell. Such vectors may be obtained commercially, including those provided by Clontech Laboratories, Inc. (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Bioiabs (Beverly, Mass.) And Promega. (Madison, Wis.). The term "operably linked" means that the regulatory sequences required for expression of the coding sequence are placed in the DNA molecule at appropriate positions such that when joined to the coding sequence, it retains the appropriate reading phase for the purpose of its expression. This same definition is sometimes applied for the arrangement of coding sequences and transcriptional controlling elements (e.g., promoters, enhancers, and terminating elements) in the expression vector. An exogenous coding region is typically flanked by operably linked regulatory regions that regulate expression of the exogenous coding region in a transformed cell (may be microorganism, plant or animal). A typical regulatory region operably linked to an exogenous coding region includes a promoter, that is, a nucleic acid fragment that can cause transcription of exogenous coding regions, positioned at the 5 'region of the exogenous coding region. The present invention is not limited to the use of any promoter, they may be inducible, constitutive and tissue specific. Preferably, the promoter of the present invention is from the group of cotton fiber gene promoters, which may be, but is not limited to, E6, H6S, Rac13, LTP, ACP, Expansin, CAP, Annexin, FbL2A and actin 2.

The promoter may contain enhancer elements. An enhancer is a DNA sequence that can stimulate promoter activity. It may be an innate promoter element or a heterologous element inserted to increase the level and / or tissue specificity of a promoter. "Constitutive promoters" refer to those who drive gene expression in all tissues at all times. "Tissue-specific" or "development-specific" promoters are those that drive gene expression almost exclusively in specific tissues, such as leaves, roots, stems, flowers, fruits or seeds, or at specific stages of development in a tissue, as at the beginning or end of embryogenesis.

As described above, the term "expression vectors" may comprise an inducible promoter operably linked to a nucleic acid sequence encoding the pesticidal protein of the present invention. "Inducible" promoters may direct expression of a polynucleotide to which they are operatively linked, at a specific tissue or stage of development or in response to environmental conditions. In one aspect of the invention, expression vectors comprise a tightly regulated inducible promoter operably linked to a nucleic acid molecule encoding a pesticidal protein. Such an expression vector may further comprise a selection marker gene (e.g., a gene encoding an antibiotic resistance conferring protein) operably linked to a constitutive promoter or a tightly regulated inducible promoter. Depending on the application, it may benefit the expression of the nucleic acid sequence encoding a pesticide protein through an inducible insect pest promoter. In one aspect of the present invention it may be advantageous to use promoters that are expressed locally or near the pest infection site.

In one aspect of the invention, the promoter is a plant expressed promoter. As used herein, the term "plant expressed promoter" means a DNA sequence that is capable of initiating and / or controlling transcription in a plant cell. This includes any promoter of plant origin; any promoter of non-plant origin capable of directing the synthesis of the gene present in the Agrobaterium T-DNA; tissue-specific or organ-specific promoters, including but not limited to seed-specific promoters (WO8903887), primordial organ-specific promoters (as mentioned in US20030175783, An, YQ, Huang, S., McDowell, JM, McKin Ney, E. C, Meagher, RB, Conserved expression of the Arabidopsis ACT1 and ACT3 actin subclass in organ primordia and mature pollen. The Plant Celi 8, 5-30, 1996), stem-specific promoters (as mentioned in US20030175783, Keller, B., Sauer, N., Lamb, CJ, Glycine-rich cell wall proteins in bean: Gene structure and association of the protein with the vascular system (EMBO J. 7: 3625-3633, 1988), leaf-specific promoters (as mentioned in patent application US20030175783, Hudspeth, R.L, Greece, JW, Structure and expression of the maize gene encoding the phosphoenolpyruvate carboxylase involved in C ₄ photosynthesis. Plant Mol Biol 12: 579-589, 1989 ), mesophil-specific promoters o, root-specific promoters (as mentioned in US20030175783, Keller, B., Lamb, CJ, Specific expression of a novel cell wall hydroxyproline-rich glycoprotein gene in lateral root initiation. Devi genes. 3: 1639-1646, 1989), tuber-specific promoters (as mentioned in US20030175783, Keil, ML, Sanchez-Serrano, JJ, Willmitzer, L., Both wound-inducible and tuberculosis expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family EMBO J. 8: 1323: 1330, 1989), vascular tissue specific promoters (as mentioned in patent application US20030175783, Peleman J., Saito, K., Cottyn, B., Engler, G., Seurinck, J., Van Montagu, M., Inze, D., Structure and expression analyzes of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene 84: 359-369 (1989), stamen-specific promoters (WO8910396, W09213956), dehiscence zone-specific promoters (W09713865); and the like.

A "leader sequence" or "signal sequence" in the present invention means a nucleic acid sequence which, when operatively linked to a nucleic acid molecule, allows secretion of the product from the nucleic acid molecule. The leader sequence is preferably located in the 5 'region of the nucleic acid molecule. Preferably, the leader sequence is obtained from the same gene as the promoter used to direct transcription of the nucleic acid molecule, or is obtained from the gene where the nucleic acid molecule is derived. Preferably the present invention utilizes the signal sequence from a Brazilian cotton cultivar.

The transcription termination signal and polyadenylation region of the present invention includes, but is not limited to, SV40 termination signal, HSV TK adenylation signal, Agrobacterium tumefasciens (NOS) nopaline synthase gene termination signal, octopine synthase gene termination signal, CaMV 19S and 35S gene termination signal, corn alcohol dehydrogenase gene termination signal, mannopine synthase gene termination signal, beta-phaseoline gene termination signal, ssRUBISCO gene termination signal, sucrose synthase gene termination signal, virus termination signal attacking Trifolium subterranean (SCSV), Aspergillus nidulans trpC gene termination signal, and the like.

The terms "polypeptide", "peptide" and "protein" are used interrelated to refer to a polymer of amino acid residues. The terms apply to amino acid polymers where one or more amino acid residue is an artificial chemical analog of a corresponding naturally occurring amino acid as well as to naturally occurring amino acid polymers.

Polypeptides of the invention may be produced either by a nucleic acid described herein or by the use of standard molecular biology techniques. For example, a truncated protein of the invention may be produced by expressing a recombinant nucleic acid of the invention in an appropriate host cell, or alternatively by combining procedures such as protease digestion and purification.

The term "substantially pure" refers to preparations comprising at least 50-60% by weight of the component of interest (e.g., nucleic acid, oligonucleotide, polypeptide, protein, etc.). More preferably, the preparation comprises at least 75 wt%, and more preferably 90-99 wt% of the component of interest. Purity is measured by methods appropriate for the component of interest (e.g. mass spectrometry and the like).

The term "isolated protein" or "isolated and purified protein" is sometimes used in the present invention. This term refers to a protein produced by the expression of an isolated nucleic acid molecule of the present invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins to which it could be naturally associated as it exists in its "substantially pure" form. The term "isolated" does not exclude synthetic or artificial mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity of the protein, and which may be present, for example, in incomplete purification, addition of stabilizers, or combined in, for example, in an agriculturally acceptable composition.

The term "agriculturally acceptable carrier" refers to solutions in which a pesticidal protein or nucleic acid sequence encoding a pesticidal protein may be maintained without altering the functional properties of the protein molecule described herein for agricultural use. The vehicles used in the present invention may be liquid or solid. Liquid carriers which may be used to form compositions using recombinant protein of the present invention may be, but are not limited to water or organic solvents such as polyols, esters, methylene chloride, alcohol, or vegetable oil. Other components that may be included in the formulation include humectants, preservatives, thickeners, antimicrobial agents, antioxidants, emulsifiers, film-forming polymers and mixtures thereof. Humectants may include polyols, sugars (such as molasses), glycols and hygroscopic salts. Glass membranes, film-forming polymers include rosin gum, latex, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyethylene, polyvinyl acetate, and mixtures thereof. Additional optional additives include methyl, methacrylate, and mixtures thereof.

The terms "peptide analog" or "mutant analog" mean a natural or mutant analog of a protein, comprising a series of linear or discontinuous fragments of that protein and in which it may have one or more amino acids replaced with another amino acid (s). (s) and may have their biological activity altered, aided, increased or decreased compared to the native or non-mutated parental protein.

The term "biological activity" refers to a function or group of functions performed by a molecule in a biological context (ie, an in vitro organism or substitute or some other similar model). For entomotoxic proteins, biological activity is characterized by physicochemical properties such as structuring in highly hydrophobic domains capable of forming oligomers, and biological membrane affinity, causing their destruction. This membrane affinity can be caused by the presence of specific receptors as well as by the simple chemical interaction between them.

As used herein, the term "impacting pest insects" refers to the effect of changing on insects feeding, growth, and / or behavior at any stage of development, including, but not limited to: killing the insect; slow growth; prevent capacity reproductive; anti-eating activity; and the like.

