RU2816526C2 - Engineered pesticide proteins and plant pest control methods - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to a chimeric insecticide protein, which is toxic for a pest related to Lepidoptera. Disclosed is a polynucleotide coding said protein; an expression cassette and a cell containing said polynucleotide; an expression vector comprising said expression cassette; transgenic plant, part thereof or transgenic seed containing said cell.

EFFECT: invention makes it possible to effectively control a pest related to lepidoptera.

11 cl, 3 dwg, 12 tbl, 7 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to engineered pesticidal proteins and the nucleic acid molecules that encode them, as well as compositions and methods for controlling plant pests.

BACKGROUND OF THE INVENTION

[0002] Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming soil bacterium that is distinguished by its ability to produce crystalline inclusions that are particularly toxic to certain orders and species of plant pests, including insects, but are harmless to plants and other non-target organisms. For this reason, compositions containing Bacillus thuringiensis strains or insecticidal proteins thereof can be used as environmentally acceptable insecticides for the control of agriculturally important insect pests or insect vectors of various human or animal diseases.

[0003] Crystal (Cry) proteins from Bacillus thuringiensis have strong insecticidal activity against predominantly lepidopteran, dipteran and coleopteran insect pests. These proteins have also demonstrated activity against pests from the orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga and pests from the orders Acari, as well as other groups of invertebrates such as Nemathelminthes, Platyhelminthes and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, N.Y.). These proteins were initially divided into classes CryI - CryVI, based primarily on their insecticidal activity. The main classes were Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and Nematode-specific (V) and (VI). The proteins were then divided into subfamilies, and proteins with a higher degree of relatedness within each family were assigned letters to denote the section, such as: CryIA, CryIB, CryIC, etc. Even more closely related proteins within each section were given names, such as CryIC(a), CryIC(b), etc.

[0004] The expressions "Cry toxin" and "delta endotoxin" have been used interchangeably with the expression "Cry protein". Current nomenclature of Cry proteins and genes is based on amino acid sequence homology rather than specificity for target insects (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In this more common classification, each toxin is assigned a unique name, including a primary rank (Arabic numeral), secondary rank (capital letter), tertiary rank (lowercase letter), and quaternary rank (another Arabic numeral). In the modern classification, Roman numerals have been replaced by Arabic numerals in the primary rank. For example, "CryIA(a)" according to the old nomenclature is now called "CryIAa" according to the modern nomenclature. According to Ibrahim et al. (2010, Bioeng. Bugs, 1:31-50) Cry toxins have been divided into six major classes according to their insect host specificity and include: group 1 Lepidoptera (eg, CryI, Cry9 and Cry15); group 2 - lepidoptera and diptera (for example, Cry2); group 3 - Coleoptera (Cry3, Cry7 and Cry8); group 4 - dipterans (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19 and Cry20); group 5 - Lepidoptera and Coleoptera (Cry1I); and group 6 - nematodes (Cry6). The toxins CryII, Cry2, Cry3, Cry10, and Cry11 (73–82 kDa) are unique because they appear to result from natural truncations of the larger CryI and Cry4 proteins (130–140 kDa).

[0005] Cry proteins are globular protein molecules that accumulate as protoxins in crystalline form during the sporulation stage of Bt. Once eaten by a pest, the crystals typically dissolve to release protoxins, the size of which can vary, for example from 130-140 kDa in the case of Cry proteins, active against many lepidopterans, such as Cry1 and Cry9, and 60-80 kDa in the case of Cry3 proteins , active against Coleoptera, and Cry2 proteins, active against Lepidoptera/Diptera. Once the crystals dissolve in the susceptible insect, the released protoxins are processed by proteases in the insect's gut, such as trypsin and chymotrypsin, to form the protease-resistant Cry protein toxin core. This proteolytic processing involves the removal of amino acids from different sites on the various Cry protoxins. For example, 130-140 kDa Cry protoxins are typically activated by proteolytic removal of an N-terminal 25-30 amino acid peptide and approximately half of the remaining protein at the C terminus, resulting in the formation of a mature Cry toxin of approximately 60-70 kDa. Protoxins 60–80 kDa in size, such as Cry2 and Cry3, are also processed, but not to the same extent as larger protoxins. Smaller protoxins typically have the same or more amino acids removed from the N-terminus than larger protoxins, but fewer amino acids are removed from the C-terminus. For example, proteolytic activation of Cry2 family members typically involves the removal of approximately 40-50 N-terminal amino acids. Many Cry proteins are quite toxic to certain target insects, but many have narrow spectra of activity.

[0006] The toxin portions of Cry proteins typically contain five conserved sequence blocks and three conserved structure domains (see, eg, de Maagd et al. (2001) Trends Genetics 17:193-199). The first domain with a conserved structure, called domain I, typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three beta-pleated sheets arranged in a Greek key configuration, while domain III consists of two antiparallel beta-pleated sheets in a 'jelly-roll' structure (de Maagd et al., 2001, supra ). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.

[0007] Five conserved sequence blocks are numbered CB1 to CB5 from the N-terminus to the C-terminus of the Cry protein (Hofte & Whitely, 1989, Microbiol. Rev. 53:242-255). Conserved block 1 (CB1) contains approximately 29 amino acids, conserved block 2 (CB2) contains approximately 67 amino acids, conserved block 3 (CB3) contains approximately 48 amino acids, conserved block 4 (CB4) contains approximately 10 amino acids, and conserved block 5 (CB5) contains approximately 12 amino acids. The sequences upstream and downstream of these five conserved blocks are highly variable and are thus designated "variable regions" V1-V6. Domain I of Bt delta endotoxin typically contains variable region 1, conserved box 1, variable region 2, and the N-terminal amino acids of conserved box 2. Domain II typically contains approximately 15 C-terminal amino acids of conserved box 2, variable region 3, and approximately 10 N -terminal amino acids of conserved box 3. Domain III typically contains approximately 38 C-terminal amino acids of conserved box 3, variable region 4, conserved box 4, variable region 5, and conserved box 5. Among other delta endotoxins, the lepidopteran-active toxins Cry1 have a variable region 6 with approximately 1-3 amino acids lying in domain III.

[0008] Numerous commercially valuable plants, including common crops, are susceptible to attack by plant pests, including insect pests and nematode pests, resulting in significant reductions in crop yield and quality. For example, plant pests are a major factor in yield loss in the world's important crops. Damage caused by invertebrate pests, including insects, costs the United States approximately $8 billion annually. Insect pests also become a burden for vegetable and fruit growers, producers of ornamental flowers, as well as homestead owners.

[0009] Insect pests are controlled primarily through the intensive application of chemical pesticides, which act by inhibiting insect growth, inhibiting feeding or reproduction, or causing mortality. Biological pest control agents, such as strains of Bacillus thuringiensis expressing pesticide toxins such as Cry proteins, have also been applied to crop plants with satisfactory results, providing an alternative or complement to chemical pesticides. The genes encoding some of these Cry proteins have been isolated and their expression in heterologous hosts, such as transgenic plants, has been shown to provide another tool for the control of economically important insect pests.

[0010] Thus, good insect control can be achieved, but continued use of certain chemical and biological control methods increases the likelihood of insect pests developing resistance to such control measures. This has been partially mitigated through various resistance control practices, but there remains a need to develop new and effective pest control products. Particularly needed are control agents that can target a broader range of economically important insect pests and/or that provide effective control of lines of insects that may or are becoming resistant to existing insect control agents.

SHORT DESCRIPTION

[0011] The present invention provides nucleic acids, polypeptides, compositions and methods for imparting pesticidal activity (eg, insecticidal activity) to bacteria, plants, plant cells, tissues and seeds. In particular, the present invention provides novel chimeric pesticide proteins (eg, chimeric insecticidal proteins), optionally with altered or increased activity compared to the parent molecule.

[0012] In embodiments, the chimeric proteins of the present invention are toxic to economically important insect pests (eg, by inhibiting the ability of the insect pests to survive, grow, and/or reproduce), especially insect pests that infest plants. For example, in embodiments, the chimeric insecticidal proteins of the present invention can be used to control one or more economically important lepidopteran pests, including, but not limited to, armyworm (Agrotis ipsilon), corn borer (Ostrinia nubilalis), armyworm (Spodoptera frugiperdd), American corn armyworm (Helicoverpa zed), cane moth (Diatraea saccharalis), velvet bean armyworm (Anticarsia gemmatalis), soybean armyworm (Chrysodeixis includes), southwestern corn moth (Diatraea grandiosella), western bean armyworm (Richia albicosta ), tobacco bollworm (Heliothis virescens), eastern corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), yellow rice moth (Chilo suppressalis), pink stem moth (Sesamia calamistis), rice moth (Cnaphalocrocis medinalis), etc. . In embodiments, the chimeric insecticidal protein has activity against the fall armyworm insect pest that is resistant to the Vip3A protein and/or the Cry1F protein.

[0013] Accordingly, as one aspect, the present invention provides a chimeric insecticidal protein that is toxic to a lepidopteran insect pest, comprising, in an N-terminal to C-terminal direction: (a) the N-terminal region of a first Cry1 protein which is optionally the N-terminal region of the BT-0029 protein of SEQ ID NO: 2 or an amino acid sequence that is substantially identical thereto fused to (b) the C-terminal region of another Cry1 protein; wherein the transition position between the first Cry1 protein and the other Cry1 protein is in conserved block 3. In illustrative embodiments, the other Cry1 protein is a Cry1F protein (e.g., Cry1Fa), Cry1G, Cry1I (e.g., Cry1Ia, or Cry1If, such as BT-0022) , Cry1C (eg Cry1Ca) or Cry1K.

[0014] In embodiments, the chimeric insecticidal protein of the present invention has insecticidal activity against the insect pest Spodoptera frugiperda or a population of insect pests with resistance to the Vip3A protein and/or the Cry1F protein.

[0015] As a further aspect, the present invention provides a nucleotide sequence encoding the chimeric insecticidal proteins of the present invention, and expression cassettes and vectors containing the same. In embodiments, the polynucleotide is codon-optimized for expression in a plant (eg, a monocotyledonous plant such as maize or a dicotyledonous plant such as soybean).

[0016] As a further aspect, the present invention provides a transgenic cell (e.g., a transgenic plant cell such as a dicot cell or a monocot cell, or a transgenic bacterial cell), a transgenic plant part, a transgenic plant crop, and a transgenic plant seed that comprise a nucleotide sequence , expression cassette, vector and/or chimeric insecticidal protein of the present invention.

[0017] As yet another aspect, the present invention provides transgenic plants comprising a plant cell, plant part, nucleotide sequence, expression cassette, vector and/or chimeric insecticidal protein of the present invention.

[0018] As a further aspect, there are seeds from which the transgenic plants of the present invention are obtained and seeds obtained using the transgenic plants of the present invention.

[0019] Also provided are assembled products derived from transgenic plants of the present invention, wherein the assembled product optionally contains the nucleotide sequence, expression cassette, vector and/or chimeric insecticidal protein of the present invention. Additionally provided are processed products derived from the harvested products of the present invention, wherein the harvested product optionally contains the nucleotide sequence, expression cassette, vector and/or chimeric insecticidal protein of the present invention. In embodiments, the harvested product or processed product contains the chimeric insecticidal protein of the present invention and has increased resistance to an insect pest (eg, a lepidopteran insect pest).

[0020] As another aspect, the present invention provides an insecticidal composition comprising a chimeric insecticidal protein of the present invention and an agriculturally acceptable carrier.

[0021] In addition, the present invention provides as a further aspect a method for producing a transgenic plant with increased resistance to an insect pest (eg, a lepidopteran insect pest). In embodiments, the method includes introducing into a plant a polynucleotide, expression cassette, or vector of the present invention, wherein the chimeric insecticidal protein is expressed in the plant, thereby producing a plant with increased resistance to the insect pest. The introduction step optionally includes: (i) transforming a plant cell with a polynucleotide, expression cassette or vector and regenerating the transgenic plant; or (ii) crossing a first plant containing the polynucleotide, expression cassette or vector with a second plant. In embodiments, the method further includes obtaining seed from the transgenic plant. In embodiments, the method further comprises producing a progeny plant from a transgenic plant, wherein the progeny plant comprises a polynucleotide, expression cassette, or vector, expresses a chimeric insecticidal protein, and has increased resistance to an insect pest.

[0022] As another aspect, the present invention provides a method for producing a transgenic plant with increased resistance to an insect pest (for example, a lepidopteran insect pest), the method comprising: (a) sowing a seed containing a polynucleotide cassette expression or vector of the present invention; and (b) growing a transgenic plant from a seed, where the transgenic plant contains a polynucleotide, expression cassette or vector, and produces a chimeric insecticidal protein, and has increased resistance to an insect pest. In embodiments, the method further comprises: (c) collecting seed from the transgenic plant of (b), wherein the collected seed contains a polynucleotide, an expression cassette, a vector, and/or a chimeric insecticidal protein. Optionally, the seed has increased resistance to an insect pest (eg, a lepidopteran insect pest).

[0023] Additionally, as another aspect, the present invention provides a method for obtaining a seed. In embodiments, the method includes: (a) producing a transgenic plant that contains a polynucleotide, expression cassette, or vector of the present invention; and (b) collecting seed from the transgenic plant of (a), wherein the collected seed contains a polynucleotide, expression cassette, or vector, and/or chimeric insecticidal protein of the present invention. Optionally, the seed has increased resistance to an insect pest (eg, a lepidopteran insect pest).

[0024] The present invention further provides a method for producing a hybrid plant seed. In illustrative embodiments, the method includes: (a) crossing a first inbred plant that is a transgenic plant containing a polynucleotide, expression cassette, or vector of the present invention with another inbred plant that may or may not contain a polynucleotide, expression cassette, or vector of the present invention. the present invention; and (b) allowing the formation of a hybrid seed. In embodiments, the hybrid seed comprises a polynucleotide, expression cassette, or vector, and/or chimeric insecticidal protein of the present invention. Optionally, the seed has increased resistance to an insect pest (eg, a lepidopteran insect pest).

[0025] As another aspect, the present invention provides a method for controlling an insect pest (e.g., a lepidopteran insect pest such as cutworm), the method comprising delivering to the pest or its habitat a composition, containing an effective amount of a chimeric insecticidal protein or insecticidal composition of the present invention. In embodiments, the method is a method for controlling a lepidopteran insect pest (eg, armyworm) that is resistant to the Vip3A protein and/or the Cry1F protein.

[0026] Accordingly, as another aspect, the present invention provides a method for reducing the development of resistance to the Vip3A protein and/or the Cry1F protein in a population of a target lepidopteran insect pest (eg, cutworm). In embodiments, the method includes delivering to the target population or its habitat a transgenic plant containing (i) a polynucleotide, expression cassette or vector of the present invention and (ii) a polynucleotide containing a nucleotide sequence encoding a Vip3A protein and/or a nucleotide sequence encoding the protein Cry1F; wherein the chimeric insecticidal protein and the Vip3A protein and/or the Cry1F protein are produced in the transgenic plant.

[0027] The present invention is also directed to methods of using the polynucleotides of the present invention, for example, in DNA constructs or expression cassettes or vectors for transformation and expression in organisms, including plants and microorganisms such as bacteria. The nucleotide or amino acid sequences may be native or synthetic sequences that have been engineered for expression in an organism such as a plant or bacteria. In addition, the present invention is directed to methods for producing the insecticidal proteins of the present invention and to methods of using the polynucleotide sequences and insecticidal proteins, for example, in microorganisms to control insects, or in transgenic plants to provide protection against insect damage.

[0028] Another aspect of the present invention includes insecticidal compositions and formulations containing the chimeric insecticidal proteins of the present invention, and methods of using the compositions or formulations to control insect populations, for example, by applying the compositions or formulations to insect-infested areas, or as a preventative treatment insect-susceptible areas or plants to provide protection against insect pests. Optionally, the compositions or compositions of the present invention may, in addition to the chimeric insecticidal protein of the present invention, contain other pesticidal agents, such as chemical pesticides, other pesticidal proteins or dsRNA, for example, to increase or enhance the ability of the composition or composition to provide insect control and/or or to control insect resistance to pesticides.

[0029] The compositions and methods of the present invention are useful for the control of insect pests that attack plants, in particular crop plants. The compositions of the present invention are also useful for detecting the presence of a chimeric insecticidal protein or a nucleic acid encoding it in commercial products or transgenic organisms.

[0030] The present invention also provides methods for using the chimeric insecticidal proteins, nucleic acids, transgenic plants, plant parts, seeds and insecticidal compositions of the present invention, for example, to control an insect pest, such as a lepidopteran pest.

[0031] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, vector, or host cell of the present invention to produce an insecticidal composition for controlling an insect pest (eg, a lepidopteran insect pest).

[0032] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, or vector of the present invention to produce a transgenic seed, wherein the transgenic seed grows into a transgenic plant with increased resistance to an insect pest.

[0033] As another aspect, the present invention also contemplates the use of a transgenic plant of the present invention to produce a transgenic seed, which is not necessarily a hybrid seed.

[0034] In embodiments, the present invention provides a method of using a chimeric insecticidal protein, polynucleotide, expression cassette, vector, transgenic plant, or insecticidal composition of the present invention to prevent the development of resistance in a population of a target lepidopteran insect pest to the Vip3A protein and /or Cry1F protein.

[0035] These and other features, aspects and advantages of the present invention will become better understood with reference to the following detailed description and claims.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0036] Figures 1A and 1B show the amino acid sequence alignment of full-length BT-0029 (SEQ ID NO: 2), BT-0022 (SEQ ID NO: 1), Cry1Fa (SEQ ID NO: 8) and Cry1Ka (SEQ ID NO: 12). Core domain III and conserved block 3 (CB3) are indicated. Identical amino acids in aligned sequences are shaded.

[0037] Figures 2A and 2B show an amino acid sequence alignment of full-length BT-0029 (SEQ ID NO: 2) with exemplary BT-0029 chimeras: Bt29-Bt22 (SEQ ID NO: 3), Bt29-1Fa (SEQ ID NO: 9 ) and Bt29-1Fa (SEQ ID NO: 13). Core domain III and conserved block 3 (CB3) are indicated. The domain III core indicated in the figure is derived from the second Cry protein. Identical amino acids in aligned sequences are shaded.

[0038] Figures 3A, 3B, 3C and 3D show the full-length chimera Bt29-Bt22 (SEQ ID NO: 3) aligned with a series of chimeras Bt29-Bt22, Bt29-Bt22Tr1 (SEQ ID NO: 20), Bt29-Bt22Tr2 (SEQ ID NO: 21), Bt29-Bt22Tr3 (SEQ ID NO: 22), Bt29-Bt22Tr4 (SEQ ID NO: 23), Bt29-Bt22Tr5 (SEQ ID NO: 24) and Bt29-Bt22Tr6 (SEQ ID NO: 25), with C-terminal truncations within the tail of protoxin BT-0029.

BRIEF DESCRIPTION OF SEQUENCES IN THE LIST OF SEQUENCES

[0039] SEQ ID NO: 1 is the amino acid sequence of the BT-0022 protein.

[0040] SEQ ID NO: 2 is the amino acid sequence of the BT-0029 protein.

[0041] SEQ ID NO: 3 is the amino acid sequence of the Bt29-Bt22 chimera. The sequence of BT-0022 is amino acids 459-597.

[0042] SEQ ID NO: 4 is the nucleotide sequence encoding the Bt29-Bt22 chimera of SEQ ID NO: 3.

[0043] SEQ ID NO: 5 is an exemplary maize optimized sequence encoding the Bt29-Bt22 chimera of SEQ ID NO: 3.

[0044] SEQ ID NO: 6 is an exemplary maize optimized sequence encoding the Bt29-Bt22 chimera of SEQ ID NO: 3.

[0045] SEQ ID NO: 7 is an exemplary maize optimized sequence encoding the Bt29-Bt22 chimera of SEQ ID NO: 3.

[0046] SEQ ID NO: 8 is the amino acid sequence of full-length Cry1Fa.

[0047] SEQ ID NO: 9 is the amino acid sequence of the Bt29-1Fa chimera. The sequence of Cry1Fa is amino acids 459-597.

[0048] SEQ ID NO: 10 is the nucleotide sequence encoding the Bt29-1Fa chimera of SEQ ID NO: 9.

[0049] SEQ ID NO: 11 is an exemplary maize optimized sequence encoding the Bt29-1Fa chimera of SEQ ID NO: 9.

[0050] SEQ ID NO: 12 is the amino acid sequence of full-length Cry1Ka.

[0051] SEQ ID NO: 13 is the amino acid sequence of the Bt29-1Fa chimera. The sequence of Cry1Ka is amino acids 459-597.

[0052] SEQ ID NO: 14 is the nucleotide sequence encoding the Bt29-1Fa chimera of SEQ ID NO: 13.

[0053] SEQ ID NO: 15 is the amino acid sequence of the Bt29-1Fav2 chimera.

[0054] SEQ ID NO: 16 is the nucleotide sequence encoding the Bt29-1Fa chimera of SEQ ID NO: 15.

[0055] SEQ ID NO: 17 is the amino acid sequence of full-length Cry1Ca.

[0056] SEQ ID NO: 18 is the amino acid sequence of the Bt29-1Ca chimera. The sequence of Cry1Ca is amino acids 459-597.

[0057] SEQ ID NO: 19 is the nucleotide sequence encoding the Bt29-1Ca chimera of SEQ ID NO: 18.

[0058] SEQ ID NO: 20 is the amino acid sequence of the Bt29-Bt22Tr1 protein.

[0059] SEQ ID NO: 21 is the amino acid sequence of the Bt29-Bt22Tr2 protein.

[0060] SEQ ID NO: 22 is the amino acid sequence of the Bt29-Bt22Tr3 protein.

[0061] SEQ ID NO: 23 is the amino acid sequence of the Bt29-Bt22Tr4 protein.

[0062] SEQ ID NO: 24 is the amino acid sequence of the Bt29-Bt22Tr5 protein.

[0063] SEQ ID NO: 25 is the amino acid sequence of the Bt29-Bt22Tr6 protein.

[0064] SEQ ID NO: 26 is the amino acid sequence of the BT29BT22-TL22v1 protein.

[0065] SEQ ID NO: 27 is the amino acid sequence of the BT29BT22-TL22v2 protein.

[0066] SEQ ID NO: 28 is the amino acid sequence of the BT29BT22-TL22v3 protein.

[0067] SEQ ID NO: 29 is the amino acid sequence of the BT29BT22-TL22v4 protein.

[0068] SEQ ID NO: 30 is the amino acid sequence of the BT29BT22-TL22v5 protein.

[0069] SEQ ID NO: 31 is the amino acid sequence of the BT29BT22-TL22v6 protein.

[0070] SEQ ID NO: 32 is the amino acid sequence of the BT291FaTr1 protein.

[0071] SEQ ID NO: 33 is the amino acid sequence of the BT291FaTr2 protein.

[0072] SEQ ID NO: 34 is the amino acid sequence of the BT291FaTr3 protein.

[0073] SEQ ID NO: 35 is the amino acid sequence of the BT291FaTr4 protein.

DETAILED DESCRIPTION

[0074] This description is not intended to be a detailed listing of all the different ways in which the present invention may be implemented or all features that may be added to the present invention. For example, features illustrated with respect to one embodiment may be included in other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, it is contemplated by the present invention that in some embodiments of the present invention, any feature or combination of features set forth herein may be eliminated or omitted. Moreover, numerous variations and additions to the various embodiments provided herein will be apparent to those skilled in the art in light of the present disclosure, which does not depart from the spirit of the present invention. Accordingly, the following descriptions are intended to illustrate some specific embodiments of the present invention and not to exhaustively define all of their transformations, combinations and variations.

[0075] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention relates. The terminology used herein in describing the present invention is intended to describe specific embodiments only and is not intended to limit the present invention.

[0076] All publications, patent applications, patents and other references cited herein are incorporated by reference in their entirety to explain the concepts of the sentence and/or paragraph in which the reference is provided.

[0077] The nucleotide sequences provided herein are presented in a 5'-3' direction from left to right and are represented using the standard code for representing nucleic bases as set forth in 37 CFR §§1.821-1.825 and the ST standard. 25. World Intellectual Property Organization (WIPO), for example: adenine (A), cytosine (C), thymine (T) and guanine (G).

[0078] Likewise, amino acids are designated using the ST.25 WIPO standard, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys ; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; l), leucine (Leu; L), lysine (Lys ; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), series (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0079] Unless the context indicates otherwise, it is specifically intended that the various features of the present invention described herein may be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be eliminated or omitted. For purposes of illustration, if the specification states that a composition contains components A, B, and C, it is specifically intended that any of A, B, or C, or a combination thereof, may be omitted and discarded individually or in any combination.

Definitions

[0080] As used in the description of the present invention and the accompanying claims, the singular number forms also include the plural forms unless the context clearly indicates otherwise.

[0081] As used herein, the expression “and/or” refers to and covers any and all possible combinations of one or more of the respective listed elements, as well as the absence of combinations when interpreted as an alternative (“or”).

[0082] The expression "about" as used herein when referring to a measurable quantity, such as a dosage or period of time, etc., is intended to cover changes in a specified amount of ±20%, ±10%, ±5%, ±1 %, ±0.5% or even ±0.1%. As used herein, phrases such as "from about X to Y" mean "from about X to about Y", and phrases such as "from about X to Y" mean "from about X to about Y".

[0083] As used herein, phrases such as “X to Y” should be interpreted to include X and Y unless the context indicates otherwise.

[0084] By "activity" of an insecticidal protein of the present invention, it is meant that the insecticidal protein functions as an orally active insect control agent that produces a toxic effect, for example, by inhibiting the ability of an insect pest to survive, grow and/or reproduce (e.g. causing morbidity and/or mortality), and/or is capable of disrupting and/or inhibiting insect feeding, which may or may not cause insect death. Thus, when the insecticidal protein of the present invention is delivered to an insect, the result is typically morbidity and/or mortality of the insect, and/or the insect reduces or stops feeding on the source that makes the insecticidal protein available to the insect.

[0085] In the context of the present invention, a "chimeric" protein is a protein created by fusing all or part of at least two different proteins. In embodiments of the present invention, the chimeric protein is a chimeric Cry protein comprising all or a portion of two different Cry proteins fused together into one polypeptide. A "chimeric insecticidal protein" is a chimeric protein that has insecticidal activity (as described herein).

[0086] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. In embodiments, the RNA is then translated to produce a protein.

[0087] As used herein, the term “codon-optimized” nucleotide sequence means a nucleotide sequence of a recombinant, transgenic, or synthetic polynucleotide in which the codons are selected to reflect the bias toward certain codons that may be observed in a host cell or organism. Typically, this is done in a manner that preserves the amino acid sequence of the polypeptide encoded by the codon-optimized nucleotide sequence. In certain embodiments, the nucleotide sequence is codon-optimized for the cell (eg, animal, plant, fungal, or bacterial cell) in which the construct will be expressed. For example, in a construct that will be expressed in a plant cell, the entire sequence or portions thereof may be codon optimized for expression in the plant. See, for example, US Pat. No. 6,121,014. In embodiments, the polynucleotides of the present invention are codon-optimized for expression in a plant cell (eg, a dicot cell or a monocot cell) or a bacterial cell.

[0088] "Control" of insect pests means inhibiting, through toxic action, the ability of insect pests to survive, grow, feed and/or reproduce, and/or limiting insect-related damage or death of a crop plant caused by the insect pest, and/or protecting the maximum yield potential of a crop caused by a pest when grown in the presence of the pest. “Controlling” an insect pest may or may not mean killing the insect, although in embodiments of the present invention, “controlling” an insect means killing the insect.

[0089] The term "contents", "comprises" or "comprising" when used herein indicates the presence of the set forth features, integers, steps, operations, elements or components, but does not exclude the presence or addition of one or more other features, integers numbers, stages, operations, elements, components and/or groups thereof.

[0090] As used herein, the transitional phrase "essentially consisting of" (and grammatical variations thereof) means that the scope of the claim should be interpreted to cover the specified materials or steps listed in the claim "and those which are not substantially change the main and new characteristic(s) of the claimed invention. Thus, the term "essentially consisting of" when used in the claims of the present invention is not intended to be interpreted as equivalent to the term "comprising".

[0091] In the context of the present invention, "corresponding" or "corresponds" means that when the amino acid sequences of modified or homologous proteins are aligned with each other, the amino acids that "correspond to" certain enumerated positions in the modified or homologous protein are those that align with these positions in the reference protein, but are not necessarily at these exact numerical positions relative to the particular reference amino acid sequence of the present invention. For example, if SEQ ID NO: 2 (BT-0029) is the reference sequence and is aligned with SEQ ID NO: 1 (BT-0022), as in Figure 1, the sequence TLEAVT immediately following domain III in SEQ ID NO: 1 (BT-0022), “corresponds” to the sequence TFEAEY immediately following domain III in SEQ ID NO: 2 (BT-0029).

[0092] As used herein, the term “Cry protein” means an insecticidal crystalline delta endotoxin type protein from Bacillus thuringiensis. The term “Cry protein” may refer to the protoxin form or any insecticidal active fragment or toxin thereof, including the partially processed form and the mature toxin form (eg, lacking the N-terminal peptidyl moiety and/or the C-terminal tail of the protoxin).

[0093] As used herein, the expression "delivery" or "deliver" (and grammatical variations) of a composition or insecticidal protein means that the composition or insecticidal protein comes into contact with the insect, promoting oral ingestion of the composition or insecticidal protein, resulting in a toxic effect and insect control. The composition or insecticidal protein can be delivered in a variety of known ways, including, but not limited to, expression in a transgenic plant, formulated protein composition(s), sprayable protein composition(s), a matrix with bait or any other protein delivery system known in the art.

[0094] The term “domain” refers to a set of amino acids conserved at certain positions along the length of the sequence alignment of evolutionarily related proteins. While amino acids at other positions of homologs may differ, amino acids that are highly conserved at certain positions indicate amino acids that are likely to be essential for protein structure, stability, or function. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified group of polypeptides.

[0095] “Insect control effective amount” means a concentration of insecticidal protein that provides toxic inhibition of the ability of insects to survive, grow, feed, and/or reproduce, or limits insect-related damage or loss of crop yield. "Insect control effective amount" may or may not mean killing the insect, although in embodiments it means killing the insect.

[0096] As used herein, the term “expression cassette” means a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest, such as a polynucleotide encoding an insecticidal protein of the present invention, in a suitable host cell, comprising a promoter operably linked with a polynucleotide of interest that is operably linked to the termination signal. The "expression cassette" also typically contains additional polynucleotides to facilitate proper translation of the polynucleotide of interest. The expression cassette may also contain other polynucleotides that are not associated with expression of the polynucleotide of interest, but which are present due to the appropriate restriction sites to extract the cassette from the expression vector. In embodiments, at least one of the components in the expression cassette may be heterologous (ie, foreign) to at least one of the other components (eg, a heterologous promoter operably linked to the polynucleotide of interest). An expression cassette may also be a sequence that occurs naturally but has been produced in recombinant form suitable for heterologous expression. However, typically the expression cassette is heterologous to the host, i.e. the expression cassette (or even the polynucleotide of interest) does not occur naturally in the host cell and has been introduced into the host cell or its progenitor cell by a transformation process or a mating process. Expression of the polynucleotide(s) of interest in an expression cassette is typically under the control of a promoter. In the case of a multicellular organism such as a plant, the promoter may also be specific or preferred for a particular tissue or organ or developmental stage (as described in more detail herein). Once transformed into a plant, the expression cassette or fragment thereof may also be referred to as an "inserted polynucleotide" or "insertion polynucleotide".

[0097] "Gene" is defined herein as a unit of heredity containing one or more polynucleotides that occupies a specific location on a chromosome or plasmid, and that contains the genetic instructions for a specific characteristic or trait found in an organism.

[0098] As used herein, the term “gut protease” refers to a protease that occurs naturally in the digestive tract of an insect. This protease is usually involved in the digestion of ingested proteins. Examples of alimentary canal proteases include trypsin, which typically cleaves peptides from the C-terminal portion of lysine (K) or arginine (R) residues, and chymotrypsin, which typically cleaves peptides from the C-terminal portion of phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0099] As used herein, the term “heterologous” means foreign, exogenous, non-native and/or not naturally occurring. In embodiments, a “heterologous” polynucleotide or polypeptide is a polynucleotide or polypeptide not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence or polypeptide. In embodiments, the nucleotide sequence is heterologous to another sequence to which it is operably linked, for example, a promoter may be heterologous (ie, foreign) to the operably linked coding sequence.

[00100] As used herein, “homologous” means native. For example, a homologous nucleotide sequence or an amino acid sequence means a nucleotide sequence or an amino acid sequence that is naturally associated with the host cell into which it is inserted, a homologous promoter sequence is a promoter sequence that is naturally associated with a coding sequence, and the like. .

[00101] The terms "increase", "increase", "increased", "boost", "elevated", "enhancement" and "improvement" (and grammatical variations thereof) and similar terms as used herein describe increased pest control plants, for example, by contacting the plant with a polypeptide of the present invention (such as, for example, by transgenic expression or topical application methods). This increase in control may be relative to the level of control of the plant pest in the absence of the polypeptide of the present invention (eg, a plant that does not transgenically express the polypeptide or is not locally treated with the polypeptide). Thus, in embodiments, the terms "increase", "increase", "increased", "raise", "elevated", "enhancement" and "improvement" (and grammatical variations thereof) and similar terms may indicate an increase of at least at least approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to a suitable control (e.g. plant, part of a plant, plant cell that is not in contact with the polypeptide of the present invention).

[00102] The term "insecticidal" as used herein is defined as a toxic biological activity capable of controlling an insect pest, optionally, but preferably by killing it.

