CHIMERIC INSECTICIDAL PROTEIN AND GENE CODING THEREFORE

Abstract: The present invention describes the design and construction of a chimeric insecticidal protein by joining the 5' portion of a synthetic maize optimized cry1B gene (SFLIB) to the 3' end of a full-length synthetic maize optimized cry1A(b) gene to generate a full-length hybrid cry1B gene (hyFLIB). When the chimeric insecticidal protein gene is expressed in transgenic maize from both PEPC and pith promoters, insecticidal activity is observed in transgenic maize tissue against European corn borer (Ostrinia nubilalis). An additional aspect of the invention is recombinant, biologically pure microbial strains transformed with the hyFLIB gene which can be used in entomocidal formulations for the control of Lepidopteran insects. Yet another aspect of the invention is plants transformed with the toxin gene or active fragments thereof, particularly where the transforming sequences have been optimized for expression in maize.

Description: CHIMERIC INSECTICIDAL PROTEIN AND GENE CODING THEREFORE Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore- forming bacteria. Unlike other very closely related species of Bacillus such as B. cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, on account of its crystalline structure, is generally referred to also as a crystalline body. This crystalline body is composed of insecticidally active crystalline protoxin proteins, the so-called δ-endotoxins. These protein crystals are responsible for the toxicity to insects of Bacillus thuringiensis. The δ-endotoxin does not exhibit its insecticidal activity until after oral intake of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects. The δ-endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity with respect to certain target insects, especially with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against these larvae. A further advantage in using δ-endotoxins of Bacillus thuringiensis resides in the fact that the toxins are harmless to humans, other mammals, birds and fish. With the introduction of genetic engineering and the new possibilities resulting from it, the field of Bacillus thuringiensis toxins has received a fresh impetus. For example, it is known that many naturally-occurring strains possess more than one insect toxin protein, which may account for a wide spectrum of insecticidal activity of those strains. However, with the ability to genetically transform Bacillus it is possible to create recombinant strains which may contain a chosen array of insect toxin genes obtained by isolation and cloning from naturally-occurring sources. Such recombinant strains can be made to provide whatever spectrum of insecticidal activity might be desired for a particular application, based upon a knowledge of the insecticidal activity of individual toxin proteins. Furthermore, it is also possible to create recombinant toxin proteins which have a chosen combination of functions designed to enhance the degree of insecticidal activity against a particular insect or insect class, or to expand the spectrum of insects against which the toxin protein is active. The various naturally-occurring insecticidal crystal proteins from Bacillus thuringiensis have been classified based upon their spectrum of activity and sequence similarity. The - 2 - classification put forth by Hofte and Whiteley, Microbiol. Rev. 53: 242-255 (1989) placed the then known insecticidal crystal proteins into four major classes. Generally, the major classes are defined by the spectrum of activity, with the Cryl proteins active against Lepidoptera, Cryll proteins active against both Lepidoptera and Diptera, Crylll proteins active against Coleoptera, and CrylV proteins active against Diptera. Within each major class, the δ-endotoxins are grouped according to sequence similarity. The Cryl proteins are typically produced as 130-140 kDa protoxin proteins which are proteolytically cleaved to produce active toxin proteins about 60-70 kDa. The active portion of the δ-endotoxin resides in the NH 2 -terminal portion of the full-length molecule. Hόfte and Whiteley, supra, classified the then known Cryl proteins into six groups, IA(a), IA(b), IA(c), IB, IC, and ID. Since then, proteins classified as CrylE, CrylF, CrylG, and CrylX have also been characterized. It is well known that many δ-endotoxin proteins from Bacillus thuringiensis are actually expressed as protoxins. To be insecticidal, these protoxins must first be ingested by the insect, solubilized in the alkaline environment of the insect gut and are proteolytically converted by proteases into a toxic core fragment (Hofte and Whiteley, Microbiol. Rev. 53: 242-255 (1989)). For δ-endotoxin proteins of the Cryl class, the toxic core fragment is localized in the N-terminal half of the protoxin, whereas the C-terminal portion of the protoxin is proteolytically cleaved to form an active toxin. Chimeric insecticidal proteins can be constructed having novel sequences not found in nature by combining the toxin portion from one δ-endotoxin with the protoxin portion of a different δ-endotoxin. The invention thus provides: - synthetic insecticidal proteins and their genes which have been optimized for expression in plants and - methods of protecting plants against insect pests comprising expressing in said plant a nucleotide sequence