The terms "pesticidal activity" and "insecticidal activity" are used synonymously to refer to the activity of an organism or a substance (eg a protein) that can be measured by, but not limited to, pest mortality, loss pest weight, pest repellency, and other behaviors and physical changes of a pest after feeding and exposure for an appropriate period of time. Thus, the impact of pesticide activity must have at least one measurable parameter of pest suitability. For example, "pesticidal proteins" are proteins that trigger pesticidal activity by themselves or in combination with other proteins. Endotoxins and δ-endotoxins are pesticide proteins. Other examples of pesticidal proteins include, for example, pentin-1 and jaburetox.

The term "effective amount of pesticide" refers to an amount of a substance or organism that has pesticidal activity when present in the pest environment. For each substance or organism, the effective amount of pesticide is determined empirically for each pest affected in a specific environment. Similarly the term "effective amount of pesticide" may be used to refer to an "effective amount of pesticide" when a pest is an insect pest.

The term "recombinantly engineered" or "engineered" refers to the use of recombinant DNA technology to generate (engineer) a change in protein structure based on an understanding of its mechanism of action, and amino acids may be introduced, deleted or deleted. replaced.

The term "DNA shuffling" is used to describe a method employed in in vitro directed molecular evolution to generate variants of a single gene sequence, or two or more homologous gene sequences by recombination of randomly generated fragments with retrieving modified sequences and thereby modifying amino acid residues in the protein encoded by the mutant analog. The term "Phage display surface protein presentation" refers to a bacteriophage-fused protein expression and interaction system that allows cells, tissues, or organs to be scanned for receptor-ligand pairs. these ligands are proteins that bind to receptors present on the target under study.

As used herein, the term "mutated nucleotide sequence" or "mutation" or "mutagenized nucleotide sequence" refers to a nucleotide sequence that has been mutated or altered to contain to contain one or more nucleotide residues (e.g. : base pairs) that are not present in the wild type or in the unchanged sequence. Such mutagenesis or alteration consists of one or more additions, deletions, or substitutions or reallocation of nucleic acid residues.

The term "analog" or "mutant" is used to identify a gene that has been altered by mutation and makes it different from wild type or normal population variation.

As used herein, the term "enhancement of insecticidal activity" or "enhancement of pesticidal activity" characterizes a polypeptide or δ-endotoxin of the invention that has improved coleopteran pesticidal activity over other δ-endotoxins that are effective against insects. To measure the improvement of pesticide or insecticide activity, a demonstration of at least 10% increase in toxicity against the target insect should be required, and more preferably 20%, 25%, 30%, 35%, 40%, 45%. , 50%, 60%, 70%, 100%, 200% or a greater increase in toxicity with respect to the insecticidal activity of existing Cry8 δ-endotoxins that are active against the same insect.

The term "toxin" or "endotoxin" is related to a polypeptide which exhibits toxic insecticidal activity. It is known in the prior art that naturally occurring δ-endotoxins are synthesized by B. thuríngiensis, which sporulate by releasing protein crystalline inclusion containing δ-endotoxin.

For a particular interest of the invention, the following were optimized: sequences encoding pesticidal proteins of this invention. As used herein, the terms "optimized nucleotide sequences" or "synthetic sequences" refer to nucleic acids that are optimized for expression in a particular organism. Optimized nucleotide sequences include those sequences that have been so modified that the GC content of the nucleotide sequence becomes altered. Such modification of the nucleotide sequence may or may not comprise a coding region. Where the modified nucleotide sequence comprises a coding region, changes in GC content may be made in view of another genetic phenomenon, such as the preference of a codon for a particular organism or the tendency of GC content in the genome. coding region.

In some embodiments of the invention, where the optimized nucleotide sequence comprises the coding region, the change in GC content does not result in a change in the protein encoded by the nucleotide sequence. In other embodiments, the change in GC content results in changes in encoded protein that may be changes in conserved amino acids that may not significantly alter the function of the encoded protein. The GC content of a nucleotide sequence may differ from the native nucleotide sequence by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. , or 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% , 44%, 45%, 46%, 47%, 48%, 49%, or 50% or more. So the GC content of an optimized nucleotide sequence can be 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% or greater.

One skilled in the art is aware that advances in the field of molecular biology such as site-specific or random mutagenesis, polymerase chain reaction (PCR) methodology and Protein engineering experts have an extensive collection of workable tools and protocols for use to alter or engineer both amino acid sequences and disguised genetic sequences of proteins of agricultural interest. Thus, the pesticidal proteins of the invention may be altered in a number of ways, including amino acid substitution, deletions, truncations, and insertions. Methods for such manipulations are generally known in the prior art. For example, a variant amino acid sequence of the pesticidal protein of the present invention may be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleic acid changes are well described in the state of the art.

Synthetic gene design was based on the original gene sequence, including the N-terminal portion of the protein with the three domains responsible for insecticidal activity. In the design of the synthetic gene, 262 base pairs were modified, resulting in the elimination of 25 possible polyadenylation signals, 17 instability motifs, 95 rarely used codons in plants and an increase in G-C content from 35.6 to 43: 8%. The final protein sequence of the synthetic gene is identical to the original sequence, ie remained unchanged.

It is understood that the polypeptides of the invention may be produced either by expression of a nucleic acid described herein or by the use of standard molecular biology techniques.

Pesticide proteins are known to be oligomeric and vary in molecular weight, number of residues, peptide components, activities against particular pests, and other characteristics. However, by the methods described herein, proteins active against a variety of pests can be isolated and characterized. The pesticidal proteins of the invention may be used in combination with δ-endotoxins Bt or other insecticidal proteins to enhance action on the target insect. Moreover, the use of pesticidal proteins of the present invention in combination with Bt δ-endotoxins or other insecticidal principles of a different nature may have particular utility for preventing and / or managing resistance. the insect. Other insecticidal principles include, but are not limited to protease inhibitors (both serine and cysteine), lectins, alpha-amylases, and peroxidases.

The invention also relates to microorganisms transformed with at least one nucleic acid of the present invention, a chimeric gene comprising the nucleic acid, or an expression vector comprising the chimeric gene. Preferably, the microorganism is one that multiplies in plants. More preferably, the microorganism is a root colonizing bacterium. One embodiment of the present invention relates to an encapsulated pesticidal protein comprising a transformed microorganism comprising at least one pesticidal protein of the invention.

The invention also provides pesticidal compositions comprising a transformed organism of the invention. Preferably the transformed microorganism is present in the pesticidal composition in an effective pesticidal amount, together with an acceptable carrier vehicle. The invention also comprises pesticidal compositions comprising an isolated protein of the invention alone or in combination with a transformed organism of the invention and / or an encapsulated pesticidal protein of the invention in an effective insecticidal amount together with an acceptable carrier.

The invention also provides a method of increasing the range of the target insect by the use of the inventive pesticide proteins in combination with at least one second pesticide protein that is different from the inventive pesticide protein. Any pesticidal protein known in the art may be used in the method of the present invention. Such pesticidal proteins include, but are not limited to Bt δ-endotoxins, protease inhibitors, lectins, alpha amylases, acyl lipid hydrolases, and peroxidases.

The invention also comprises transgenic or transformed plants comprising at least one nucleotide sequence of the invention. Preferably, the plant is stably transformed with a chimeric gene comprising at least one nucleotide sequence of the invention operably linked to a promoter that directs expression in plant cells. As used herein, the term "transgenic plants" or "transformed plants" refers to a plant comprising within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated into the genome of a transgenic plant so that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated within the genome alone or as part of a recombinant vector.

As used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant part, or plant genotype that has been altered by the presence of heterologous nucleic acid including those initially altered as well as those created by sexual crossing or sexual propagation of the sexual transgenic.

The term "plants" refers to photosynthetic organisms, both eukaryotes and prokaryotes, where the term "developed plants" refers to eukaryotic plants. The term refers to whole plants, plant organs (e.g., leaves, stems, roots, flowers, among others), seeds, plant cells, and progenies thereof. Parts of transgenic plants are also included within the scope of the invention comprising, for example, plant cells, protoplasts, tissues, corns, embryos as well as flowers, eggs, stems, fruits, leaves, roots originating from transgenic plants or their previously transformed progeny. with a DNA molecule of the invention and therefore consisting of at least part of the transgenic cells, are also object of the present invention. The nucleic acids of the invention may be used to confer desired treatments on essentially any plant. Thus, the invention has use over various plant species, including species of the genera Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Coconuts, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Pragana, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigon Triticum, Vicia, Vitis, Vigna, and Zea. Particularly the present invention relates to cotton plants transformed with the nucleotide sequences of the present invention as well as fragments and derivatives thereof, more specifically transformed Gossypium hirsutum plants.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, for example, monocotyledons or dicotyledons, targets of transformation. Viable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome are well described in the state of the art and may be, but are not limited to techniques such as electroporation and microinjection of plant cell protoplasts, or construction may be introduced directly into plant tissue using ballistic methods such as bombardment of DNA-coated particles.