[00103] A nucleic acid sequence is “isocodontal” with respect to a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

[00104] In illustrative embodiments, the nucleic acid molecules, polynucleotides, or polypeptides of the present invention are “isolated.” An "isolated" nucleic acid molecule, polynucleotide or protein, etc. is a nucleic acid molecule, polynucleotide or protein, etc., which is no longer found in its natural environment. The isolated nucleic acid molecule, polynucleotide or protein of the present invention may be in purified form or may be in a recombinant host, such as a transgenic bacterium or a transgenic plant. In embodiments, the isolated nucleic acid molecule, nucleotide sequence, or polypeptide is in purified form, that is, at least partially separated from at least some other components of the naturally occurring organism or virus, such as structural components of the cell or virus or other polypeptides, or nucleic acids usually found associated with a polynucleotide. In other embodiments, the "isolated" nucleic acid molecule, nucleotide sequence, or polypeptide may exist in a non-natural environment, such as a recombinant host cell. Thus, for example, with respect to nucleotide sequences, the term “isolated” may mean that the nucleotide sequence is separated from the chromosome and/or cell in which it naturally occurs. A polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs and is then inserted into a genetic environment, chromosome, and/or cell in which it does not naturally occur (e.g., another cell). host, different regulatory sequences and/or different genomic position compared to those found in nature). Accordingly, recombinant nucleic acid molecules, nucleotide sequences, and the polypeptides they encode are “isolated” because they, through human intervention, exist separately from their natural environment and, therefore, are not natural products, however, in some embodiments, they can be introduced into a recombinant cell -the owner and exist in it. In illustrative embodiments, the isolated nucleic acid molecule, isolated nucleotide sequence, and/or isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% pure. , 80%, 90%, 95%, or more.

[00105] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are often highly conserved portions of domains, but may also comprise only part of a domain or be located outside a conserved domain (if all amino acids in a motif extend beyond a particular domain).

[00106] A “native” or “wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, “wild-type mRNA” is mRNA that occurs naturally in or is endogenous to the body.

[00107] The terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “oligonucleotide,” and “polynucleotide” are used interchangeably herein unless the context otherwise indicates and refer to a heteropolymer of nucleotides. These terms include, without limitation, DNA and RNA molecules, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and RNA, plasmid DNA, mRNA, antisense RNA, and RNA/DNA hybrids, any of which may be linear or branched, single-stranded or double-stranded, or combinations thereof. In case dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used to pair antisense sequences, dsRNA and ribozymes. For example, polynucleotides that contain the C-5-propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications can also be made, such as modification of the phosphodiester backbone or the 2'-hydroxy group of the ribose RNA sugar. In embodiments, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “oligonucleotide,” or “polynucleotide” refer to DNA.

[00108] As used herein, the expressions “operably related” or “operably related” mean that the elements are functionally related to each other and are also typically physically related. Thus, as used herein, the expressions “operably linked” or “operably linked” refer to nucleotide sequences within a single nucleic acid molecule that are operably linked. Thus, the first nucleotide sequence having a functional relationship with the second nucleotide sequence means a situation where the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a nucleotide sequence if the promoter affects the transcription or expression of the nucleotide sequence. Those skilled in the art will appreciate that regulatory sequences (eg, a promoter) need not be contiguous to the nucleotide sequence to which they are operably linked as long as the regulatory sequences function to control its expression. Thus, for example, intervening untranslated yet transcribed sequences may be present between a promoter and a nucleotide sequence, and the promoter may still be considered to be “operably linked” or “operably linked” to the nucleotide sequence.

[00109] As used herein, the term “plant” refers to any plant at any stage of development.

[00110] Any plant (or grouping of plants, for example, into a genus or higher order of classification) can be used in the practice of the present invention, including angiosperms or gymnosperms, monocotyledons or dicotyledons.

[00111] Typical plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including but not limited to Indica and/or Japonica varieties), canola (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tobacum), potato (Solanum tuberosum), peanut (Arachis hypogaed), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee tree (Cofea spp.), coconut palm (Cocos nucifera), pineapple (Ananas comosus), citrus trees ( Citrus spp.), cocoa tree (Theobroma cacao), tea plant (Camellia sinensis), banana (Musa spp.), avocado (Persea americand), fig tree (Ficus casicd), guava (Psidium guajava), mango (Mangifera indica) , olive (Olea europaea), melon tree (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet species (Beta vulgaris), apple (Malus pumila), blackberry (Rubus ), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum (Prunus domestica), pear tree (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oat species (Avena sativa), barley (Hordium vulgare), vegetables, ornamental plants, conifers, and lawn grass species (for example, for ornamental purposes, for recreation or for fodder purposes), and biomass-producing grass species (e.g. switchgrass and miscanthus).

[00112] Vegetables include, but are not limited to, nightshade species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), carrot species (Caucus carotd), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant ( Solarium melongena), asparagus (Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), pea species (Lathyrus spp.), members of the genus Cucurbita such as Hubbard squash (C. hubbard ), butternut squash (C. moschata), common pumpkin (C. rero), gourd pumpkin (C. crookneck), C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C lundelliana and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis) and cantaloupe (C. meld).

[00113] Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), rose species (Rosa spp.), tulip species (Tulipa spp.), narcissus species (Narcissus spp.), spp. petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettia (Euphorbia pulcherima) and chrysanthemum.

[00114] Conifers that can be used in the practice of the present invention include, for example, pines such as lodgepole pine (Pinus taeda), Elliott pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and radiata pine (Pinus radiata); Menzies pseudotsuga (Pseudotsuga menziesii); Canadian hemlock (Tsuga canadensis); gray spruce (Picea glauca); evergreen sequoia (Sequoia sempervirens); firs such as sweet fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as arborvitae (Thuja plicata) and Nootka cypress (Chamaecyparis nootkatensis).

[00115] Lawn grasses include, but are not limited to, members of the genus Zoysia, members of the genus Agrostis, fescue species, bluegrass species, members of the genus Stenotaphrum, members of the genus Pigweed, buffalo grass species, chaff species, and cocksfoot species.

[00116] Also included are plants that serve primarily as laboratory models, such as Arabidopsis.

[00117] A "plant cell" is the structural and physiological unit of a plant, containing a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or cultured cell, or as part of a higher organization unit, such as, for example, plant tissue, a plant organ, or an entire plant.

[00118] “Plant cell culture” means the culture of plant units, such as, for example, protoplasts, cells in cell culture, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[00119] “Plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, eggs, zygotes, seeds, cuttings, cell or tissue cultures, or any other parts or products of a plant.

[00120] A "plant organ" is a distinct and visually structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[00121] As used herein, the expression "plant part" includes, without limitation, embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers and/or plant cells, including those plant cells which are intact in plants and/or plant parts, plant protoplasts, plant tissues, plant tissue cultures, plant calli, accumulations of plant material, and the like.

[00122] The expression "plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any plant tissue in planta or in culture is contemplated. The term includes, without limitation, whole plants, plant organs, plant seeds, tissue culture, and any group of plant cells organized into structural or functional units. The use of this term in combination with or without any specific type of plant tissue listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[00123] "Polynucleotide of interest" refers to any polynucleotide that, when transferred into an organism, such as a plant, provides the organism with a desired characteristic, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in the production process, production of commercially valuable enzymes or metabolites or altered reproductive capacity, etc.

[00124] By “part” or “fragment” of a polypeptide of the present invention is meant an amino acid sequence of reduced length relative to the reference amino acid sequence of the polypeptide of the present invention. Such a portion or fragment of the present invention may, when appropriate, be included in a larger polypeptide of which it is a part (eg, a tagged or fusion protein). In embodiments, the "part" or "fragment" substantially retains insecticidal activity (e.g., at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or even 100% activity of the full-length protein or even has greater insecticidal activity than the full-length protein).

[00125] The terms "protein", "peptide" and "polypeptide" are used interchangeably herein.

[00126] The term “promoter” refers to a polynucleotide, typically located upstream (5') of a coding polynucleotide, that controls the expression of the coding polynucleotide, allowing recognition by RNA polymerase and other factors necessary for proper transcription.

[00127] As used herein, the term “protoplast” refers to an isolated plant cell without a cell wall or with only portions of a cell wall.

[00128] As used herein, the term “recombinant” refers to a form of nucleic acid (eg, DNA or RNA) or protein or organism that would not normally occur in nature, and which as such was created through human intervention. As used herein, a “recombinant nucleic acid molecule” (and similar terms) is a nucleic acid molecule containing a combination of polynucleotides that do not naturally occur together and are the result of human intervention, for example, a nucleic acid molecule that consists of a combination of at least two polynucleotides heterologous to each other, or a nucleic acid molecule synthesized artificially and containing a polynucleotide that differs from the polynucleotide that would normally exist in nature, or a nucleic acid molecule that contains a transgene artificially introduced into the genomic DNA of a cell - host, and associated flanking DNA of the host cell genome. An example of a recombinant nucleic acid molecule is a DNA molecule resulting from the insertion of a transgene into the genomic DNA of a plant, which may ultimately result in the expression of a recombinant RNA molecule or protein in that organism. In embodiments, a “recombinant” protein is a protein that does not normally exist in nature or is present in a non-naturally occurring environment and is expressed from a recombinant nucleic acid molecule. As used herein, the term “recombinant plant” is a plant that would not normally exist in nature and is the result of human intervention and contains a recombinant polynucleotide (eg, a transgene or heterologous nucleic acid molecule inserted into its genome). As a result of this genome change, the recombinant plant is clearly different from its wild-type relative.

[00129] The terms "reduce", "reduced", "reduce", "reduce", "reduce" and "suppress" (and grammatical variations thereof) and similar terms as used herein refer to a decrease in survival, growth and/or or propagation of a plant pest, for example, by contacting the plant with a polypeptide of the present invention (such as, for example, by transgenic expression or topical application methods). This reduction in survival, growth and/or reproduction may be relative to the level observed in the absence of the polypeptide of the present invention (eg, a plant that does not transgenically express the polypeptide or is not subjected to topical treatment with the polypeptide). Thus, in embodiments, the terms “reduce,” “reduced,” “reduce,” “reduce,” “reduce,” and “suppress” (and grammatical variations thereof) and similar terms mean a reduction of at least about 5%, 10 %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to a plant that does not come into contact with the polypeptide of the present invention (eg, a plant that does not transgenically express the polypeptide or is not locally treated with the polypeptide). In illustrative embodiments, the reduction results in no or substantially no (i.e., a small amount, e.g., less than about 10%, less than about 5%, or even less than about 1%) detectable survival, growth, and/or reproduction of the pest plants.

[00130] "Regulatory element" refers to a nucleotide sequence involved in controlling the expression of a polynucleotide. Examples of regulatory elements include promoters, termination signals, and nucleotide sequences that facilitate correct translation of the polynucleotide.

[00131] As used herein, the term “selectable marker” means a nucleotide sequence that, when expressed, results in a distinctive phenotype of the plant, plant part, and/or plant cell expressing the marker, and thus provides the ability to distinguish such transformed plants, plant parts and/or plant cells from those that do not have a marker. Such a nucleotide sequence may encode either a selectable or a screenable marker depending on whether the marker confers a trait that can be selected for by chemical means, for example, by using a selective agent (e.g., an antibiotic, herbicide, etc.) , or whether the marker is simply a trait that can be identified through observation or testing, such as screening (eg, a trait identified at the R locus).

[00132] As used herein, “specific activity” refers to the amount of protein required to produce an insecticidal effect. Therefore, if the first protein has a higher specific activity than the second protein, this means that a smaller amount of the first protein is needed compared to the amount of the second protein to produce an insecticidal effect on the same percentage of insects.

[00133] The phrase "substantially identical" in the context of two nucleic acid sequences or two amino acid sequences refers to two or more sequences or subsequences that are at least about 50% identical at nucleotide or amino acid residues when compared and aligned for maximum consistency, which is determined using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences are characterized by at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater nucleotide or amino acid residue identity. In certain embodiments, significant identity exists within a sequence region that is at least about 50 residues, 100 residues, 150 residues, 200 residues, 250 residues, 300 residues, 350 residues, 400 residues, or more in length. In further embodiments, sequences are substantially identical when they are identical throughout the entire length of the coding regions.

[00134] “Identity” or “percentage identity” refers to the degree of identity between two nucleic acid sequences or amino acid sequences. In sequence comparison, one sequence usually serves as the reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequence are specified, if necessary, and the software parameters of the sequence comparison algorithm are specified. The sequence comparison algorithm then uses the specified software parameters to calculate the percentage sequence identity for the test sequence(s) relative to the reference sequence.

[00135] When comparing sequences, one sequence typically serves as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequence are specified, if necessary, and the software parameters of the sequence comparison algorithm are specified. The sequence comparison algorithm then uses the specified software parameters to calculate the percentage sequence identity for the test sequence(s) relative to the reference sequence.

[00136] Optimal alignment of sequences for comparison can be performed, for example, using the local homology search algorithm according to Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), using the Needleman-Wunsch homologous alignment algorithm, J. Mol. Biol. 48: 443 (1970), using the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by software implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by visual inspection (see generally Ausubel et al., below).

[00137] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). The software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves initially identifying highest similarity sequence pairs (HSPs) by identifying short "words" of length W in the query sequence that either match or satisfy some positive threshold T when aligned with a "word" of the same length in the database sequence . T is called the adjacent “word” threshold (Altschul et al., 1990). These initial matches of adjacent "words" act as seeds to begin searches to discover longer HSPs containing them. “Word” matches are then extended in both directions along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, aggregate scores are calculated using the parameters M (reward point awarded for a pair of matching residues; always >0) and N (penalty point awarded for mismatched residues; always <0). In the case of amino acid sequences, a substitution matrix is used to calculate the cumulative score. Extension of "word" matches in each direction stops when the alignment's cumulative score decreases by an amount X from its maximum achieved value, with the cumulative score falling to zero or lower due to the accumulation of one or more residue alignments with negative scores, or when the end of one is reached from sequences. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of alignment. BLASTN (for nucleotide sequences) defaults to a word length (W) of 11, an expected value (E) of 10, a threshold of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, BLASTP uses a default word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 substitution matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[00138] In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the lowest cumulative probability (P(N)), which is a measure of the likelihood that matches between two nucleotide or amino acid sequences will be observed by chance. For example, a test nucleic acid sequence is considered to be similar to a reference sequence if the lowest overall probability when comparing the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[00139] Another widely used and established computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain the percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two sequences being compared have different lengths, then the number of matches is divided by the shorter of the two lengths. For example, if proteins of 200 and 400 amino acids had 100 amino acid matches, then they are 50 percent identical given the shorter sequence. If the length of the shorter sequence is less than 150 bases or 50 amino acids, then the number of matches is divided by 150 (for nucleobases) or 50 (for amino acids) and multiplied by 100 to obtain the percent identity.

[00140] Two nucleotide sequences can also be considered substantially identical if the two sequences hybridize to each other under stringent conditions. In illustrative embodiments, two nucleotide sequences that are considered substantially identical hybridize to each other under very stringent conditions.

[00141] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a nucleic acid will selectively hybridize to a target sequence to a detectably higher degree than to other sequences (e.g., at least 2-fold higher compared to a non-target sequence), and may not necessarily substantially exclude binding to non-target sequences. The stringent conditions are sequence dependent and will vary under different circumstances. By controlling the stringency of hybridization conditions and/or washing, target sequences can be identified that can be up to 100% complementary to the reference nucleotide sequence. Alternatively, conditions of moderate or even low stringency may be used to provide some sequence mismatch to determine lower levels of sequence similarity. For example, those skilled in the art will appreciate that to function as a primer or probe, the nucleic acid sequence only needs to be sufficiently complementary to the target sequence to substantially bind to it, thereby forming a stable double-stranded structure under the conditions employed. Thus, primers or probes can be used under conditions of high, moderate or even low stringency. Likewise, conditions of low or moderate stringency may be preferable for the detection of homologous, orthologous and/or paralogous sequences having lower degrees of sequence identity than those that would be identified under very stringent conditions.

[00142] As used herein, the terms “complementary” or “complementarity” (and similar terms) refer to the natural binding of polynucleotides under salt and temperature conditions that permit it, by base pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules can be partial, in which only some of the nucleotides bind, or it can be complete, in which there is absolute complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands significantly influences the efficiency and strength of hybridization between molecules. As used herein, the term “substantially complementary” (and similar terms) means that two nucleic acid sequences are at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% complementary. , 95%, 96%, 97%, 98%, 99% or more. Alternatively, the term “substantially complementary” (and similar terms) may mean that two nucleic acid sequences can hybridize together under high stringency conditions (as described herein).

[00143] As used herein, "specifically" or "selectively" hybridizes (and similar terms) refers to the binding, duplexing, or hybridization of a molecule to a particular target nucleic acid sequence under stringent conditions when that sequence is present in a complex mixture (eg , total cellular DNA or RNA) with essentially the exclusion of non-target nucleic acids or even without detectable binding, duplex formation or hybridization with non-target sequences. The specifically or selectively hybridizing sequences are typically at least about 40% complementary, and not necessarily substantially complementary or even completely complementary (ie, 100% identical).

[00144] For DNA-DNA hybrids, T _m is approximately calculated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-84 (1984): T _m =81.5°C+16.6 (log M)+0.41 (% GC>0.61 (% formamide) - 500/l; where M is the molarity of the solution of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % Formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. T _m is the temperature (at a given ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matching probe. T _m is reduced by approximately 1°C for every 1% mismatch; thus, T _m , hybridization and/or wash conditions can be adjusted to hybridize with sequences with the desired degree of identity. For example, if sequences with >90% identity are sought, T _m can be reduced by 10° C. In general, stringent conditions are chosen to be approximately 5° C lower than the melting temperature (T _m ) for a specific sequence and its complementary sequence at a specific ionic strength and pH. However, under very stringent conditions, hybridization and/or washing at the melting temperature ( _Tm ) or 1, 2, 3 or 4°C below the melting temperature ( _Tm ) can be used; under conditions of moderate stringency, hybridization and/or washing can be used at a temperature of 6, 7, 8, 9 or 10 ° C below the melting temperature (T _m ); under conditions of low stringency, hybridization and/or washing can be used at a temperature of 11, 12, 13, 14, 15 or 20°C below the melting temperature ( _Tm ). If the required degree of mismatch results in a T _m below 45° C. (aqueous solution) or 32° C. (formamide solution), then the SSC concentration may optionally be increased to allow a higher temperature to be used. A comprehensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993); Current Protocols in Molecular Biology, Chapter 2, edited by Ausubel, et al. Greene Publishing and Wiley-Interscience, New York (1995); and Green & Sambrook in Molecular Cloning, A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[00145] Typically, severe conditions are those where the salt concentration is less than about 1.5 M Na ions, typically the Na ion (or other salt) concentration is about 0.01-1.0 M at about pH 7 .0 - pH 8.3, and the temperature is at least about 30°C for short probes (eg, 10-50 nucleotides) and at least about 60°C for longer probes (eg, more than 50 nucleotides). Severe conditions can also be achieved by adding destabilizing agents such as formamide or Denhardt's solution (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml water). Exemplary low stringency conditions include hybridization using a buffer solution of 30%-35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and a wash in 1X-2X SSC (20X SSC=3.0M NaCl/0 .3 M trisodium citrate) at 50°C-55°C. Exemplary moderate stringency conditions include hybridization in 40-45% formamide, 1 M NaCl, 1% SDS at 37°C and wash in 0.5X-1X SSC at 55°C-60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and wash in 0.1X SSC at 60°C-65°C. Additional non-limiting example of high stringency conditions include hybridization in 4X SSC, 5X Denhardt's solution, 0.1 mg/ml boiled salmon milk DNA, 25 mM Na phosphate at 65°C and wash in ODC SSC, 0.1% SDS at 65°C WITH. Another example of high stringency hybridization conditions includes hybridization in 7% SDS, 0.5 M NaPO ₄ , 1 mM EDTA at 50°C with a 2X SSC wash, 0.1% SDS at 50°C, alternatively with a 1X SSC wash , 0.1% SDS at 50°C, alternatively, with a wash in 0.5X SSC, 0.1% SDS at 50°C or, alternatively, with a wash in 0.1X SSC, 0.1% SDS at 50° C, or also with washing in ODC SSC, 0.1% SDS at 65°C. Those skilled in the art will appreciate that specificity generally depends on the washes after hybridization, with the ionic strength and temperature of the final wash solution being important factors.

[00146] In this case, nucleic acids that do not hybridize with each other under stringent conditions are essentially identical if the proteins they encode are essentially identical (for example, due to degeneracy of the genetic code).

[00147] Another indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive with the protein encoded by the second nucleic acid. Thus, the protein is typically substantially identical to the second protein, for example, if the two proteins differ only by conservative substitutions.

[00148] As used herein, if a modified polypeptide or fragment (and the like) "substantially retains" insecticidal activity, this means that the modified polypeptide or fragment retains at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or even 100% of the insecticidal activity of the reference protein or has even greater insecticidal activity.

[00149] “Synthetic” refers to a nucleotide sequence containing bases or structural feature(s) not present in the natural sequence. For example, an artificial sequence encoding a protein of the present invention that is more similar in G+C content and normal codon distribution to the genes of dicotyledonous or monocotyledonous plants is considered synthetic.

[00150] As used herein, a protein that is "toxic" to an insect pest is an orally active insect control agent that kills the pest insect, causes a reduction in the growth and/or reproduction of the insect pest, and/or is capable of disrupting or inhibit the insect's feeding, the latter two of which may or may not cause the insect's death. When the protein of the present invention is delivered to an insect or the insect comes into contact with the protein, the result is typically the death of the insect, the growth and/or reproduction of the insect is slowed, and/or the insect reduces or stops feeding on the source that makes the toxic protein available to insect.

[00151] The terms “toxin moiety” and “toxin moiety” are used interchangeably herein to refer to a fragment or portion of a longer (e.g., full-length) chimeric insecticidal protein of the present invention, wherein the “toxin moiety” or “toxin moiety” retains insecticidal activity . For example, it is known in the art that native Cry proteins are expressed as protoxins, which are processed at the N-terminus and C-terminus to produce the mature toxin. In embodiments, the "toxin fragment" or "toxin portion" of the chimeric insecticidal protein of the present invention is truncated at the N-terminus and/or the C-terminus. In embodiments, the "toxin fragment" or "toxin portion" is truncated at the N-terminus to remove part or all of the N-terminal peptidyl fragment and optionally contains at least about 400, 425, 450, 475, 500, 510, 520, 530, 540 , 550, 560, 570, 580, or 590 contiguous amino acids of a chimeric insecticidal protein specifically described herein, or an amino acid sequence that is substantially identical thereto. Thus, in embodiments, the “toxin fragment” or “toxin portion” of the chimeric insecticidal protein is N-terminally truncated (e.g., to eliminate part or all of the peptidyl fragment), e.g., N-terminal truncation of one amino acid or more than one amino acid, e.g. up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids. In embodiments, the "toxin fragment" or "toxin portion" of the chimeric insecticidal protein is C-terminally truncated (e.g., to eliminate part or all of the protoxin tail), e.g., C-terminal truncation of one amino acid or more than one amino acid, e.g., to 2 , 3, 4, 5, 6, 7, 8, 9, 10, 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, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 , 450, 475, 500, 525, 550, 560 or more amino acids. In embodiments, a “toxin moiety” or “toxin moiety” includes domains 1 and 2 and core domain III (eg, as shown in Figure 2). In embodiments, a “toxin fragment” or “toxin moiety” is a mature (ie, processed) toxin (eg, Cry toxin).

[00152] "Transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism. In specific embodiments, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest (eg, a plant cell).

[00153] As used herein, the terms “transformed” and “transgenic” refer to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host genome, or the nucleic acid molecule may also be present as an extrachromosomal molecule. Such an extrachromosomal molecule may be autoreplicating. "Transformed" or "transgenic" cells, tissues or plants are meant to include not only the end product of the transformation process, but also their progeny containing the heterologous nucleic acid molecule. A "non-transformed" or "non-transgenic" host refers to a wild-type organism, such as a bacterium or plant, that does not contain a heterologous nucleic acid molecule.

[00154] The term “vector” refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell. The vector contains a nucleic acid molecule containing nucleotide sequence(s) to be transferred, delivered or administered.

Chimeric insecticidal proteins

[00155] The present invention provides novel chimeric insecticidal proteins comprising at least one region of a first Cry protein (eg, BT-0029 protein [SEQ ID NO: 2] and substantially identical variants thereof). In embodiments, the present invention provides a chimeric insecticidal protein comprising a region of two or more different Cry proteins. In embodiments, the resulting chimeric insecticidal protein is characterized by increased activity against one or more insect pests (e.g., enhanced activity or activity against a new target pest) and/or a different mode of action against one or more insect pests compared to one or more more (or even all) of the original proteins. In illustrative embodiments, the chimeric insecticidal protein is a chimera comprising regions from two different Cry proteins (eg, Cry1), and the chimera has increased insecticidal activity against one or more insect pests compared to both parent proteins.

[00156] In embodiments, the chimeric insecticidal proteins of the present invention may provide new modes of action against one or more target insect pests. For example, a chimeric insecticidal protein may have insecticidal activity against insect pests or colonies that are generally resistant to the insecticidal activity of another agent (e.g., an insecticidal protein, including, but not limited to, a Bt protein such as a Cry protein or a Vip protein). In embodiments, the parent proteins themselves are not insecticidal active or are only weakly active against resistant insect pests or colonies, which may indicate that the chimera has a new mode of action compared to the parent proteins. For example, if the parent proteins are active against the target insect pest ("susceptible") but not against the resistant counterpart, and the chimeric insecticidal protein is active against the resistant insect pest, this indicates that the chimeric insecticidal protein is toxic to resistant pest thanks to a new mode of action.

[00157] Accordingly, in embodiments of the present invention, there is provided a chimeric insecticidal protein that is toxic to an insect pest (e.g., a lepidopteran insect pest), wherein the chimeric insecticidal protein comprises a region from the BT-0029 protein (SEQ ID NO : 2) or a polypeptide that is substantially identical to a region from the BT-0029 protein. In embodiments, the chimeric insecticidal protein comprises the N-terminal region of the first Cry1 protein, which is optionally the BT-0029 protein (SEQ ID NO: 2), or a polypeptide comprising an amino acid sequence that is substantially identical to the N-terminal region of the BT-0029 protein. In embodiments, the N-terminal region of the first Cry protein is fused to the C-terminal region from another Cry protein (eg, another Cry1 protein) to form a chimeric insecticidal protein (eg, a chimeric Cry insecticidal protein).

[00158] In illustrative embodiments, the C-terminal region from another Cry protein may be the C-terminal region of another Cry1 protein or a polypeptide comprising an amino acid sequence that is substantially identical to the C-terminal region from another Cry1 protein.

[00159] Thus, in an illustrative embodiment, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists, in an N-terminal to C-terminal direction, of the following: (a) the N-terminal region of a first Cry protein (e.g. , the Cry1 protein), which is optionally the N-terminal region of the BT-0029 protein, or an amino acid sequence that is substantially identical to the N-terminal region of the BT-0029 protein fused to (b) the C-terminal region of another Cry protein (e.g., another protein Cry1).

[00160] In exemplary embodiments, another Cry protein (e.g., a Cry1 protein) is selected based on an alignment of a C-terminal region from another Cry protein and a corresponding region from a first Cry protein (e.g., a Cry1 protein), e.g., see exemplary alignments in the figures 1A and 1B. In embodiments, the other Cry protein is selected such that the amino acid sequence of the C-terminal region of the other Cry protein is substantially identical to the corresponding region of the first Cry protein. In illustrative embodiments, the amino acid sequence of the C-terminal region of another Cry protein (e.g., a Cry1 protein) is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the corresponding C-terminal region from the first Cry protein. Methods for aligning and determining the identity of amino acid sequences in the aligned region are well known to those skilled in the art.

[00161] In embodiments, the other Cry1 protein includes, but is not limited to, a Cry1F protein (e.g., a Cry1Fa protein or a Cry1Fb protein), a Cry1G protein (e.g., a Cry1Ga, Cry1Gb, or Cry1Gc protein), a Cry1I protein (e.g., a Cry1Ia protein, a Cry1Ib protein, Cry1Ic protein, Cry1Id protein, Cry1Ie protein, Cry1If protein or Cry1Ig protein), Cry1K (eg Cry1Ka protein) or Cry1C (eg Cry1Ca protein). In embodiments, the other Cry1 protein is a Cry1 If protein, optionally a BT-0022 protein (SEQ ID NO: 1). In embodiments, the other Cry1 protein is the Cry1Fa protein (SEQ ID NO: 8). In embodiments, the other Cry1 protein is the Cry1K protein (SEQ ID NO: 12). In embodiments, the other Cry1 protein is the Cry1Ca protein (SEQ ID NO: 17).

[00162] The terms "N-terminal region" and "C-terminal region" do not necessarily indicate that the majority of the N-terminal or C-terminal amino acids (e.g., N-terminus or C-terminus), respectively, of the full-length protein are included in region. For example, those skilled in the art are well aware that Cry protoxins are processed from both the N-terminus and the C-terminus to produce a mature (ie, processed) toxin. Thus, in embodiments, the "N-terminal region" and/or the "C-terminal region" excludes some or all of the processed portions of the protoxin such that the chimeric insecticidal protein comprises the mature toxin protein (e.g., domains I, II, and III of the protein Cry) lacking part or all of the N-terminal peptidyl fragment and/or the C-terminal tail of the protoxin, or a polypeptide that is substantially identical to the mature toxin protein. In embodiments, the chimeric insecticidal protein comprises a peptidyl moiety and/or a protoxin tail. In embodiments, the chimeric insecticidal protein does not contain a peptidyl moiety or protoxin tail, i.e. corresponds to a mature processed toxin.

[00163] In embodiments, the N-terminal region of the first Cry1 protein comprises, consists essentially of, or consists of a polypeptide corresponding to the amino acid sequence from approximately positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 or 40 to approximately position 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 , 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 4 74 , 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 4 99 or 500 SEQ ID NO: 2 (full size BT-0029) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00164] In embodiments, the N-terminal region of the first Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 1 to about position 430, 431, 432, 433, 434, 435, 436, 437, 438 , 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 4 63 , 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 4 88 , 489, 490 or 491 SEQ ID NO: 2.

[00165] In an illustrative embodiment, the N-terminal region of the first Cry1 protein comprises, consists of, or consists of an amino acid sequence corresponding to the amino acid sequence from about position 1 to about amino acid position 458 of SEQ ID NO: 2.

[00166] In illustrative embodiments, the C-terminal region of the second Cry1 protein comprises, consists essentially of, or consists of a polypeptide corresponding to the amino acid sequence from approximately positions 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459 , 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484 , 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 to approximately position 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 587, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612 , 613, 614, 615, 616, 617, 618, 619 or 620 SEQ ID NO: 8 (full-length Cry1Fa) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00167] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 464 to about position 580, 581, 582, 583, 584, 585, 586, 587, 588 , 589, 590, 591, 592, 593, 594, 595, 596, 587, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 6 13 , 614, 615, 616, 617, 618, 619 or 620 SEQ ID NO: 8.

[00168] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445 , 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 4 60 , 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 4 85 , 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 to approximately position 602 SEQ ID NO: 8.

[00169] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 464 to about position 602 of SEQ ID NO: 8.

[00170] In illustrative embodiments, the C-terminal region of the second Cry1 protein comprises, consists essentially of, or consists of a polypeptide corresponding to the amino acid sequence from approximately positions 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504 , 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529 , 530, 531, 532, 533, 534, or 535 to approximately position 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632 , 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654 or 655 SEQ ID NO: 1 (full size BT-0022) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00171] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 498 to about position 615, 616, 617, 618, 619, 620, 621, 622, 623 , 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 6 48 , 649, 650, 651, 652, 653, 654 or 655 SEQ ID NO: 1.

[00172] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480 , 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 5 05 , 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 5 30 , 531, 532, 533, 534, or 535 to approximately position 636 SEQ ID NO: 1.

[00173] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 498 to about position 636 of SEQ ID NO: 1.

[00174] In illustrative embodiments, the C-terminal region of the second Cry1 protein contains, consists essentially of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506 , 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531 , 532, 533, 534, 535, 536 or 537 to approximately position 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632 , 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656 or 657 SEQ ID NO: 12 (full-length Cry1Ka) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00175] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 590, 591, 592, 593, 594, 595, 596, 597, 598 , 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 6 23 , 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 6 48 , 649, 650, 651, 652, 653, 654, 655, 656 or 657 SEQ ID NO: 12.

[00176] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 , 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 5 07 , 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 5 32 , 533, 534, 535, 536, 537 to approximately position 597 SEQ ID NO: 12.

[00177] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482 , 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 5 07 , 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 5 32 , 533, 534, 535, 536, 537 to approximately position 610 SEQ ID NO: 12.

[00178] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 597 SEQIDNO: 12.

[00179] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 500 to about position 610 of SEQ ID NO: 12.

[00180] In illustrative embodiments, the C-terminal region of the second Cry1 protein contains, consists essentially of, or consists of a polypeptide corresponding to the amino acid sequence from approximately positions 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459 , 460, 461, 462, 463, 464, 465, 466 or 467 to approximately position 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, or 617 from SEQ ID NO: 17 ( full-length Cry1Ca) and any combination of lower and higher positions, as if each such combination were specifically set forth herein.

[00181] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 467 to about position 580, 581, 582, 583, 584, 585, 586, 587, 588 , 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 6 13 , 614, 615, 616 or 617 SEQIDNO: 17.

[00182] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from approximately position 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445 , 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 4 60 , 461, 462, 463, 464, 465, 466, or 467 to approximately position 617 SEQ ID NO: 17.

[00183] In embodiments, the C-terminal region of the second Cry1 protein comprises, consists of, or consists of a polypeptide corresponding to the amino acid sequence from about position 467 to about position 617 of SEQ ID NO: 17.

[00184] As is well known in the art, Cry proteins from Bt have 5 conserved sequence domains (conserved block [CB] 1-5]) separated by more variable regions (Hofte & Whitely, 1989, Microbiol. Rev. 53:242 -255), and three domains with conserved structure (domains I, II and III) (de Maagd et al., 2001, Trends Genetics 17:193-199). Figures 1A and IB show the alignment of the Cry proteins BT-0029, BT-0022, Cry1Fa, and Cry1Ka with CB3 and domain III labeled. Those skilled in the art can use the generally known sequence and structure information available for Cry proteins to create a chimeric insecticidal Cry protein in accordance with the present invention, for example, to select suitable transition region(s) between two Cry proteins. In embodiments, the transition region is located in a conservative block, for example, in CB3. The term "in a conservative block" includes positions at each end of the conservative block. To illustrate this, the term “in CB3” includes the position immediately before (for example, between the methionine and phenylalanine residues within the sequence VPMFSW in the sequence BT-0029 in Figure 2A) and immediately after (for example, between the threonine and asparagine residues within the sequence RRTNVG in sequence BT-0029 in Figure 2A) conserved block 3. Figures 2A and 2B show exemplary chimeric insecticidal proteins in accordance with the present invention with a transition region at CB3.