to produce said anti-insect protein or applying said anti-insect protein to plants In particular we provide herein DNA molecules and proteins as mentioned before, wherein • the DNA molecule comprises a nucleotide sequence encoding a chimeric insecticidal protein of 1200 to 1300 amino acids, wherein said insecticidal protein comprises a crylB core N-terminal toxin portion of 600 to 1000 amino acids and a ylA(b) C-terminal protoxin portion of 300 to 600 amino acids - 3 - • said insecticidal protein comprises a cry B core N-terminal toxin portion comprising about two thirds of the amino acids of the protein and a crylA(b) C-terminal protoxin portion comprising about one third of the amino acids of the protein • wherein said nucleotide sequence encodes a chimeric insecticidal protein of 1227 amino acids, wherein said insecticidal protein comprises a c/ylB core N-terminal toxin portion of 845 amino acids and a c/ylA(b) C-terminal protoxin portion of 382 amino acids, as set forth in SEQ ID NO:1 • the DNA molecule comprises additionally a promoter operably linked to the nucleotide sequence encoding the chimeric insecticidal protein • said promoter is selected from the group consisting of a PEPC promoter, pith promoter, MTL promoter, and ubiquitin promoter • the insecticidal protein set forth in SEQ ID NO:2. The invention further provides methods of protecting plants against insect pests comprising • expressing in said plant a nucleotide sequence as mentioned before to produce an anti- insect protein • applying to said plant the insecticidal protein mentioned herein before In particular, the invention provides a method to protect plants against insect pests, wherein the insect pest is caused by the European corn borer. The invention further provides transgenic plants expressing the DNA molecules as mentioned before to protect plants against insect pests. In particular, the invention provides plants as mentioned hereinbefore, wherein the plant is a maize plant. The invention also provides recombinant hosts transformed to express a chimeric insecticidal protein comprising a nucleotide sequence as mentioned hereinbefore wherein • the recombinant host is selected from bacteria, algae, and fungi • the recombinant host is selected from bacteria known to occupy the phytosphere. • the recombinant host is Bacillus thuringiensis. The invention further provides entomocidal composition for the control of Lepidopteran insects comprising a recombinant host or the insecticidal protein of as mentioned before or a combination thereof in an insecticidally effective amount together with an agriculturally acceptable carrier and, optionally, any other agricultural adjuvant. It is another object of the present invention to provide synthetic Bt insecticidal protein genes to maximize the expression of Bt proteins in a plant, preferably in a maize plant. It is one feature of the present invention that a synthetic Bt insecticidal protein gene is constructed using the most preferred maize codons, except for alterations necessary to provide ligation sites for construction of the full synthetic gene. Another object of the present invention is a chimeric insecticidal protein comprising a N- terminal toxin portion of the CrylB protein and the C-terminal protoxin portion of the CrylA(b) protein. In a preferred embodiment, a chimeric insecticidal protein comprises the amino acid sequence encoded by the 5' portion of a synthetic maize optimized cyiB gene (SFLIB) fused to the amino acid sequence encoded by the 3' portion of a synthetic maize optimized crylA(b) gene. This protein is encoded by a full-length hybrid c/ylB gene and is designated "hyFLIB". In a particularly preferred embodiment, the chimeric insecticidal protein is that set forth in SEQ ID NO: 2, and is encoded by the coding sequence set forth in SEQ ID NO: 1. An additional aspect of the invention is recombinant, biologically pure microbial strains transformed with the hyFLIB gene which can be used in entomocidal formulations for the control of Lepidopteran insects. Yet another aspect of the invention are plants transformed with the toxin gene or active fragments thereof, particularly where the transforming sequences have been optimized for expression in maize. According to the above objects, we have synthesized Bt insecticidal crystal protein genes in which the codon usage has been altered in order to increase expression in plants, particularly maize. However, rather than alter the codon usage to resemble a maize gene in terms of overall codon distribution, we have optimized the codon usage by using the codons which are most preferred in maize (maize preferred codons) in the synthesis of the synthetic gene. The optimized maize preferred codon usage is effective for expression of high levels of the Bt insecticidal protein. This might be the result of maximizing the amount of Bt insecticidal protein translated from a given population of messenger RNAs. The synthesis of a Bt gene using maize preferred codons also tends to eliminate fortuitous processing sites that might occur in the native coding sequence. The expression of this synthetic gene is significantly higher in maize cells than that of the native insecticidal protein Bt gene. Preferred synthetic, maize optimized DNA sequences of the present invention derive from the protein encoded by the