Microinjection techniques are known in the state of the art and well described in scientific and patent literature (Zhou, G., Wang, J., Zeng, Y., Huang, J., Qian, S., Liu, G., Introduction of exogenous DNA into cotton embryos, Meth in Enzymol., 101, 433-448, 1983) (as mentioned in US patent application 4743548). Introduction of gene constructs using polyethylene glycol precipitates is described in Paszkowski et al. (Paszkowski, J., Shillito, RD, Saul, M., Mandák, V., Hohn, T. Hohn, B., Tryptus , I., Direct gene transfer to plants, Embo J. 3: 2717-2722, 1984) (as mentioned in US20020 52501). Electroporation techniques are described in Fromm et al (Fromm, ME, Taylor, LP Walbot, V., Expression of genes electroporated into monocot and dicot plant cells. Proc. Natl. Acad. Sci. USA 82: 5824, 1985) (as mentioned in US20020152501). Ballistic transformation techniques are described in Klein et al (Klein, TM, Wolf, ED, Wu, R., Sanford, J. C, High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70-73, 1987) (as mentioned in US20020152501).

Alternatively, the gene constructs may be combined with appropriate T-DNA flanking regions and introduced into a conventional vector, the host Agrobacterium tumefaciens. The virulence function of the host Agrobacterium tumefaciens will direct the insertion of the gene constructs and adjacent marker into the plant cell DNA when the cell is infected with the bacterium. Agrobacterium tumefaciens-mediated transformation techniques, including disarmament and the use of binary vectors, are well described in the scientific literature (as mentioned in US Patent Application 20020152501, Horsch, RB, Fraley, RT, Rogers, SG, Sanders, PR, Lloyd). , A., Hoffmann, N. Inheritance of functional foreign genes in plants Science 233: 496-498, 1984, and Fraley, RT, Rogers, SG, Horsch, RB, Sanders, PR, Flick, JS, Adams, SP, Bittner, M.L., Brand, L.A., Fink, C.L., Fry, JS, Galluppi, GR, Goldberg, SB, Hoffmann, N.L., Woo, SC Expression of bacterial genes in plant cells Proc. Natl. Acad. Sci. USA 80: 4803, 1983).

Transformed plant cells derived from any of the transformation techniques described above can be cultured to regenerate an entire plant having the transformed genotype and then the desired phenotype, such as insect resistance. Such regeneration techniques rely on the manipulation of certain phytohormones in tissue culture growth media, typically containing a biocidal and / or herbicidal marker, which must be introduced together with the desired nucleotide sequence. Plant regeneration from protoplast culture is described in Evans et al (Evans, DE, and Bravo, JE, Protoplasts Isolation and Culture, Handbook of Plant Celi Culture, Vol. 1, 124-176, MacMillilan Publishing Company, New York , 1983); and Binding 1985 (Binding, H., Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985) (as mentioned in US20020 52501). Regeneration can also be achieved through plant calli, explants, organs, or part thereof. Such regeneration techniques are generally described. in Klee et al (Klee, H., Horsch, R., Rogers, S., Agrobacterium-mediated plant transformation and its applications for plant biology. Ann. See. Of Plant Phys. 38: 467-486, 1987 (as mentioned in US20020 52501).

Genes encoding pesticide proteins are known to be used to transform pathogens to insects. Such organisms include baculoviruses, fungi, protozoa, bacteria, and nematodes.

A gene encoding a pesticidal protein of the invention may be introduced via a viable vector into a microbial host, and said host may be applied to the environment, or to plants, or to animals. The term "introduced" in the context of inserting a nucleic acid into a cell means "transfection" or "transformation" or "transduction" and includes the incorporation of a nucleic acid into a prokaryotic or eukaryotic cell where the nucleic acid may be be incorporated into the cell genome (eg, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (eg, transfected mRNA).

Host microorganisms have been known to occupy the "phytosphere" (phylloplane, philosopher, rhizosphere, and / or rhizoplane) of one or more cultivars of selected interest (s). These microorganisms of interest are selected to be able to successfully compete in a particular environment with wild microorganisms, providing stable maintenance and expression of the gene expressing the pesticidal protein, and desirably improving the protection of the pesticide from environmental degradation and inactivation.

Such microorganisms include, but are not limited to bacteria, algae and fungi. Particularly the microorganisms include bacteria such as Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acabacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoces, and Alcalige-,. , for example, Saccharomyces, Cryp- W 201

36

tococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are bacterial species of the phytosphere such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xylland and Azotobacter levir sp. such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.

There are several viable methods for introducing a gene expressing the pesticidal protein into a host microorganism under conditions that permit the maintenance and stable expression of the gene. For example, expression vectors may be constructed containing the nucleotide sequence of interest operably linked to regulatory transcriptional and translational signals for expression of the nucleotide sequence. When an internal homologous nucleotide sequence of the organism meets the sequence in the expression vector, there may be a recombination between them and the gene encoding the pesticidal protein will integrate into the host organism's genome stably.

Viable host cells, where cells containing the pesticidal protein will be treated to prolong the activity of the pesticidal protein in the cell when the treated cell is applied to the target pest environment, may include prokaryotic or eukaryotic cells, usually being limited to those cells that do not. produce toxic substances in higher organisms. However, organisms that produce toxic substances in higher organisms may be used as long as the toxin is unstable or the level of application sufficiently low to avoid any possibility of mammalian host toxicity. Particularly the hosts are less developed prokaryotes and eukaryotes such as fungi. Examples of prokaryotes, both gram negative and gram positive, include, but are not limited to Enterobacteriaceae, such as Escheridi. Chia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as Photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudodonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among eukaryotes can be exemplified fungi such as Phycomycetes and Ascomycetes which include yeast such as Saccharomyces and Schizosaccharomyces; and Basidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.

Features of particular interest in selecting a host cell for the purpose of producing the pesticidal protein include the ease of introducing the pesticidal protein gene into the expression system, expression efficiency, protein stability in the host, and the presence of genetic capabilities. auxiliaries. Features of interest for the use of a pesticidal microcapsule include protective quality for the pesticide, such as cell wall thickness, pigmentation, and intracellular packaging or inclusion body formation; leaf affinity; absence of mammalian toxicity; attractiveness for ingestion by pests; ease of killing and fixing without harming the toxin; and the like. Other considerations include ease of formulation and handling, economy, storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., And Sporobolomyces spp., Philoplane organisms such as Pseudomonas spp., Erwinia spp., And Flavobacterium spp., And other organisms, including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cevisia, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like.

In the present invention, a transformed microorganism (containing the coding sequence of the pesticidal protein of the invention) or an isolated pesticidal protein may be formulated as an acceptable carrier vehicle within a pesticidal composition, which may be, for example, a suspension, a solution, an emulsion, a powder, a dispersible granule, a moistened powder, an emulsified concentrate, an aerosol, an impregnated granule, an adjuvant, a covered capsule, and also encapsulations in, for example, polymeric substances.

Such compositions disclosed herein may be obtained by the addition of a surface active agent, an inert carrier vehicle, a preservative, a humectant, a food stimulant, an attractant, an encapsulant agent, a binder, an emulsifier, a dye, a UV (ultraviolet) protector, a buffer, a flow agent or fertilizer, micronutrient donors, or other preparations that influence plant growth. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscides, acaricides, plant growth regulators, crop supports, and fertilizers may be combined with carrier vehicles, surfactants or adjuvants commonly used in formulations, or other components used to facilitate handling and application to a particular target pest. Viable fillers and adjuvants may be solid or liquid and correspond to substances ordinarily employed in a formulation technology, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, binders, or fertilizers. The active ingredients of the present invention are usually applied in the form of compositions and may be applied to the cultivated area, plants, or seeds to be treated. For example, the compositions of the present invention may be applied to grains in preparations or during storage in a grain silo, for example. The compositions of the present invention may be applied simultaneously or in succession with other compounds. Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention containing at least one of the pesticidal proteins produced by the bacterial strains of the present invention include, but are not limited to foliar application, seed coverage, and application in ground. The number of applications and rate of application will depend on the intensity of the infestation by the corresponding pest. Examples of inert materials include, but are not limited to inorganic minerals such as kaolin, phyllylicates, carbonates, sulfates, phosphates, or botanical materials such as cork, corn kernel powder, peanut pasta, rice pasta, walnut shell.

The compositions of the present invention may be in a viable form for direct application or as a concentrate of the primary composition which requires dilution with a viable amount of water or other diluent prior to application. The pesticidal concentration will vary depending on the nature of the particular formulation, specifically whether it is a concentrate or will be used directly. The composition may contain from 1 to 98% of a liquid or solid inert carrier, and 0 to 50%, preferably from 0.1% to 50% of a surfactant. These compositions will be administered at a labeled rate for commercial products, preferably about 0.01 lb - 5.0 lb per acre when in dry form and about 0.01 pts - 10 lb per acre when in liquid form.