[00185] With reference to Figure 2A, in embodiments the transition is located at a position corresponding to the position immediately before amino acid residue 1 or immediately after amino acid residue 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, 50 or 51, or any combination thereof in CB3. In embodiments, the transition is located within a region (including residues at each end of the region) corresponding to the region from amino acid residue 2 to amino acid residue 7, 8, 9 or 10 of CB3, within the region corresponding to the region from amino acid residue 11 to amino acid residue 14, 15, 16, 17 or 18 of CB3, within the region corresponding to the region from amino acid residue 19 to amino acid residue 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 of CB3, and/or within the region, corresponding to the region from amino acid residue 37, 38 or 39 to amino acid residue 51, and any combination of such regions. In illustrative embodiments, the transition is located at a position within a region corresponding to amino acid residue 19 to amino acid residue 51, amino acid residue 19 to amino acid residue 33, amino acid residue 19 to amino acid residue 28, amino acid residue 19 to amino acid residue 27, or from amino acid residue 19 to amino acid residue 26 in CB3. In embodiments, the transition position is located at a specific site between amino acid residue 18 and amino acid residue 19 in CB3 (eg, specifically between valine and isoleucine in the sequence DPDVITQ in BT-0029, SEQ ID NO: 2).

[00186] Chimeric insecticidal proteins can also be defined in terms of structural domains derived from each of the parent molecules. For example, in embodiments, the N-terminal region of the first Cry protein comprises domain I of the first Cry protein and all or substantially all of domain II. In embodiments, the C-terminal region of the other Cry protein comprises all or substantially all of domain III of the other Cry protein. Those skilled in the art will appreciate that there is some variability in determining the exact location of the ends of the structural domains of the Cry protein and the linker regions between domains, for example, the location of the end of the linker region between domains II and III and the beginning of domain III, although the core of domain III is readily identifiable by those skilled in the art. region (eg, according to the location of core domain III, as shown in Figure 1A and Figure 2A). In embodiments, the transition between the N-terminal region of the first Cry protein and the C-terminal region derived from another Cry protein is located in CB3.

[00187] In illustrative embodiments, the C-terminal region of another Cry protein (e.g., a Cry1 protein) is selected based on an alignment of domain III (e.g., core domain III as shown in Figure 1 A) from another Cry protein and the corresponding domain III sequence from the first Cry protein (eg, a Cry1 protein such as BT-0029), for example, see the exemplary alignments in Figures 1A and 1B with domain III specifically noted. In embodiments, the other Cry protein is selected such that the amino acid sequence of domain III of the other Cry protein is substantially identical to the corresponding region of domain III of the first Cry protein (eg, BT-0029). In illustrative embodiments, the amino acid sequence of the domain III region of another Cry protein (e.g., a Cry1 protein) is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of the corresponding region of domain III from the first Cry protein. Methods for aligning and determining the identity of amino acid sequences in the aligned region are well known to those skilled in the art.

[00188] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of (a) an amino acid sequence of any of amino acids 1-597 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-610 of SEQ ID NO: 15, or amino acids 1-617 of SEQ ID NO: 18, or a toxin fragment from any of SEQ ID NO: 3, 9, 13, 15 or 18; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00189] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of (a) an amino acid sequence of any of amino acids 1-603 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-615 of SEQ ID NO: 15 or SEQ ID NO: 18, or a toxin fragment thereof; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00190] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of (a) the amino acid sequence of any of amino acids 1-607 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-620 of SEQ ID NO: 15 or SEQ ID NO: 18, or a toxin fragment thereof; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00191] In specific embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of (a) an amino acid sequence of any of amino acids 1-610 of SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 13, or amino acids 1-620 of SEQ ID NO: 15 or SEQ ID NO: 18, or a toxin fragment thereof; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a).

[00192] As will be understood by those skilled in the art, native Cry toxins are expressed as protoxins that are processed to produce a mature toxin that is processed by cleavage of the N-terminal peptidyl moiety and the C-terminal tail of the protoxin. The N-terminal peptidyl moiety and/or C-terminal tail of the protoxin may function to enhance the stability and/or insecticidal activity of the Cry toxin. In embodiments of the present invention, the chimeric insecticidal protein comprises all or a portion of the N-terminal peptidyl fragment and/or tail of the protoxin. In embodiments, the chimeric insecticidal protein does not contain the complete N-terminal peptidyl fragment and/or the complete protoxin tail. In embodiments, the chimeric insecticidal protein does not contain an N-terminal peptidyl moiety and/or protoxin tail, i.e. corresponds to a mature processed toxin.

[00193] In embodiments, the N-terminal peptidyl fragment is derived from a Cry protein (eg, from a first Cry protein, which is not necessarily a Cry1 protein). In other embodiments, the N-terminal peptidyl fragment is heterologous to the first Cry protein, for example, not derived from the Cry protein and/or is partially or completely synthetic. In embodiments, the peptidyl moiety contains at least about 5, 6, 7, 8, 9, 10, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids, optionally from the first Cry protein, including without limitation the first Cry1 protein (e.g., BT-0029, SEQ ID NO: 2) . In embodiments, the peptidyl moiety comprises amino acids from about amino acid 1 to about amino acid 5, 6, 7, 8, 9, 10, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 of a first Cry protein, for example a Cry1 protein such as BT-0029.

[00194] In embodiments, the chimeric insecticidal protein of the present invention comprises a C-terminal protoxin tail region of the Bt Cry protein, including modifications of the native Cry protein protoxin tails that are substantially identical to the native Cry protein protoxin tail. In embodiments, the protoxin tail is derived from the lepidopteran-active Cry protein. In embodiments, the protoxin tail is not derived from the Cry protein and/or is partially or completely synthetic. In embodiments, the Cry protein is heterologous to the first Cry protein and/or another Cry protein. In embodiments, the protoxin tail is derived from a Cry1 protein, e.g., BT-0029, BT-0022, Cry1F (e.g., Cry1Fa), Cry1I (e.g., Cry1Ia or Cry1If), Cry1K (e.g., Cry1Ka), or Cry1C (e.g., Cry1Ca), or it is a polypeptide that is essentially identical to the protoxin tail (or fragment thereof) of any of the above. The protoxin tail region may contain the entire Cry protein protoxin tail or any portion thereof. In embodiments, the protoxin tail region contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 38, 40, 45, 50 or more contiguous amino acids from the Cry protein protoxin tail (eg, the Cry1 protoxin tail, such as BT-0029), for example, as shown for the various Cry proteins in Figure 1A. In embodiments, the protoxin tail contains at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 38, 40, 45, 50 or more contiguous amino acids starting at amino acid position 598 of SEQ ID NO: 2 (BT-0029) or the corresponding region from another Cry protein, such as the Cry1 protein (eg, BT-0022, Cry1I, Cry1F, Cry1K or Cry1C protein). In embodiments, the protoxin tail comprises amino acids 598-1169, or 598-652, or 598-636, or 598-622, or 598-610, or 598-607, or 598-603, or 598-600 from SEQ ID NO: 2 (BT-0029) or the corresponding region from another Cry protein (see, for example, the alignment of BT-0029 with BT-0022, Cry1Fa and Cry1Ka in Figures 1A and 1B). In some embodiments, the chimeric insecticidal protein comprises any of SEQ ID NO: 20-25 or SEQ ID NO: 32-35. In other embodiments, the protoxin tail comprises amino acids 637-715, or 637-691, or 637-675, or 637-661, or 637-649, or 637-646, or 637-642 from SEQ ID NO: 1 (BT- 0022). In yet other embodiments, the chimeric insecticidal protein of the present invention contains, consists essentially of, or consists of any of SEQ ID NOs: 26-31.

[00195] Accordingly, in embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of (a) the amino acid sequence of any of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO : 15, SEQ ID NO: 18, SEQ ID NO: 20-35, or a toxin fragment thereof; or (b) an amino acid sequence that is substantially identical to the amino acid sequence of (a). In optional embodiments, the chimeric insecticidal protein of the present invention comprises, consists essentially of, or consists of an amino acid sequence of any of the amino acids of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO: 20-35.

[00196] Those skilled in the art will appreciate that the chimeric insecticidal proteins of the present invention may further contain other functional domains and/or peptide tags, for example, a peptide tag at the N-terminus and/or the C-terminus. For example, it may be useful to express the chimeric insecticidal protein with a peptide tag that can be recognized by a commercially available antibody (eg, a FLAG motif) or with a peptide tag that facilitates purification (eg, by adding a poly-His tag) and/or detection. Alternatively, an epitope can be introduced into the chimeric protein to facilitate the generation of antibodies that specifically recognize the modified chimeric protein to distinguish the modified chimeric protein from the unmodified chimera and/or the parent protein(s). For example, one or more amino acids can be replaced in an antigenic loop of a native sequence to create a new epitope. In one embodiment, the antigenic loop is located in a non-conserved region outside domain I of the native Cry protein. In embodiments, the antigenic loop is not a loop involved in recognition of a receptor in the insect gut by the Cry protein, and/or is not involved in activation of the Cry protein by a protease. In other embodiments, the chimeric protein can be modified to increase its stability, for example, by fusing maltose binding protein (MBP) or glutathione S-transferase to the polypeptide. As another alternative, the fusion protein may contain a reporter group.

[00197] Chimeric insecticidal proteins that are modified by introducing or removing a protease processing site at a suitable position(s) to promote or eliminate proteolytic cleavage by the protease of insects, plants and/or microorganisms are also within the scope of the present invention. In embodiments, the modified chimeric insecticidal protein substantially retains insecticidal activity. In embodiments, the stability and/or insecticidal activity of such modified chimeric proteins is increased compared to a chimeric insecticidal protein that does not contain such modification to introduce/eliminate a protease cleavage site.

[00198] Thus, the present invention covers polypeptides having amino acid sequences that are substantially identical to the sequences specifically disclosed herein and toxin fragments thereof. It will be understood that the chimeric insecticidal proteins specifically disclosed herein will generally tolerate modifications in the amino acid sequence and substantially retain biological activity (eg, insecticidal activity). Such modifications include insertions, deletions (including truncations at either end), and substitutions of one or more amino acids, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155 or more amino acid substitutions, deletions and/or insertions.

[00199] To identify polypeptides substantially identical to the chimeric insecticidal proteins specifically disclosed herein, amino acid substitutions can be based on any characteristic known in the art, including the relative similarity or difference of amino acid side chain substituents, such as their hydrophobicity , hydrophilicity, charge, size, etc.

[00200] For example, when identifying amino acid sequences encoding insecticidal polypeptides other than those specifically disclosed herein, the amino acid hydrophobicity index may be taken into account. The importance of amino acid hydrophobicity indices in ensuring the coordinated biological function of a protein is generally known in the art (see Kyte and Doolittle, (1982) J. Mol. Biol. 157:105; incorporated herein by reference in its entirety). It is assumed that the relative hydrophobic characteristic of an amino acid contributes to the secondary structure of the resulting protein, which in turn determines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[00201] Each amino acid was assigned a hydrophobicity index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, Id.), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); series (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[00202] Accordingly, the hydrophobicity index of an amino acid (or amino acid sequence) can be taken into account when modifying chimeric polypeptides specifically disclosed herein.

[00203] It is also known in the art that amino acid substitutions can be made based on hydrophilicity. US Pat. No. 4,554,101 states that the highest average local hydrophilicity of a protein, which is determined by the hydrophilicity of its adjacent acids, correlates with the biological property of the protein.

[00204] As detailed in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to the amino acid residues: arginine (+3.0); lysine (.+-.3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); series (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[00205] Thus, the hydrophilicity of an amino acid (or amino acid sequence) can be taken into account when identifying additional insecticidal polypeptides beyond those specifically disclosed herein.

[00206] The chimeric insecticidal proteins of the present invention, including modifications and chimeric polypeptide toxin fragments specifically disclosed herein, can be produced by any suitable method known in the art, typically by modifying nucleic acid coding sequences. Methods for Manipulating and Modifying Nucleic Acids to achieve the desired modification are well known in the art. In addition, gene editing techniques can also be used to produce the chimeric insecticidal protein of the present invention or to introduce further modifications therein.

[00207] As another approach, the polypeptide to be modified can be expressed in a host cell that exhibits a high rate of base misincorporation during DNA replication, such as XL-1 Red (Stratagene, La Jolla, Calif.). After propagation in such strains, DNA can be isolated (for example, by preparing plasmid DNA or by PCR amplification and cloning the resulting PCR fragment into a vector), expressing mutant protein sequences in a strain that does not produce mutations, and identifying mutated genes with insecticidal activity. activity, for example, by performing an assay to test insecticidal activity. In exemplary methods, the protein is mixed and used in feeding assays. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such tests may involve exposing the plants to one or more pests and determining the plants' ability to survive or cause the pests to die. Examples of mutations that lead to increased toxicity can be found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.

[00208] In embodiments, the chimeric insecticidal protein (including substantially similar polypeptides and toxin fragments) of the present invention is isolated. In embodiments, the chimeric insecticidal protein (including substantially similar polypeptides and toxin fragments) of the present invention is a recombinant protein.

[00209] The chimeric insecticidal proteins of the present invention are characterized by insecticidal activity against lepidopteran pests. In embodiments, the chimeric insecticidal protein is active against one or more of the following non-limiting examples of a lepidopteran pest: Ostrinia spp., such as O. nubilalis (corn borer) and/or O. furnacalis (eastern corn borer); Plutella spp., such as P. xylostella (cabbage moth); Spodoptera spp., such as S. frugiperda (grass armyworm), S. littoralis (Egyptian cotton armyworm), S. ornithogalli (yellow-striped armyworm), S. praefica (western yellow armyworm), S. eridania (southern armyworm), and/or S. exigua (small cutworm); Agrotis spp., such as A ipsilon, A. segetum, A. gladiaria, and/or A. orthogonia; Striacosta spp., such as S. albicosta (western bean cutworm); Helicoverpa spp., such as. H, zea (corn armyworm), N. punctigera (Australian cotton bollworm) and/or H. armigera (cotton bollworm); Heliothis spp., such as H., virescens (tobacco budworm); Diatraea spp., such as D. grandiosella (southwest corn borer) and/or D. saccharalis (sugarcane stalk borer); Trichoplusia spp., such as T. ni (cabbage cutworm); Sesamia spp., such as S. nonagroides (Mediterranean corn borer) and/or S. calamistis (pink cutworm); Pectinophora spp., such as P. gossypiella (pink bollworm); Cochylis spp., such as C. hospes (striped sunflower moth); Manduca spp., such as M. sexta (tobacco hawkmoth) and/or M. quinquemaculata (five-spotted hawkmoth); Elasmopalpus spp., such as E. lignosellus (small corn stalk borer); Pseudoplusia spp., such as P. includens (soybean armyworm); Anticarsia spp., such as A. gemmatalis (velvet bean scoop); Plathypena spp., such as P. scabra (clover armyworm); Pieris spp., such as P. brassicae, Papaipema spp., such as P. nebris (Papaipema nebris); Pseudaletia spp. such as P. unipuncta (common meadow cutworm); Peridroma spp., such as P. saucia (daisy armyworm); Keiferia spp., such as K. lycopersicella (tomato pinworm); Artogeia spp., such as A. rapae; Phthorimaea spp., such as P. operculella (potato moth); Chrysodeixis spp., such as C. includes (soybean armyworm); Feltia spp., such as P. ducens (feltia jaculifera caterpillar); Chilo spp., such as C. suppressalis (yellow rice moth), Cnaphalocrocis spp., such as C. medinalis (rice moth), or any combination of the above.

[00210] Optionally, the chimeric insecticidal protein has increased activity against one or more lepidopteran pests compared to one or more parent molecules (eg, the first Cry protein and the other Cry protein). In embodiments, the chimeric insecticidal protein has increased activity against one or more lepidopteran pests compared to BT-0029. In embodiments, the chimeric insecticidal protein has increased activity against one or more lepidopteran pests compared to BT-0022, Cry1Fa, Cry1Ia, Cry1If, Cry1Ka, or Cry1C.

[00211] In embodiments, the chimeric insecticidal protein has increased activity against armyworm (Spodoptera frugiperda) compared to one or more parent molecules (eg, the first Cry protein and the other Cry protein). In embodiments, the chimeric insecticidal protein has increased activity against armyworm compared to BT-0029. In embodiments, the chimeric insecticidal protein has increased activity against armyworm compared to BT-0022, Cry1Fa, Cry1Ia, Cry1If, Cry1Ka and/or Cry1Ca. In accordance with the above embodiments, the chimeric insecticidal protein may optionally have insecticidal activity against an insect pest or armyworm colony that is resistant to another insecticidal agent, including another insecticidal protein (such as, for example, the Bf protein). In embodiments, the chimeric insecticidal protein has enhanced activity against a fall armyworm colony that is resistant to a Vip3A protein (e.g., Vip3Aa, including but not limited to the maize transformant MIR162) or a Cry1F protein (e.g., Cry1Fa, including but not limited to the maize transformant TC1507). In embodiments, the chimeric insecticidal protein has increased activity against resistant armyworm compared to one or more parent molecules, for example, BT-0029, BT-0022, Cry1Fa, Cry1Ia, Cry1If, Cry1Ka and/or Cry1Ca.

[00212] The present invention also includes antibodies that specifically bind to the chimeric insecticidal protein of the present invention. The antibody may optionally be a monoclonal antibody or a polyclonal antiserum. In embodiments, the antibody is selective for the chimeric protein and does not bind to one or more parent molecules (eg, BT-0029, BT-0022, Cry1Fa, etc.) and can be used to distinguish between the chimeric protein and the parent protein. Such antibodies can be obtained using standard immunological techniques for obtaining polyclonal antiserum and, if necessary, immortalizing the antibody-producing cells of the immunized host to obtain sources of monoclonal antibodies. Techniques for preparing antibodies to any substance of interest are well known, for example as described in Harlow and Lane (1988. Antibodies a laboratory manual, pp. 726. Cold Spring Harbor Laboratory) and in Goding (Monoclonal Antibodies: Principles & practice. 1986. Academic Press, Inc., Orlando, FL). The present invention also includes an insecticidal protein that cross-reacts with an antibody, in particular a monoclonal antibody, raised against one or more chimeric insecticidal proteins of the present invention.

[00213] Antibodies of the present invention are useful, for example, in immunoassays to determine the amount or presence of a chimeric insecticidal protein of the present invention or an antigenically related polypeptide, for example, in a biological sample. Such assays are also useful in preparing compositions containing one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide, while ensuring quality control. In addition, antibodies can be used to evaluate the efficiency of recombinant production of one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide, as well as to screen expression libraries for the presence of a nucleotide sequence encoding one or more chimeric insecticidal proteins of the present invention or an antigenically related polypeptide. Antibodies are further useful as affinity ligands for the purification or isolation of any one or more proteins of the present invention or antigenically related polypeptide.

Nucleic acids, expression cassettes and vectors

[00214] As a further aspect, the present invention provides nucleic acids encoding the polypeptides of the present invention, including modified polypeptides and toxin fragments, as described herein.

[00215] In some embodiments, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that contains, consists essentially of, or consists of the following: (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 20-35, or a toxin fragment thereof; (b) a nucleotide sequence encoding an amino acid sequence that is substantially identical to the amino acid sequence from (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence of (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequences of (a), (b) or (c) due to degeneracy of the genetic code.

[00216] In embodiments, the nucleic acid molecule contains a nucleotide sequence that contains, is substantially composed of, or consists of the following: (a) the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 16 or a toxin encoding fragment thereof; (b) a nucleotide sequence that is substantially identical to the nucleotide sequence from (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence of (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequences of (a), (b) or (c) due to degeneracy of the genetic code. Optionally, the nucleotide sequence contains, consists of, or consists of the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 or SEQ ID NO: 16.

[00217] In embodiments, the nucleotide sequence is a partially or entirely synthetic sequence, for example, that has been codon-optimized for expression in a host organism, such as a bacterium host or a plant host (e.g., a transgenic monocot host or a transgenic dicot host). host plant). Non-limiting examples of nucleotide sequences that are codon optimized for expression in the maize plant include SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 11.

[00218] In illustrative embodiments, the nucleotide sequences of the present invention are modified and/or optimized for expression in transgenic plants. For example, although in many cases microbial genes can be expressed at high levels in plants without modification, low levels of expression in transgenic plants may be due to microbial nucleotide sequences having codons that are not preferred by the plant. As is known in the art, living organisms have certain preferences regarding the frequency of codon use, and therefore the codons of the nucleotide sequences described in this invention can be changed in accordance with the preferences characteristic of plants, while maintaining the amino acids encoded by them. It is also known in the art that high level expression in plants, such as corn plants, is best achieved with coding sequences that have at least about 35% GC content, or at least about 45%, or at least about 50% or at least about 60%. Microbial nucleotide sequences that have low GC content may be poorly expressed in plants. Although certain nucleotide sequences can be properly expressed in both monocot and dicot plant species, the sequences can be modified to accommodate codon preferences and GC content preferences for monocots or dicots, as these preferences have been shown to differ (Murray et al Nucl Acids Res 17:477-498 (1989)). Additionally, in embodiments, the nucleotide sequence is modified to remove illegal splice sites that may cause transcript truncation. Such modifications of nucleotide sequences can be performed using well-known techniques of site-directed mutagenesis, PCR and the construction of synthetic genes using methods described, for example, in US patent No. 5625136; 5500365 and 6013523.

[00219] In some embodiments, the present invention provides synthetic coding sequences or polynucleotide obtained according to the procedure disclosed in US Pat. No. 5,625,136. In this procedure, the preferred codons for maize are used, i.e. one codon that most often codes for a given amino acid in maize. The maize preferred codon encoding a particular amino acid can be determined, for example, based on known maize gene sequences. For example, codon frequency data in maize for 28 genes from maize plants are found in Murray et al., Nucleic Acids Research 17:477-498 (1989). It is known that codons optimized for expression in a plant of one species will also function in plants of other species, but probably not to the same extent as in the plant species for which the codons were optimized. Likewise, nucleotide sequences can be optimized for expression in any plant. It should be understood that all or any part of the nucleotide sequence may be optimized or synthetic. That is, the polynucleotide may contain a nucleotide sequence that is partly a native sequence and partly a codon optimized sequence.

[00220] In illustrative embodiments, the polynucleotide of the present invention is an isolated polynucleotide. In embodiments, the polynucleotide of the present invention is a recombinant polynucleotide.

[00221] In embodiments, the present invention further provides a nucleic acid molecule comprising a polynucleotide operably linked to a promoter (eg, heterologous to the promoter). Promoters may include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters. In specific aspects, a promoter useful in the present invention is a promoter capable of initiating transcription of a nucleotide sequence in a plant cell, such as a monocot (eg, maize or rice) or dicot (eg, soybean, cotton) plant cell.

[00222] In embodiments, the heterologous promoter is a promoter that allows expression in a plant (eg, expressed in a monocot or expressed in a dicot). For example, without limitation, the promoter driving expression in a plant can be selected from: ubiquitin, Cestrum orange virus, maize TrpA, OsMADS 6, maize histone H3, 5'-UTR of bacteriophage T3 gene 9, maize sucrose synthetase 1, maize alcohol dehydrogenase 1 , corn light harvesting complex, corn heat shock protein, maize mtl, pea carboxylase small subunit RuBP, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine-rich protein 1, potato patatin, lectin , 35S CaMV and the small subunit S-E9 of RuBP carboxylase.

[00223] Although many promoters from dicots have been shown to function in monocots and vice versa, in embodiments, dicot promoters are selected for expression in dicots and promoters from monocots are selected for expression in monocots. However, there are no restrictions regarding the origin of the selected promoters; it is sufficient that they are functional in relation to controlling the expression of nucleotide sequences in the desired cell.

[00224] The choice of promoter may vary depending on the temporal and spatial requirements for expression, as well as depending on the host cell to be transformed. Thus, for example, the expression of the nucleotide sequences of the present invention can occur in any plant and/or part of a plant (for example, in leaves, in pedicels or stems, in ears, in inflorescences (for example, ears, panicles, ears, etc. .), in roots, seeds and/or seedlings, etc.). For example, when expression in a particular tissue or organ is desired, a tissue-specific or tissue-preferred promoter (eg, root-specific/-preferred promoter) can be used. In contrast, if expression in response to a stimulus is desired, a promoter inducible by certain stimuli or chemicals can be used. If constant expression at a relatively stable level in all cells of the plant is desired, a constitutive promoter may be selected.

[00225] Promoters useful in accordance with the present invention include, but are not limited to, promoters that drive expression of a nucleotide sequence constitutively, promoters that drive expression upon induction, and promoters that drive expression in a tissue-specific or developmental stage-specific manner. These different types of promoters are known in the art.

[00226] Suitable constitutive promoters include, for example, the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. W004081173A2); the maize Ubi 1 promoter (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); 19S CaMV (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant J November; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); maize histone H3 (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); actin 2 (An et al., Plant J. 10(1); 107-121, 1996), constitutive CT2 root tip promoter (SEQ ID NO: 1535; see also PCT application No. IL/2005/000627) and synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those described in US patent No. 5659026, 5608149; 5608144; 5604121; 5569597: 5466785; 5399680; 5268463 and 5608142.

[00227] Tissue-specific or tissue-preferred promoters useful for expression of the polypeptides of the present invention in plants, optionally in maize, include those that drive expression in root, pith, leaf or pollen. Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [such as those described, for example, in Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed-preferred promoters (for example, from seed-specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), promoters of Brazil nut albumin (Pearson et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), glutelin (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts 221: 43-47, 1987), zein (Matzke et al., Plant Mol Biol, 143. 323-32 1990), napA (Stalberg, et al., Planta 199: 515-519, 1996), wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], endosperm-specific promoters (e.g. wheat LMW and HMW , glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat gliadins a, b and g (EMBO3:1409-15, 1984), barley ltrl promoter, hordein B1, C, D barley (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), barley DOF (Mena et al., The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33 promoter, rice globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), alpha globulin REB/ Rice OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR family gene promoter (Plant J 12: 235-46, 1997), sorghum gamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], germ-specific promoters (e.g. rice OSH1; Sato et al., Proc. Nati. Acad. Sci. USA , 93: 8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998), specific for flowers, promoters such as AtPRP4, chalcone synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217 :240-245; 1989), apetala-3 and promoters specific for plant reproductive tissues (eg, OsMADS promoters; US Patent Publication No. 2007/0006344).

[00228] Examples of promoters suitable for preferential expression in green tissue include many promoters that regulate genes involved in photosynthesis, many of which have been cloned from both monocots and dicots. One such promoter is the PEPC promoter of the maize phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Another root-specific expression promoter is that described by de Framond (FEBS 290:103-106 (1991) or US Pat. No. 5,466,785). Another promoter useful in the present invention is the stem-specific promoter described in US Pat. No. 5,625,136, which naturally drives the expression of the maize trpA gene.

[00229] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 5'-UTR gene 9 promoter and other promoters disclosed in US Pat. No. 7,579,516. Other promoters useful in accordance with the present invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene (Kti3) promoter.

[00230] In some embodiments of the present invention, inducible promoters may be used. Thus, for example, chemically regulated promoters can be used to modulate gene expression in a plant through the use of an exogenous chemical regulator. Regulation of the expression of the nucleotide sequences of the present invention through chemically regulated promoters ensures the synthesis of the polypeptides of the present invention only when the crop plants are treated with inducing chemicals. Depending on the purpose, the promoter may be a chemical-inducible promoter, where application of a chemical induces expression of a gene, or a chemical-repressible promoter, where application of a chemical inhibits expression of a gene. Examples of such technology for chemical induction of gene expression are described in detail in the publication of application EP 0332104 and US patent No. 5614395.

[00231] Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the o Tobacco PR-1a, which is activated by salicylic acid (eg, PR1a systems), steroid-dependent promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421- 10425 and McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) WE. Gen. Genet. 221, 229-237 , and US Pat. Nos. 5,814,618 and 5,789,156), Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (eg, PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant Jr. 11:605-612), and promoters of the ecdysone-inducible system.

[00232] Other non-limiting examples of inducible promoters include ABA and turgor pressure inducible promoters, auxin binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), UDP-glucose flavonoid glycosyltransferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150) and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) PlantMol. Biol 29:1293-1298; Martinez et al (1989) J Mol Biol 208:551-565 and Quigley et al (1989) J Mol Evol 29:412-421). Also included are benzenesulfonamide-inducible (US Pat. No. 5,364,780) and alcohol-inducible systems (International Patent Application Publications Nos. WO 97/06269 and WO 97/06268) and glutathione S-transferase gene promoters. Likewise, any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol can be used. 7:168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108. Other chemically inducible promoters useful for driving the expression of the nucleotide sequences of the present invention in plants are disclosed in US Pat. No. 5,614,395. Chemical induction of gene expression is also detailed in EP 0332104 (from Ciba-Geigy) and US Patent No. 5,614,395.

[00233] Another category of promoters useful in the present invention are wound-inducible promoters. Numerous promoters have been described that are expressed at sites of wounding as well as at sites of phytopathogen infection. Ideally, such a promoter should be activated locally at sites of insect infestation, and thus insecticidal proteins accumulate exclusively in cells where the synthesis of insecticidal proteins is necessary to destroy the attacking insect pest. Examples of promoters of this type include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993) and Warner et al. Plant J 3:191-201 (1993).

[00234] In embodiments, the nucleic acid of the present invention may comprise, consist essentially of, or be comprised of an expression cassette, or may be contained in an expression cassette.

[00235] An expression cassette containing a polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. An expression cassette may also be a sequence that occurs naturally but has been produced in a recombinant form suitable for heterologous expression. However, as a rule, the expression cassette is heterologous to the host, i.e. the particular nucleic acid sequence in the expression cassette does not occur naturally in the host cell and had to be introduced into the host cell or host cell ancestor by a transformation event.

[00236] In addition to promoters operably linked to the nucleotide sequences of the present invention, the expression cassette of the present invention may also contain other regulatory elements. Regulatory elements include, but are not limited to, enhancers, nitrones, translation control leader sequences, termination signals, and polyadenylation signal sequences. Examples of suitable transcription termination signals are available and known in the art (eg, tml from CaMV, E9 from rbcS). In the context of the present invention, any available terminator known to function in plants can be used.

[00237] Many other sequences can be introduced into the expression cassettes described in the present invention. They provide sequences that have been shown to enhance expression, such as intronic sequences (eg from Adhl and bronzel) and viral leader sequences (eg from TMV, MCMV and AMV).

[00238] To more efficiently initiate translation, the sequences adjacent to the initiation methionine can be modified. For example, they can be modified by including sequences known to be effective in plants. Joshi has proposed a suitable consensus sequence for plants (NAR 15:6643-6653 (1987)), and Clonetech offers an additional translation initiator consensus sequence (catalog 1993/1994, p. 210). These consensus sequences are suitable for use with the nucleotide sequences of the present invention. The sequences are inserted into constructs containing nucleotide sequences upstream and inclusive of ATG (with the second amino acid remaining unmodified) or alternatively upstream and inclusive of GTC located downstream of ATG (with the possibility of modifying the second amino acid of the transgene).

[00239] In embodiments, it may be advantageous to target expression of the polypeptides of the present invention to a specific cellular location in a plant cell. In some cases, localization in the cytosol may be desirable, while in other cases, localization in a specific subcellular organelle may be preferred. Any mechanism for targeting gene products, for example in plants, can be used to practice the present invention, and such mechanisms are known to exist in plants, and the sequences regulating the operation of such mechanisms have been described in some detail. Sequences that mediate targeting of gene products to other cellular compartments have been characterized. For example, amino-terminal sequences may cause the targeting of a protein of interest to a cellular compartment such as the vacuole, mitochondria, peroxisome, protein bodies, endoplasmic reticulum, chloroplast, starch granule, amyloplast, apoplast, or plant cell wall (eg, Unger et. al. Plant Molec Biol 13: 411-418 (1989) Rogers et al (1985) Proc Natl Acad Sci USA 82: 6512-651 US Patent No. 7102057 WO 2005/096704 Optionally, the signal sequence may be an N-terminal signal sequence from waxy, an N-terminal signal sequence from gamma-zein, a starch binding domain, a C-terminal starch binding domain, a chloroplast targeting sequence that ensures delivery of the mature protein to the chloroplast (Comai et. al. (1988) J. Biol. Chem. 263: 15104-15109; van den Broeck, et. al. (1985) Nature 313: 358-363; US patent No. 5639949) or the signal sequence for secretion from aleurone layer cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). In addition, amino-terminal sequences together with carboxy-terminal sequences mediate targeting of gene products to the vacuole and can be used in the present invention (Shinshi et. al. (1990) Plant Molec. Biol. 14: 357-368). In one embodiment, the selected signal sequence includes a known cleavage site, and the design of the fusion takes into account any amino acids downstream of the cleavage site(s) required for cleavage. In some cases, this requirement can be met by adding a small number of amino acids between the cleavage site and the ATG of the transgene or, alternatively, by replacing some amino acids within the transgene sequence. These design techniques are well known in the art and are equally applicable to any cellular compartment.

[00240] It will be appreciated that the cellular targeting mechanisms described above can be used not only in combination with their cognate promoters, but also in combination with heterologous promoters, such that a specific cellular targeting task is carried out under the transcriptional control of a promoter characterized by an expression pattern different from that of the promoter from which the targeting signal is derived.

[00241] The expression cassette of the present invention may also include a nucleotide sequence for a selectable marker that can be used to select a transformed plant, plant part, or plant cell. Many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[00242] Examples of selectable markers include, but are not limited to, the nucleotide sequence encoding neo or nptII, which confer resistance to kanamycin, G418, and the like. (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) that confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase, such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application No. 154204); a nucleotide sequence encoding methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding dalapon dehalogenase, which confers resistance to dalapon; a nucleotide sequence encoding mannose-6-phosphate isomerase (also called phosphomannose isomerase (PMI)), which confers the ability to metabolize mannose (US Pat. No. 5,767,378 and US Pat. No. 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph that confers resistance to hygromycin. One skilled in the art will be able to select an appropriate selectable marker for use in the expression cassette of the present invention.