Embodiments of the present invention may be effective against a variety of pests. For purposes of the present invention, pests include, but are not limited to, insects, fungi, bacteria, nematodes, mites, protozoan pathogens, animal parasites, and the like. Pests of particular interest are pest insects, particularly pest insects that cause significant damage to agricultural plants. "Pest insects" are insects and other similar pests such as, for example, insects of the orders Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermapterra, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, especially Anthonomus grandis, Diabrotica virgifera and Lepidoptera. Pest insects of the present invention from most cultivars include, but are not limited to Corn: Ostrinia nubilalis, Agrotis ipsilon, Helicoverpa zea, Spodoptera frugiperda, Diatraea grandiosella, Elasmopalpus lignosellus, Diatraea saccharalis, Diabrotica longgifera virgera virgifera virgifera virgifera - cornis barberi, Diabrotica undecimpunctata howardi, Melanotus spp., Cyclocephala borealis, Cyclocephala immaculata, PopilHa japonica, Chaetocnema pulicaria, Sphenophorus maidis, Rhopalosiphum maidis, Anuraphis maidira-dicis, Blissus leucopterus leucopterus, Melanoplus femurrubrum, Melanoplus sanguinipes, Hylemya platura, Agromyza parvicornis, Anaphothrips obscrurusus, Solenopsis, Solenopsis; Sorghum: Chilo partellus, Spodopera frugiperda, Helicoverpa zea, Elasmopalpus lignosellus, Underground feltia, Phyllophaga crinita, Eleodes, Conoderus, and Aeolus spp. leucopterus, Contarinia sorghicola, Tetranychus cinnabarinus, Tetranychus urticae; Wheat: Pseudaletia unipunctata, Spodoptera frugiperda, Elasmopalpus lignosellus, Agrotis orthogonia, Elasmopalpus lignosellus, Oulema melanopus, Hypera punctata. mosellana, Meromyza americana, Hylemya coarctata, Frankliniella fusca, Cephus cinctus, Aceria tulipae; Sunflower: C-ylindrocupturus adspersus, Smicronyx fulus, Smicronyx sordidus, Suleima helianthana, Homoeosoma electellum, Zygogramma exclamationis, Bothyrus gibbosus, Neolasioptera murtfeldtiana; Cotton: Heliothis virescens, apple caterpillar; Helicoverpa zea, corn cob caterpillar; Small Spodoptera, cartridge caterpillar; Pectinophora gossypiella, pink caterpillar; Anthonomus grandis, cotton boll; Aphis gossypii, cotton aphid; Pseudostomoscelis seriatus, cotton jumping flea; Trialeurodes abutilonea, whitefly Bemisia tabaci; Melanoplus femurrubrum, grasshopper; Melanoplus differentialis, grasshopper; Thrips tabaci, smoke thrips; Franklinkiella fusca, tripods; Tetranychus cinnabarinus, red mite; Tetranychus urticae, guinea fowl; Rice: Diatraea saccharalis, Spodoptera frugiperda, Helicoverpa zea, Colaspis brunnea, Lissorhoptrus oryzophilus, Sitophilus oryzae, Nephotettix nigropictus, Blissus leucopterus leucopterus, Acrosternum hilare; Soybeans: Pseudoplusia includens, Anticarsia gemmatalis, Plathypena scabra, Ostrinia nubilalis, Agrotis ipsilon, Spodoptera exigua, Heliothis virescens, Helicoverpa zea, Epilachna varivestis, Myzus persicae, Empoasca fabae, Melanoplum, Melanoplumum, Melanoplumum ylemya platura, Sericothrips variabilis, Thrips tabaci, Tetranychus turkestani, Tetranychus urticae; Barley: Ostrinia nubilalis, Agrotis ipsilon, Schizaphis graminum, Blissus leucopterus leucopterus; Acrosternum hilare, Euschistus servus, Jylemya platura, Mayetiola destructor, Petrobia latens; Canola: Vrevoryne brassicae, Phyllotreta cruciferae, Phyllotreta striolata, Phyllotreta nemorum, Meligethes aeneus, Meligethes rufímanus, Meligethes nigrescens, Meligethes canadianus, and Meligethes viridescens; Potato, Leptinotarsa delinlineata.

The examples below are set forth to further illustrate and elucidate the invention and may not be construed as limiting the present invention.

EXAMPLES

Usual molecular biology techniques (eg, bacterial transformation and electrophoresis on nucleic acid agarose gel) are described by commonly used terms. Details of practicing such techniques are described in Sambrook et al (Sambrook, J., Russell, D.W., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. 1989).

Example 1 - Selection of Bacillus thuringiensis strain S811 from the Embrapa Germplasm Bank Genetic and Biotechnological Resources.

In a previous paper by Silva-Werneck et al. (Monerat, RG, Silva, SF, Silva-Werneck, OJ Catalog of Bacillus Bacteria Germplasm Bank. Brasilia: Embrapa-Cenargen, Documents 60, 65 p. 2001), several strains belonging to the Bank were identified and characterized. of Microbial Germplasm of Embrapa Genetic Resources and Biotechnology. Among these, strain S811 was selected due to its high entomotoxic activity against Coleopteran insects, such as Anthonomus grandis. Toxicity was assessed by selective bioassays using the total protein extract of Bacillus thuringiensis S811 as substrate.

To obtain crude protein extract the strain was grown in culture medium nutrient broth (MCCN; nutrient broth 8 g / l, yeast extract 1 g / l and 1 g / l monobasic potassium phosphate) at 30 ° C under a stirring of 200 rpm. After 12 hours of cultivation, with the culture in the vegetative phase and after 48 - 72 hours with complete sporulation, the genetic material and the crude protein extract can be obtained, respectively.

Example 2 - Identification, isolation and characterization of the cry8 gene from Bacillus thuringiensis strain S811.

Total DNA extraction from Bacillus thuringiensis S811 was performed according to the CTAB protocol (2% CTAB, 0.2% β-mercaptoethanol). After 12 hours of cultivation, 30 mL of the culture in the vegetative phase were centrifuged at 5000 rpm for 20 minutes. The pellet was frozen in liquid nitrogen and macerated following the protocol descriptions described by Romano, E. (Romano, E. Plant DNA Extraction. In: Plant Genetic Transformation Manual. ACM Brasileiro & VTC Carneiro (Eds Embrapa Genetic Resources and Biotechnology, Brasília, 1998). The final product was dried and resuspended in 50 µl Milli-Q water and then stored at -20 ° C.

To identify Cry toxin coding genes in strain S811, the PCR (Polymerase Chain Reaction) technique was used. Amplifications were performed using specific oligonucleotides for detection of cryl subgroup genes (Cerón, J.; Covarrubias, L .; Quintero, R .; Ortiz, A.; Ortiz, M .; Landa, L.; Bravo, A. PCR analysis of the insecticidal cryl crystal family genes from Bacillus thuringiensis Appl Environ Microbiol, 60, 353-356, 1994 Cerón, J. Ortiz, A. Quintero, R. Giiereca, L. ., Bravo, A. Specific PCR primers directed to identify cryl and cryol genes within a Bacillus thuringiensis strain collection. Appl. Environ. Microbiol., 61, 3826-3831, 1995) and cry8 (Bravo, A .; Sarabia, S. ; Lopez, L.; Ontiveros, H.; Abarca, C.; Ortiz, A.; Ortiz, M.; Lina, L.; Villalobos, F.; Pena, G.; Nunez-Valdez, ME; Soberon, M.; Quintero; , R. Characterization of cry Genes in a Mexican Bacillus thuringiensis Strain Collection (Appl. Environ. Microbiol., V. 64, pp. 4965-4972, 1998). PCR reaction conditions containing the cryl group oligonucleotides were described by Cerón et al (Ce- Rón, J .; Covarrubias, L; Quintero, R .; Ortiz, A .; Ortiz, M .; Aranda, E .; Lina, L., Bravo, A. PCR analysis of the insecticidal cryl crystal family genes from Bacillus thuringgiensis. Appl. Environ. Microbiol., 60, 353-356, 1994) and PCR reaction conditions containing cry8 group oligonucleotides have been described by Bravo et al (Bravo, A.; Sarabia, S.; Lopez, L.; Ontiveros, H. ; Abarca, C.; Ortiz, A.; Ortiz, ML; Lina, L.; Villalobos, FJ; Pena, G.; Nunez-Valdez, ME; Soberon, M.; Quintero, R. Characterization of cry Genes in a Mexican Bacillus thuringgiensis Strain Collection, Appl Environ, Microbiol., V. 64, pp. 4965-4972, 1998). All reactions were performed in 25 pL volumes containing 2.5 pg total DNA, 10 mM Tris-HCI pH 8.4, 2 mM Mg-Cl ₂ , 50 mM KCI, 200 mM of each dNTP (deoxy ribonucleotides triphosphate), 500 nM of each oligonucleotide and 0.1 U / pL of Taq DNA polymerase for each DNA sample. Amplification was performed in a thermal cycler (MasterCicle Gradient Eppendorf) under the following conditions: previous denaturation at 94 ° C for 2 minutes, repetition and 30 cycles at 94 ° C for 45 seconds (denaturation), oligonucleotide annealing for 45 seconds ( specific temperature for each oligonucleotide), 72 ° C for 2 minutes (DNA polymerase extension) and at the end a final extension of 72 ° C for 5 minutes. PCR amplified fragments were separated and visualized on 0.8% agarose gel. DNA fragments were excised from the gel and purified using GeneClean kit (Bio101 System) and quantified by spectrophotometry. The purified fragments were then ligated into 50 ng of commercial vector pGEMT-easy (PROMEGA) at a 3: 1 molar ratio (insert.vector) with 4 U / pL T4 DNA ligase and 1 X buffer at the final volume of 15 µl. pL The generated vectors were used to transform competent Escherichia coii cells by electroporation. Positive clones were identified by colony PCR and had their plasmid DNA extracted. The plasmid DNAs obtained were sequenced in an ABI automated sequencer using general T7, SP6, reverse and universal oligonucleotides (Nag, DK, Huang, HV and Berg, DE Bidirectional Nucleotide Sequencing: Transposon Tn5seq1 as a Mobile Source of Primer Sites, Gene 64, 135-145.1988). The sequences obtained were compared to the Database sequences (GeneBank and SwissProt) by the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple alignment of the clone sequences obtained was performed with the most similar sequences deposited in the Database (GeneBank) by CLUSTALW (http://www.ebi.ac.uk/clustalw/) (Thompson, JD, DG Higgins and TJ Gibson CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice Nucleic Acids Res, v.22, n.22, Nov 11, p.4673-4680 1994).

The first PCR amplification reaction with the cry8 family-specific oligonucleotides, Bravo et al, 1998, resulted in a 442 bp fragment (Figure 1) corresponding to the 5 'end of a new cry8 family gene ( SEQ ID NO.1). In order to obtain the complete cry8 gene sequence, two rounds of amplification were performed by the TAIL-PCR (Asymmetric Thermal Interlacing Chain Polymerase Reaction) technique (Liu, Y .; Whittier, RF Thermal asymmetric interlaced PCR). : Automatic amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking Genomics, v. 25, pp. 674-681, 1995) (Figure 2). This technique is an application of the PCR technique that allows the isolation of DNA segments adjacent to known sequences, using the genomic DNA of the organism. The technique utilizes sequential specific oligonucleotides along with degenerate small arbitrary oligonucleotides to thermally control the relative amplification efficiency of specific and unspecific products. By interspersing high and low stringency cycles, specific products are preferably amplified over non-specific products.

Briefly, three PCR sequential reactions are performed using specific oligonucleotides derived from previously amplified one-sided sequences (Bravo, A.; Sarabia, S.; Lopez, L.; Ontiveros, H.; Abarca, C.; Ortiz, A ; Ortiz, M .; Lina, L.; Villalobos, FJ; Pena, G .; Nunez-Valdez, ME; Soberon, M .; Quintero, R. Characterization of cry. Genes in a Mexican Bacillus thuringiensis Strain Collection. Appl. Environ. Microbiol., V. 64, p. 4965-4972, 1998) and eight arbitrary oligonucleotides from the other (Liu, Y .; Whittier, RF Thermal asymmetric interlaced PCR: Automated amplification and sequencing of insert fragments from P1 and YAC clones for chromosome walking. Genomics, v. 25 , pp. 674-681, 1995). Fragments were amplified, cloned, sequenced and analyzed under the same conditions as described above for initial gene identification.

The final product, obtained by the TAIL-PCR technique, contains 2688 bp amplified (SEQ ID NO: 1) and encodes a new 896 amino acid δ-endotoxin (SEQ ID NO: 2). Analysis of the nucleotide sequence by the BLASTn program, using the NCBI filed patent database as the search pattern, identified the sequence of the present invention as corresponding to the Cry8 family, having more than 90% identity as shown in the alignments ( Figures 18, 19, 20 and 21).

Analyzes of the predicted protein sequence of the new Cry8 gene show the presence of the three characteristic structural domains of δ-endotoxins in their N-terminal portion. The analyzes also show the presence of an additional 240 amino acids of the C-terminal extension of the new δ-endotoxin (Figure 3).

Alignment of the amino acid sequences of the present invention with other patented proteins of the Cry8 family shows that the new δ-endotoxin differs 80% from other amino acid sequences, having about 150 conserved amino acids (Figure 19).

When compared to other δ-endotoxins, the new Cry8 δ-endotoxin was more similar to the cry8Aa gene (53% identity and 67% similarity), followed by the cry8Ba (53% identity and 66% similarity) and cry8Ca (49%) genes. identity and 65% similarity). Figure 4 shows a dendogram of the alignment of the new Cry8 toxin with the other Cry8 toxins deposited in the database to date. In Figure 4 we can observe the high identity between the toxins. The scale indicates that in the represented space there is at least 0.1 aa exchange.

N-terminal and C-terminal ends of new δ-endotoxin Cry8Ka1 have high identity with other Cry8 δ-endotoxins, while the three structural domains are less conserved, particularly the second and third domains (Table 1), which are involved in receptor binding, suggesting new insecticidal activities / specificities for the isolated gene.

Table 1. Mean identity between the Cry8Ga δ-endotoxin domains and the corresponding domains in other Cry8 δ endotoxin domains.

Cry8Ka1

N-terminal Domain I Domain II Domain III C-terminal

Cry8Aa 87.7% 48.6% 29.8% 35.5% 90.7%

Cry8Ba 89.8% 47.7% 31.3% 37.6% 91.1%

Cry8Ca 73.5% 57.7% 31.6% 33.3% 68.6%

Example 3 - Construction of the expression vector containing the new cry8Ka1 gene and obtaining the recombinant toxin.

For expression of the heterologous protein we used the commercial expression vector pET101-D / TOPO (Invitrogen - Figure 5). The vector was acquired in its linearized form with one abrupt and one cohesive end, complementary to the amplified gene insert end. In this system, the PCR product is directly cloned by the addition of the four base pairs of the sense orienting oligonucleotide. The cohesive end of the cloning vector (GTGG) invades the 5 ^' end of the PCR product, annealing with the four added bases (CACC) and stabilizes the PCR product in the correct orientation. Topoisomerase then cleaves the spare portion of the PCR product for binding to be effective (Figure 6). The inserts can be cloned in this way with 90% efficiency.

To amplify the cry8 gene with complementary ends, oligonucleotides based on the initiation codon (ATG) of the genes were designed, with addition of the CACC sequence in the 5 'region of the oligonucleotide in "sense" orientation, according to the system manufacturer's instructions. pET Directional TOP cloning (Invitrogen). The antisense oligonucleotide does not have the stop codon as it is soon after the polyhistidine tail (Figure 5). These oligonucleotides were then used in a 25 μΙ_ final volume PCR reaction containing 400 nM of each oligonucleotide, 200 mM dNTPs, 1 X pfu enzyme buffer, 2.5 U pfu DNA polymerase (Stratagene) and 10 ng of cry genes cloned into pGEMT-easy vector (Invitrogen). Amplification was performed in a thermocycler (Mastercycler Gradient - Eppendorf) under the following conditions: previous denaturation at 94 ° C for 1.5 minutes a repetition of 30 cycles at 94 ° C for 1 minute (denaturation); 55 ° C for 1 minute (oligonucleotide annealing) and 72 ° C for 2 minutes (DNA polymerase extension) and at the end a 72 ° C extension for 5 minutes.

The generated product was then subjected to a ligation reaction under the following conditions: 10 ng PCR product, 200 mM NaCl, 10 mM MgCl 2, 1 μΐ_ of the pET101 vector. The mixture was incubated at room temperature 25 ° C for 30 minutes. Competent E. coli TOP10 cells were transformed with 3 µl of the ligation system (10 ng) by thermal shock. For this procedure, the 10 ng DNA was mixed to 200 µl of competent cells and the mixture incubated on ice for 30 min. Thermal shock was performed for 3 minutes at 42 ° C. The cells were immediately transferred to ice and subsequently 500 μΙ of SOC culture medium (2% tryptone; 0.5% yeast extract; 0.05% NaCl; 2.5 mM KCI; 20 mM MgCl2) were added. Subsequently, cells were inoculated into 10 mL Luria-Bertani agar culture medium containing 100 μΜ ampicillin / mL and grown for 16 hours at 37 ° C. For positive clones, a colony PCR was performed, which used the transformed bacteria DNA as template and the same conditions described for gene cloning. Positive clones were then inoculated into 5 mL Luria-Bertani agar medium containing 100 μΜ ampicillin / mL.

For expression of the new gene, the generated plasmids were transformed by thermal shock into Escherichia coli BL21 (DE) Star cells (Invitrogen). 10 ng of the pET101 / cry8Ka1 vectors were added in 200 µl of competent cells and the mixture was incubated on ice for 30 minutes. The thermal shock was performed for 3 min at 42 ° C and then Cell mixture was placed on ice. Then 250 μΙ SOC was added and incubated for 30 minutes at 37 ° C and 200 rpm shaking. After this time the cells were inoculated in 10 ml LB-amp medium and grown for 16 hours. This culture was then used as a pre-inoculum for expression. For each 100 mL of Luria-Bertoni medium was added to 5 mL of the pre-inoculum. The material was incubated at 37 ° C with 200 rpm shaking. After the culture reached ϋθβοο between 0.6-0.8 the inducer (IPTG) was added at a concentration of 1 m and the culture remained at 37 ° C for a further 16 hours to obtain the recombinant Cr 8Ka1 toxin. Determining the optimal culture conditions for better yield of recombinant protein expression, cells were inoculated in 500 ml volumes. After 18 h of cultivation, the amount of cells was centrifuged for 10 minutes at 4000 g and the supernatant was discarded. The cell pellet was resuspended in 10 mL lysis buffer (50 mM phosphate buffer pH 7.8; 300 mM NaCl, 10% glycerol, 0.5% triton X-100 whether or not containing 2 mg / mL li - sozyme) and cells were ultrasonically disrupted (3 X 5 min). The lysed product was then centrifuged for 15 min at 10,000 g. The supernatant was then collected, quantified by the methodology described by Lowry et al. (Lowry, OH, NJ Rosebrough, AL Farr and RJ Randall. Protein measurement with the Folin phenol reagent. J Biol Chem, v.193, no.1, Nov, p.265-275. 1951).