[00243] Additional selectable markers include, but are not limited to, the nucleotide sequence encoding β-glucuronidase or uidA (GUS), which encodes an enzyme for which various chromogenic substrates are known; nucleotide sequence of the R-locus, which encodes a product that regulates the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac," pp. 263-282 , in Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates are known (eg, PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE, which encodes catechol dioxytenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which chromogenic substrates exist; a nucleotide sequence encoding luciferase (lux), which allows detection by bioluminescence (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which can be used in the detection of calcium-sensitive bioluminescence (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One skilled in the art can select a suitable selectable marker for use in the expression cassette of the present invention.

[00244] In some embodiments, the expression cassette of the present invention may also contain polynucleotides that encode other desired features in addition to the chimeric insecticidal proteins of the present invention. Examples of such other polynucleotides include polynucleotides that encode a polypeptide or dsRNA for other desired feature(s) of interest. Such expression cassettes containing "stacked" traits can be used, for example, to produce plants, plant parts or plant cells having the desired phenotype with stacked traits (ie, molecular stacking). Such packaged combinations in plants can also be created using other methods, including, but not limited to, cross-breeding plants using any conventional technique (ie, breeding package). When "packaging" by genetic transformation of plants, nucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant containing one or more desired traits can be used as a target for introducing additional traits through subsequent transformation. Additional nucleotide sequences can be introduced according to the co-transformation protocol simultaneously with the nucleotide sequence, nucleic acid molecule, nucleic acid construct or composition of the present invention provided by any combination of expression cassettes. For example, if two nucleotide sequences are introduced, they can be inserted into separate cassettes (trans), or they can be inserted into one cassette (cis). Expression of polynucleotides can be driven by the same promoter or by different promoters. In addition, it is known that polynucleotides can be packaged at a desired location in the genome using a site-specific recombination system. See, for example, international patent application publications Nos. WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855 and WO 99/25853.

[00245] In illustrative embodiments, the expression cassette may also contain an additional sequence encoding one or more polypeptides or double-stranded RNA (dsRNA) molecules that provide an agronomic trait of interest (e.g., an agronomic trait that primarily benefits a seed company, agricultural producer, or grain processor). The polypeptide of interest may be any polypeptide encoded by the nucleotide sequence of interest. Non-limiting examples of polypeptides of interest that are suitable for production in plants include those that confer agronomically important traits such as herbicide tolerance (also sometimes referred to as “herbicide tolerance”), virus resistance, pathogenic bacterial resistance, resistance insect resistance, nematode resistance or fungal resistance. See, for example, US Pat. Nos. 5,569,823; 5304730; 5495071; 6,329,504 and 6,337,431. In embodiments, the polypeptide of interest may also be one that increases plant vigor or yield (including traits that enable a plant to grow under varying temperatures, soil conditions, and levels of sunlight and precipitation), or one that which makes it possible to identify a plant exhibiting a trait of interest (eg, selectable marker, seed coat color, etc.). A variety of polypeptides of interest, as well as methods for introducing these polypeptides into a plant, are described, for example, in US patent No. 4761373; 4769061; 4810648; 4940835; 4975374; 5013659;5162602;5276268; 5304730; 5495071; 5554798; 5561236; 5569823; 5767366; 5879903, 5928937; 6084155; 6329504 and 6337431; and US Patent Application Publication No. 2001/0016956. See also on the World Wide Web at lifesci.Sussex.ac.ukyhome/Neil_Crickmore/Bt/.

[00246] In some embodiments of the present invention, polynucleotides that confer resistance/tolerance to a growth cone or meristem inhibitory herbicide, such as an imidazolinone or a sulfonylurea, may also be used. Exemplary polynucleotides in this category encode mutant ALS and AHAS enzymes, as described, for example, in US Pat. Nos. 5,767,366 and 5,928,937. US Pat. Nos. 4,761,373 and 5,013,659 cover plants resistant to various imidazolinone or sulfonamide herbicides. US Patent No. 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a mutant glutamine synthetase (GS) that is resistant to inhibition by herbicides known to inhibit GS, such as phosphinothricin and methionine sulfoximine. US Pat. No. 5,162,602 discloses plants that are resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. Resistance is imparted by altered acetyl-coenzyme A carboxylase (ACCase).

[00247] Polypeptides encoded by nucleotide sequences that confer resistance to glyphosate are also suitable for the present invention. See, for example, US Pat. No. 4,940,835 and US Pat. No. 4,769,061. US Pat. No. 5,554,798 discloses transgenic maize plants resistant to glyphosate, the resistance of which is conferred by the altered 5-enolpyruvyl-3-phosphoshikimate synthase (EPSP) gene.

[00248] Polynucleotides encoding resistance to phosphonic compounds such as glufosinate ammonium or phosphinothricine, as well as pyridinoxy or phenoxypropionic acids and cyclohexanone are also suitable. See European Patent Application No. 0242246. See also US Patent Nos. 5879903, 5276268 and 5561236.

[00249] Other suitable polynucleotides include those encoding resistance to photosynthetic inhibitory herbicides such as triazine and benzonitrile (nitrilase gene). See US Patent No. 4810648. Additional suitable polynucleotides encoding herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are polynucleotides that confer resistance to protox enzyme inhibitors or that provide increased resistance to plant diseases; increased tolerance to adverse environmental conditions (types of abiotic stress), including but not limited to drought, excessive cooling, excessive heating, or excessive soil salinity, or extreme acidity or alkalinity; and changes in plant structure or development, including changes in the timing of development. See, for example, US Patent Application Publication No. 2001/0016956 and US Patent No. 6084155.

[00250] Additional suitable polynucleotides include those encoding pesticidal (eg, insecticidal) polypeptides. These polypeptides can be obtained in amounts sufficient to control, for example, insect pests (ie, in amounts that provide insect control). In embodiments, the polypeptide is a lepidopteran-active polypeptide, a coleopteran-active polypeptide, a hemiptera-active polypeptide, and/or a dipteran-active polypeptide, or any combination thereof. It is believed that the amount of pesticide polypeptide produced in a plant necessary to control insects or other pests may vary depending on the cultivar, type of pest, environmental factors, and the like. Polynucleotides useful for imparting additional resistance to insects or pests include, for example, those encoding toxins identified in Bacillus organisms. Polynucleotides containing nucleotide sequences encoding Bacillus thuringiensis (Bt) Cry proteins from several subspecies have been cloned and recombinant clones have been found to be toxic to the larvae of Lepidoptera, Diptera and Coleoptera insects. Examples of such Bt insecticidal proteins include Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, Cry9C, etc., as well as vegetative insecticidal proteins such as Vip1, Vip2, Vip3, etc., and any combinations of the above Bt insecticidal proteins. A complete list of Bt proteins can be found on the World Wide Web in the Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[00251] In embodiments, the additional polypeptide is an insecticidal polypeptide derived from a source other than Bt, including, but not limited to, alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase , lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus spp. insecticidal protein (such as X nematophila or X. bovienii), Photorhabdus spp. insecticidal protein (such as P. luminescens or P. asymobiotica), Brevibacillus insecticidal protein spp. (such as B. laterosporous), insecticidal protein of Lysinibacillus spp. (such as L. sphearicus), insecticidal protein of Chromobacterium spp. (such as C. subtsugae or C. piscinae), insecticidal protein of Yersinia spp. (such as Y. entomophagd), insecticidal protein of Paenibacillus spp. (such as P. propylaea), insecticidal protein of Clostridium spp. (such as C. bifermentans), Pseudomonas spp. (such as P. fluorescens) and lignin.

[00252] Polypeptides suitable for production in plants further include those polypeptides that enhance or otherwise facilitate the conversion of harvested plants or plant parts into a commercially usable product, including, for example, providing increased or altered carbohydrate content or distribution, improved fermentability properties, increased oil content, increased protein content, improved digestibility or increased nutraceutical content, for example, increased phytosterol content, increased tocopherol content, increased stanol content or increased vitamin content. Polypeptides of interest also include, for example, those that cause or contribute to the reduction of an undesirable component in the harvested crop, such as phytic acid or sugar degrading enzymes. By “causing” or “promoting” is meant that the polypeptide of interest can directly or indirectly promote the presence of the trait of interest (eg, by increasing the breakdown of cellulose by the heterologous enzyme cellulase).

[00253] In some embodiments, the polypeptide helps improve the digestibility of a food or feed. Xylanases are hemicellulose-degrading enzymes that enhance the breakdown of plant cell walls, resulting in better utilization of plant nutrients by animals. This leads to increased growth rates and feed conversion. It is also possible to reduce the viscosity of feeds containing xylan. Heterologous production of xylanases in plant cells may also contribute to the conversion of lignocellulose to fermentable sugars during industrial processing.

[00254] Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized (see, for example, US Pat. No. 5,437,992; Coughlin et al. (1993) "Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases" Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; US Patent Publication No. 2005/0208178; and PCT Publication WO 03/16654). In particular, three specific xylanases (XYL-I, XYL-II and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) EnzymeMicrob. Technol. 14:566; Torronen et al. (1992) Bio/ Technology 10:1461; and Xu et al. (199%) Appl. Microbiol. Biotechnol. 49:718).

[00255] In other embodiments, a polypeptide useful for the present invention may be a polysaccharide-degrading enzyme. Plants of the present invention that produce such an enzyme can be used to obtain, for example, fermentable raw materials for biological processing. In some embodiments, enzymes useful for the fermentation process include alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycosyltransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolyzing enzyme, and others glucoamylase.

[00256] Polysaccharide-degrading enzymes include starch-degrading enzymes such as alpha-amylases (EC 3.2.1.1), glucuronidases (EC 3.2.1.131); exo-1,4-alpha-B-glucanases such as amyloglucosidases and glucoamylase (EC 3.2.1.3), beta-amylases (EC 3.2.1.2), alpha-glucosidases (EC 3.2.1.20) and other exo-amylases; enzymes that break down starch, such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), etc.; b) cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-beta-D-glucanase (EC 3.2.1.39), beta-glucosidase (EC 3.2.1.21); c) L-arabinases such as endo-1,5-alpha-L-arabinase (EC 3.2.1.99), alpha-arabinosidases (EC 3.2.1.55) and the like; d) galactanases such as endo-1,4-beta-D-galactanase (EC 3.2.1.89), endo-1,3-beta-B-galactanase (EC 3.2.1.90), alpha-galactosidase (EC 3.2.1.22 ), beta-galactosidase (EC 3.2.1.23), etc.; e) mannanases such as endo-1,4-beta-D-mannanase (EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), alpha-mannosidase (EC 3.2.1.24) and the like; f) xylanases such as endo-1,4-beta-xylanase (EC 3.2.1.8), beta-D-xylosidase (EC 3.2.1.37), 1,3-beta-B-xylanase and the like; and g) other enzymes such as alpha-L-fucosidase (EC 3.2.1.51), alpha-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7), etc. P. In one embodiment, the alpha-amylase is a synthetic alpha-amylase, Amy797E, described in US Pat. No. 8,093,453.

[00257] Additional enzymes that can be used in accordance with the present invention include proteases, such as fungal and bacterial proteases. Fungal proteases include, but are not limited to, those derived from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei. In some embodiments, the polypeptides of the present invention may be cellobiohydrolase (CBH) enzymes (EC 3.2.1.91). In one embodiment, the cellobiohydrolase enzyme may be CBH1 or CBH2.

[00258] Other enzymes useful in accordance with the present invention include, but are not limited to, hemicellulases such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (for example, E.C.3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha-1,6-glucosidases (for example, E.C.3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73) and acetylxylanesterase (EC 3.1.1.72); and cutinases (eg E.C.3.1.1.74).

[00259] Double-stranded RNA (dsRNA) molecules useful in the present invention include, but are not limited to, those that suppress gene expression in a target pest (eg, insect). In embodiments, the dsRNA targets a gene of a lepidopteran, coleopteran, hemiptera, or dipteran insect pest, or any combination of the above. As used herein, the phrase “gene suppression” is intended to collectively refer to any well-known methods of reducing the levels of protein produced by transcription of a gene into mRNA and subsequent translation of the mRNA. It is also intended that gene suppression means a decrease in protein expression from a gene or coding sequence, including post-transcriptional gene suppression and transcriptional suppression. Post-transcriptional gene suppression is due to homology of all or part of the mRNA transcribed from the gene or coding sequence targeted for suppression and the corresponding double-stranded RNA used for suppression, and means a significant and measurable reduction in the amount of mRNA available in the cell for binding by ribosomes. The transcribed RNA can be in the sense orientation to produce an effect called cosuppression, in the antisense orientation to produce an effect called antisense suppression, or in both orientations to produce a dsRNA to produce an effect called RNA interference (RNAi). Transcriptional suppression is caused by the presence in the cell of dsRNA, a means of suppressing a gene, characterized by significant sequence identity with the promoter DNA sequence or its complementary strand, to produce an effect called promoter suppression in trans. Gene suppression may be effective against a native plant gene associated with a trait, for example, to provide plants with reduced levels of the protein encoded by the native gene or increased levels of the altered metabolite. Gene suppression may also be effective against target genes in plant pests that may ingest or come into contact with plant material containing agents to provide gene suppression specifically designed to inhibit or suppress one or more homologous or complementary sequences in cells of the pest. Such suppression target genes may encode an essential protein, the main function of which is selected from the group consisting of muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, cell membrane potential maintenance, amino acid biosynthesis, amino acid degradation , sperm formation, pheromone synthesis, pheromone perception, antenna formation, wing formation, limb formation, development and differentiation, egg formation, larval maturation, formation of digestive enzymes, hemolymph synthesis, maintaining a constant composition of hemolymph, transmission of nerve impulses, cell division, energy metabolism , development and differentiation, respiration and apoptosis.

[00260] In embodiments, the nucleic acids of the present invention may further comprise, consist of, or consist of a vector. In embodiments, the polynucleotides and expression cassettes of the present invention are contained in a vector. Vectors for use in the transformation of plants and other organisms are well known in the art. Non-limiting examples of common classes of vectors include plasmid, phage vector, phagemid vector, cosmid vector, fosmid, bacteriophage, artificial chromosome or viral vector. In embodiments, the vector is a plant vector, for example, for use in plant transformation. In embodiments, the vector is a bacterial vector, for example, for use in transforming bacteria. Suitable vectors for plants, bacteria and other organisms are known in the art.

Transgenic plants, plant parts, plant cells, seeds

[00261] The present invention also includes a transgenic host cell other than a human cell that contains a polynucleotide, nucleic acid molecule, expression cassette, vector or polypeptide of the present invention. A transgenic host cell other than a human cell may include, but is not limited to, a plant cell (including a monocot cell and/or a dicot cell), a yeast cell, a bacterial cell, or an insect cell. Therefore, in some embodiments of the present invention, a bacterial cell selected from the genus Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius is provided , Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc or Alcaligenes. Thus, for example, as insect biological control agents, chimeric insecticidal proteins of the present invention can be produced by expressing a polynucleotide encoding them in a bacterial cell. For example, in some embodiments, a Bacillus thuringiensis cell is provided containing a polynucleotide encoding a chimeric insecticidal protein of the present invention.

[00262] In embodiments, the transgenic plant cell is a monocot plant cell or a dicot plant cell. In further embodiments, the dicotyledonous plant cell is a soybean cell, a sunflower cell, a tomato cell, a cabbage cell, a cotton cell, a sugar beet cell, and a tobacco cell. In further embodiments, the monocot cell is a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, and a wheat cell. Embodiments of the present invention provide a collection of dicotyledonous plant cells or monocotyledonous plant cells containing a polynucleotide expressing the chimeric insecticidal protein of the present invention. In embodiments, the cells are collectively located adjacent to form an apoplast and are grown in natural sunlight. In embodiments, the transgenic plant cell cannot regenerate into a whole plant.

[00263] In embodiments of the present invention, the insecticidal protein of the present invention is expressed in a higher organism, such as a plant. In this case, transgenic plants expressing effective amounts of the insecticidal protein protect themselves from plant pests such as insect pests. When an insect begins to feed on such a transgenic plant, it also takes up the expressed insecticidal protein. This may keep the insect from boring further into the plant tissue or may even harm or kill the insect. In embodiments, a polynucleotide of the present invention is inserted into an expression cassette, which is then stably integrated into the plant genome. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus.

[00264] In some embodiments of the present invention, the transgenic plant cell comprising a nucleic acid molecule or polypeptide of the present invention is a cell of a plant part, a plant organ, or a plant crop (each as described herein), including, but not limited to, a root, a leaf , seed, flower, fruit, pollen cell, plant organ or culture, etc., or callus cell or culture.

[00265] The transgenic plant or plant cell in accordance with the present invention may be a monocotyledonous or dicotyledonous plant or plant cell and includes, but is not limited to, corn (maize), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beets, cotton, sugar cane, oilseed rape, alfalfa, tobacco, peanuts, vegetables (including sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, carrots, eggplant, cucumber , radish, spinach, potatoes, tomato, asparagus, onion, garlic, melon, pepper, celery, pumpkin, butternut squash, pumpkin, etc.), fruit crops (including apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, etc.) and a specialized plant or plant cell (such as Arabidopsis), or a woody plant or plant cell (such as coniferous and/or deciduous trees). In embodiments, the plant or plant cell of the present invention is a crop plant or plant cell, such as a plant or plant cell of maize, sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybean, sugar beet, sugar cane, tobacco, barley, oilseed rape, etc.

[00266] The present invention further provides a transgenic plant portion of the present invention. Optionally, the plant part contains the chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00267] The present invention further provides a seed of a transgenic plant of the present invention or a seed from which a transgenic plant of the present invention is obtained. Optionally, the seed contains the chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00268] Additional embodiments of the present invention include harvested products derived from transgenic plants, plant parts or seeds of the present invention, as well as a processed product derived from the harvested product. The harvested product may be a whole plant or any part of a plant, as disclosed herein. Thus, in some embodiments, non-limiting examples of the harvested product include a seed, fruit, flower or part thereof (e.g., anther, stigma, etc.), leaf, stem, and the like. In other embodiments, the processed product includes, but is not limited to, flour, meal, oil, starch, grain, and the like obtained from the harvested seed or other part of the plant of the present invention. Optionally, the collected product or processed product contains the chimeric insecticidal protein of the present invention and/or a nucleic acid encoding it.

[00269] In other embodiments, the present invention provides an extract from a transgenic plant or plant part of the present invention, wherein optionally the extract contains a chimeric insecticidal protein of the present invention and/or a nucleic acid encoding the same. Extracts from plants or plant parts can be prepared according to procedures well known in the art (see de la Torre et al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic Acids Res. 22(9): 1772-1773 (1994); Lipton et al., Food Agric. Immun. 12:153-164 (2000)).

[00270] The chimeric insecticidal protein may function in a plant part, a plant cell, a plant organ, a seed, a harvested product, a processed product or extract, and the like. as an insect control agent. In other words, the chimeric insecticidal protein can continue to perform the insecticidal function that it had in the transgenic plant. The nucleic acid may function to express the chimeric insecticidal protein. As an alternative to encoding the insecticidal protein of the present invention, the nucleic acid may function to detect a transgenic plant part, plant cell, plant organ, seed, harvested product, processed product or extract of the present invention.

[00271] In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is hemizygous for the polynucleotide or expression cassette of the present invention. In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is homozygous for the polynucleotide or expression cassette of the present invention.

[00272] In embodiments, the transgenic plant, plant part, plant cell, plant organ, seed, harvested product, processed product, or extract has increased resistance to one or more insect pests (e.g., a lepidopteran pest such as armyworm ) compared to a suitable control that does not contain a nucleic acid encoding the insecticidal protein of the present invention.

Plant transformation

[00273] Plant transformation procedures are well known and accepted in the art and are described in the literature in all respects. Non-limiting examples of plant transformation methods include transformation by bacterial (e.g., Agrobacterium)-mediated nucleic acid delivery, virus-mediated nucleic acid delivery, silicon carbide or nucleic acid microneedles-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, or any other electrical, chemical, physical (mechanical) or biological mechanism , which results in the introduction of a nucleic acid into a plant cell, including any combination thereof. General guidance on a variety of plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy- Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[00274] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are generally suitable, while for direct gene transfer (eg, by particle bombardment, etc.) any vector is suitable. in this case, linear DNA containing only the construct of interest can be used. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). In the case of both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) performed with a selectable marker, which may be a positive selection agent (e.g., phosphomannose isomerase) that confers resistance to an antibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide (eg glyphosate or glufosinate). However, the choice of selectable marker is not critical to the present invention.

[00275] Agrobacterium-mediated transformation is a method widely used for plant transformation due to its high transformation efficiency and its wide applicability to many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying foreign DNA of interest into the appropriate Agrobacterium strain, which may depend on the host Agrobacterium strain's vir gene set located either on a coresident Ti plasmid or on the chromosome (Uknes et al (1993) Plant Cell 5:159-169). Transfer of the recombinant binary vector into Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector with an E. coli helper strain carrying a plasmid that is capable of mobilizing the recombinant binary vector into the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred into Agrobacterium by nucleic acid transformation (Hofgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[00276] Dicotyledons as well as monocotyledons can be transformed using Agrobacterium. Methods for Agrobacterium-mediated transformation of rice include well-known methods for transformation of rice, such as those described in any of the following: European Patent Application EP 1198985 Al, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22(3):491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), the disclosures of which are incorporated herein by reference as if set forth in their entirety. In the case of maize transformation, the preferred method is as described or in Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996), or Frame et al. (Plant Physiol 129(1): 13-22, 2002), the disclosures of which are incorporated herein by reference as if set forth in their entirety. These methods are also described by way of example in c. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or construct to be expressed are preferably cloned into a vector that is suitable for transformation of Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, such as model plants such as Arabidopsis, or crops such as, for example, tobacco plants, for example by dipping crushed leaves or chopped leaves into an Agrobacteria solution and then culturing them in a suitable medium. Plant transformation by Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known in particular from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[00277] Plant transformation with recombinant Agrobacterium typically involves co-cultivating Agrobacterium with plant explants and is carried out according to methods well known in the art. The transformed tissue is regenerated on a selective medium containing an antibiotic or herbicide resistance marker between the bordering T-DNA sequences of the binary plasmid.

[00278] As previously discussed, another method of transforming plants, plant parts and plant cells involves introducing inert or biologically active particles into plant tissues and cells. See, for example, US Patent Nos. 4,945,050, 5,036,006 and 5,100,792. In general, this method involves introducing inert or biologically active particles into plant cells under conditions effective to penetrate the outer surface of the cell and allow for incorporation into the interior. When using inert particles, the vector can be introduced into a cell by coating the particles with a vector containing the nucleic acid of interest. Alternatively, the cell or cells may be surrounded by the vector such that the vector follows the particle into the cell. Biologically active particles (eg, a dried yeast cell, a dried bacterium, or a bacteriophage, each containing one or more nucleic acids to be introduced) can also be incorporated into plant tissue.

[00279] In other embodiments, the polynucleotide of the present invention can be directly introduced into the plastid genome by transformation. The main advantage of plastid transformation is that plastids are usually capable of expressing bacterial genes without significant modification, and plastids are capable of expressing multiple open reading frames under the control of a single promoter. Plastid transformation technology is described in detail in US Patent Nos. 5451513, 5545817 and 5545818, PCT Application No. WO 95/16783 and McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing sections of cloned plastid DNA flanking a selectable marker, along with the gene of interest, into a suitable target tissue, for example, using biolistics or protoplast transformation (eg, calcium chloride or PEG-mediated transformation). The flanking areas are 1-1.5 in size, i.e. called targeting sequences, promote homologous recombination with the plastid genome and thus ensure the replacement or modification of specific regions of the plastome. First, point mutations in the chloroplast 16S rRNA and rps12 genes, conferring resistance to spectinomycin or streptomycin, can be used as a selectable transformation marker (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci USA 87, 8526-8530; Staub, J.M., and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allows the creation of a plastid-targeted vector for the introduction of foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606). A significant increase in the frequency of transformation was achieved by replacing recessive rRNA or r-protein genes that provide resistance to antibiotics with a dominant selectable marker, the bacterial aadA gene, encoding the enzyme aminoglycoside 3'-adenyltransferase, which neutralizes spectinomycin (Svab, Z., and Maliga, P (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker was successfully used to transform the plastid genome of the green alga Chlamydomonas reinhardtii with high frequency (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present invention. Typically, it takes about 15-20 cycles of cell division after transformation to reach a state in which all plastids are identical. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number compared to genes expressed in the nucleus, allowing expression levels that can easily exceed 10% of the total amount of soluble vegetable proteins. In one embodiment, the polynucleotide of the present invention can be inserted into a plastid-targeting vector and introduced into the plastid genome of the desired host plant by transformation. Thus, it is possible to obtain plants that are homoplastic with respect to plastid genomes containing the nucleotide sequence of the present invention, capable of expressing the polynucleotide at a high level.

[00280] Methods for selecting transformed transgenic plants, plant cells or plant tissue cultures are generally accepted in the art and can be used in the methods of the present invention provided herein. For example, the recombinant vector of the present invention may also include an expression cassette containing a nucleotide sequence of a selectable marker that can be used to select a transformed plant, plant part or plant cell.

[00281] Additionally, as is well known in the art, whole transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts using any of a variety of known techniques. Regeneration of plants from plant cells, plant tissue culture or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I.R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

[00282] In addition, the above-described gene properties provided by genetic engineering techniques in the transgenic seeds and plants, plant parts or plant cells of the present invention can be transmitted through sexual reproduction or vegetative growth and therefore can be maintained and inherited descendant plants. Typically, maintenance and inheritance are carried out using known agricultural methods designed to suit specific purposes, such as harvesting, sowing or cultivating.

[00283] Therefore, the polynucleotide can be introduced into a plant, plant part, or plant cell using any of a variety of methods well known in the art, as described above. Therefore, no particular method of introducing one or more polynucleotides into a plant is followed, but rather any method that ensures stable integration of one or more polynucleotides into the plant genome can be used. If more than one polynucleotide is desired to be introduced, the corresponding polynucleotides can be assembled as parts of a single nucleic acid molecule or as separate nucleic acid molecules and can be located within the same or different nucleic acid molecules. Accordingly, introduction of polynucleotides into a cell of interest can be accomplished during a single transformation event, during separate transformation events, or, for example, into plants as part of a crossing protocol.

[00284] Once a desired polynucleotide is introduced into a particular plant species by transformation, using traditional propagation techniques it can be transferred within that species or into other varieties of the same species, particularly including commercial varieties.

Insecticidal compositions

[00285] In some embodiments, the present invention provides an insecticidal composition comprising a chimeric insecticidal protein of the present invention in an agriculturally acceptable carrier. As used herein, the expression "agriculturally acceptable carrier" may include a natural or synthetic, organic or inorganic material that is combined with an active protein to facilitate its application to or in a plant or part thereof. Examples of agriculturally acceptable carriers include, but are not limited to, powders, dusts, pellets, granules, aerosols, emulsions, colloids and solutions. Agriculturally acceptable carriers further include, but are not limited to, inerts, dispersants, surfactants, adjuvants, tackifiers, adhesives, binders, or combinations thereof, which can be used in formulations used in agriculture. Such compositions can be applied in any manner by which pesticide proteins or other pest control agents are brought into contact with pests. Accordingly, the compositions can be applied to the surfaces of plants or plant parts, including seeds, leaves, flowers, stems, tubers, roots, and the like. In other embodiments, a plant that produces the insecticidal protein of the present invention in planta provides an agriculturally acceptable carrier for the expressed insecticidal protein. In embodiments, the compositions and agriculturally acceptable carriers of the present invention exclude transgenic plants.

[00286] In further embodiments, the insecticidal composition comprises a bacterial cell or transgenic bacterial cell of the present invention, wherein the bacterial cell or transgenic bacterial cell produces an insecticidal protein of the present invention. Such an insecticidal composition can be prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, pelleting or concentrating a Bacillus thuringiensis (Bt) cell culture, including a transgenic Bt culture. In embodiments, the composition of the present invention may contain at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30% , at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least 70%, at least about 80%, at least about 90%, according to at least about 95%, at least about 97%, or at least 99% by weight of the polypeptide of the present invention. In additional embodiments, the composition contains from about 1% to about 99% by weight of the insecticidal protein of the present invention.

[00287] The insecticidal proteins of the present invention can be used in combination with other pest control agents to increase the range of target pests and/or to prevent or control insect resistance. In addition, the use of the insecticidal proteins of the present invention in combination with an insecticidal agent that has a different mechanism of action or targets a different receptor in the insect's digestive tract is particularly useful in preventing and/or controlling the emergence of resistance in insects.

[00288] Therefore, in some embodiments of the present invention, a composition is provided that provides control of one or more plant pests (e.g., an insect pest such as a lepidopteran insect pest, a coleopteran insect pest, an insect pest hemiptera and/or a dipteran insect pest), wherein the composition contains a first pest control agent that is a chimeric insecticidal protein of the present invention, and at least a second pest control agent that is different from the first agent for pest control. In other embodiments, the composition is a composition for topical application to a plant. In yet other embodiments, the composition is a transgenic plant. In additional embodiments, the composition is a combination comprising a composition and a transgenic plant to which it is applied. In some embodiments, the composition comprises a first pest control agent that is a chimeric insecticidal protein of the present invention if the transgenic plant contains a second pest control agent. In other embodiments, the composition comprises a second pest control agent if the transgenic plant contains a first pest control agent that is a chimeric insecticidal protein of the present invention.

[00289] In some embodiments, the second pest control agent may be one or more of a chemical pesticide such as an insecticide, a Bacillus thuringiensis (Bt) insecticidal protein, and/or an insecticidal agent from a non-Bt source, including, but not limited to, an insecticidal protein Xenorhabdus, Photorhabdus insecticidal protein, Brevibacillus laterosporus insecticidal protein, Bacillus sphaericus insecticidal protein, protease inhibitor (both serine and cysteine types), lectin, alpha-amylase, peroxidase, cholesterol oxidase, or double-stranded RNA (dsRNA) molecule.

[00290] In other embodiments, the second pest control agent is one or more chemical pesticides, which is not necessarily a seed coating. Non-limiting examples of chemical pesticides include pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics. In other embodiments, the chemical pesticide is one or more of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binphenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl , chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diophenolan, emamectin, endosulfan, esfenvalerate, ethiprole, phenoticarb, fenoxycarb, fenpropathrin, fenproximate, fenvalerate , fipronil, flonicamid, flucitrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonofos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathione, methomyl, methoprene, methoxy chlorine, monocrotophos, methoxyfenozide, nithiazine, novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate, fosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifene (BSN 2060) , sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap sodium, tralomethrine, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, quinomethionate, chlorobenzilate , cyhexatin, dicofol, dienochlor, etoxazole, phenazaquin , phenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridabene and tebufenpyrad. In another embodiment, the chemical pesticide is selected from one or more of cypermetrin, cigalotrin, digitalrin and beta-cyphlutrin, Esfenvalerat, Fenvalerata, Tralometrin, Phenotikarb, Metomil, Oxyle, Thiodicarb, Clotianidine, Imidaclopride, Tiaculopride, Indoxicarb, Spin-Proside, Abamectin, Averktin, Averex Ektina , emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, diophenolan, pyriproxyfen, pymetrozine and amitraz.

[00291] In additional embodiments, the second pest control agent may be one or more of any number of Bacillus thuringiensis insecticidal proteins, including, but not limited to, Cry protein, vegetative insecticidal protein (VIP), and insecticidal chimeric variants of any of the above insecticidal proteins . In other embodiments, the second pest control agent is a Cry protein selected from: Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg , Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry lib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja, Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2 Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7 ВБ, Cry 7Ca, Cry7Cb, Cry7Da , Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8 Da, Cry8Db, Cry8Ea, Cry8Fa , Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, Cry8Kb, Cry8La, Cry8Ma, Cry8Na, Cry8Pa, Cry8Qa, Cry8Ra, Cry8Sa, Cry8Ta, Cry9Aa, Cry9Ba, Cry9Bb, Cry9Ca, Cry9Da, Cry9Db, Cry9Dc , Cry9Ea, Cry9Eb, Cry9Ec , Cry9Ed, Cry9Ee, Cry9Fa, Cry9Ga, Cry10Aa, Cry1IAa, Cry1IBa, Cry1IBb, Cry12Aa, Cry13Aa, Cry14Aa, Cry14Ab, Cry15Aa, Cry16Aa, Cry17Aa, Cry18Aa, Cry18Ba, Cry18Ca, Cry19 Aa, Cry19Ba, Cry19Ca, Cry20Aa, Cry20Ba, Cry21Aa, Cry21Ba , Cry21Ca, Cry21Da, Cry21Ea, Cry21Fa, Cry21Ga, Cry21Ha, Cry22Aa, Cry22Ab, Cry22Ba, Cry22Bb, Cry23Aa, Cry24Aa, Cry24Ba, Cry24Ca, Cry25Aa, Cry26Aa, Cry27Aa, Cry28Aa, C ry29Aa, Cry29Ba, Cry30Aa, Cry30Ba, Cry30Ca, Cry30Da, Cry30Db , Cry30Ea, Cry30Fa, Cry30Ga, Cry31Aa, Cry31Ab, Cry31Ac, Cry31Ad, Cry32Aa, Cry32Ab, Cry32Ba, Cry32Ca, Cry32Cb, Cry32Da, Cry32Ea, Cry32Eb, Cry32Fa, Cry32Ga, Cry32Ha, C ry32FIb, Cry32Ia, Cry32Ja, Cry32Ka, Cry32La, Cry32Ma, Cry32Mb , Cry32Na, Cry320a, Cry32Pa, Cry32Qa, Cry32Ra, Cry32Sa, Cry32Ta, Cry32Ua, Cry33Aa, Cry34Aa, Cry34Ab, Cry34Ac, Cry34Ba, Cry35Aa, Cry35Ab, Cry35Ac, Cry35Ba, Cry3bAa, Cry3 7Aa, Cry38Aa, Cry39Aa, Cry40Aa, Cry40Ba, Cry40Ca, Cry40Da , Cry41Aa, Cry41Ab, Cry41Ba, Cry42Aa, Cry43Aa, Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc, Cry44Aa, Cry45Aa, Cry46Aa Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry6OBa, Cry61Aa, Cry62Aa, Cry6ZAa, Cry64Aa, Cry65Aa, Cry66Aa, Cry 67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, Cry73Aa, or any combination of the above. In embodiments, the Cry protein is Cry1Fa, for example, as present in the maize transformant TC1507.

[00292] In additional embodiments, the second pest control agent is one or more vegetative insecticidal proteins Vip3 selected from Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3 Aa12, Vip3Aa13, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17, Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa 29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Abl, Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3, Vip3Ag1, Vip3Ag2, Vip3 Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1, Vip3Ba2, Vip3Bbl, Vip3Bb2 , Vip3Bb3, or any combination of the above. In embodiments, the Vip3 protein is Vip3Aa (US Patent No. 6137033), for example, as presented in the maize transformant MIR162 (US Patent No. 8232456; US Patent No. 8455720 and US Patent No. 8618272).

[00293] In embodiments, the second pest control agent may be obtained from sources other than P. thuringiensis. For example, the second pest control agent may be alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, insecticidal protein of Xenorhabdus spp. (such as X nematophila or X. bovienii), insecticidal protein of Photorhabdus spp. (such as P. luminescens or P. asymobiotica), insecticidal protein of Brevibacillus spp. (such as P. laterosporous), insecticidal protein of Lysinibacillus spp. (such as P. sphearicus), insecticidal protein of Chromobacterium spp. (such as C. subtsugae or C. piscinae), insecticidal protein of Yersinia spp. (such as Y. entomophagd), insecticidal protein of Paenibacillus spp. (such as P. propylaed), insecticidal protein of Clostridium spp. (such as C. bifermentans), Pseudomonas spp. (such as P. fluorescens) and lignin. In other embodiments, the second agent may be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) from Photorhabdus, Xenorhabus, Serratia or Yersinia. In other embodiments, the insecticidal protein may be an ADP-ribosyltransferase derived from an insecticidal bacterium such as Photorhabdus ssp. In other embodiments, the insecticidal protein may be a VIP protein, such as VIP1 or VIP2 from B. cereus. In yet other embodiments, the insecticidal protein may be a binary toxin derived from insecticidal bacteria, such as ISP1A and ISP2A from B. laterosporous or BinA and BinB from L. sphaericus. In still other embodiments, the insecticidal protein may be engineered or may be a hybrid or chimera of any previous insecticidal proteins.

[00294] In some embodiments, the second pesticidal agent may be non-proteinaceous, for example, an interfering RNA molecule, such as dsRNA, which can be expressed transgenically or applied as part of a composition (eg, using topical application methods). Typically, interfering RNA contains at least an RNA fragment corresponding to the target gene, a spacer sequence and a second RNA fragment that is complementary to the first, due to which a double-stranded RNA structure can be formed. RNA interference (RNAi) occurs when the body recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments approximately 19-24 nucleotides in length, called small interfering RNA (siRNA). The siRNAs then spread or are carried throughout the body, including through cell membranes, where they hybridize with mRNA (or other RNAs) and cause RNA hydrolysis. Interfering RNAs are recognized by the RNA interference silencing complex (RISC), into which an effector strand (or “guide strand”) of RNA is introduced. This guide strand acts as a template for recognizing and degrading duplex sequences. This process is repeated each time the siRNA hybridizes with its complementary target RNA, effectively preventing the translation of such mRNAs, and thus silencing the expression of the specific genes from which the mRNAs were transcribed. Interfering RNAs are known in the art for use in insect control (see, for example, publication WO2013/192256, incorporated herein by reference). Interfering RNA, intended for use in insect control, produces a non-naturally occurring double-stranded RNA that uses the native RNAi pathways of the insect to trigger the silencing of target genes, which can result in cessation of feeding and/or growth and can lead to the death of the insect. pest The interfering RNA molecule may confer insect resistance against the same target pest as the protein of the present invention, or may specifically target a different pest. The target plant pest may feed by chewing, sucking, or piercing. Interfering RNAs are known in the art to be useful for insect control. In embodiments, a dsRNA useful for insect control is described in U.S. Provisional Patent Application Nos. 62/371259, 62/371261, or 62/371262, filed August 5, 2016. In embodiments, a dsRNA useful for insect control is described in patents US No. 92388223, 9340797 or 8946510. In embodiments, dsRNA useful for insect control is described in US patent applications No. 12/868994, 13/831230, 14/207313 or 14/207318. In other embodiments, the interfering RNA may confer resistance against a plant pest other than an insect, such as a nematode pest or a virus pest.

[00295] In still further embodiments, a first insect control agent that is a chimeric insecticidal protein of the present invention and a second pest control agent are coexpressed in the transgenic plant. Such co-expression of more than one pesticide component in the same transgenic plant can be achieved by producing a plant containing and expressing nucleic acid sequences encoding insect control agents through genetic engineering techniques. For example, coexpression of more than one pesticide in the same transgenic plant can be achieved by producing a single recombinant vector containing the coding sequences of more than one pesticide in a so-called "molecular package" and genetically engineering a plant to contain and express all these pesticides in the transgenic plant. Such molecular packages can also be obtained using minichromosomes, which are described, for example, in US patent No. 7235716. Alternatively, plant parent 1 may be genetically engineered to express the chimeric insecticidal protein of the present invention. A second plant, Parent 2, may be genetically engineered to express a second pest control agent. By crossing Parent 1 with Parent 2, offspring plants are produced that express both insect control agents from Parents 1 and 2.

[00296] In other embodiments, the present invention provides a trait-packaged transgenic plant that is resistant to plant pest infestation, comprising a nucleic acid sequence (e.g., DNA) encoding a dsRNA for suppressing a major gene in the target pest, and a nucleic acid sequence (e.g., DNA) encoding the chimeric insecticidal protein of the present invention exhibiting insecticidal activity against the target pest. It has previously been reported that dsRNAs are ineffective against certain lepidopteran pests (Rajagopol et al. 2002. J. Biol. Chem. 277:468–494) likely due to high midgut pH, which impairs dsRNA stability. . Therefore, in some embodiments, in which the target pest is a lepidopteran pest, the chimeric insecticidal protein of the present invention acts to temporarily lower midgut pH, which stabilizes the simultaneously ingested dsRNA, making the dsRNA effective in silencing the target genes.

[00297] Transgenic plants or seeds containing and/or expressing an insecticidal protein of the present invention can also be treated with an insecticide or insecticidal seed coating, which are described in US Pat. Nos. 5,849,320 and 5,876,739. In embodiments, wherein as an insecticide or insecticidal coating for seeds, and the transgenic plant or seed of the present invention is active against the same target insect, for example a lepidopteran pest (e.g. armyworm), the combination is useful (i) in a method of further enhancing the activity of the composition of the present invention in relation to the target insect and/or (ii) in a method of preventing the development of resistance to the composition of the present invention by providing another mechanism of action in relation to the target insect. Thus, embodiments of the present invention provide a method for increasing control of a lepidopteran insect population comprising providing a transgenic plant or seed of the present invention and applying to the plant or seed an insecticide or an insecticidal seed coating directed to the transgenic plant or seed according to the present invention. the present invention.

[00298] Even if the insecticide or insecticidal seed coating is active against another insect, the insecticide or insecticidal seed coating is useful for extending the range of insect control, for example, by adding an insecticide or insecticidal seed coating that is active in against Coleopteran insects, the transgenic seed of the present invention, which in some embodiments has activity against Lepidopteran insects, wherein the resulting coated transgenic seed provides control of both Lepidopteran and Coleopteran insect pests.

Methods for producing and using chimeric insecticidal proteins, nucleic acids and transgenic plants

[00299] The present invention also covers methods for producing a transgenic plant that is resistant to insects (eg, resistant to lepidopteran insects). In illustrative embodiments, the method includes introducing into a plant a polynucleotide, expression cassette, or vector of the present invention containing a nucleotide sequence that encodes a chimeric insecticidal protein of the present invention (including toxin fragments and modified forms that are substantially identical to the polypeptides specifically disclosed herein ), wherein the nucleotide sequence is expressed in a plant to produce a chimeric insecticidal protein of the present invention, thereby rendering the plant resistant to an insect pest and providing an insect-resistant transgenic plant (for example, compared to a suitable control plant, such as a plant that does not contains a polynucleotide, expression cassette or vector of the present invention and/or does not express a polypeptide of the present invention).

[00300] In embodiments, a method of introducing a polynucleotide, expression cassette or vector of the present invention into a plant includes first transforming a plant cell using the polynucleotide, expression cassette or vector and regenerating a transgenic plant therefrom, wherein the transgenic plant contains the polynucleotide, expression cassette or vector and expresses the chimeric insecticidal protein of the present invention.

[00301] Alternatively or additionally, the introduction step may include crossing a first plant containing a polynucleotide, expression cassette, or vector with a second plant (e.g., a plant different from the first plant, e.g., a plant that does not contain the polynucleotide, expression cassette, or vector) and optionally providing a progeny plant that contains a polynucleotide, expression cassette, or vector and expresses the chimeric insecticidal protein of the present invention, thereby resulting in increased resistance to at least one insect pest. Thus, a transgenic plant of the present invention encompasses a plant that is the direct result of a transformation event and its descendant (of any generation) that contains a polynucleotide, expression cassette or vector and optionally expresses a chimeric insecticidal protein resulting in increased resistance to at least one insect pest.

[00302] The present invention further provides a method for detecting a transgenic plant of the present invention, comprising detecting the presence of a polynucleotide, expression cassette, vector or chimeric insecticidal protein of the present invention in the plant (or plant cell, plant part, etc. derived therefrom ) and thereby identifying the plant as a transgenic plant of the present invention based on the presence of a polynucleotide, expression cassette, vector or chimeric insecticidal protein of the present invention.

[00303] The present invention further provides a method for producing a transgenic plant with increased resistance to at least one insect pest (for example, at least one lepidopteran pest), comprising: planting a seed containing a polynucleotide, expression cassette or vector of the present invention, and growing a transgenic plant from the seed, wherein the transgenic plant contains a polynucleotide, expression cassette or vector and produces a chimeric insecticidal protein.

[00304] In embodiments, transgenic plants produced using the methods of the present invention contain a polynucleotide, expression cassette, or vector of the present invention. In embodiments, the transgenic plant produced using the methods of the present invention contains a chimeric insecticidal protein of the present invention and optionally has increased resistance to at least one insect pest.

[00305] The methods for producing a transgenic plant described herein optionally include the additional step of collecting seed from the transgenic plant, wherein the seed contains a polynucleotide, expression cassette, or vector and produces a chimeric insecticidal protein. Optionally, the seed provides an additional transgenic plant that contains a polynucleotide, expression cassette, or vector and produces a chimeric insecticidal protein and thus has increased resistance to at least one insect pest.

[00306] The present invention further provides plant parts, plant cells, plant organs, plant crops, seeds, plant extracts, harvested products and processed products of transgenic plants obtained using the methods of the present invention.

[00307] As a further aspect, the present invention also provides a method for producing a seed, the method comprising producing a transgenic plant that contains a polynucleotide, expression cassette, or vector of the present invention, and collecting seed from the transgenic plant, wherein the seed contains a polynucleotide, expression cassette , vector and produces a chimeric insecticidal protein. Optionally, the seed provides an additional transgenic plant that contains a polynucleotide, expression cassette, or vector and produces a chimeric insecticidal protein and thus has increased resistance to at least one insect pest. In illustrative embodiments, the step of producing a transgenic plant includes planting a seed that produces a transgenic plant.

[00308] The present invention further provides a method for producing a hybrid plant seed, the method comprising crossing a first inbred plant, which is a transgenic plant containing a polynucleotide, expression cassette or vector of the present invention and optionally expressing a chimeric insecticidal protein of the present invention, with another inbred plant (eg, an inbred plant that does not contain the polynucleotide, expression cassette or vector of the present invention) and allowing the formation of a hybrid seed. Optionally, the method further includes collecting the hybrid seed. In embodiments, the hybrid seed comprises a polynucleotide, expression cassette, or vector of the present invention and, in embodiments, may further comprise a chimeric insecticidal protein of the present invention and have enhanced resistance to an insect pest. In embodiments, the hybrid seed produces a transgenic plant that contains a polynucleotide, expression cassette, or vector of the present invention, expresses a chimeric insecticidal protein of the present invention, and has increased resistance to at least one insect pest.

[00309] In some embodiments, the transgenic plant of the present invention is resistant to at least one lepidopteran insect pest (as described herein). In embodiments, the transgenic plant provides control of an insect pest or armyworm colony that is resistant to the Vip3A protein (e.g., the Vip3Aa protein, for example, which is expressed in the maize transformant MIR162) and/or Cry1F (for example, the Cry1Fa protein, which is expressed in the maize transformant TC1507 ).

[00310] In further embodiments, a method of controlling at least one insect pest (eg, at least one lepidopteran insect pest such as cutworm) includes providing a chimeric insecticidal protein of the present invention. In embodiments, the method includes delivering (eg, oral delivery) to the pest or its habitat an effective amount of a chimeric insecticidal protein of the present invention. Typically, the polypeptide is ingested orally by the insect for effectiveness. However, the chimeric insecticidal protein can be delivered to an insect using many known methods. Methods of delivering protein to an insect by oral route include, but are not limited to, providing the protein (1) in a transgenic plant, wherein the insect eats (absorbs) one or more parts of the transgenic plant, thereby consuming a polypeptide expressed in the transgenic plant; (2) in a formulated protein(s) composition(s) that can be applied, for example, to or included in an insect culture medium; (3) in a protein composition(s) that can be applied to a surface, for example by spraying the surface of a plant part, and which is then ingested by an insect due to the insect eating one or more sprayed plant parts; (4) in a bait matrix; or (5) using any other protein delivery system known in the art. Thus, any method of oral delivery to an insect can be used to deliver the toxic proteins of the present invention. In some specific embodiments, the chimeric insecticidal protein of the present invention is delivered to an insect orally, such as when the insect eats one or more parts of a transgenic plant of the present invention.

[00311] In other embodiments, the insecticidal protein of the present invention is delivered orally to an insect, wherein the insect eats one or more plant parts sprayed with a composition containing the insecticidal protein of the present invention. Delivery of the composition of the present invention to the surface of a plant can be accomplished using any method of applying compounds, compositions, formulations, or the like. on the surface of plants, known to specialists in the art. Some non-limiting examples of delivery to or contact with a plant or part thereof include spraying, dusting, sprinkling, spraying, misting, misting, scattering, drenching, injecting into soil, introducing into soil, wetting (e.g., treating roots, soil ), dipping, watering, coating, absorption into leaves or stems, inter-row application or seed treatment, etc. and their combinations. These and other procedures for bringing a plant or part thereof into contact with the compound(s), composition(s) or composition(s) are well known to those skilled in the art.

[00312] In additional embodiments, the present invention provides a method for controlling a lepidopteran insect pest that is resistant to the Vip3A protein (e.g., the Vip3Aa protein, e.g., which is expressed by the maize transformant MFR162) and/or the Cry1F protein (e.g., the Cry1Fa protein, e.g. which is expressed by the maize transformant TC1507), comprising delivering to the lepidopteran insect or its habitat an effective amount of the chimeric insecticidal protein or composition of the present invention. In illustrative embodiments, the resistant insect pest is a resistant insect pest or armyworm colony.

[00313] In other embodiments, the present invention provides a method for preventing the development of resistance in a population of a target lepidopteran insect pest to the Vip3A protein (for example, the Vip3Aa protein, for example, which is expressed by the maize transformant MIR162) and/or Cry1F (for example, the Cry1Fa protein , for example, which is expressed by the maize transformant TC1507) expressed in a transgenic plant, the method comprising delivering to the target population a transgenic plant containing a polynucleotide that contains a nucleotide sequence encoding the Vip3A protein and/or a nucleotide sequence encoding the Cry1F protein; and a polynucleotide, expression cassette or vector of the present invention expressing the chimeric insecticidal protein of the present invention. In some embodiments, the target lepidopteran insect pest is a cutworm. According to the above embodiments, the transgenic plant may comprise a selection package of two or more insecticidal traits, a molecular package of two or more insecticidal traits, or a combination thereof.

[00314] In some embodiments, the present invention includes a method of providing a farmer with a means of controlling an insect pest (e.g., a lepidopteran insect pest such as cutworm), the method comprising supplying or selling plant material, such as a seed, to the farmer wherein the plant material contains a polynucleotide, an expression cassette, a vector capable of expressing the chimeric insecticidal protein of the present invention. In embodiments, the plant material comprises a chimeric insecticidal protein of the present invention and optionally has increased resistance to at least one insect pest. In embodiments, the plant material is a seed, and the plant grown from the seed contains a polynucleotide, expression cassette, or vector of the present invention, expresses a chimeric insecticidal protein of the present invention, and has increased resistance to at least one insect pest.

[00315] In addition to providing compositions, the present invention provides methods for producing a chimeric insecticidal protein that is toxic to a lepidopteran pest. Such a method involves culturing a transgenic host cell, other than a human cell, that contains a polynucleotide, expression cassette, or vector of the present invention that expresses a chimeric insecticidal protein of the present invention, under conditions under which the host cell produces a chimeric insecticidal protein that is toxic to pest belonging to Lepidoptera. In some embodiments, the transgenic host cell, other than a human cell, is a plant cell. In some other embodiments, the plant cell is a maize cell. In other embodiments, the conditions under which the plant or maize cell is grown include natural sunlight. In other embodiments, the transgenic host cell other than a human cell is a bacterial cell. In yet other embodiments, the transgenic host cell, other than a human cell, is a yeast cell.

[00316] In some embodiments, the methods of the present invention provide control of at least one lepidopteran insect pest, including, without limitation, one or more of the following: Ostrinia spp., such as O. nubilalis (corn borer) and/or O. furnacalis (eastern corn borer); Plutella spp., such as P. xylostella (cabbage moth); Spodoptera spp., such as S. frugiperda (grass armyworm), S. littoralis (Egyptian cotton armyworm), S. ornithogalli (yellow-striped armyworm), S. praefica (western yellow armyworm), S. eridania (southern armyworm), and/or S. exigua (small cutworm); Agrotis spp., such as L. ipsilon, A. segetum, L. gladiaria, and/or A. orthogonia; Striacosta spp., such as S. albicosta (western bean cutworm); Helicoverpa spp., such as N. zea (maize armyworm), N. punctigera (Australian cotton bollworm) and/or N. armigera (cotton bollworm); Heliothis spp., such as H., virescens (tobacco budworm); Diatraea spp., such as P. grandiosella (southwest corn borer) and/or D. saccharalis (sugarcane stalk borer); Trichoplusia spp., such as T. ni (cabbage cutworm); Sesamia spp., such as S. nonagroides (Mediterranean corn borer) and/or S. calamistis (pink cutworm); Pectinophora spp., such as P. gossypiella (pink bollworm); Cochylis spp., such as C. hospes (striped sunflower moth); Manduca spp., such as M. sexta (tobacco hawkmoth) and/or M. quinquemaculata (five-spotted hawkmoth); Elasmopalpus spp., such as P. lignosellus (small corn stalk borer); Pseudoplusia spp., such as P. includens (soybean armyworm); Anticarsia spp., such as A gemmatalis (velvet bean armyworm); Plathypena spp., such as P. scabra (clover armyworm); Pieris spp., such as P. brassicae, Papaipema spp., such as P. nebris (Papaipema nebris); Pseudaletia spp., such as P. unipuncta (common fall armyworm); Peridroma spp., such as P. saucia (daisy armyworm); Keiferia spp., such as K. lycopersicella (tomato pinworm); Artogeia spp., such as A garae; Phthorimaea spp., such as P. operculella (potato moth); Chrysodeixis spp., such as C. includes (soybean armyworm); Feltia spp., such as P. due ens (feltia jaculifera caterpillar); Chilo spp., such as C. suppressalis (yellow rice moth), Cnaphalocrocis spp., such as C. medinalis (rice moth), or any combination of the above. In embodiments, the methods of the present invention provide control of an insect pest or armyworm colony that is resistant to the Vip3A protein (e.g., the Vip3Aa protein, for example, which is expressed in the maize transformant MIR162) and/or Cry1F (e.g., the Cry1Fa protein, for example, which expressed in the maize transformant TC1507).

[00317] The present invention also provides for the use of chimeric insecticidal proteins, nucleic acids, transgenic plants, plant parts, seeds and insecticidal compositions of the present invention, for example, to control an insect pest, such as a lepidopteran pest (as described herein). document).

[00318] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, vector, or host cell of the present invention to produce an insecticidal composition for controlling an insect pest (eg, a lepidopteran insect pest).

[00319] In embodiments, the present invention provides a method of using a polynucleotide, expression cassette, or vector of the present invention to produce a transgenic seed, wherein the transgenic seed grows into a transgenic plant with increased resistance to an insect pest.

[00320] As another aspect, the present invention also contemplates the use of a transgenic plant of the present invention to produce a transgenic seed, which is not necessarily a hybrid seed.

[00321] In embodiments, the present invention provides a method of using a chimeric insecticidal protein, polynucleotide, expression cassette, vector, transgenic plant, or insecticidal composition of the present invention to prevent the development of resistance in a population of a target lepidopteran insect pest to the Vip3A protein and /or Cry1F protein (each as described herein).

[00322] The claims of the present invention include, without limitation, the following:

[00323] 1. A chimeric insecticidal protein that is toxic to a lepidopteran insect pest, comprising, in an N-terminal to C-terminal direction: (a) an N-terminal region of the first Cry1 protein, which is an N-terminal region the BT-0029 protein of SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical thereto, fused to (b) the C-terminal region of another Cry1 protein; where the transition position between the first Cry1 protein and another Cry1 protein is in conserved block 3.

[00324] 2. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1F, Cry1G, Cry1I, or Cry1K protein.

[00325] 3. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1Fa protein.

[00326] 4. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is a Cry1 1a or a Cry1If protein.

[00327] 5. The chimeric insecticidal protein of claim 1, wherein the other Cry1 protein is the BT-0022 protein of SEQ ID NO: 1.

[00328] 6. The chimeric insecticidal protein of claim 1, wherein the C-terminal region of the other Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from positions 464 to 602 of SEQ ID NO: 8 or an amino acid sequence that is at least 80% identical to her.

[00329] 7. The chimeric insecticidal protein of claim 1, wherein the C-terminal region of the other Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from positions 498 to 636 of SEQ ID NO: 1 or an amino acid sequence that is at least 80% identical to her.

[00330] 8. The chimeric insecticidal protein according to any one of claims. 1-7, wherein the N-terminal region of the first Cry1 protein contains an amino acid sequence corresponding to the amino acid sequence from positions 1 to 458 of SEQ ID NO: 2 or an amino acid sequence that is at least 90% identical thereto.

[00331] 9. The chimeric insecticidal protein according to any one of claims. 1-8, wherein the chimeric insecticidal protein further contains at the N-terminus a peptidyl moiety, which is cleaved from the protoxin after ingestion by a lepidopteran insect pest.

[00332] 10. The chimeric insecticidal protein according to any one of claims. 1-9, wherein the chimeric insecticidal protein further comprises at the C-terminus a protoxin tail from the Cry protein, which is cleaved from the protoxin after ingestion by a lepidopteran insect pest.

[00333] 11. The chimeric insecticidal protein according to any one of claims. 1-10, wherein the chimeric insecticidal protein contains (a) an amino acid sequence from positions 1 to 597 of SEQ ID NO: 3 or a toxin fragment thereof, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a) .

[00334] 12. The chimeric insecticidal protein according to any one of claims. 1-11, wherein the chimeric insecticidal protein contains (a) the amino acid sequence of SEQ ID NO: 3 or a toxin fragment thereof, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a).

[00335] 13. The chimeric insecticidal protein according to any one of claims. 1-10, wherein the chimeric insecticidal protein contains (a) an amino acid sequence from positions 1 to 597 of SEQ ID NO: 9 or a toxin fragment thereof, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a) .

[00336] 14. The chimeric insecticidal protein according to any one of claims. 1-10 or claim 13, wherein the chimeric insecticidal protein contains (a) the amino acid sequence of SEQ ID NO: 9 or a toxin fragment thereof, or (b) an amino acid sequence that is at least 80% identical to the amino acid sequence of (a) .

[00337] 15. The chimeric insecticidal protein according to any one of claims. 1-14, wherein the chimeric insecticidal protein contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 9.

[00338] 16. The chimeric insecticidal protein according to any one of claims. 1-15, wherein the chimeric insecticidal protein has insecticidal activity against one or more of Spodoptera frugiperda (fall armyworm), Chrysodeixis includes (soybean armyworm), Diatraea saccharalis (sugarcane stalk borer), Diatraea grandiosella (southwestern corn moth), and Agrotis ipsilon (upsilon cutworm).

[00339] 17. The chimeric insecticidal protein according to any one of claims. 1-16, wherein the chimeric insecticidal protein has insecticidal activity against an insect pest or colony of Spodoptera frugiperda with resistance to the Vip3A protein and/or the Cry1F protein.

[00340] 18. A polynucleotide containing a nucleotide sequence encoding a chimeric insecticidal protein according to any one of claims. 1-17.

[00341] 19. The polynucleotide of claim 18, wherein the polynucleotide is codon optimized for expression in a plant.

[00342] 20. The polynucleotide of claim 18 or 19, wherein the polynucleotide contains a nucleotide sequence that contains (a) the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 , SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 14 or a toxin-encoding fragment thereof; (b) a nucleotide sequence that is substantially identical to the nucleotide sequence from (a); (c) a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence of (a) or (b); or (d) a nucleotide sequence that differs from the nucleotide sequence of (a), (b) or (c) due to degeneracy of the genetic code.

[00343] 21. The polynucleotide of claim 18 or 19, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 14.

[00344] 22. A nucleic acid molecule containing a polynucleotide according to any one of claims. 18-21, operably linked to a heterologous promoter.

[00345] 23. The nucleic acid molecule of claim 22, wherein the heterologous promoter is a promoter for expression in a plant.

[00346] 24. A vector containing a polynucleotide according to any one of claims. 18-21 or a nucleic acid molecule according to claim 22 or claim 23.

[00347] 25. A transgenic cell containing a polynucleotide according to any one of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23, or a vector according to claim 24.

[00348] 26. The transgenic cell of claim 25, wherein the cell is a transgenic bacterial cell.

[00349] 27. The transgenic cell of claim 25, wherein the cell is a transgenic plant cell.

[00350] 28. The transgenic plant cell of claim 27, wherein the plant cell is: (i) a monocot cell, optionally a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, or a wheat cell; or (ii) a dicot cell, optionally a soybean cell, a sunflower cell, a tomato cell, a cabbage cell, a cotton cell, a sugar beet cell, or a tobacco cell.

[00351] 29. A transgenic plant comprising a transgenic plant cell according to claim 27 or claim 28.

[00352] 30. The transgenic plant of claim 29, wherein the plant is: (i) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugarcane plant, or a wheat plant; or (ii) a dicotyledonous plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cultivated cabbage plant, a cotton plant, a sugar beet plant, or a tobacco plant.

[00353] 31. The transgenic plant of claim 28 or 29, wherein the transgenic plant contains an additional nucleotide sequence encoding a second insect control agent.

[00354] 32. A transgenic plant part from a transgenic plant according to any one of claims. 29-31, where the transgenic plant part contains a chimeric insecticidal protein.

[00355] 33. Transgenic seed of a transgenic plant according to any one of claims. 29-31.

[00356] 34. A harvested product obtained from a transgenic plant according to any one of claims. 29-31, a transgenic plant part according to claim 32 or a transgenic seed according to claim 33, where the harvested product contains a chimeric insecticidal protein.

[00357] 35. A processed product derived from the harvested product of claim 34, wherein the processed product is flour, meal, oil, starch, or a product derived from any of the foregoing.

[00358] 36. An insecticidal composition containing a chimeric insecticidal protein according to any one of paragraphs. 1-17 and an agriculturally acceptable carrier.

[00359] 37. The insecticidal composition of claim 36, wherein the agriculturally acceptable carrier is a powder, dust, pellet, granule, aerosol, emulsion, colloid or solution.

[00360] 38. The insecticidal composition of claim 36 or 37, wherein the composition is a dried, lyophilized, homogenized, extracted, filtered, centrifuged and/or precipitated composition and/or is a bacterial culture concentrate.

[00361] 39. The insecticidal composition according to any one of claims. 36-38, where the composition contains a transgenic bacterial cell that produces a chimeric insecticidal protein.

[00362] 40. The insecticidal composition according to any one of claims. 36-39, wherein the composition contains from about 1% to about 99% by weight of the chimeric insecticidal protein.

[00363] 41. The insecticidal composition according to any one of claims. 36-40, where the composition contains a second insect control agent.

[00364] 42. A method for producing a transgenic plant with increased resistance to lepidopteran insect pests, the method comprising introducing into the plant a polynucleotide according to any one of claims. 18-21, the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24, wherein the chimeric insecticidal protein is expressed in a plant, thereby producing a transgenic plant with increased resistance to an insect pest.

[00365] 43. The method of claim 42, wherein the step of introducing includes (i) transforming a plant cell with a polynucleotide, nucleic acid molecule or vector and regenerating the transgenic plant or (ii) crossing a first plant containing the polynucleotide, nucleic acid molecule or vector with second plant.

[00366] 44. The method of claim 42 or 43, wherein the method further comprises producing a progeny plant from a transgenic plant, wherein the progeny plant contains a polynucleotide, nucleic acid molecule, or vector and is characterized by increased resistance to an insect pest.

[00367] 45. A method for producing a transgenic plant with increased resistance to a lepidopteran insect pest, the method comprising: (a) sowing a seed containing a polynucleotide according to any one of claims. 18-21, the nucleic acid molecule of claim 22 or claim 23, or the vector of claim 24 and (b) growing a transgenic plant from a seed, wherein the transgenic plant contains a polynucleotide, nucleic acid molecule or vector and produces a chimeric insecticidal protein.

[00368] 46. The method of claim 45, wherein the method further comprises (c) collecting seed from the transgenic plant of (b), wherein the collected seed contains the chimeric insecticidal protein.

[00369] 47. A method for producing a seed, the method comprising (a) producing a transgenic plant that contains a polynucleotide according to any one of claims. 18-21, the nucleic acid molecule of claim 22 or 23, or the vector of claim 24, and (b) collecting seed from the transgenic plant of (a), wherein the collected seed contains the chimeric insecticidal protein.

[00370] 48. A method for producing a hybrid plant seed, the method comprising: (a) crossing a first inbred plant that is a transgenic plant containing a polynucleotide according to any one of claims. 18-21, the nucleic acid molecule of claim 22 or 23, or the vector of claim 24, with another inbred plant, and (b) allowing the formation of a hybrid seed.

[00371] 49. A method for controlling a lepidopteran insect pest, the method comprising delivering to the pest or its habitat a composition containing an effective amount of a chimeric insecticidal protein according to any one of claims. 1-17 or an insecticidal composition according to any one of paragraphs. 36-41.

[00372] 50. A method for controlling a lepidopteran insect pest resistant to a Vip3A protein and/or a Cry1F protein, the method comprising delivering an insecticidal protein of any type to the body of the lepidopteran resistant insect pest or to its habitat. from paragraphs. 1-17 or an insecticidal composition according to any one of paragraphs. 36-41.

[00373] 51. A method for reducing the development of resistance to the Vip3A protein and/or the Cry1F protein in a population of a target lepidopteran insect pest, the method comprising delivering to the target population or its habitat a transgenic plant containing (i) a polynucleotide at any of paragraphs. 18-21, a nucleic acid molecule according to claim 22 or claim 23 or a vector according to claim 24, and (ii) a polynucleotide containing a nucleotide sequence encoding the Vip3A protein and/or a nucleotide sequence encoding the Cry1F protein; wherein the chimeric insecticidal protein and the Vip3A protein and/or the Cry1F protein are produced in the transgenic plant.

[00374] 52. The method according to any one of paragraphs. 42-51, wherein the lepidopteran insect pest includes one or more of Spodoptera frugiperda, Chrysodeixis includes soybean armyworm, Diatraea saccharalis, Diatraea grandiosella, and Diatraea grandiosella. and Agrotis ipsilon (upsilon cutworm).

[00375] 53. The method according to any one of paragraphs. 42-52, wherein the lepidopteran insect pest includes Spodoptera frugiperda, which is resistant to the Vip3A protein and/or the Cry1F protein.

[00376] 54. The method according to any one of paragraphs. 42-53, where the plant is: (i) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugarcane plant or a wheat plant; or (ii) a dicotyledonous plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cultivated cabbage plant, a cotton plant, a sugar beet plant, or a tobacco plant.

[00377] 55. The method according to any one of paragraphs. 42-54, where the plant is a maize plant.

[00378] The present invention will now be described with reference to the following examples. Those skilled in the art will appreciate that these examples do not limit the scope of the claims of the present invention, but rather are intended to be illustrative of certain embodiments. Other embodiments of the present invention may be practiced without departing from the spirit and scope of the present invention, the scope of which is defined by the present disclosure and the appended claims.

EXAMPLES

Example 1. Detection of the chimeric protein BT-0029, Bt29-Bt22, with insecticidal activity against the armyworm

[00379] Two lepidopteran-active proteins from Bacillus thuringiensis (Bt), BT-0022 (SEQ ID NO: 1; the closest known member of the Cry family is Cry1If) and BT-0029 (SEQ ID NO: 2; the closest known member of the Cry family - Cry1Gb), were disclosed in PCT/US16/038947.

[00380] As shown in Table 1 below, the insect spectrum data for BT-0022 and BT-0029 indicate that BT-0022 has no activity against FAW, Spodoptera frugiperda, and BT-0029 has only weak activity against FAW. Other lepidopteran insect pests tested were corn borer (ECB; Ostrinia nubilalis), fall armyworm (BCW; Agrotis ipsilon), fall armyworm (CEW; Helicoverpa zed), sugarcane stalk borer (SCB; Diatraea saccharalis ), southwestern corn borer (SWCB; Diatraea grandiosella), western bean borer (WBCW; Striacosta albicosta), soybean borer (SBL; Pseudoplusia includens), velvet bean borer (VBC; Anticarsia gemmatalis), and tobacco budworm (TBW; Heliothis virescens ).