In order to obtain the purified recombinant toxin, the obtained supernatant was subjected to nickel (Ni) affinity chromatography using 5 ml of the Ni-NTA (nitrilotriacetic acid-nickel) resin, with the ability to retain 5 -10 mg of recombinant polyhistidine tail protein. The resin was then packaged on a glass column and equilibrated with 4 column volumes with equilibration solution (50 mM sodium phosphate buffer pH 7.8; 300 mM NaCl and 10 mM imidazole). The sample was added (not exceeding the total resin capacity) and the non-retained portion reserved and quantified for analysis. Excess material was removed with the addition of 3 column volumes of equilibration buffer. Washing was performed with 6 column volumes of wash solution (50 mM phosphate buffer pH 7.8; 300 mM NaCl and 20 mM imidazole). The protein was eluted with two column volumes of elution buffer (50mM phosphate buffer pH 7.8; 300mM NaCl and 250mM imidazole). The eluted material was then dialyzed against 15 mM carbonate buffer (1.59 g Na ₂ CO ₃ and 2.93 g NaHCO ₃ ), quantified by Lowry and subjected to one-dimensional 12% polyacrylamide gel electrophoresis (Figure 7). Figure 7 shows the whole process of expression and purification of the new δ-endotoxin in 12% polyacrylamide gel.

Example 4 - Selective bioassays against cotton weed for determination of entomotoxic activity of new recombinant δ-endotoxin Cry8Ka1.

In order to verify the activity of recombinant toxins, selective bioassays were performed against the pest insects of interest. The selective bioassays were performed according to Praça eí al (Praça, LB, Batista, A.C, Martins, ES, Siqueira, CB, Dias, DGS, Gomes, ACMM, Falcão, R., Monnerat, GR Strains Of Bacillus thurigiensis Effective Against Insects Of The Orders Lepidoptera, Coleoptera And Diptera Brasília: Embrapa-Cenargen 2004, Vol 39, No 1, p 11-16), incorporating 50 pg / mL, 100 pg / mL, 200 pg / ml of the new recombinant toxin Cry8Ka1 in 5 ml artificial diet (at 50 ° C), poured into NUNC 6-well Cell Plates. After solidification of the diet, 15 holes of approximately 0.6 mm ² were drilled, where neonate larvae (one per hole) were inserted, and the reading was performed on the seventh day (Monnerat, RG, Dias, SC, Oliveira Neto, OB, Noble, SD, Silva-Werneck, OJ and Sa, MFG of Mass Creation of the Cotton Bug Anthonomus Grandis In Laboratory Brasilia: Embrapa-Cenargen, 2000. 4p. The bioassays were repeated three times and used as positive control cultures of Bacillus thuringiensis S811 strain containing the native cry8 gene and, as negative control, 15 mM carbonate buffer.

As external control to the experiment, bioassays were performed against neonate larvae of Spodoptera frugiperda. The bioassays showed significant toxic activity of Bacillus thuringiensis culture expressing native Cry8Ka1 toxin as well as pure recombinant toxin on Anthonomus grandis (Figure 8). Thus, the entiotoxic activity of the cry8Ka1 gene was confirmed.

Example 5 - Generation of mutant genes, analogous to the native cry8Ka1 gene, highly effective in controlling Anthonomus grandis by DNA shuffling technique.

The construction of a recombinant gene library analogous to the new cry8 gene is an important biotechnological strategy, contributing significantly to plant breeding programs via genetic transformation for transgenic generation. This technology provides a variety of new molecules with potential use in plant transformation for target insect control as well as improved insecticidal activity of new proteins encoded by recombinant genes. This factor becomes even more relevant when we consider the low expression levels of these heterologous proteins in genetically transformed plants.

Once the entomotoxic activity of the new Cry8Ka1 δ-endotoxin against the pest insect Anthonomus grandis was confirmed, we proceeded to the strategy of obtaining in vitro new genes, analogous to the cry8 gene, encoding the same Cry8Ka1 toxin. For this, the cry8Ka1 native gene was re-amplified by PCR with specific gene sequence-specific oligonucleotides, which contain the restriction enzyme sequence Sfi \ (5 ^' GGCCNN NNNGGCC3 ^' ) - These oligonucleotides were designed in our laboratory using the native cry8Ka1 gene sequence is cast and the 5 ^' and 3 ^' ends of the native gene introduced into the sequence of the enzyme in question (5 'oligonucleotide: Sfil F - 5 ^' CCCGGCCCAGGC GGCCGACCACGCGTATCGA 3 ^' and 3' oligonucleotide: Sfil R - 5 ^' CCCGGCCGGCCT GGCCGTTCAAGGAACCGTT 3 ^' ). These oligonucleotides were then used in a 25 µl final-volume PCR reaction containing 300 nM of each specific oligonucleotide, 200 nM dNTPs, 1 X taq enzyme buffer (PHT), 1 U DNA polymerase. (PHT) and 400 ng of cry8Ka1 DNA (active. Amplification was performed Thermocycler (Mastercycler Gradient - Eppendorf) under the following conditions: prior denaturation at 95 ° C for 5 minutes; a repeat of 29 cycles at 95 ° C for 40 seconds (denaturation), 45 ° C for 40 seconds (oligonucleotide annealing) and 72 ° C for 40 seconds (DNA polymerase extension) and at the end a 72 ° C extension for 2 minutes

The reaction generated a 2000 bp (base pair) product which was subjected to 1% agarose gel electrophoresis at 100 Volts for 90 minutes. The gene fragment was excised and eluted from the agarose gel using the Geneclean ^® II kit (Qbiogene). 100 µg of the new DNA product (Sfillcry8Ka1 / Sfil) was digested with the enzyme Sfil for 24 hours at 50 ° C. The enzyme digestion product was subjected to 1% agarose gel electrophoresis, excised and eluted from the gel. In the end, approximately 40pg of the new Sfil-digested DNA product (Sfil / cry8Ka1 / Sfíl) was obtained.

Following the protocol of the DNA scrambling technique described by Stemmer, WPCe / a., (Stemmer, WPC Rapid evolution of a protein in vitro. By DNA Shuffling. Nature. London, 1994, Vol. 370, pp. 389-391; Zhao, H. and Arnold, FH Functional and nonfunctional mutations distinguished by random recombination of homologous genes (Proc. Natl. Acad. Sci. USA., 1997, Vol. 94, p. 7997 - 8000), digestion was performed with the 10gp DNAsel nuclease of the new Sfil digested DNA product (Sfil / cry8Ka1 / Sfil). The reaction was conducted in the enzyme's own 10 U buffer and stopped by the addition of 26 mM EDTA (4-acetic acid 2-amino ethylene). After this step, the gene product is completely fragmented into small 30 to 50 bp gene pieces, which were purified with the High Pure PCR Product Purification ^® Kit (Roche). The purified fragments were used in a PCR reaction which followed the following conditions: 00 ng of the DNAsel digested pure product, 1 X Taq Platinum Buffer, 2.5 mM dNTPs, 0.5 mM MgSO ₄ , 2.5 U of Taq Platinum High Fidelity DNA Polymerase. The PCR reaction was performed in a thermocycler (Mastercycler Gradient - Eppendorf) under the following conditions: previous denaturation at 95 ° C for 2 minutes, a repetition of 43 cycles at 95 ° C for 1 minute (denaturation); 44 ° C for 1 min. (fragment annealing) and 72 ° C for 1 minute with an addition of 5 seconds per cycle (DNA polymerase extension) and at the end a 72 ° C extension for 7 minutes.

This DNA scrambling reaction is conducted without the addition of oligonucleotides, which ultimately generates an amount of fragments of varying sizes. This new product is then used in the second PCR reaction as a template under the following conditions: 1/3 of the volume of the first reaction product (template), 1 X Taq Platinum Buffer, 0.2 mM dNTPs, 0.8 μΜ of specific oligonucleotides Sf / IF and Sf / ΔR, 2mM MgSO ₄ and 25 U in the 1: 1 Taq Platinum High Fidelity (Invitrogen) / Taq PHT mixture. The amplification reaction was performed in a thermocycler (Mastercycler Gradient - Eppendorf) under the following conditions: previous denaturation at 95 ° C for 2 minutes, a repetition of 10 cycles at 95 ° C for 30 seconds (denaturation); 45 ° C for 30 seconds (fragment annealing), 72 ° C for 1 minute (DNA polymerase extension), another repeat of 14 cycles at 95 ° C for 30 seconds (denaturation), 43 ° C for 30 seconds (DNA annealing) 72 ° C for 42 seconds (DNA polymerase extension) with an increase of 20 seconds per cycle and at the end a 72 ° C extension for 7 minutes.