[00381] A protein engineering approach was used in an attempt to increase the activity of BT-0029 against FAW. Using BT-0029 as a template, six engineered proteins were created by replacing domain III (DIII) from BT-0029 with the ID domain from another Cry protein. Table 2 illustrates six chimeric proteins with information on their domain composition. Table 3 provides sequence information for the full-length proteins.

[00382] The cDNA encoding engineered proteins were synthesized at Genewiz (South Plainfield, NJ) and cloned into a Bacillus thuringiensis (Bt) expression vector containing the Cry1 Ac promoter and no terminator. Plasmid DNAs were introduced into Bt strain AB227 using an electroporation-mediated transformation procedure. All six engineered proteins were obtained as crystalline proteins in Bt. Crystalline proteins were purified from cultures and dissolved in a buffer containing 50 mM Na ₂ CO ₃ , pH 11.0, 2 mM DTT. Each soluble protein was then evaluated in an insect biological assay.

[00383] Briefly, the insecticidal activity of the chimeric proteins was tested using an artificial diet-based insect bioassay in which dissolved crystalline proteins were placed on the surface of an artificial insect diet at a final concentration of 2-3.2 μg/cm ² . Buffer (50 mM Na ₂ CO ₃ , pH 11.0, 2 mM DTT) used to dissolve crystalline Bt proteins served as a negative control. Full-length Cry1Fa protein (SEQ ID NO: 8) was used as a positive control for FAW activity. Each chimeric protein was tested in duplicate. Insect mortality was assessed on day 7.

[00384] The results are shown in Table 4. When the chimeric proteins were tested in an insect bioassay, the Bt29-Bt22 chimera (SEQ ID NO: 3) showed strong insecticidal activity against FAW, while the other two chimeric proteins (Bt29-Bt67 and Bt29-3A) showed only minor activity against the pest. The Bt29-1Ab chimera did not demonstrate any detectable activity against FAW.

[00385] This test did not produce enough protein from the Bt29-Bt51 and Bt29-8D chimeras to evaluate insecticidal activity.

Example 2. Insecticidal activity of chimeric proteins VT-0029 against various lepidopteran pests

[00386] The four chimeric proteins described in Example 1 (Bt29-3A, Bt29-Bt22, Bt29-1Ab and Bt29-Bt67) were tested for their insecticidal activity against the following lepidopteran pests using methods known in the art methods of biological analyzes with artificial diet: sugarcane stalk borer (SCB; Diatraea saccharlis), southwestern corn borer (SWCB; Diatraea grandiosella), soybean armyworm (SBL; Pseudoplusia includens), corn armyworm (CEW; Helicoverpa zed), tobacco leaf roller (TBW; Heliothis virescens), bollworm (BCW; Agrotis ipsilon) and corn borer (ECB; Ostrinia nubilalis).

[00387] As shown in Table 5, all four chimeric proteins tested retained the potent activity of native BT-0029 against SBL. Bt29-Bt22 and Bt29-Bt67 had weak SCB activity comparable to native BT-0029, whereas Bt29-3A and Bt29-1Ab essentially lost all SCB activity. Slightly increased activity against SWCB was observed in Bt29-Bt67 compared to full-length BT-0029. Fairly consistent with the results in Table 1 for native BT-0029, only weak activity against BCW was observed for Bt29-3A, Bt29-Bt22, and Bt29-Bt67, and none of the chimeras had activity against CEW, TBW, or ECB.

Example 3. Bt29-1F has insecticidal activity against armyworm

[00388] To explore additional chimeric proteins, a second set of chimeric proteins, BT-0029, was developed and tested (Table 6). Bt29-1Fa is a chimeric protein in which domain III from BT-0029 is replaced by domain III from Cry1Fa. The full-length amino acid sequence of Cry1Fa is provided as SEQ ID NO: 8 (see also Genbank accession number AAB00376.1). The amino acid sequence of the Bt29-1Fa chimera is shown as SEQ ID NO: 9, where the sequence of Cry1Fa is amino acids 459-597.

[00389] Bt29-1Fa is a chimeric protein in which domain III of BT-0029 is replaced with domain III of Cry1Ka. The full-length amino acid sequence of Cry1Ka is provided as SEQ ID NO: 12 (see also Genbank accession number AAB00376.1). The amino acid sequence of the Bt29-1Ka chimera is also shown as SEQ ID NO: 13, where the Cry1Ka sequence is amino acids 459-597. The second chimeric protein variant Bt29-1Fa (referred to herein as Bt29-1Fav2) was generated by moving the transition site between the Cry1Ka sequence and the BT-0029 sequence at the C-terminus of domain III from amino acid 598 (the transition in the first Bt 29-1Ka chimera) to amino acid 610. The amino acid sequence of the Bt29-1Fav2 chimera is shown as SEQ ID NO: 15, where the Cry1Ka sequence is amino acids 459-609.

[00390] Bt29-1Ca is a chimeric protein in which domain III of Bt-0029 is replaced with domain III of Cry1Ca. The full-length amino acid sequence of Cry1Ca is provided as SEQ ID NO: 17 (see also Genbank accession number AF362020.1). The amino acid sequence of the Bt29-1Ca chimera is shown as SEQ ID NO: 18, where the Cry1Ca sequence is amino acids 467-617.

[00391] The insecticidal activity of the chimeras Bt29-1Fa (SEQ ID NO: 9), Bt29-1Fa (SEQ ID NO: 13, Bt29-1Fav2 (SEQ ID NO: 16) and Bt29-1Ca (SEQ ID NO: 18) was studied with using an artificial diet-based insect bioassay in which dissolved proteins were placed on the surface of an artificial insect diet at a final concentration of 2 μg/cm ² Buffer (50 mM Na ₂ CO ₃ , pH 10.5, 2 mM DTT) , used to solubilize Bt crystalline proteins, served as a negative control. Full-length Cry1Fa protein (SEQ ID NO: 8) was used as a positive control for activity against FAW. Each chimeric protein was tested in triplicate. Insecticidal activity was assessed as effective mortality at 7 th day (larvae that are stunted and die are assessed as effectively dead).

[00392] As shown in Table 7, the chimeric protein designated Bt29-1Fa exhibited potent activity against FAW.

[00393] This test did not produce enough Bt29-1Fa protein to evaluate in a biological assay. Surprisingly, the protein derived from the Bt29-1Fav2 chimera was active against armyworm (see Table 7).

Example 4. Bt29-Вт22 and Bt29-1Fa have insecticidal activity against cutworm resistant to Cry1F

[00394] To determine whether the toxicity of the BT-0029 chimeric proteins to FAW is dependent on a mode of action (MOA) other than Cry1Fa, two chimeric proteins, Bt29-Bt22 and Bt29-1Fa, were prepared as described in Example 1 and crystallized proteins were isolated and purified. Purified crystals were dissolved in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11.2 mM DTT) and the purity of the dissolved protein preparation was monitored using a Bio-Rad Experion system (BioRad, Hercules, CA).

[00395] The purified proteins were tested for effectiveness against the FAW strain, which is resistant to the insecticidal toxin Cry1Fa. Diet coating assays were performed with varying doses of each purified toxin (125, 500, and 2000 ng/ ^cm2 ) essentially as described in Example 1. Vip3A protein (positive control) was dissolved in PBS and the other proteins in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT). The two negative control treatments were PBS and Buffer 1. A FAW population that is susceptible to Cry1Fa or Vip3A was also tested, i.e. susceptible line FAW. Each protein was tested in duplicate. Insecticidal activity was assessed as effective mortality on day 7 (larvae that are stunted and die are counted as effectively dead).

[00396] As shown in Table 8, susceptible FAW larvae were controlled by Cry1Fa, Vip3A, Bt29-Bt22 and Bt29-1Fa even at the lowest concentration. In contrast, the Cry1F-resistant FAW line was not controlled with any tested dose of Cry1Fa, demonstrating that the line is resistant to this toxin (Table 9). Surprisingly, both Bt29-Bt22 and Bt29-1Fa were very effective in controlling Cry1F-resistant FAW even at a very low dose of 125 iG/cm as shown in Table 9, indicating that the mode of action of these two chimeric proteins against resistant FAW differs from the mode of action of the Cry1Fa protein.

Example 5. Bt29-Bt22 and Bt29-1Fa have insecticidal activity against Vip3A-resistant armyworm

[00397] To determine whether the toxicity of the BT-0029 chimeric proteins depends on a MOA other than the Vip3A protein, Bt29-Bt22 and Bt29-1Fa proteins were prepared as described in Example 1, and the crystalline proteins were isolated and purified. Purified crystals were dissolved in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT) and the purity of the solubilized protein preparation was monitored using a Bio-Rad Experion system (BioRad, Hercules, CA).

[00398] The purified proteins were tested for effectiveness against the FAW strain, which is resistant to the insecticidal toxin Vip3A. Diet-coverage assays were performed with varying doses of each purified toxin (125, 500, and 2000 ng/ ^cm2 ) essentially as described in Example 1. Vip3A protein was dissolved in PBS and the other proteins in buffer 1 (50 mM Na ₂ CO ₃ /NaHCO ₃ , pH 11, 2 mM DTT). The two negative control treatments were PBS and buffer 1. Cry1Fa proteins were used as a positive control for the Vip3A-resistant FAW line. Each protein was tested in duplicate. Insecticidal activity was scored as effective mortality on day 7 (larvae that are stunted and die are scored as effectively dead).

[00399] The Vip3 A-resistant FAW line was not controlled with any test dose of Vip3 A demonstrating that the line is resistant to this toxin (Table 10). In contrast, both Bt29-Bt22 and Bt29-1Fa showed a high degree of efficacy against Vip3A-resistant FAW, indicating that the mode of action of these two chimeric proteins against resistant FAW is different from the mode of action of the Vip3A protein.

Example 6. Truncations in the protoxin tail region

[00400] It is well known that the protoxin form of the Bt Cry proteins is processed at both the N- and C-terminus to produce the mature toxin. Six C-terminally truncated forms of the Bt29-Bt22 chimera (SEQ ID NO: 3), which contains the tail region of the BT-0029 protoxin, were prepared as shown in Figure 3. All truncated variants are referred to herein as Bt29-Bt22Tr1 ( SEQ ID NO: 20), Bt29-Bt22Tr2 (SEQ ID NO: 21), Bt29-Bt22Tr3 (SEQ ID NO: 22), Bt29-Bt22Tr4 (SEQ ID NO: 23), Bt29-Bt22Tr5 (SEQ ID NO: 24) , Bt29-Bt22Tr6 (SEQ ID NO: 25), domain III from BT-0022 (terminating PVTA) and at least the first 6 amino acids of the BT-0029 protoxin tail (TFEAEY) were retained. Those skilled in the art will appreciate that the end of domain III and the start of the protoxin tail have not been precisely defined for BT-0029 or the chimeras described herein. Core domain III shown in Figures 1A and 1B is based on an alignment with other Cry proteins in which the domain III region has been defined.

[00401] The truncated forms of Bt29-Bt22Tr1, -Tr2, -Tr4 and -Tr5 all exhibited FAW activity assessed as growth inhibition (not lethality) in insect bioassays when tested as E. coli lysates. The Bt29-Bt22Tr1 and -Tr4 forms were the most promising, demonstrating the greatest activity in this assay (67% and 75% growth inhibition, respectively).

[00402] A second full-length Bt29-Bt22 chimera was produced in which the protoxin tail region was derived from BT-0022 (Cry1I) instead of BT-0029 (Cry1G). Six C-terminally truncated forms of this full-length chimera, provided herein as SEQ ID NOs: 26-31, were prepared and tested against armyworm and soybean armyworm. Cry1Fa was used as a positive control. The results are shown in Table 11.

[00403] Four truncated forms of Bt29-1Fa (SEQ ID NO: 9) that contain the tail region of the BT-0029 protoxin, represented herein as B129-1FaTr1 (SEQ ID NO: 32), Bt29-1FaTr2 (SEQ ID NO: 33), Bt29-1FaTr3 (SEQ ID NO: 34) and Bt29-1FaTr4 (SEQ ID NO: 35), were produced in the E. coli expression system, and the lysates were tested against armyworm. The results are shown in Table 12.

Example 7. Expression and activity of chimeric proteins Bt29-Bt22 and Bt29-Cry1Fa in maize plants

[00404] Prior to expression in plants, synthetic polynucleotides containing the nucleotide sequence encoding the Bt29-Bt22 and Bt29-1Fa chimera were synthesized using an automated gene synthesis platform (Genscript, Inc., Piscataway, NJ). In this example, a first and second expression cassette were prepared containing the maize ubiquitin promoter (Ubil) operably linked to a chimeric sequence encoding Bt29-Bt22 or Bt29-1Fa, which was operably linked to the maize ubiquitin terminator, and a third expression cassette was prepared containing the Ubil promoter , operably linked to the phosphomannose isomerase (PMI) coding sequence, which is operably linked to the Ubi terminator. PMI expression allows positive selection of transgenic plants on mannose. For plant transformation, the first and third expression cassettes and the second and third expression cassettes were cloned into a suitable vector for Agrobacterium-mediated transformation of maize.

[00405] Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798,803. Briefly, Agrobacterium strain LBA4404 (pSBl) containing an expression vector expressing Bt29-Bt22 or Bt29- Cry1Fa, grown on solid YEP medium (yeast extract (5 g/l), peptone (10 g/l), NaCl (5 g/l), 15 g/l agar, pH 6.8) for 2-4 days at 28°C. Approximately 0.8 X 10 ⁹ Agrobacterium cells were suspended in LS-inf medium supplemented with 100 μM As. The bacteria were pre-induced in this medium for approximately 30-60 minutes.

[00406] Immature embryos of a maize inbred line were excised from 8-12 day old ears and transferred to liquid LS-inf+100 µM As. The embryos were rinsed once with fresh infection medium. The Agrobacterium solution was then added and the embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred with the scutellum side up onto LSAs medium and cultured in the dark for two to three days. Subsequently, approximately 20-25 embryos per Petri dish were transferred to LSDc medium supplemented with cefotaxime (250 mg/L) and silver nitrate (1.6 mg/L) and cultured in the dark at approximately 28°C for 10 days.

[00407] Immature embryos forming embryogenic callus were transferred to LSD1M0.5S medium. Selection of crops on this medium was carried out for approximately 6 weeks, with a subculturing stage carried out after approximately 3 weeks. Surviving calli were transferred to Regl medium supplemented with mannose. After culture in light (16 hours light/8 hours dark), green tissues were then transferred to Reg2 medium without growth regulators and incubated for approximately 1-2 weeks. Seedlings were transferred to Magenta GA-7 containers (Magenta Corp, Chicago, IL) containing Reg3 medium and grown in the light. After approximately 2-3 weeks, the plants were tested by PCR for the presence of PMI genes as well as the chimeric Bt cry gene. Plants that showed positive results in the PCR analysis were transferred to the greenhouse for further evaluation.

[00408] Transgenic plants were evaluated for copy number (determined by Taqman assay), protein expression level (determined by ELISA), and effectiveness against insect species of interest in leaf-excision bioassays. Specifically, plant tissue (leaf or stigma) was excised from single-copy transformants (stages V3 - V4) and infected with newborn larvae of the target pest, then incubated at room temperature for 5 days. Leaf discs from transgenic plants expressing each chimeric Bt protein were tested against the armyworm (Spodoptera frugiperda, FAW).

[00409] The results confirm that the transgenic plants express the chimeric proteins of the present invention and are active against insect pests. Protein expression in transgenic transformants for the Bt29-Bt22 chimera was approximately 25-125 ng/mg total soluble protein (TSP), and for the Bt29-1Fa chimera was approximately 25-290 ng/mg TSP. Approximately 90% and 92% of transgenic transformants expressing the Bt29-Bt22 and Bt29-1Fa chimeras, respectively, resulted in 100% mortality and suppressed growth of fall armyworm larvae.

[00410] The examples presented above clearly illustrate the advantages of the present invention. Although the present invention has been described in terms of specific details of certain embodiments thereof, it is not intended that such details should be construed as limiting the scope of the claimed invention except and to the extent that they are included in the appended claims.

--->

LIST OF SEQUENCES

<110> ZINGENTA PARTITIONS AG

<120> DESIGNED PESTICIDE PROTEINS AND CONTROL METHODS

PLANT PESTS

<130> 81090-WO-REG-ORG-P-1

<150>US 62/432,909

<151> 2016-12-12

<160> 35

<170> PatentIn version 3.5

<210> 1

<211> 715

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Sequence of BT-0022 assembled from data analysis

in relation to the Bacillus genome.

<400> 1

Met Lys Ser Lys Asn Gln Asn Met His Gln Ser Leu Ser Asn Asn Ala

1 5 10 15

Thr Val Asp Lys Asn Phe Thr Gly Ser Leu Glu Asn Asn Thr Asn Thr

20 25 30

Glu Leu Gln Asn Phe Asn His Glu Gly Ile Glu Pro Phe Val Ser Val

35 40 45

Ser Thr Ile Gln Thr Gly Ile Gly Ile Ala Gly Lys Ile Leu Gly Asn

50 55 60

Leu Gly Val Pro Phe Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile

65 70 75 80

Leu Gly Glu Leu Trp Pro Lys Gly Lys Ser Gln Trp Glu Ile Phe Met

85 90 95

Glu His Val Glu Glu Leu Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg

100 105 110

Asn Lys Ala Leu Ala Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val

115 120 125

Tyr His Glu Ser Leu Glu Ser Trp Ile Lys Asn Arg Asn Asn Thr Arg

130 135 140

Thr Arg Ser Val Val Lys Ser Gln Tyr Ile Thr Leu Glu Leu Met Phe

145 150 155 160

Val Gln Ser Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu

165 170 175

Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg

180 185 190

Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Asp Ser Glu Ile

195 200 205

Ser Thr Phe Tyr Asn Arg Gln Val Glu Arg Thr Ser Asp Tyr Ser Asp

210 215 220

His Cys Thr Lys Trp Phe Asp Thr Gly Leu Asn Arg Leu Lys Gly Ser

225 230 235 240

Asn Ala Glu Ile Trp Val Lys Tyr Asn Gln Phe Arg Arg Asp Met Thr

245 250 255

Leu Met Val Leu Asp Leu Val Ala Leu Phe Gln Ser Tyr Asp Thr His

260 265 270

Met Tyr Pro Ile Lys Thr Thr Ala Gln Leu Thr Arg Glu Val Tyr Thr

275 280 285

Asn Ala Ile Gly Thr Val His Pro His Pro Ser Phe Thr Ser Thr Thr

290 295 300

Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Val

305 310 315 320

Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile Tyr

325 330 335

Ser Leu Leu Ser Arg Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly

340 345 350

Gly His Lys Leu Glu Phe Arg Thr Ile Gly Gly Thr Leu Asn Thr Ser

355 360 365

Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Pro Phe

370 375 380

Thr Ser Arg Asp Ile Tyr Arg Thr Glu Ser Leu Ala Gly Leu Asn Leu

385 390 395 400

Phe Leu Thr Gln Pro Val Asn Gly Val Pro Arg Val Asp Phe His Trp

405 410 415

Lys Phe Val Thr His Pro Ile Ala Ser Asp Asn Phe Tyr Tyr Pro Gly

420 425 430

Tyr Ala Gly Ile Gly Thr Gln Leu Gln Asp Ser Glu Asn Glu Leu Pro

435 440 445

Pro Glu Thr Thr Gly Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu

450 455 460

Ser His Ile Gly Leu Ile Ser Ala Ser His Val Lys Ala Leu Val Tyr

465 470 475 480

Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile His Ser Asp

485 490 495

Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly

500 505 510

Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg

515 520 525

Arg Thr Ser Gly Gly Pro Phe Ala Phe Ser Asn Val Asn Leu Asp Trp

530 535 540

Asn Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr

545 550 555 560

Asn Leu Arg Met Tyr Val Thr Ile Ala Gly Glu Arg Ile Phe Ala Gly

565 570 575

Gln Phe Asn Lys Thr Met Asn Thr Gly Asp Pro Leu Thr Phe Gln Ser

580 585 590

Phe Ser Tyr Ala Thr Ile Asp Thr Ala Phe Thr Phe Pro Thr Lys Ala

595 600 605

Ser Ser Leu Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val

610 615 620

Tyr Val Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala

625 630 635 640

Val Thr Asp Leu Glu Arg Ala Gln Lys Ala Val His Glu Leu Phe Thr

645 650 655

Ser Thr Asn Pro Gly Gly Leu Lys Thr Asp Val Ala Lys Asp His Tyr

660 665 670

Thr Asn Thr Ile Ser Lys Ser Val Gln Ser Val Phe Arg Cys Arg Cys

675 680 685

Ser Glu Arg Thr Arg Ile Tyr Arg Trp Gly Tyr Pro Ser Lys Lys Glu

690 695 700

Tyr Trp Tyr Ile Trp Gly Tyr Thr Ser Lys Tyr

705 710 715

<210> 2

<211> 1169

<212> PROTEIN

<213> Artificial sequence

<220>

<223> BT-0029 sequence assembled from data analysis

in relation to the Bacillus genome.

<400> 2

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Ile Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Val Gly Ser Phe

485 490 495

Gly Asp Met Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg

500 505 510

Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Tyr Thr Asn

515 520 525

Ile Asn Gly Thr Thr Ile Asn Ile Gly Asn Phe Ser Ser Thr Met Asp

530 535 540

Ser Gly Asp Asp Leu Gln Tyr Gly Arg Phe Arg Val Ala Gly Phe Thr

545 550 555 560

Thr Pro Phe Thr Phe Ser Asp Ala Met Ser Thr Phe Thr Ile Gly Ala

565 570 575

Phe Ser Phe Ser Ser Asn Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe

580 585 590

Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser

645 650 655

Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu

660 665 670

Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp

675 680 685

Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys

690 695 700

Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr

705 710 715 720

Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg

725 730 735

Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr

740 745 750

Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr

755 760 765

Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly

770 775 780

Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp

785 790 795 800

Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe

805 810 815

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly

820 825 830

Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu

835 840 845

Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu

850 855 860

Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys

865 870 875 880

Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val

885 890 895

Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr

900 905 910

Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg

915 920 925

Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala

930 935 940

Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr

945 950 955 960

Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser

965 970 975

Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His

980 985 990

Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

995 1000 1005

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1010 1015 1020

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1025 1030 1035

Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1040 1045 1050

Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1055 1060 1065

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1070 1075 1080

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1085 1090 1095

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1100 1105 1110

Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1115 1120 1125

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1130 1135 1140

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1145 1150 1155

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 3

<211> 1169

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Chimeric protein Bt29-Bt22.

<400> 3

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser

645 650 655

Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu

660 665 670

Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp

675 680 685

Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys

690 695 700

Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr

705 710 715 720

Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg

725 730 735

Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr

740 745 750

Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr

755 760 765

Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly

770 775 780

Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp

785 790 795 800

Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe

805 810 815

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly

820 825 830

Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu

835 840 845

Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu

850 855 860

Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys

865 870 875 880

Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val

885 890 895

Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr

900 905 910

Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg

915 920 925

Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala

930 935 940

Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr

945 950 955 960

Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser

965 970 975

Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His

980 985 990

Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

995 1000 1005

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1010 1015 1020

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1025 1030 1035

Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1040 1045 1050

Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1055 1060 1065

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1070 1075 1080

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1085 1090 1095

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1100 1105 1110

Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1115 1120 1125

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1130 1135 1140

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1145 1150 1155

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 4

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Coding sequences for the Bt29-Bt22 chimeric protein.

<400> 4

atggagatta ataatcagaa ccaatgtgtc ccttataatt gtttgaataa tcctgaaagc

60

gagatattaa acgttgcaat ttttagtagc gaacaggtag cagaaattca cttaaagatc

120

acgcgcttaa ttttagagaa ttttttacca ggtgggagtt ttgcattcgg cttatttgat

180

ttaatatggg ggatttttaa tgaagatcaa tggagcgcat ttcttcggca ggtagaagaa

240

ttaattaatc aaaggataac ggaattcgca agagggcaag caattcagag actagtaggg

300

tttggaagga gttatgatga atatatttta gcactaaaag aatgggaaaa cgatcctgat

360

aacccagctt caaaggaaag agtgcgcact cgatttcgga caactgatga tgccttgcta

420

accggtgttc ctcttatggc aattccaggt tttgaattag ctactttatc tgtttatgct

480

caatcagcca atctacattt agccctatta agagatgctg tattttttgg ggagagatgg

540

ggattgacac aaacaaatat aaatgattta tatagtagat taaaaaactc cattcgtgat

600

tatacaaatc attgtgttcg tttttataat ataggtttag ggaatttaaa tgttataaga

660

ccagagtatt accgtttcca aagagaatta acaatatctg tcttagatct tgtagctctt

720

tttccaaatt acgatatccg aacatatcca ataccaacta aaagtcaatt aacaagagaa

780

atttatacag atccgattat ttcacctggt gcacaggcag gttatactct tcaagatgtt

840

ttgagagaac cacaccttat ggacttttta aaccgactta ttatttatac tggtgagtat

900

cgcggaattc gtcactgggc aggacatgaa gtagaatcta gtagaacagg tatgatgact

960

aatataagat ttcctttgta tggaacagcc gcaacagcag aaccaacacg atttataact

1020

cctagtactt ttcctggtct taatttattt tatagaacat tatcagctcc tatttttaga

1080

gatgaaccgg gagctaatat tattattaga tatagaacga gtttggtgga aggagtagga

1140

tttattcaac caaataacgg tgaacagctt tacagagtga gaggaacatt agattctctt

1200

gatcaattac cacttgaggg tgagagtagt ctaactgaat atagtcatcg attatgccat

1260

gttagatttg cgcaatcatt gaggaatgca gaacctttag attatgcaag ggttccgatg

1320

ttttcttgga cacatcgtag tgcaacccct acaaatacaa ttgatccaga tgtcatcacc

1380

caaataccgt tagtaaaagc acataccctt cagtcaggta ctactgttgt aaaagggcca

1440

gggtttacag gtggagatat cctccgacga actagtggag gaccatttgc ttttagtaat

1500

gttaatttag actggaactt gtcacaaaga tatcgtgcta gaatacgcta tgcttctact

1560

actaatctaa gaatgtacgt aacgattgca ggggaacgaa tttttgctgg tcaatttaat

1620

aaaacaatga atactggtga tccattaaca ttccaatctt ttagttacgc aactattgat

1680

acagcattta cattcccaac gaaagcgagc agcttgactg taggtgctga tacttttagc

1740

tcaggtaatg aagtttatgt agatagattt gaattgatcc cagttactgc aacatttgag

1800

gcagaatatg atttagagaa agctcagaaa gcggtgaatg cgctgtttac ttcttccaat

1860

caaatcgggt taaaaacaga tgtgacggac tatcatattg ataaagtatc caatctagtt

1920

gagtgtttat cagatgaatt ttgtctagat gaaaagcgag aattgtccga gaaagtcaaa

1980

catgcgaagc gactctgtga tgagcggaat ttacttcaag atccaaactt cagaggcatc

2040

aatagacaac cagaccgtgg ttggagagga agtacggata ttaccatcca aggaggat

2100

gacgtattca aagagaatta cgttacgcta ccgggtacct ttgatgagtg ctatccaacg

2160

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga

2220

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacat

2280

gaaacagtaa atgtgccagg tacgggttcc ttatggccgc tttcagccca aagtccaatc

2340

ggaaagtgtg gagaaccgaa tcgatgtgcg acacaccttg aatggaatcc tgacttagat

2400

tgttcgtgta gggatggaga aaagtgtgcc catcattcgc atcatttctc cttagacatt

2460

gatgtaggat gtacagacct aaatgaggac ctaggtgtat gggtgatctt taagattaag

2520

acgcaagatg gtcatgcgag actaggaaat ctagaatttc tcgaagagaa accattagta

2580

ggagaagcgc tagctcgtgt gaagagagcg gagaaaaaat ggagagacaa acgcgaaaaa

2640

ttggaattgg aaacaaatat tgtttataaa gaggcaaaaa aatctgtaga tgctttattt

2700

gtgaactctc aatatgatag attacaagcg gatacgaata tcgcgataat tcatgcggca

2760

gataaacgcg ttcatagcat tcgagaagca tatcttccag agttgtctgt aattccgggt

2820

gtaaatgcag ctatttttga agaattagag ggacgtattt tcacagccta ctctctatat

2880

gatgcgagaa atgtcattaa aaatggcgat ttcaataatg gcttatcatg ctggaacgtg

2940

aaagggcatg tagatgtaga agaacagaac aaccatcgtt cggtccttgt tgttccagaa

3000

tgggaagcag aagtgtcaca agaggttcgt gtctgtccag gtcgtggcta tatccttcgt

3060

gttacagcgt acaaagaggg atatggagag ggctgtgtaa cgattcatga gatcgaagac

3120

aatacagacg aactgaaatt cagcaactgt gtagaagagg aaatatatcc aaacaacacg

3180

gtaacgtgta atgattatac tgcgactcaa gaagaatatg agggtacgta cacttctcgt

3240

aatcgaggat atgacggagc ctatgaaagc aattcttctg taccagctga ttatgcatca

3300

gcctatgaag aaaaagcgta tacagatgga agaagagaca atacttgtga atctaacaga

3360

ggatatgggg attacacacc actaccagct ggctatgtga caaaagaatt agagtacttc

3420

ccagaaaccg ataaggtatg gattgagatt ggagaaacgg aaggaacatt tatcgtggac

3480

agcgtggaat tactccttat ggaggaatag

3510

<210> 5

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized maize coding sequence for

chimeric protein Bt29-Bt22.

<400> 5

atggaaatca acaaccagaa ccagtgcgtg ccgtacaact gcctcaacaa ccccgagtcc

60

gagatcctga acgtggccat cttctccagc gagcaggtcg cggagatcca cctcaagatc

120

acgcgcctga tcctcgagaa cttcctgccg ggcggctcct tcgctttcgg cctgttcgac

180

ctcatctggg gcatcttcaa cgaggaccag tggagcgcgt tcctcaggca ggtggaggag

240

ctgatcaacc agcgcatcac ggagttcgcc aggggccagg ctatccagcg gctggtgggc

300

ttcggcaggt cctacgacga atacatcctg gccctcaagg agtggggagaa cgaccccgac

360

aacccggcca gcaaggagcg cgtgaggacc cgcttcagga ccaccgacga cgctctcctg

420

acgggcgtcc ccctcatggc tatcccgggc ttcgagctgg ccaccctctc ggtgtacgct

480

cagtcggcca acctgcacct cgccctcctg cgggacgctg tgttcttcgg cgagaggtgg

540

ggcctgaccc aaaccaacat caacgacctc tactccaggc tgaagaacag catccgcgac

600

tacacgaacc actgcgtgcg cttctacaac atcggcctgg gcaacctcaa cgtcatcagg

660

ccggaatact accgcttcca gagggagctg accatcagcg tgctggacct cgtcgccctg

720

ttccccaact acgacatccg cacgtacccg atccccacca agtcccagct cacgagggag

780

atctacaccg acccgatcat ctcgccgggc gcccaggccg gctacaccct gcaggacgtc

840

ctgagggagc cccacctgat ggacttcctg aacaggctca tcatctacac cggcgagtac

900

aggggcatca ggcactgggc gggccacgag gtggagtcca gcaggacggg catgatgacc

960

aacatccgct tcccgctcta cggcaccgcg gccacggccg agccaacccg cttcatcacg

1020

ccgtccacct tccccggcct gaacctcttc tacaggaccc tgtcggctcc catcttccgc

1080

gacgagccgg gcgcgaacat catcatccgc tacaggacct ccctcgtgga gggcgtcggc

1140

ttcatccagc cgaacaacgg cgagcagctg taccgcgtga ggggcacgct ggacagcctg

1200

gaccagctcc cactggaggg cgagtccagc ctcaccgagt actcgcacag gctgtgccac

1260

gtcaggttcg cccagagcct caggaacgcg gagcccctgg actacgccag ggtgcccatg

1320

ttcagctgga cccacaggtc ggctaccccc accaacacca tcgacccaga cgtgatcacg

1380

cagatcccgc tcgtcaaggc ccacaccctg cagtcgggca ccaccgtggt caagggcccc

1440

ggcttcacgg gcggcgacat cctgaggagg acctccggcg gcccattcgc cttcagcaac

1500

gtcaacctcg actggaacct gtcccagcgc tacagggcgc gcatcaggta cgccagcacc

1560

acgaacctgc gcatgtatgt gaccatcgcg ggcgagagga tcttcgccgg ccagttcaac

1620

aagacgatga acaccggcga cccgctcacc ttccagtcct tcagctacgc gacgatcgac

1680

accgccttca cgttccccac gaaggcctcc agcctgaccg tgggcgccga caccttctcc

1740

agcggcaacg aggtctacgt ggaccgcttc gagctgatcc cggtgacggc gaccttcgag

1800

gccgagtacg acctggagaa ggcccagaag gcggtcaacg ccctcttcac ctccagcaac

1860

cagatcggcc tgaagacgga cgtgaccgac taccacatcg acaaggtgtc caacctcgtc

1920

gagtgcctga gcgacgagtt ctgcctcgac gagaagaggg agctgtccga gaaggtcaag

1980

cacgccaagc gcctctgcga cgagaggaac ctcctgcagg acccgaactt caggggcatc

2040

aaccgccagc cggacagggg ctggaggggc agcaccgaca tcaccatcca gggcggcgac

2100

gacgtgttca aggagaacta cgtcacgctc ccgggcacct tcgacgagtg ctaccccacg

2160

tacctgtacc agaagatcga cgagtccaag ctcaaggcct acacccgcta cgagctgagg

2220

ggctacatcg aggacagcca ggacctcgag atctacctga tccgctacaa cgcgaagcac

2280

gagacggtga acgtccccgg cacgggctcc ctgtggcccc tctcggctca gtcgccgatc

2340

ggcaagtgcg gcgagcccaa caggtgcgcc acccacctcg agtggaaccc ggacctggac

2400

tgctcctgcc gggacggcga gaagtgcgct caccactccc accacttcag cctggacatc

2460

gacgtgggct gcacggacct caacgaggac ctgggcgtgt gggtcatctt caagatcaag

2520

acgcaggacg gccacgctag gctgggcaac ctcgagttcc tggaggagaa gccgctggtg

2580

ggcgaggctc tggccagggt caagagggcg gagaagaagt ggcgcgacaa gagggagaag

2640

ctggagctgg agacgaacat cgtctacaag gaggccaaga agtccgtgga cgcgctcttc

2700

gtcaacagcc agtacgacag gctgcaggcg gacaccaaca tcgccatcat ccacgccgcg

2760

gacaagcgcg tgcactccat cagggaggcc tacctccccg agctgagcgt gatcccgggc

2820

gtcaacgctg ccatcttcga ggagctggag ggccgcatct tcaccgccta ctccctgtac

2880

gacgcgagga acgtcatcaa gaacggcgac ttcaacaacg gcctcagctg ctggaacgtg

2940

aagggccacg tggacgtcga ggagcagaac aaccaccgct cggtgctggt ggtccccgag

3000

tgggaggctg aggtcagcca ggaggtgcgc gtctgcccgg gcaggggcta catcctccgc

3060

gtgaccgcgt acaaggaggg ctacggcgag ggctgcgtca cgatccacga gatcgaggac

3120

aacaccgacg agctgaagtt ctccaactgc gtggaggagg agatctaccc gaacaacacg

3180

gtcacctgca acgactacac ggccacccag gaggagtacg agggcacgta cacgtcgagg

3240

aacagggggct acgacggcgc ttacgagtcc aacagctcgg tgccggccga ctacgctagc

3300

gcgtacgagg agaaggccta cacggacggc cgcagggaca acacctgcga gtcgaacagg

3360

ggctacggcg actacacgcc gctcccggcc ggctacgtga ccaaggagct ggagtacttc

3420

ccggagacgg acaaggtctg gatcgagatc ggcgagacgg agggcacctt catcgtggac

3480

agcgtcgagc tgctgctcat ggaggagtag

3510

<210> 6

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized maize coding sequence for

chimeric protein BT29-BT22.

<400> 6

atggaaatca acaaccagaa ccagtgcgtg ccgtacaact gccttaacaa ccccgagtcc

60

gagatcctga acgtggccat cttctccagc gagcaggtcg cggagatcca cctcaagatc

120

acgcgcctga tcctcgagaa cttcctgccg ggcggctcct tcgctttcgg cctgttcgac

180

ctcatctggg gcatcttcaa cgaggaccag tggagcgcgt tcctcaggca ggtggaggag

240

ctgatcaacc agcgcatcac ggagttcgcc aggggccagg ctatccagcg gctggtgggc

300

ttcggcaggt cctacgacga atacatcctg gccctcaagg agtggggagaa cgaccccgac

360

aacccggcca gcaaggagcg cgtgaggacc cgcttcagga ccaccgacga cgctctcctg

420

acgggcgtcc ccctcatggc tatcccgggc ttcgagctgg ccaccctctc ggtgtacgct

480

cagtcggcca acctgcacct cgccctcctg cgggacgctg tgttcttcgg cgagaggtgg

540

ggcctgaccc aaaccaacat caacgacctc tactccaggc tgaagaacag catccgcgac

600

tacacgaacc actgcgtgcg cttctacaac atcggcctgg gcaacctcaa cgtcatcagg

660

ccggaatact accgcttcca gagggagctg accatcagcg tgctggacct cgtcgccctg

720

ttccccaact acgacatccg cacgtacccg atccccacca agtcccagct cacgagggag

780

atctacaccg acccgatcat ctcgccgggc gcccaggccg gctacaccct gcaggacgtc

840

ctgagggagc cccacctgat ggacttcctg aacaggctca tcatctacac cggcgagtac

900

aggggcatca ggcactgggc gggccacgag gtggagtcca gcaggacggg catgatgacc

960

aacatccgct tcccgctcta cggcaccgcg gccacggccg agccaacccg cttcatcacg

1020

ccgtccacct tccccggcct gaacctcttc tacaggaccc tgtcggctcc catcttccgc

1080

gacgagccgg gcgcgaacat catcatccgc tacaggacct ccctcgtgga gggcgtcggc

1140

ttcatccagc cgaacaacgg cgagcagctg taccgcgtga ggggcacgct ggacagcctg

1200

gaccagctcc cactggaggg cgagtccagc ctcaccgagt actcgcacag gctgtgccac

1260

gtcaggttcg cccagagcct caggaacgcg gagcccctgg actacgccag ggtgcccatg

1320

ttcagctgga cccacaggtc ggctaccccc accaacacca tcgacccaga cgtgatcacg

1380

cagatcccgc tcgtcaaggc ccacaccctg cagtcgggca ccaccgtggt caagggcccc

1440

ggcttcacgg gcggcgacat cctgaggagg acctccggcg gcccattcgc cttcagcaac

1500

gtcaacctcg actggaacct gtcccagcgc tacagggcgc gcatcaggta cgccagcacc

1560

acgaacctgc gcatgtatgt gaccatcgcg ggcgagagga tcttcgccgg ccagttcaac

1620

aagacgatga acaccggcga cccgctcacc ttccagtcct tcagctacgc gacgatcgac

1680

accgccttca cgttccccac gaaggcctcc agcctgaccg tgggcgccga caccttctcc

1740

agcggcaacg aggtctacgt ggaccgcttc gagctgatcc cggtgacggc gaccttcgag

1800

gccgagtacg acctggagaa ggcccagaag gcggtcaacg ccctcttcac ctccagcaac

1860

cagatcggcc tgaagacgga cgtgaccgac taccacatcg acaaggtgtc caacctcgtc

1920

gagtgcctga gcgacgagtt ctgcctcgac gagaagaggg agctgtccga gaaggtcaag

1980

cacgccaagc gcctctgcga cgagaggaac ctcctgcagg acccgaactt caggggcatc

2040

aaccgccagc cggacagggg ctggaggggc agcaccgaca tcaccatcca gggcggcgac

2100

gacgtgttca aggagaacta cgtcacgctc ccgggcacct tcgacgagtg ctaccccacg

2160

tacctgtacc agaagatcga cgagtccaag ctcaaggcct acacccgcta cgagctgagg

2220

ggctacatcg aggacagcca ggacctcgag atctacctga tccgctacaa cgcgaagcac

2280

gagacggtga acgtccccgg cacgggctcc ctgtggcccc tctcggctca gtcgccgatc

2340

ggcaagtgcg gcgagcccaa caggtgcgcc acccacctcg agtggaaccc ggacctggac

2400

tgctcctgcc gggacggcga gaagtgcgct caccactccc accacttcag cctggacatc

2460

gacgtgggct gcacggacct caacgaggac ctgggcgtgt gggtcatctt caagatcaag

2520

acgcaggacg gccacgctag gctgggcaac ctcgagttcc tggaggagaa gccgctggtg

2580

ggcgaggctc tggccagggt caagagggcg gagaagaagt ggcgcgacaa gagggagaag

2640

ctggagctgg agacgaacat cgtctacaag gaggccaaga agtccgtgga cgcgctcttc

2700

gtcaacagcc agtacgacag gctgcaggcg gacaccaaca tcgccatcat ccacgccgcg

2760

gacaagcgcg tgcactccat cagggaggcc tacctccccg agctgagcgt gatcccgggc

2820

gtcaacgctg ccatcttcga ggagctggag ggccgcatct tcaccgccta ctccctgtac

2880

gacgcgagga acgtcatcaa gaacggcgac ttcaacaacg gcctcagctg ctggaacgtg

2940

aagggccacg tggacgtcga ggagcagaac aaccaccgct cggtgctggt ggtccccgag

3000

tgggaggctg aggtcagcca ggaggtgcgc gtctgcccgg gcaggggcta catcctccgc

3060

gtgaccgcgt acaaggaggg ctacggcgag ggctgcgtca cgatccacga gatcgaggac

3120

aacaccgacg agctgaagtt ctccaactgc gtggaggagg agatctaccc gaacaacacg

3180

gtcacctgca acgactacac ggccacccag gaggagtacg agggcacgta cacgtcgagg

3240

aacagggggct acgacggcgc ttacgagtcc aacagctcgg tgccggccga ctacgctagc

3300

gcgtacgagg agaaggccta cacggacggc cgcagggaca acacctgcga gtcgaacagg

3360

ggctacggcg actacacgcc gctcccggcc ggctacgtga ccaaggagct ggagtacttc

3420

ccggagacgg acaaggtctg gatcgagatc ggcgagacgg agggcacctt catcgtggac

3480

tcagtcgagc tgctgctcat ggagagtag

3510

<210> 7

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized maize coding sequence for

chimeric protein Bt29-Bt22.

<400> 7

atggagatca acaaccagaa ccagtgcgtg ccgtacaact gccttaacaa ccccgagtcc

60

gagatcctga acgtggccat cttctccagc gagcaggtcg cggagatcca cctcaagatc

120

acgcgcctga tcctcgagaa cttcctgccg ggcggctcct tcgctttcgg cctgttcgac

180

ctcatctggg gcatcttcaa cgaggaccag tggagcgcgt tcctcaggca ggtggaggag

240

ctgatcaacc agcgcatcac ggagttcgcc aggggccagg ctatccagcg gctggtgggc

300

ttcggcaggt cctacgacga gtacatcctg gccctcaagg agtgggagaa cgaccccgac

360

aacccggcca gcaaggagcg cgtgaggacc cgcttcagga ccaccgacga cgctctcctg

420

acgggcgtcc ccctcatggc tatcccgggc ttcgagctgg ccaccctctc ggtgtacgct

480

cagtcggcca acctgcacct cgccctcctg cgggacgctg tgttcttcgg cgagaggtgg

540

ggcctgaccc agacgaacat caacgacctc tactccaggc tgaagaacag catccgcgac

600

tacacgaacc actgcgtgcg cttctacaac atcggcctgg gcaacctcaa cgtcatcagg

660

ccggagtact accgcttcca gagggagctg accatcagcg tgctggacct cgtcgccctg

720

ttccccaact acgacatccg cacgtacccg atcccaacca agtcccagct cacgagggag

780

atctacaccg acccgatcat ctcgccgggc gcccaggccg gctacaccct gcaggacgtc

840

ctgagggagc cccacctgat ggacttcctg aacaggctca tcatctacac cggcgagtac

900

aggggcatca ggcactgggc gggccacgag gtggagtcca gcaggacggg catgatgacc

960

aacatccgct tcccgctcta cggcaccgcg gccacggccg agccaacccg cttcatcacg

1020

ccgtccacct tccccggcct gaacctcttc tacaggaccc tgtcggctcc catcttccgc

1080

gacgagccgg gcgcgaacat catcatccgc tacaggacct ccctcgtgga gggcgtcggc

1140

ttcatccagc cgaacaacgg cgagcagctg taccgcgtga ggggcacgct ggacagcctg

1200

gaccagctcc cactggaggg cgagtccagc ctcaccgagt actcgcacag gctgtgccac

1260

gtcaggttcg cccagagcct caggaacgcg gagcccctgg actacgccag ggtgcccatg

1320

ttcagctgga cccacaggtc ggctaccccc accaacacca tcgacccaga cgtgatcacg

1380

cagatcccgc tcgtcaaggc ccacaccctg cagtcgggca ccaccgtggt caagggcccc

1440

ggcttcacgg gcggcgacat cctgaggagg acctccggcg gcccattcgc cttcagcaac

1500

gtcaacctcg actggaacct gtcccagcgc tacagggcgc gcatcaggta cgccagcacc

1560

acgaacctgc gcatgtatgt gaccatcgcg ggcgagagga tcttcgccgg ccagttcaac

1620

aagacgatga acaccggcga cccgctcacc ttccagtcct tcagctacgc gacgatcgac

1680

accgccttca cgttccccac gaaggcctcc agcctgaccg tgggcgccga caccttctcc

1740

agcggcaacg aggtctacgt ggaccgcttc gagctgatcc cggtgacggc gaccttcgag

1800

gccgagtacg acctggagaa ggcccagaag gcggtcaacg ccctcttcac ctccagcaac

1860

cagatcggcc tgaagacgga cgtgaccgac taccacatcg acaaggtgtc caacctcgtc

1920

gagtgcctga gcgacgagtt ctgcctcgac gagaagaggg agctgtccga gaaggtcaag

1980

cacgccaagc gcctctgcga cgagaggaac ctcctgcagg acccgaactt caggggaatc

2040

aaccgccagc cggacagggg ctggaggggc agcaccgaca tcaccatcca gggcggcgac

2100

gacgtgttca aggagaacta cgtcacgctc ccgggcacct tcgacgagtg ctaccccacg

2160

tacctgtacc agaagatcga cgagtccaag ctcaaggcct acacccgcta cgagctgagg

2220

ggatacatcg aggacagcca ggacctcgag atctacctga tccgctacaa cgcgaagcac

2280

gagacggtga acgtccccgg cacgggctcc ctgtggcccc tctcggctca gtcgccgatc

2340

ggcaagtgcg gcgagcccaa caggtgcgcc acccacctcg agtggaaccc ggacctggac

2400

tgctcctgcc gggacggcga gaagtgcgct caccactccc accacttcag cctggacatc

2460

gacgtgggct gcacggacct caacgaggac ctgggcgtgt gggtcatctt caaaatcaag

2520

acgcaggacg gccacgctag gctgggcaac ctcgagttcc tggaggagaa gccgctggtg

2580

ggcgaggctc tggccagggt caagagggcg gagaagaagt ggcgcgacaa gagggagaag

2640

ctggagctgg agacgaacat cgtctacaag gaggccaaga agtccgtgga cgcgctcttc

2700

gtcaacagcc agtacgacag gctgcaggcg gacaccaaca tcgccatcat ccacgccgcg

2760

gacaagcgcg tgcactccat cagggaggcc tacctccccg agctgagcgt gatcccgggc

2820

gtcaacgctg ccatcttcga ggagctggag ggccgcatct tcaccgccta ctccctgtac

2880

gacgcgagga acgtcatcaa gaacggcgac ttcaacaacg gcctcagctg ctggaacgtg

2940

aagggccacg tggacgtcga ggagcagaac aaccaccgct cggtgctggt ggtccccgag

3000

tgggaggctg aggtcagcca ggaggtgcgc gtctgcccgg gcaggggata catcctccgc

3060

gtgaccgcgt acaaggaggg ctacggcgag ggctgcgtca cgatccacga gatcgaggac

3120

aacaccgacg agctgaagtt ctccaactgc gtggaggagg agatctaccc gaacaacacg

3180

gtcacctgca acgactacac ggccacccag gaggagtacg agggcacgta cacgtcgagg

3240

aacagggggct acgacggcgc ttacgagtcc aacagctcgg tgccggccga ctacgctagc

3300

gcgtacgagg agaaggccta cacggacggc cgcagggaca acacctgcga gtcgaacagg

3360

ggctacggcg actacacgcc gctcccggcc ggctacgtga ccaaggagct ggagtacttc

3420

ccggagacgg acaaggtctg gatcgagatc ggcgagacgg agggcacctt catcgtggac

3480

tcagtcgagc tgctgctcat ggagagtag

3510

<210> 8

<211> 1174

<212> PROTEIN

<213> Bacillus thuringiensis

<400> 8

Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu

20 25 30

Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe

35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln

65 70 75 80

Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr

85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu

100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val

115 120 125

Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val

145 150 155 160

Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe

165 170 175

Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn

180 185 190

Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr

195 200 205

Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp

210 215 220

Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp

225 230 235 240

Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln

245 250 255

Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu

260 265 270

Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu

275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe

290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu

305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr

325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro

340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly

355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln

370 375 380

Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile

385 390 395 400

Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro

420 425 430

Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile

450 455 460

Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr

465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu

500 505 510

Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu

515 520 525

Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe

530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser

545 550 555 560

Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser

565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile

580 585 590

Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr

595 600 605

Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile

610 615 620

Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln

625 630 635 640

Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu

645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp

660 665 670

Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln

675 680 685

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly

690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp

705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu

725 730 735

Lys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln

740 745 750

Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val

755 760 765

Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro

770 775 780

Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp

785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His

805 810 815

His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu

820 825 830

Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp

835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu

850 855 860

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg

865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu

885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln

900 905 910

Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg

915 920 925

Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro

930 935 940

Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr

945 950 955 960

Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe

965 970 975

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu

980 985 990

Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala

995 1000 1005

Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile

1010 1015 1020

Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val

1025 1030 1035

Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser

1040 1045 1050

Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys

1055 1060 1065

Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr

1070 1075 1080

Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser

1085 1090 1095

Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr

1100 1105 1110

Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly

1115 1120 1125

Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu

1130 1135 1140

Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr

1145 1150 1155

Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu

1160 1165 1170

Glu

<210> 9

<211> 1169

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Constructed chimeric protein BT29-Cry1Fa1.

<400> 9

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser

645 650 655

Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu

660 665 670

Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp

675 680 685

Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys

690 695 700

Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr

705 710 715 720

Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg

725 730 735

Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr

740 745 750

Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr

755 760 765

Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly

770 775 780

Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp

785 790 795 800

Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe

805 810 815

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly

820 825 830

Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu

835 840 845

Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu

850 855 860

Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys

865 870 875 880

Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val

885 890 895

Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr

900 905 910

Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg

915 920 925

Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala

930 935 940

Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr

945 950 955 960

Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser

965 970 975

Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His

980 985 990

Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

995 1000 1005

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1010 1015 1020

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1025 1030 1035

Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1040 1045 1050

Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1055 1060 1065

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1070 1075 1080

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1085 1090 1095

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1100 1105 1110

Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1115 1120 1125

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1130 1135 1140

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1145 1150 1155

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 10

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Coding sequence BT29-Cry1Fa1.

<400> 10

atggagatta ataatcagaa ccaatgtgtc ccttataatt gtttgaataa tcctgaaagc

60

gagatattaa acgttgcaat ttttagtagc gaacaggtag cagaaattca cttaaagatc

120

acgcgcttaa ttttagagaa ttttttacca ggtgggagtt ttgcattcgg cttatttgat

180

ttaatatggg ggatttttaa tgaagatcaa tggagcgcat ttcttcggca ggtagaagaa

240

ttaattaatc aaaggataac ggaattcgca agagggcaag caattcagag actagtaggg

300

tttggaagga gttatgatga atatatttta gcactaaaag aatgggaaaa cgatcctgat

360

aacccagctt caaaggaaag agtgcgcact cgatttcgga caactgatga tgccttgcta

420

accggtgttc ctcttatggc aattccaggt tttgaattag ctactttatc tgtttatgct

480

caatcagcca atctacattt agccctatta agagatgctg tattttttgg ggagagatgg

540

ggattgacac aaacaaatat aaatgattta tatagtagat taaaaaactc cattcgtgat

600

tatacaaatc attgtgttcg tttttataat ataggtttag ggaatttaaa tgttataaga

660

ccagagtatt accgtttcca aagagaatta acaatatctg tcttagatct tgtagctctt

720

tttccaaatt acgatatccg aacatatcca ataccaacta aaagtcaatt aacaagagaa

780

atttatacag atccgattat ttcacctggt gcacaggcag gttatactct tcaagatgtt

840

ttgagagaac cacaccttat ggacttttta aaccgactta ttatttatac tggtgagtat

900

cgcggaattc gtcactgggc aggacatgaa gtagaatcta gtagaacagg tatgatgact

960

aatataagat ttcctttgta tggaacagcc gcaacagcag aaccaacacg atttataact

1020

cctagtactt ttcctggtct taatttattt tatagaacat tatcagctcc tatttttaga

1080

gatgaaccgg gagctaatat tattattaga tatagaacga gtttggtgga aggagtagga

1140

tttattcaac caaataacgg tgaacagctt tacagagtga gaggaacatt agattctctt

1200

gatcaattac cacttgaggg tgagagtagt ctaactgaat atagtcatcg attatgccat

1260

gttagatttg cgcaatcatt gaggaatgca gaacctttag attatgcaag ggttccgatg

1320

ttttcttgga cacatcgtag tgcaacccct acaaatacaa ttgatccaga tgtcatcacc

1380

caaataccgt tagtaaaagc acatacactt cagtcaggta ctactgttgt aagagggccc

1440

gggtttacgg gaggagatat tcttcgacga acaagtggag gaccatttgc ttatactatt

1500

gttaatataa atgggcaatt accccaaagg tatcgtgcaa gaatacgcta tgcctctact

1560

acaaatctaa gaatttacgt aacggttgca ggtgaacgga tttttgctgg tcaatttaac

1620

aaaacaatgg ataccggtga cccattaaca ttccaatctt ttagttacgc aactattaat

1680

acagctttta cattcccaat gagccagagt agtttcacag taggtgctga tacttttagt

1740

tcagggaatg aagtttatat agacagattt gaattgattc cagttactgc aacatttgag

1800

gcagaatatg atttagagaa agctcagaaa gcggtgaatg cgctgtttac ttcttccaat

1860

caaatcgggt taaaaacaga tgtgacggac tatcatattg ataaagtatc caatctagtt

1920

gagtgtttat cagatgaatt ttgtctagat gaaaagcgag aattgtccga gaaagtcaaa

1980

catgcgaagc gactctgtga tgagcggaat ttacttcaag atccaaactt cagaggcatc

2040

aatagacaac cagaccgtgg ttggagagga agtacggata ttaccatcca aggaggat

2100

gacgtattca aagagaatta cgttacgcta ccgggtacct ttgatgagtg ctatccaacg

2160

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga

2220

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacat

2280

gaaacagtaa atgtgccagg tacgggttcc ttatggccgc tttcagccca aagtccaatc

2340

ggaaagtgtg gagaaccgaa tcgatgtgcg acacaccttg aatggaatcc tgacttagat

2400

tgttcgtgta gggatggaga aaagtgtgcc catcattcgc atcatttctc cttagacatt

2460

gatgtaggat gtacagacct aaatgaggac ctaggtgtat gggtgatctt taagattaag

2520

acgcaagatg gtcatgcgag actaggaaat ctagaatttc tcgaagagaa accattagta

2580

ggagaagcgc tagctcgtgt gaagagagcg gagaaaaaat ggagagacaa acgcgaaaaa

2640

ttggaattgg aaacaaatat tgtttataaa gaggcaaaaa aatctgtaga tgctttattt

2700

gtgaactctc aatatgatag attacaagcg gatacgaata tcgcgataat tcatgcggca

2760

gataaacgcg ttcatagcat tcgagaagca tatcttccag agttgtctgt aattccgggt

2820

gtaaatgcag ctatttttga agaattagag ggacgtattt tcacagccta ctctctatat

2880

gatgcgagaa atgtcattaa aaatggcgat ttcaataatg gcttatcatg ctggaacgtg

2940

aaagggcatg tagatgtaga agaacagaac aaccatcgtt cggtccttgt tgttccagaa

3000

tgggaagcag aagtgtcaca agaggttcgt gtctgtccag gtcgtggcta tatccttcgt

3060

gttacagcgt acaaagaggg atatggagag ggctgtgtaa cgattcatga gatcgaagac

3120

aatacagacg aactgaaatt cagcaactgt gtagaagagg aaatatatcc aaacaacacg

3180

gtaacgtgta atgattatac tgcgactcaa gaagaatatg agggtacgta cacttctcgt

3240

aatcgaggat atgacggagc ctatgaaagc aattcttctg taccagctga ttatgcatca

3300

gcctatgaag aaaaagcgta tacagatgga agaagagaca atacttgtga atctaacaga

3360

ggatatgggg attacacacc actaccagct ggctatgtga caaaagaatt agagtacttc

3420

ccagaaaccg ataaggtatg gattgagatt ggagaaacgg aaggaacatt tatcgtggac

3480

agcgtggaat tactccttat ggaggaatag

3510

<210> 11

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized maize coding sequence BT29-

Cry1Fa1.

<400> 11

atggaaatca acaaccagaa ccagtgcgtg ccgtacaact gcctcaacaa ccccgagtcc

gagatcctga acgtggccat cttctccagc gagcaggtcg cggagatcca cctcaagatc

120

acgcgcctga tcctcgagaa cttcctgccg ggcggctcct tcgctttcgg cctgttcgac

180

ctcatctggg gcatcttcaa cgaggaccag tggagcgcgt tcctcaggca ggtggaggag

240

ctgatcaacc agcgcatcac ggagttcgcc aggggccagg ctatccagcg gctggtgggc

300

ttcggcaggt cctacgacga atacatcctg gccctcaagg agtggggagaa cgaccccgac

360

aacccggcca gcaaggagcg cgtgaggacc cgcttcagga ccaccgacga cgctctcctg

420

acgggcgtcc ccctcatggc tatcccgggc ttcgagctgg ccaccctctc ggtgtacgct

480

cagtcggcca acctgcacct cgccctcctg cgggacgctg tgttcttcgg cgagaggtgg

540

ggcctgaccc aaaccaacat caacgacctc tactccaggc tgaagaacag catccgcgac

600

tacacgaacc actgcgtgcg cttctacaac atcggcctgg gcaacctcaa cgtcatcagg

660

ccggaatact accgcttcca gagggagctg accatcagcg tgctggacct cgtcgccctg

720

ttccccaact acgacatccg cacgtacccg atccccacca agtcccagct cacgagggag

780

atctacaccg acccgatcat ctcgccgggc gcccaggccg gctacaccct gcaggacgtc

840

ctgagggagc cccacctgat ggacttcctg aacaggctca tcatctacac cggcgagtac

900

aggggcatca ggcactgggc gggccacgag gtggagtcca gcaggacggg catgatgacc

960

aacatccgct tcccgctcta cggcaccgcg gccacggccg agccaacccg cttcatcacg

1020

ccgtccacct tccccggcct gaacctcttc tacaggaccc tgtcggctcc catcttccgc

1080

gacgagccgg gcgcgaacat catcatccgc tacaggacct ccctcgtgga gggcgtcggc

1140

ttcatccagc cgaacaacgg cgagcagctg taccgcgtga ggggcacgct ggacagcctg

1200

gaccagctcc cactggaggg cgagtccagc ctcaccgagt actcgcacag gctgtgccac

1260

gtcaggttcg cccagagcct caggaacgcg gagcccctgg actacgccag ggtgcccatg

1320

ttcagctgga cccacaggtc ggctaccccc accaacacca tcgacccaga cgtgatcacg

1380

cagatcccgc tcgtcaaggc ccacaccctg cagtcgggca ccaccgtggt ccgcggccca

1440

ggcttcaccg gcggcgacat cctgaggagg acgagcggcg gccccttcgc ttacaccatc

1500

gtcaacatca acggccagct gccacagagg tacagggcgc gcatcaggta cgcctccacc

1560

acgaacctgc gcatctacgt gaccgtggcg ggcgagagga tcttcgccgg ccagttcaac

1620

aagacgatgg acaccggcga cccgctgacc ttccagtcct tcagctacgc gacgatcaac

1680

accgccttca cgttccccat gagccagtcc agcttcaccg tgggcgccga cacgttctcc

1740

agcggcaacg aggtgtacat cgaccgcttc gagctgatcc cggtgacggc gaccttcgag

1800

gccgagtacg acctggagaa ggcccagaag gcggtcaacg ccctcttcac ctccagcaac

1860

cagatcggcc tgaagacgga cgtgaccgac taccacatcg acaaggtgtc caacctcgtc

1920

gagtgcctga gcgacgagtt ctgcctcgac gagaagaggg agctgtccga gaaggtcaag

1980

cacgccaagc gcctctgcga cgagaggaac ctcctgcagg acccgaactt caggggcatc

2040

aaccgccagc cggacagggg ctggaggggc agcaccgaca tcaccatcca gggcggcgac

2100

gacgtgttca aggagaacta cgtcacgctc ccgggcacct tcgacgagtg ctaccccacg

2160

tacctgtacc agaagatcga cgagtccaag ctcaaggcct acacccgcta cgagctgagg

2220

ggctacatcg aggacagcca ggacctcgag atctacctga tccgctacaa cgcgaagcac

2280

gagacggtga acgtccccgg cacgggctcc ctgtggcccc tctcggctca gtcgccgatc

2340

ggcaagtgcg gcgagcccaa caggtgcgcc acccacctcg agtggaaccc ggacctggac

2400

tgctcctgcc gggacggcga gaagtgcgct caccactccc accacttcag cctggacatc

2460

gacgtgggct gcacggacct caacgaggac ctgggcgtgt gggtcatctt caagatcaag

2520

acgcaggacg gccacgctag gctgggcaac ctcgagttcc tggaggagaa gccgctggtg

2580

ggcgaggctc tggccagggt caagagggcg gagaagaagt ggcgcgacaa gagggagaag

2640

ctggagctgg agacgaacat cgtctacaag gaggccaaga agtccgtgga cgcgctcttc

2700

gtcaacagcc agtacgacag gctgcaggcg gacaccaaca tcgccatcat ccacgccgcg

2760

gacaagcgcg tgcactccat cagggaggcc tacctccccg agctgagcgt gatcccgggc

2820

gtcaacgctg ccatcttcga ggagctggag ggccgcatct tcaccgccta ctccctgtac

2880

gacgcgagga acgtcatcaa gaacggcgac ttcaacaacg gcctcagctg ctggaacgtg

2940

aagggccacg tggacgtcga ggagcagaac aaccaccgct cggtgctggt ggtccccgag

3000

tgggaggctg aggtcagcca ggaggtgcgc gtctgcccgg gcaggggcta catcctccgc

3060

gtgaccgcgt acaaggaggg ctacggcgag ggctgcgtca cgatccacga gatcgaggac

3120

aacaccgacg agctgaagtt ctccaactgc gtggaggagg agatctaccc gaacaacacg

3180

gtcacctgca acgactacac ggccacccag gaggagtacg agggcacgta cacgtcgagg

3240

aacagggggct acgacggcgc ttacgagtcc aacagctcgg tgccggccga ctacgctagc

3300

gcgtacgagg agaaggccta cacggacggc cgcagggaca acacctgcga gtcgaacagg

3360

ggctacggcg actacacgcc gctcccggcc ggctacgtga ccaaggagct ggagtacttc

3420

ccggagacgg acaaggtctg gatcgagatc ggcgagacgg agggcacctt catcgtggac

3480

agcgtcgagc tgctgctcat ggaggagtag

3510

<210> 12

<211> 1215

<212> PROTEIN

<213> Bacillus thuringiensis

<400> 12

Met Asn Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser

1 5 10 15

Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Pro Asp

20 25 30

Ala Arg Ile Glu Asp Ser Leu Cys Val Ala Glu Gly Asn Asn Ile Asp

35 40 45

Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Ser Ile Ala Gly

50 55 60

Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser

65 70 75 80

Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro

85 90 95

Trp Glu Ile Phe Met Glu His Val Glu Gln Ile Val Arg Gln Gln Gln

100 105 110

Ile Thr Asp Ser Val Arg Asp Thr Ala Ile Ala Arg Leu Glu Gly Leu

115 120 125

Gly Arg Gly Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp

130 135 140

Asn Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Arg Glu Arg Tyr Ile

145 150 155 160

Ala Leu Glu Leu Asp Ile Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg

165 170 175

Asn Glu Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu

180 185 190

His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly

195 200 205

Met Ser Ser Ala Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr

210 215 220

Thr Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn Thr Gly Leu

225 230 235 240

Asn Arg Leu Arg Gly Thr Thr Ala Glu Thr Trp Val Arg Tyr Asn Gln

245 250 255

Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe

260 265 270

Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Pro Thr Thr Ala Gln Leu

275 280 285

Thr Arg Glu Val Tyr Thr Asp Pro Asn Gly Val Val Ala Gly Pro Asn

290 295 300

Asn Ser Trp Phe Arg Asn Gly Ala Ser Phe Ser Ala Ile Glu Asn Ala

305 310 315 320

Ile Ile Arg Gln Pro His Leu Tyr Asp Phe Leu Thr Asn Leu Thr Ile

325 330 335

Tyr Thr Arg Arg Ser Gln Val Gly Thr Thr Ile Met Asn Leu Trp Ala

340 345 350

Gly His Arg Ile Thr Phe Asn Arg Ile Gln Gly Gly Ser Thr Ser Glu

355 360 365

Met Val Tyr Gly Ala Ile Thr Asn Pro Val Ser Val Ser Asp Ile Pro

370 375 380

Phe Val Asn Arg Asp Val Tyr Arg Thr Val Ser Leu Ala Gly Gly Leu

385 390 395 400

Gly Ser Leu Ser Gly Ile Arg Tyr Gly Leu Thr Arg Val Asp Phe Asp

405 410 415

Met Ile Phe Arg Asn His Pro Asp Ile Val Thr Gly Leu Phe Tyr His

420 425 430

Pro Gly His Ala Gly Ile Ala Thr Gln Val Lys Asp Ser Asp Thr Glu

435 440 445

Leu Pro Pro Glu Thr Thr Glu Gln Pro Asn Tyr Arg Ala Phe Ser His

450 455 460

Leu Leu Ser His Ile Ser Met Gly Pro Thr Thr Gln Asp Val Pro Pro

465 470 475 480

Val Tyr Ser Trp Thr His Gln Ser Ala Asp Arg Thr Asn Thr Ile Asn

485 490 495

Ser Asp Arg Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln

500 505 510

Ser Gly Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile

515 520 525

Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Phe Ser Asn Val Asn Leu

530 535 540

Asp Phe Asn Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser

545 550 555 560

Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe

565 570 575

Ala Gly Gln Phe Asp Lys Thr Met Asp Ala Gly Ala Pro Leu Thr Phe

580 585 590

Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Glu

595 600 605

Arg Ser Ser Ser Leu Thr Ile Gly Ala Asp Thr Phe Ser Ser Gly Asn

610 615 620

Glu Val Tyr Val Asp Arg Phe Glu Leu Ile Gln Val Thr Ala Thr Phe

625 630 635 640

Glu Ala Glu Ser Asp Leu Glu Arg Ala Arg Lys Ala Val Asn Ala Leu

645 650 655

Phe Thr Ser Thr Asn Pro Arg Gly Leu Lys Thr Asp Val Thr Asp Tyr

660 665 670

His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe

675 680 685

Cys Leu Asp Lys Lys Arg Glu Leu Leu Glu Glu Val Lys Tyr Ala Lys

690 695 700

Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Thr Phe Thr Ser

705 710 715 720

Ile Ser Gly Gln Thr Asp Arg Gly Trp Ile Gly Ser Thr Gly Ile Ser

725 730 735

Ile Gln Gly Gly Asp Asp Ile Phe Lys Glu Asn Tyr Val Arg Leu Pro

740 745 750

Gly Thr Val Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp

755 760 765

Glu Ser Gln Leu Lys Ser Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile

770 775 780

Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys

785 790 795 800

His Glu Thr Leu Ser Val Pro Gly Thr Glu Ser Pro Trp Pro Ser Ser

805 810 815

Gly Val Tyr Pro Ser Gly Arg Cys Gly Glu Pro Asn Arg Cys Ala Pro

820 825 830

Arg Ile Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Tyr Gly Glu

835 840 845

Lys Cys Val His His Ser His His Phe Ser Leu Asp Ile Asp Val Gly

850 855 860

Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile

865 870 875 880

Lys Thr Gln Asp Gly His Ala Lys Leu Gly Asn Leu Glu Phe Ile Glu

885 890 895

Glu Lys Pro Leu Leu Gly Lys Ala Leu Ser Arg Val Lys Arg Ala Glu

900 905 910

Lys Lys Trp Arg Asp Lys Tyr Glu Lys Leu Gln Leu Glu Thr Lys Arg

915 920 925

Val Tyr Thr Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asp Ser

930 935 940

Gln Tyr Asp Lys Leu Gln Ala Asn Thr Asn Ile Gly Ile Ile His Gly

945 950 955 960

Ala Asp Lys Gln Val His Arg Ile Arg Glu Pro Tyr Leu Ser Glu Leu

965 970 975

Pro Val Ile Pro Ser Ile Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly

980 985 990

His Ile Phe Lys Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys

995 1000 1005

Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly

1010 1015 1020

His Val Asp Val Gln Gln Asn His His Arg Ser Val Leu Val Leu

1025 1030 1035

Ser Glu Trp Glu Ala Glu Val Ser Gln Lys Val Arg Val Cys Pro

1040 1045 1050

Asp Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr

1055 1060 1065

Gly Glu Gly Cys Val Thr Ile His Glu Phe Glu Asp Asn Thr Asp

1070 1075 1080

Val Leu Lys Phe Arg Asn Phe Val Glu Glu Glu Val Tyr Pro Asn

1085 1090 1095

Asn Thr Val Thr Cys Asn Asp Tyr Thr Thr Asn Gln Ser Ala Glu

1100 1105 1110

Gly Ser Thr Asp Ala Cys Asn Ser Tyr Asn Arg Gly Tyr Glu Asp

1115 1120 1125

Gly Tyr Glu Asn Arg Tyr Glu Pro Asn Pro Ser Ala Pro Val Asn

1130 1135 1140

Tyr Thr Pro Thr Tyr Glu Glu Gly Met Tyr Thr Asp Thr Gln Gly

1145 1150 1155

Tyr Asn His Cys Val Ser Asp Arg Gly Tyr Arg Asn His Thr Pro

1160 1165 1170

Leu Pro Ala Gly Tyr Val Thr Leu Glu Leu Glu Tyr Phe Pro Glu

1175 1180 1185

Thr Glu Gln Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe

1190 1195 1200

Ile Val Gly Ser Val Glu Leu Leu Leu Met Glu Glu

1205 1210 1215

<210> 13

<211> 1169

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Constructed chimeric protein BT29-Cry1Ka1.