Thus, the original gene is reconstituted with modifications in its nucleotide structure, either by introducing, deleting or replacing nucleotides. This reconstructed final product was subjected to 1% agarose gel electrophoresis at 100 Volts for 90 minutes, excised and eluted from the gel with the Geneclean ^® II Kit (Qbiogene). The purified product, approximately 25 g, was then digested with restriction enzyme Sf / I (above conditions) and subjected to 1% agarose gel electrophoresis at 100 Volts for 90 minutes. The approximate size band of the original gene (approximately 2000 bp) was excised from the gel and the DNA eluted by the Geneclean ^® II Kit (Qbiogene) (Figure 9).

The end product (population of recombinant genes) with specific adapters becomes clonable in the pCOMB3X vector (Andris-Widhopf, J.; Rader, C.; Steinberger, P.; Fuller, R., Barbas III, CF Methods for the generation of chicken monoclonal antibody fragments by Phage display. Journal of Immunological Methods, 242: 159-181, 2000). Thus, the newly reconstructed genes (analogous to the cry8Ka1 native gene) were cleaved into the vector with the aid of the enzyme T4 DNA Ligase ^® (Invitrogen) and used to transform Escherichia coli XL1-Blue ^® (Stratagene) cells via electroporation. under the following conditions: capacitance 25 uFD, resistance 200 Ω, voltage 2.5 KVolts. Transformants were then seeded in plates containing Luria-Bertani Agar and Ampicilina ^® USB culture media (100 pg / mL). After 17 hours at 37 ° C the colonies grown on selective medium indicate the titer of the library containing ⁵⁰ transformants.

This library of cry8Ka1 analogs generated by DNA shuffling and fusion protein III of filamentous phage capsule M13 (fusion phages) was then selected by the Phage Display (Barbas III, CF) surface protein presentation technique. Burton, DR, Scott, JK, Silverman, GJ Selection from antibody libraries In: Phage display - A Laboratory Manual - USA: Cold Spring Laboratory, 10.1 - 10.20, 2001) using A. grandis BBMVs as ligands (Francis, BR , Maaty, WSA, Bulla- Jr, LA Effects of Midgut-Protein-Preparative and Ligand Binding Procedures on the Toxin Binding Characteristics of BT-R1, the Common High-Affinity Receptor in Manduca Friday for CrylA Bacillus thuringiensis Toxins Applied and Environmental Microbiology June 1998, Vol. 64, No. 6, pp. 2158-2165).

Cultivation of transformed E. coli XL1-Blue cells in SB medium containing 100pg mL carbenicillin, 5pg mL ^"1 tetracycline was incubated at 37 ° C with shaking until reaching optical density A550 = 0.6-0.8. Then 1x10 ¹² pfu mL ¹ µl of helper phage (VCS 13 ^® Stratagene) was added to produce fusion phages containing cry8Ka1 analogs incubated for 2 h at 37 ° C. Kanamycin 100pg mL ^{"1 was added} , and incubation followed for 12 h at 37 ° C. Cells collected by centrifugation were stored at -20 ° C for further DNA preparation. Fusion phages were precipitated with PEG-8000 (4 % w / v) for 30 minutes on ice and after centrifugation resuspended in 2 mL of 1% (w / v) ASB (bovine serum albumin) in saline. Collected after centrifugation, fusion phage preparation is used in the selection cycles.

In the binding affinity selection procedure, fused phages were deposited in wells of a microtitre plate previously sensitized with BBMVs (100 pg pL ^" ) extracted from the gut membrane of cotton boll weevils. At each round of selection, the wells are washed with PBS-Tween solution (137 mM NaCl, 2.7 mM KCI, 12 mM Na ₂ HP0 ₄ , 1.2 mM KH ₂ P0 ₄ and 0.05% Tween 20 ^® ) and specific phages eluted at low pH are used. to transfect new E. coli cells Amplified phage particles are used in the successive selection cycle The procedure involved five wash, elution and amplification cycles Titration of the colonies collected in each cycle is done by plating colonies into dilutions. SBen agar medium containing 100 pg mL ^-1 . Colonies isolated from the amount of specific phage eluted in the fifth selection cycle (showing the highest titer and thus representing the specific phage enrichment cycle) Figure 10 were amplified with cry8Ka1 gene specific oligonucleotides. Colonies showing PCR amplification and containing approximately 2000bp (original gene size) were selected for phage expression. Figure 11.

The cry8Ka1 gene and analogous genes selected in the fifth round of selection were expressed in selective medium (1% MOPS, 2% Yeast Extract, 3% Tryptone, 100 pg mL ^"1 ampicillin, 5 pg mL ^{" 1} tetracycline , 100 pg mL ^"1 Kanamycin, pH 7.0) containing the auxiliary phage VCSM13, incubated at 18 h at 37 ° C. The culture was centrifuged and the collected phages precipitated with PEG-NaCl (20% Polyethylene-Glycol 8000, 15% Sodium Chloride) for 30 minutes on ice After centrifugation the phages were resuspended in TBS saline (5 mM Tris-HCI, 15 mM NaCI, pH 7.5), again centrifuged, collected and stored at 4 ° C for use. immediate bioassay.

The selected analogues were evaluated by selective bioassays against the larvae of Anthonomus grandis. showed greater entomotoxic activity subjected to a sequencing reaction under the following conditions: 400-800 ng of plasmid DNA containing the analogs and 4 pM of specific oligonucleotides (Sf / IR and SfH F) in a 3130 xL Genetic Analyzer (AP- PLIED BIOSYSTEMS).

In the end, four sequences analogous to the new cry8Ka1 gene were selected, which are detailed in Table 2. The new molecules generated by the cry8Ka1 recombination showed significant differences of 13.29 to 16.33% base pairs and 2.10%. to 5.11% modified amino acid residues (Table 2). For sequence analysis and classification as analogous to the cry8Ka1 native gene, these molecules were grouped by nucleotide sequence similarity following the Cry toxin nomenclature system (http://www.lifesci.sussex.ac.uk/home/ Neil Crickmore / Bt /). Due to a <5% variation between analog toxins and native Cry8Ka1 toxin, the new sequences were classified into the Cry8 toxin family, and were later named Cry8Ka2, Cry8Ka3, Cry8Ka4 and Cry8Ka5 (Table 2) (SEQ ID NO: 5-12).

Table 2. Modifications of nucleotide bases and amino acid residues generated by the DNA shuffling technique in the cry8Ka1 gene and mortality of neonates larvae of Anthonomus grandis fed with proteins expressed in the phage system.

Amino Acid Nucleotides

Modified Pairs DL 50

Modified Waste (%)

Base gene (%) (%) cry8Ka1 2001 - 666 - 36.1 cry8Ka2 1982 13.29 660 2.1 54.6 cry8Ka3 1991 13.25 663 2.84 63 cry8Ka4 1989 14.1663 2.99 50 cry8Ka5 1947 16.33 649 5.11 77.08

Despite the high number of nucleotide mutations in the generated sequences (from 13 to 16%) there were few modifications of residues. amino acids (from 2 to 5%) and deletion of amino acid residues at the 5 ^' end of the variants is also generated. The new Cry8Ka5 molecule was approximately 3 times more active than the original molecule (Cry8Ka1) exhibiting a 77% mortality in the diet-fed neonate larvae containing 6 pg protein per mL diet (Figure 12).

Example 6 - Determination of tertiary in silico structure of native Cry8Ka1 and analogous Cry8Ka5 toxins.

The tertiary structures of Cry8Ka1 toxins and Cry8Ka5 analog were predicted in silico and modeled by molecular modeling using the crystal structures of CrySBbl and Cry3A toxins (1ji6.pdb; Galitsky, N., Cody, V .; Wojtczak, A as a template). Ghosh, D.; Luft, JR; Pangborn, W. & L., L. Structure of Bacterial Insecticidal δ-endotoxin Cry3Bb1 of Bacillus thuringiensis. Acta Crystallographica, Section D, Biological Crystallography, 57: 1101-1109, 2001) and Cry3A (Idlc.pdb; Li, J .; Carral, J., Ellar, DJ. Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5A resolution. Nature, 353: 815-821, 1991) deposited in Protein framework (PDB), Multiple sequence alignment containing the template and toxin framework sequences for modeling was submitted to the Modeller Version 9.2 program (Sali A, Blundell TL: Comparative protein modeling by spatial restraints. J Mol Biol 1993, 234 (3): 77 9-815.

The models obtained by the Modeller program were analyzed for their stereochemical properties by the PROCHECK program (Laskowski RA, Macarthur MW, Moss DS, Thomton JM: Procheck - the Program to Check the Stereochemical Quality of Protein Structures. Journal of Applied Crystallography 1993, 26 : 283-291).

The Cry8Ka1 and Cry8Ka5 analog models showed the same structural skeleton, with differences observed only in the side chains of the amino acids substituted in the analog. The original and analogous Cry8Ka5 structures have the three conserved functional domains (I, II, and III), typical of Cry toxins, all mutations and / or substituted. located on the outer surface of the molecule (Figure 13).

Example 7 - Selective bioassays against cotton weed for determination of entomotoxic activity of new recombinant δ-endotoxin Cry8Ka1 and its analogues Cry8Ka2, Cr 8Ka3, Cry8Ka4 and Cry8Ka5.

Selective bioassays were performed following the same conditions previously described in example 3 of this invention. Figure 14 shows the results regarding the determined entomotoxic activities.

Example 8 - Design of a synthetic cry8Ka1 gene optimized for expression in cotton plants.

The design of the synthetic cry8Ka1 gene was based on the native cry8Ka1 gene sequence, including the three domains responsible for insecticidal activity, consisting of 666 amino acids. In the design of the synthetic cry8Ka1 gene, 262 base pairs were modified, resulting in the elimination of 25 possible polyadenylation signals, 17 instability motifs, 95 rarely used codons in plants and an increase in G-C content from 35.6 to 43.8%. The final protein sequence of the synthetic cry8Ka1 gene (SEQ ID NO: 4) is identical to the original sequence (SEQ ID NO: 2). A summary of the modifications introduced is presented in Table 3.

Table 3. Modifications made to the nucleotide sequence of the synthetic cry8Ka1 gene and the parameters taken into account for sequence modifications (SEQS ID 03 and 04).

N-terminal segment domains I,

Cry8Ka1 Synthetic Cry8Ka1 II & III

Base pairs (bp) 1998 bp = 666 aa 1998 bp = 666 aa

A 690 558

T 597 565 and 333 441

G 378 434

A + T 1287 (64.4%) 1123 (56.2%)

C + G 711 (35.6%) 875 (43.8%) modified bp 0 262 (13%)

Modified Codons 0 261 (39%) N-terminal segment domains 1,

Cry8Ka1 Synthetic Cry8Ka1 II & III

Base pairs (bp) 1998 bp = 666 aa 1998 bp = 666 aa

Reason A l 1 IA 17 0

Polyadenite Sites

26 1 putative lesson

NCG Codons 23 0

NTA 72 Codons 0

To modify the cry8Ka1 gene sequence, the TDL-PCR Direct Polymerase Reaction Mold Linkage methodology, described by Strizhov et al. (Strizhov, N .; Keller, M .; Mathur, J .; Koncz-Kálmán, K .; Bosch, D.; Prudovsky, E .; Schne, J .; Koncz, C.; Zilberstein, A; Synthetic crylC gene, encoding Bacillus thuringiensis endotoxin, confers Spodoptera resistance in alfalfa and tobacco (Proc. Natl. Acad. Sci. USA, v. 93, pp. 5012-5017, 1996)

The cry8Ka1 gene sequence was divided into three blocks named A, B and C with 595, 665 and 753 bp, respectively. Blocks A and B are delimited by a Nde I site and blocks B and C by a Spe I site. For the synthesis of block A 6 oligonucleotides ', for block B, 7 oligonucleotides' and for block C, 9 Oligonucleotides'. Oligonucleotides at the ends of each block contain unique sequences not complementary to the original gene for subsequent selective PCR amplification. Within each block there is no overlap in the sequence of oligonucleotides.

Example 9 - Construction of the cry8Ka1 synthetic gene optimized for expression in cotton plants.

Briefly, the methodology used, 'template directed binding-PCR' - TDL-PCR, described by Strizhov et al. (Strizhov, N .; Keller, M .; Mathur, J .; Koncz-Kálmán, K .; Bosch, D.; Prudovsky, E .; Schne, J .; Koncz, C.; Zilberstein, A; The synthetic crylC gene encoding Bacillus thuringiensis endotoxin, confers Spodoptera resistance in alfalfa and tobacco (Proc. Natl. Acad. Sci. USA, v. 93, pp. 15012-15017, 1996) consists of the following: following steps: (1) Sequence analysis and chemical synthesis of oligonucleotides with the modifications to be introduced; (2) Production of single stranded DNA from the gene sequence that will be used as a template in the subsequent step; (3) Ringing the oligonucleotides with the partially complementary single stranded template DNA derived from the original gene and ligating the oligos using a DNA ligase; (4) Selective amplification and synthesis of the second strand of synthetic DNA by PCR, with oligonucleotides complementary only to synthetic DNA; (5) Gene assembly, subcloning and sequencing.

Table 3 shows the modifications introduced to the nucleotide sequence of the synthetic cry8Ka1 gene. The Table also shows the parameters taken into account for sequence modifications.

Modifications of the embodiments herein related to the present invention may be envisioned by those skilled in the art to which this invention relates based on the teachings set forth herein and their Figures. Therefore, it is understood that the invention is not limited to the specifically disclosed embodiments and that any modifications and other embodiments may be included within the scope of the invention disclosed herein.

All publications and patent applications mentioned in the specification are indicative of the state of the art to which this invention belongs. All publications and patent applications are incorporated herein by reference.

Claims

An isolated nucleic acid molecule characterized in that it encodes a protein with pest insect activity wherein said molecule comprises a nucleotide sequence capable of encoding a protein having at least 95% identity to the amino acid sequence of the sequence SEQ ID NO. 2.

An isolated nucleic acid molecule according to claim 1, characterized in that the pest insect is preferably cotton boll weevil.

3. Isolated nucleic acid molecule characterized in that it has the original and / or optimized plant-expression domains I, II and III, where the nucleotide sequence comprises the sequence described in SEQ ID NO. 3

4. A gene construct comprising: a) a polynucleotide comprising the sequence described in

SEQ ID NO. 3; and

b) an active promoter operably linked to the polynucleotide defined in (a).

A vector comprising the isolated nucleic acid molecule of claim 1 or a fragment thereof.

A vector according to claim 5, characterized in that said vector is capable of promoting expression of the molecule of interest or a fragment thereof.

7. A transgenic cell characterized in that it contains a polynucleotide sequence optimized for expression in plants, wherein the polynucleotide comprises the sequence described in SEQ ID NO. 3

Transgenic cell according to claim 7, characterized in that the cell consists of a plant cell.

Transgenic cell according to claim 7, characterized in that the cell consists of a microbial cell.

10. Method for obtaining a transgenic cell, comprising the following steps: a) transforming a cell with a gene construct according to claim 4; and

b) regenerate the transformed cell containing the gene construct of interest stably inserted into its genome under optimal cell culture growth conditions; and

c) express the gene product of the construct inserted in the regenerated cell.

Method according to claim 10, characterized in that the cell is of a microorganism.

Method according to claim, characterized in that the microorganism is a root colonizing bacterium.

Method according to claim 10, characterized in that the cell is a plant cell.

14. Method for obtaining a transgenic plant comprising the following steps:

a) transforming a plant cell with a gene construct according to claim 4;

b) culturing the transformed cell containing the gene construct of interest stably inserted into its genome under optimal cell culture growth conditions; and

c) regenerate a transgenic plant expressing the product of the inserted construct from the transformed cell.

Method according to claim 14, characterized in that the plant is monocotyledonous or dicotyledonous.

Method according to claim 15, characterized in that the dicot is a cotton plant.

17. Isolated and purified polypeptide comprising the amino acid sequence as described in SEQ ID NO. 2.

Polypeptide according to claim 17, characterized in that it exhibits insecticidal activity when administered orally to susceptible insect larvae.

Polypeptide according to claim 17, characterized in exhibited insecticidal activity when fed on a diet administered orally to a Coleoptera insect larva.

Polypeptide according to claim 19, characterized in that the insect larva is a cotton boll weevil.

2 . Isolated polypeptide, characterized in that it is encoded by a nucleic deoxyribonucleic acid segment comprising the open reading frame described in SEQ ID NO. 3, from nucleotide 001 to nucleotide 2001.

Isolated polypeptide according to claims 17 and 21, characterized in that it is an δ-endotoxin.

Biodegradable pesticidal composition comprising an effective concentration of the isolated polypeptide according to claim 22 or mutant analog in an agronomically acceptable carrier vehicle.

Biodegradable pesticidal composition according to claim 23, characterized in that the acceptable carrier carrier is a transformed microorganism.

Biodegradable pesticidal composition according to claim 23, characterized in that an acceptable carrier may be a surface active agent, an inert carrier, a preservative, a humectant, a food stimulant, an attractant, an agent. encapsulant, binder, emulsifier, dye, UV (ultraviolet) protector, buffer, flow agent or fertilizer, micronutrient donors, or other preparations that influence plant growth.

Biodegradable pesticidal composition according to claim 23, characterized in that the polypeptide according to claim 22 is used in combination with δ-endotoxins Bt or other insecticidal proteins.

27. A pest control method comprising:

a) detect the occurrence of the pest in an environment; b) contacting the pest with an isolated pesticidal protein or with a composition of the invention, wherein said protein consists of the amino acid sequence described in SEQ ID NO. 4

28. Method for obtaining transgenic strains resistant to a pest insect, characterized by the following steps:

a) transforming a cultivar of interest with a gene construct according to claim 4;

b) regenerate transgenic strains containing said construct stably integrated into their genomes;

c) select the transgenic strains with the highest levels of δ-endotoxin expression of the invention.