<400> 13

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Phe Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asp Lys Thr Met Asp

530 535 540

Ala Gly Ala Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Glu Arg Ser Ser Ser Leu Thr Ile Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Gln Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser

645 650 655

Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu

660 665 670

Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp

675 680 685

Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys

690 695 700

Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr

705 710 715 720

Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg

725 730 735

Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr

740 745 750

Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr

755 760 765

Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly

770 775 780

Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp

785 790 795 800

Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe

805 810 815

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly

820 825 830

Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu

835 840 845

Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu

850 855 860

Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys

865 870 875 880

Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val

885 890 895

Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr

900 905 910

Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg

915 920 925

Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala

930 935 940

Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr

945 950 955 960

Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser

965 970 975

Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His

980 985 990

Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

995 1000 1005

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1010 1015 1020

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1025 1030 1035

Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1040 1045 1050

Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1055 1060 1065

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1070 1075 1080

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1085 1090 1095

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1100 1105 1110

Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1115 1120 1125

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1130 1135 1140

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1145 1150 1155

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 14

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Coding sequence BT29-Cry1Ka1.

<400> 14

atggagatta ataatcagaa ccaatgtgtc ccttataatt gtttgaataa tcctgaaagc

60

gagatattaa acgttgcaat ttttagtagc gaacaggtag cagaaattca cttaaagatc

120

acgcgcttaa ttttagagaa ttttttacca ggtgggagtt ttgcattcgg cttatttgat

180

ttaatatggg ggatttttaa tgaagatcaa tggagcgcat ttcttcggca ggtagaagaa

240

ttaattaatc aaaggataac ggaattcgca agagggcaag caattcagag actagtaggg

300

tttggaagga gttatgatga atatatttta gcactaaaag aatgggaaaa cgatcctgat

360

aacccagctt caaaggaaag agtgcgcact cgatttcgga caactgatga tgccttgcta

420

accggtgttc ctcttatggc aattccaggt tttgaattag ctactttatc tgtttatgct

480

caatcagcca atctacattt agccctatta agagatgctg tattttttgg ggagagatgg

540

ggattgacac aaacaaatat aaatgattta tatagtagat taaaaaactc cattcgtgat

600

tatacaaatc attgtgttcg tttttataat ataggtttag ggaatttaaa tgttataaga

660

ccagagtatt accgtttcca aagagaatta acaatatctg tcttagatct tgtagctctt

720

tttccaaatt acgatatccg aacatatcca ataccaacta aaagtcaatt aacaagagaa

780

atttatacag atccgattat ttcacctggt gcacaggcag gttatactct tcaagatgtt

840

ttgagagaac cacaccttat ggacttttta aaccgactta ttatttatac tggtgagtat

900

cgcggaattc gtcactgggc aggacatgaa gtagaatcta gtagaacagg tatgatgact

960

aatataagat ttcctttgta tggaacagcc gcaacagcag aaccaacacg atttataact

1020

cctagtactt ttcctggtct taatttattt tatagaacat tatcagctcc tatttttaga

1080

gatgaaccgg gagctaatat tattattaga tatagaacga gtttggtgga aggagtagga

1140

tttattcaac caaataacgg tgaacagctt tacagagtga gaggaacatt agattctctt

1200

gatcaattac cacttgaggg tgagagtagt ctaactgaat atagtcatcg attatgccat

1260

gttagatttg cgcaatcatt gaggaatgca gaacctttag attatgcaag ggttccgatg

1320

ttttcttgga cacatcgtag tgcaacccct acaaatacaa ttgatccaga tgtcatcacc

1380

caaataccgt tagtaaaggc gcataccctc caatcgggta caactgtagt aaaagggcca

1440

gggtttacag gaggggatat cctccgtcga acaagtggag gaccatttgc ttttagtaat

1500

gttaatctag attttaactt gtcacaaagg tatcgtgcta gaattcgtta tgcctctact

1560

actaacctaa gaatttacgt aacggttgca ggtgaacgaa tttttgctgg tcaatttgac

1620

aaaacgatgg atgctggtgc cccattaaca ttccaatctt ttagttacgc aactattaat

1680

acagctttta cattcccaga aagatcgagc agcttgacta taggtgccga tacgtttagt

1740

tcaggtaatg aagtttatgt agatagattt gaattaatcc aagttactgc aacatttgag

1800

gcagaatatg atttagagaa agctcagaaa gcggtgaatg cgctgtttac ttcttccaat

1860

caaatcgggt taaaaacaga tgtgacggac tatcatattg ataaagtatc caatctagtt

1920

gagtgtttat cagatgaatt ttgtctagat gaaaagcgag aattgtccga gaaagtcaaa

1980

catgcgaagc gactctgtga tgagcggaat ttacttcaag atccaaactt cagaggcatc

2040

aatagacaac cagaccgtgg ttggagagga agtacggata ttaccatcca aggaggat

2100

gacgtattca aagagaatta cgttacgcta ccgggtacct ttgatgagtg ctatccaacg

2160

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga

2220

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacat

2280

gaaacagtaa atgtgccagg tacgggttcc ttatggccgc tttcagccca aagtccaatc

2340

ggaaagtgtg gagaaccgaa tcgatgtgcg acacaccttg aatggaatcc tgacttagat

2400

tgttcgtgta gggatggaga aaagtgtgcc catcattcgc atcatttctc cttagacatt

2460

gatgtaggat gtacagacct aaatgaggac ctaggtgtat gggtgatctt taagattaag

2520

acgcaagatg gtcatgcgag actaggaaat ctagaatttc tcgaagagaa accattagta

2580

ggagaagcgc tagctcgtgt gaagagagcg gagaaaaaat ggagagacaa acgcgaaaaa

2640

ttggaattgg aaacaaatat tgtttataaa gaggcaaaaa aatctgtaga tgctttattt

2700

gtgaactctc aatatgatag attacaagcg gatacgaata tcgcgataat tcatgcggca

2760

gataaacgcg ttcatagcat tcgagaagca tatcttccag agttgtctgt aattccgggt

2820

gtaaatgcag ctatttttga agaattagag ggacgtattt tcacagccta ctctctatat

2880

gatgcgagaa atgtcattaa aaatggcgat ttcaataatg gcttatcatg ctggaacgtg

2940

aaagggcatg tagatgtaga agaacagaac aaccatcgtt cggtccttgt tgttccagaa

3000

tgggaagcag aagtgtcaca agaggttcgt gtctgtccag gtcgtggcta tatccttcgt

3060

gttacagcgt acaaagaggg atatggagag ggctgtgtaa cgattcatga gatcgaagac

3120

aatacagacg aactgaaatt cagcaactgt gtagaagagg aaatatatcc aaacaacacg

3180

gtaacgtgta atgattatac tgcgactcaa gaagaatatg agggtacgta cacttctcgt

3240

aatcgaggat atgacggagc ctatgaaagc aattcttctg taccagctga ttatgcatca

3300

gcctatgaag aaaaagcgta tacagatgga agaagagaca atacttgtga atctaacaga

3360

ggatatgggg attacacacc actaccagct ggctatgtga caaaagaatt agagtacttc

3420

ccagaaaccg ataaggtatg gattgagatt ggagaaacgg aaggaacatt tatcgtggac

3480

agcgtggaat tactccttat ggaggaatag

3510

<210> 15

<211> 1169

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Chimeric protein Bt29-1Kav2

<400> 15

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Phe Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asp Lys Thr Met Asp

530 535 540

Ala Gly Ala Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Glu Arg Ser Ser Ser Leu Thr Ile Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Gln Val Thr Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala

595 600 605

Arg Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser

645 650 655

Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu

660 665 670

Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp

675 680 685

Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys

690 695 700

Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr

705 710 715 720

Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg

725 730 735

Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr

740 745 750

Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr

755 760 765

Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly

770 775 780

Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp

785 790 795 800

Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe

805 810 815

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly

820 825 830

Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu

835 840 845

Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu

850 855 860

Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys

865 870 875 880

Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val

885 890 895

Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr

900 905 910

Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg

915 920 925

Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala

930 935 940

Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr

945 950 955 960

Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser

965 970 975

Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His

980 985 990

Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

995 1000 1005

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1010 1015 1020

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1025 1030 1035

Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1040 1045 1050

Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1055 1060 1065

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1070 1075 1080

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1085 1090 1095

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1100 1105 1110

Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1115 1120 1125

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1130 1135 1140

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1145 1150 1155

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1160 1165

<210> 16

<211> 3510

<212> DNA

<213> Artificial sequence

<220>

<223> Chimera Bt29-1Ka

<400> 16

atggagatta ataatcagaa ccaatgtgtc ccttataatt gtttgaataa tcctgaaagc

60

gagatattaa acgttgcaat ttttagtagc gaacaggtag cagaaattca cttaaagatc

120

acgcgcttaa ttttagagaa ttttttacca ggtgggagtt ttgcattcgg cttatttgat

180

ttaatatggg ggatttttaa tgaagatcaa tggagcgcat ttcttcggca ggtagaagaa

240

ttaattaatc aaaggataac ggaattcgca agagggcaag caattcagag actagtaggg

300

tttggaagga gttatgatga atatatttta gcactaaaag aatgggaaaa cgatcctgat

360

aacccagctt caaaggaaag agtgcgcact cgatttcgga caactgatga tgccttgcta

420

accggtgttc ctcttatggc aattccaggt tttgaattag ctactttatc tgtttatgct

480

caatcagcca atctacattt agccctatta agagatgctg tattttttgg ggagagatgg

540

ggattgacac aaacaaatat aaatgattta tatagtagat taaaaaactc cattcgtgat

600

tatacaaatc attgtgttcg tttttataat ataggtttag ggaatttaaa tgttataaga

660

ccagagtatt accgtttcca aagagaatta acaatatctg tcttagatct tgtagctctt

720

tttccaaatt acgatatccg aacatatcca ataccaacta aaagtcaatt aacaagagaa

780

atttatacag atccgattat ttcacctggt gcacaggcag gttatactct tcaagatgtt

840

ttgagagaac cacaccttat ggacttttta aaccgactta ttatttatac tggtgagtat

900

cgcggaattc gtcactgggc aggacatgaa gtagaatcta gtagaacagg tatgatgact

960

aatataagat ttcctttgta tggaacagcc gcaacagcag aaccaacacg atttataact

1020

cctagtactt ttcctggtct taatttattt tatagaacat tatcagctcc tatttttaga

1080

gatgaaccgg gagctaatat tattattaga tatagaacga gtttggtgga aggagtagga

1140

tttattcaac caaataacgg tgaacagctt tacagagtga gaggaacatt agattctctt

1200

gatcaattac cacttgaggg tgagagtagt ctaactgaat atagtcatcg attatgccat

1260

gttagatttg cgcaatcatt gaggaatgca gaacctttag attatgcaag ggttccgatg

1320

ttttcttgga cacatcgtag tgcaacccct acaaatacaa ttgatccaga tgtcatcacc

1380

caaataccgt tagtaaaagc acataccctt cagtcaggta ctactgttgt aaaagggcca

1440

gggtttacag gtggagatat cctccgacga actagtggag gaccatttgc ttttagtaat

1500

gttaatttag actttaactt gtcacaaaga tatcgtgcta gaatacgcta tgcttctact

1560

actaatctaa gaatttacgt aacggtagca ggggaacgaa tttttgctgg tcaatttgat

1620

aaaacaatgg atgcaggtgc accattaaca ttccaatctt ttagttacgc aactattaat

1680

acagcattta cattcccaga aagaagtagc agcttgacta ttggtgctga tacttttagc

1740

tcaggtaatg aagtttatgt agatagattt gaattgatcc aggttactgc aacatttgag

1800

gcagaatcag atttagagag agcacgaaaa gcggtgaatg cgctgtttac ttcttccaat

1860

caaatcgggt taaaaacaga tgtgacggac tatcatattg ataaagtatc caatctagtt

1920

gagtgtttat cagatgaatt ttgtctagat gaaaagcgag aattgtccga gaaagtcaaa

1980

catgcgaagc gactctgtga tgagcggaat ttacttcaag atccaaactt cagaggcatc

2040

aatagacaac cagaccgtgg ttggagagga agtacggata ttaccatcca aggaggat

2100

gacgtattca aagagaatta cgttacgcta ccgggtacct ttgatgagtg ctatccaacg

2160

tatttatatc aaaaaataga tgagtcgaaa ttaaaagcct atacccgcta tgaattaaga

2220

gggtatatcg aggatagtca agacttagaa atctatttaa ttcgctacaa tgcaaaacat

2280

gaaacagtaa atgtgccagg tacgggttcc ttatggccgc tttcagccca aagtccaatc

2340

ggaaagtgtg gagaaccgaa tcgatgtgcg acacaccttg aatggaatcc tgacttagat

2400

tgttcgtgta gggatggaga aaagtgtgcc catcattcgc atcatttctc cttagacatt

2460

gatgtaggat gtacagacct aaatgaggac ctaggtgtat gggtgatctt taagattaag

2520

acgcaagatg gtcatgcgag actaggaaat ctagaatttc tcgaagagaa accattagta

2580

ggagaagcgc tagctcgtgt gaagagagcg gagaaaaaat ggagagacaa acgcgaaaaa

2640

ttggaattgg aaacaaatat tgtttataaa gaggcaaaaa aatctgtaga tgctttattt

2700

gtgaactctc aatatgatag attacaagcg gatacgaata tcgcgataat tcatgcggca

2760

gataaacgcg ttcatagcat tcgagaagca tatcttccag agttgtctgt aattccgggt

2820

gtaaatgcag ctatttttga agaattagag ggacgtattt tcacagccta ctctctatat

2880

gatgcgagaa atgtcattaa aaatggcgat ttcaataatg gcttatcatg ctggaacgtg

2940

aaagggcatg tagatgtaga agaacagaac aaccatcgtt cggtccttgt tgttccagaa

3000

tgggaagcag aagtgtcaca agaggttcgt gtctgtccag gtcgtggcta tatccttcgt

3060

gttacagcgt acaaagaggg atatggagag ggctgtgtaa cgattcatga gatcgaagac

3120

aatacagacg aactgaaatt cagcaactgt gtagaagagg aaatatatcc aaacaacacg

3180

gtaacgtgta atgattatac tgcgactcaa gaagaatatg agggtacgta cacttctcgt

3240

aatcgaggat atgacggagc ctatgaaagc aattcttctg taccagctga ttatgcatca

3300

gcctatgaag aaaaagcgta tacagatgga agaagagaca atacttgtga atctaacaga

3360

ggatatgggg attacacacc actaccagct ggctatgtga caaaagaatt agagtacttc

3420

ccagaaaccg ataaggtatg gattgagatt ggagaaacgg aaggaacatt tatcgtggac

3480

agcgtggaat tactccttat ggaggaatag

3510

<210> 17

<211> 1189

<212> PROTEIN

<213> Bacillus thuringiensis

<400> 17

Met Glu Glu Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser

1 5 10 15

Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn

20 25 30

Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn

35 40 45

Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp

50 55 60

Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu

65 70 75 80

Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile

85 90 95

Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala

100 105 110

Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Ala Thr Arg Thr Arg

115 120 125

Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile

130 135 140

Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr

145 150 155 160

Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile

165 170 175

Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr

180 185 190

Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn

195 200 205

Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln Asp

210 215 220

Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu

225 230 235 240

Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile

245 250 255

Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile

260 265 270

Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe Asn

275 280 285

Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu

290 295 300

Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe

305 310 315 320

Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn

325 330 335

Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro Arg

340 345 350

Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr

355 360 365

Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg

370 375 380

Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr

385 390 395 400

Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp

405 410 415

Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala

420 425 430

Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val

435 440 445

Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro

450 455 460

Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly

465 470 475 480

Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu

485 490 495

Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile Asn

500 505 510

Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser

515 520 525

Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val

530 535 540

Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu Ile

545 550 555 560

Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn

565 570 575

Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln

580 585 590

Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp

595 600 605

Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp

610 615 620

Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn

625 630 635 640

Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val

645 650 655

Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys

660 665 670

Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu

675 680 685

Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro

690 695 700

Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp

705 710 715 720

Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Val Asp Glu

725 730 735

Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys

740 745 750

Ala Tyr Thr Arg Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp

755 760 765

Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Ile Val Asn

770 775 780

Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile

785 790 795 800

Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn

805 810 815

Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His

820 825 830

Ser His His Phe Thr Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn

835 840 845

Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly

850 855 860

His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu

865 870 875 880

Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp

885 890 895

Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala

900 905 910

Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu

915 920 925

Gln Val Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val

930 935 940

His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly

945 950 955 960

Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala

965 970 975

Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn

980 985 990

Asn Gly Leu Leu Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu

995 1000 1005

Gln Asn Asn His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala

1010 1015 1020

Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile

1025 1030 1035

Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val

1040 1045 1050

Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser

1055 1060 1065

Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys

1070 1075 1080

Asn Asn Tyr Thr Gly Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr

1085 1090 1095

Ser Arg Asn Gln Gly Tyr Asp Glu Ala Tyr Gly Asn Asn Pro Ser

1100 1105 1110

Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr

1115 1120 1125

Asp Gly Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly

1130 1135 1140

Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Asp Leu Glu

1145 1150 1155

Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr

1160 1165 1170

Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu

1175 1180 1185

Glu

<210> 18

<211> 1181

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Constructed chimeric protein Bt29-1Ca.

<400> 18

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe

485 490 495

Val Ser Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg

500 505 510

Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu

515 520 525

Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met

530 535 540

Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr

545 550 555 560

Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro

565 570 575

Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile

580 585 590

Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp

595 600 605

Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala Gln Lys Ala Val

610 615 620

Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val

625 630 635 640

Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val Glu Cys Leu Ser

645 650 655

Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys

660 665 670

His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn

675 680 685

Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr

690 695 700

Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val

705 710 715 720

Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln

725 730 735

Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Glu Leu Arg

740 745 750

Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr

755 760 765

Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp

770 775 780

Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg

785 790 795 800

Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg

805 810 815

Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile

820 825 830

Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile

835 840 845

Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu

850 855 860

Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys

865 870 875 880

Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Leu Glu

885 890 895

Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val Asp Ala Leu Phe

900 905 910

Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Ile

915 920 925

Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu

930 935 940

Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu

945 950 955 960

Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn

965 970 975

Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val

980 985 990

Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu

995 1000 1005

Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val

1010 1015 1020

Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu

1025 1030 1035

Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp Asn

1040 1045 1050

Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr

1055 1060 1065

Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu

1070 1075 1080

Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly

1085 1090 1095

Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala

1100 1105 1110

Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Thr Cys

1115 1120 1125

Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly

1130 1135 1140

Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val

1145 1150 1155

Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser

1160 1165 1170

Val Glu Leu Leu Leu Met Glu Glu

1175 1180

<210> 19

<211> 3546

<212> DNA

<213> Artificial sequence

<220>

<223> Chimera Bt29-1Ca

<400> 19

atggagatta ataatcagaa ccaatgtgtc ccttataatt gtttgaataa tcctgaaagc

60

gagatattaa acgttgcaat ttttagtagc gaacaggtag cagaaattca cttaaagatc

120

acgcgcttaa ttttagagaa ttttttacca ggtgggagtt ttgcattcgg cttatttgat

180

ttaatatggg ggatttttaa tgaagatcaa tggagcgcat ttcttcggca ggtagaagaa

240

ttaattaatc aaaggataac ggaattcgca agagggcaag caattcagag actagtaggg

300

tttggaagga gttatgatga atatatttta gcactaaaag aatgggaaaa cgatcctgat

360

aacccagctt caaaggaaag agtgcgcact cgatttcgga caactgatga tgccttgcta

420

accggtgttc ctcttatggc aattccaggt tttgaattag ctactttatc tgtttatgct

480

caatcagcca atctacattt agccctatta agagatgctg tattttttgg ggagagatgg

540

ggattgacac aaacaaatat aaatgattta tatagtagat taaaaaactc cattcgtgat

600

tatacaaatc attgtgttcg tttttataat ataggtttag ggaatttaaa tgttataaga

660

ccagagtatt accgtttcca aagagaatta acaatatctg tcttagatct tgtagctctt

720

tttccaaatt acgatatccg aacatatcca ataccaacta aaagtcaatt aacaagagaa

780

atttatacag atccgattat ttcacctggt gcacaggcag gttatactct tcaagatgtt

840

ttgagagaac cacaccttat ggacttttta aaccgactta ttatttatac tggtgagtat

900

cgcggaattc gtcactgggc aggacatgaa gtagaatcta gtagaacagg tatgatgact

960

aatataagat ttcctttgta tggaacagcc gcaacagcag aaccaacacg atttataact

1020

cctagtactt ttcctggtct taatttattt tatagaacat tatcagctcc tatttttaga

1080

gatgaaccgg gagctaatat tattattaga tatagaacga gtttggtgga aggagtagga

1140

tttattcaac caaataacgg tgaacagctt tacagagtga gaggaacatt agattctctt

1200

gatcaattac cacttgaggg tgagagtagt ctaactgaat atagtcatcg attatgccat

1260

gttagatttg cgcaatcatt gaggaatgca gaacctttag attatgcaag ggttccgatg

1320

ttttcttgga cacatcgtag tgcaacccct acaaatacaa ttgatccaga tgtcatcacc

1380

caaataccgc tggtgaaagg ctttcgcgtg tggggcggca ccagcgtgat taccggcccg

1440

ggctttaccg gcggcgatat tctgcgccgc aacacctttg gcgattttgt gagcctgcag

1500

gtgaacatta acagcccgat tacccagcgc tatcgcctgc gctttcgcta tgcgagcagc

1560

cgcgatgcgc gcgtgattgt gctgaccggc gcggcgagca ccggcgtggg cggccaggtg

1620

agcgtgaaca tgccgctgca gaaaaccatg gaaattggcg aaaacctgac cagccgcacc

1680

tttcgctata ccgattttag caacccgttt agctttcgcg cgaacccgga tattattggc

1740

attagcgaac agccgctgtt tggcgcgggc agcattagca gcggcgaact gtatattgat

1800

aaaattgaaa ttattctggc ggatgcgaca tttgaggcag aatatgattt agagaaagct

1860

cagaaagcgg tgaatgcgct gtttacttct tccaatcaaa tcgggttaaa aacagatgtg

1920

acggactatc atattgataa agtatccaat ctagttgagt gtttatcaga tgaattttgt

1980

ctagatgaaa agcgagaatt gtccgagaaa gtcaaacatg cgaagcgact ctgtgatgag

2040

cggaatttac ttcaagatcc aaacttcaga ggcatcaata gacaaccaga ccgtggttgg

2100

agaggaagta cggatattac catccaagga ggagatgacg tattcaaaga gaattacgtt

2160

acgctaccgg gtacctttga tgagtgctat ccaacgtatt tatatcaaaa aatagatgag

2220

tcgaaattaa aagcctatac ccgctatgaa ttaagagggt atatcgagga tagtcaagac

2280

ttagaaatct atttaattcg ctacaatgca aaacatgaaa cagtaaatgt gccaggtacg

2340

ggttccttat ggccgctttc agcccaaagt ccaatcggaa agtgtggaga accgaatcga

2400

tgtgcgacac accttgaatg gaatcctgac ttagattgtt cgtgtaggga tggagaaaag

2460

tgtgcccatc attcgcatca tttctcctta gacattgatg taggatgtac agacctaaat

2520

gaggacctag gtgtatgggt gatctttaag attaagacgc aagatggtca tgcgagacta

2580

ggaaatctag aatttctcga agagaaacca ttagtaggag aagcgctagc tcgtgtgaag

2640

agagcggaga aaaaatggag agacaaacgc gaaaaattgg aattggaaac aaatattgtt

2700

tataaagagg caaaaaaatc tgtagatgct ttatttgtga actctcaata tgatagatta

2760

caagcggata cgaatatcgc gataattcat gcggcagata aacgcgttca tagcattcga

2820

gaagcatatc ttccagagtt gtctgtaatt ccgggtgtaa atgcagctat ttttgaagaa

2880

ttagagggac gtattttcac agcctactct ctatatgatg cgagaaatgt cattaaaaat

2940

ggcgatttca ataatggctt atcatgctgg aacgtgaaag ggcatgtaga tgtagaagaa

3000

cagaacaacc atcgttcggt ccttgttgtt ccagaatggg aagcagaagt gtcacaagag

3060

gttcgtgtct gtccaggtcg tggctatatc cttcgtgtta cagcgtacaa agagggatat

3120

ggagaggggct gtgtaacgat tcatgagatc gaagacaata cagacgaact gaaattcagc

3180

aactgtgtag aagaggaaat atatccaaac aacacggtaa cgtgtaatga ttatactgcg

3240

actcaagaag aatatgagg tacgtacact tctcgtaatc gaggatatga cggagcctat

3300

gaaagcaatt cttctgtacc agctgattat gcatcagcct atgaagaaaa agcgtataca

3360

gatggaagaa gagacaatac ttgtgaatct aacagaggat atggggatta cacaccacta

3420

ccagctggct atgtgacaaa agaattagag tacttcccag aaaccgataa ggtatggatt

3480

gagattggag aaacggaagg aacatttatc gtggacagcg tggaattact ccttatggag

3540

gaatag

3546

<210> 20

<211> 652

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr1

<400> 20

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val

625 630 635 640

Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys

645 650

<210> 21

<211> 636

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr2

<400> 21

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu

610 615 620

Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val

625 630 635

<210> 22

<211> 622

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr3

<400> 22

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile

610 615 620

<210> 23

<211> 610

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr4

<400> 23

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala

595 600 605

Gln Lys

610

<210> 24

<211> 607

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr5

<400> 24

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys

595 600 605

<210> 25

<211> 603

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-Bt22Tr6

<400> 25

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr

595 600

<210> 26

<211> 676

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v1

<400> 26

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

116

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu Ala Val Thr Asp Leu Glu Arg Ala

595 600 605

Gln Lys Ala Val His Glu Leu Phe Thr Ser Thr Asn Pro Gly Gly Leu

610 615 620

Lys Thr Asp Val Ala Lys Asp His Tyr Thr Asn Thr Ile Ser Lys Ser

625 630 635 640

Val Gln Ser Val Phe Arg Cys Arg Cys Ser Glu Arg Thr Arg Ile Tyr

645 650 655

Arg Trp Gly Tyr Pro Ser Lys Lys Glu Tyr Trp Tyr Ile Trp Gly Tyr

660 665 670

Thr Ser Lys Tyr

675

<210> 27

<211> 644

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v2

<400> 27

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu Ala Val Thr Asp Leu Glu Arg Ala

595 600 605

Gln Lys Ala Val His Glu Leu Phe Thr Ser Thr Asn Pro Gly Gly Leu

610 615 620

Lys Thr Asp Val Ala Lys Asp His Tyr Thr Asn Thr Ile Ser Lys Ser

625 630 635 640

Val Gln Ser Val

<210> 28

<211> 634

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v3

<400> 28

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu Ala Val Thr Asp Leu Glu Arg Ala

595 600 605

Gln Lys Ala Val His Glu Leu Phe Thr Ser Thr Asn Pro Gly Gly Leu

610 615 620

Lys Thr Asp Val Ala Lys Asp His Tyr Thr

625 630

<210> 29

<211> 622

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v4

<400> 29

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu Ala Val Thr Asp Leu Glu Arg Ala

595 600 605

Gln Lys Ala Val His Glu Leu Phe Thr Ser Thr Asn Pro Gly

610 615 620

<210> 30

<211> 610

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v5

<400> 30

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu Ala Val Thr Asp Leu Glu Arg Ala

595 600 605

Gln Lys

610

<210> 31

<211> 600

<212> PROTEIN

<213> Artificial sequence

<220>

<223>BT29BT22-TL22v6

<400> 31

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr

515 520 525

Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp

545 550 555 560

Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Leu Glu

595 600

<210> 32

<211> 610

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-1FaTr1

<400> 32

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala

595 600 605

Gln Lys

610

<210> 33

<211> 607

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-1FaTr2

<400> 33

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg

595 600 605

<210> 34

<211> 603

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-1FaTr3

<400> 34

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr

595 600

<210> 35

<211> 600

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Bt29-1FaTr4

<400> 35

Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln

20 25 30

Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe

35 40 45

Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu

65 70 75 80

Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln

85 90 95

Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu

100 105 110

Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val

115 120 125

Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro

130 135 140

Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala

145 150 155 160

Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe

165 170 175

Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser

180 185 190

Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe

195 200 205

Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr

210 215 220

Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu

225 230 235 240

Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln

245 250 255

Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln

260 265 270

Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp

275 280 285

Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg

290 295 300

His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr

305 310 315 320

Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr

325 330 335

Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg

340 345 350

Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile

355 360 365

Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro

370 375 380

Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu

385 390 395 400

Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His

405 410 415

Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro

420 425 430

Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala

435 440 445

Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu

450 455 460

Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro

465 470 475 480

Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe

485 490 495

Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg

500 505 510

Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr

515 520 525

Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp

530 535 540

Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn

545 550 555 560

Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala

565 570 575

Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu

580 585 590

Ile Pro Val Thr Ala Thr Phe Glu

595 600

<---

Claims (15)

1. A chimeric insecticidal protein toxic to a lepidopteran insect pest, containing any of the sequences of SEQ ID NO: 26-31. 2. The chimeric insecticidal protein according to claim 1, where the chimeric insecticidal protein is characterized by insecticidal activity against the insect pest Spodoptera frugiperda with resistance to the Vip3A protein or the Cry1F protein. 3. A polynucleotide encoding a chimeric insecticidal protein according to claim 1, containing a nucleotide sequence encoding a chimeric insecticidal protein according to claim 1. 4. An expression cassette providing expression of the chimeric insecticidal protein according to claim 1, containing the polynucleotide according to claim 3, operably linked to a heterologous promoter. 5. The expression vector of the chimeric insecticidal protein according to claim 1, containing the expression cassette according to claim 4. 6. A transgenic cell expressing the protein of claim 1 and containing the polynucleotide of claim 3, where the transgenic cell is a bacterial cell or a plant cell. 7. Transgenic cell according to claim 6, which is a plant cell and is: (a) a monocot cell, optionally a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell or a wheat cell; or (b) a dicotyledonous plant cell, optionally a soybean cell, a sunflower cell, a tomato cell, a cabbage cell, a cotton cell, a sugar beet cell or a tobacco cell. 8. A transgenic plant that is toxic to lepidopteran insect pests, containing a transgenic plant cell according to claim 6, in which the plant is: (a) a monocotyledonous plant, optionally a barley plant, a maize plant, an oat plant, a rice plant, a sorghum plant, a sugarcane plant or a wheat plant; or (b) a dicotyledonous plant, optionally a soybean plant, a sunflower plant, a tomato plant, a cultivated cabbage plant, a cotton plant, a sugar beet plant or a tobacco plant. 9. The transgenic plant of claim 8, wherein the transgenic plant further comprises a nucleotide sequence encoding a second insect control agent. 10. A transgenic plant part that is toxic to a lepidopteran insect pest from a transgenic plant according to claim 8, where the transgenic plant part contains a chimeric insecticidal protein according to claim 1 or 2. 11. Transgenic seed, toxic to lepidopteran insect pests, from the transgenic plant according to claim 8, where the seed contains a chimeric insecticidal protein according to claim 1 or 2.

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