crylA(b) gene in Bacillus thuringiensis var. kurstaki, HD-1 ; Geiser et al., Gene. 48:109-118 (1986) or the c/ylB gene (AKA Crya4 gene) described by Blizzard and Whiteley, Nuc. Acids. Res.. 16:2723 (1988). - 5 - The following definitions are provided in order to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention. Chimeric: "chimeric" is used to indicate that a DNA sequence, such as a vector or a gene, is comprised of more than one DNA sequences of distinct origin which are fused together by recombinant DNA techniques resulting in a DNA sequence, which does not occur naturally, and which particularly does not occur in the plant or other organism to be transformed. Expression: refers to the transcription and/or translation of an endogenous gene or a transgene in plants. In the case of antisense constructs, for example, expression may refer to the transcription of the antisense DNA only. Operablv linked to/ associated with: a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence. Plant: refers to any plant, particularly to seed plants. Plant cell: structural and physiological unit of the plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ. Promoter: a DNA sequence that initiates transcription of an associated DNA sequence. The promoter region may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors. Recombinant DNA molecule: a combination of DNA sequences that are joined together using recombinant DNA technology. Recombinant DNA technology: procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Synthetic: a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic. Transformation: Introduction of a nucleic acid into a cell. In particular, the stable integration of a DNA molecule into the genome of an organism of interest. Maize preferred codon: Preferred codon refers to the preference exhibited by a specific host cell in the usage of nucleotide codons to specify a given amino acid. The preferred - 6 - codon for an amino acid for a particular host is the single codon which most frequently encodes that amino acid in that host. The maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. For example, maize codon usage for 28 genes from maize plants are listed in Table 4 of Murray et al., Nucleic Acids Research. 17:477-498 (1989), the disclosure of which is incorporated herein by reference. For instance, the maize preferred codon for alanine is GCC, since, according to pooled sequences of 26 maize genes in Murray et al., supra, that codon encodes alanine 36% of the time, compared to GCG (24%), GCA (13%), and GCT (27%). Pure maize optimized sequence: An optimized gene or DNA sequence refers to a gene in which the nucleotide sequence of a native gene has been modified in order to utilize preferred codons for maize. For example, a synthetic maize optimized Bt cr lA(b) gene is one wherein the nucleotide sequence of the native Bt c/ylA(b) gene has been modified such that the codons used are the maize preferred codons, as described above. A pure maize optimized gene is one in which the nucleotide sequence comprises 100 percent of the maize preferred codon sequences for a particular polypeptide. For example, the pure maize optimized Bt crylA(b) gene is one in which the nucleotide sequence comprises 100 percent maize preferred codon sequences and encodes a polypeptide with the same amino acid sequence as that produced by the native Bt crylA(b) gene. The pure nucleotide sequence of the optimized gene may be varied to permit manipulation of the gene, such as by altering a nucleotide to create or eliminate restriction sites. The pure nucleotide sequence of the optimized gene may also be varied to eliminate potentially deleterious processing sites, such as potential polyadenylation sites or intron recognition sites. It is recognized that "partially maize optimized," sequences may also be utilized. By partially maize optimized, it is meant that the coding region of the gene is a chimeric (hybrid), being comprised of sequences derived from a native insecticidal gene and sequences which have been optimized for expression in maize. A partially optimized gene expresses the insecticidal protein at a level sufficient to control insect pests, and such expression is at a higher level than achieved using native sequences only. Partially maize optimized sequences include those which contain at least about 5% optimized sequences. Full-length Bt Genes: Refers to DNA sequences comprising the full nucleotide sequence necessary to encode the polypeptide produced by a native Bt gene. For example, the native Bt cιy\A(b) gene is approximately 3.5 Kb in length and encodes a polypeptide which is approximately 1150 amino acids in length. A full-length synthetic crylA(b) Bt gene would be at least approximately 3.5 Kb in length. Truncated Bt Genes: Refers to DNA sequences comprising less than the full nucleotide sequence necessary to encode the polypeptide produced by a native Bt gene, but which encodes the active toxin portion of the polypeptide. For example, a truncated synthetic Bt gene of approximately 1.9 Kb encodes the active toxin portion of the polypeptide such that the protein product exhibits insecticidal activity. The present invention encompasses maize optimized coding sequences according to the teachings of U.S. Patent No. 5,625,136, herein incorporated by reference in its entirety. Also encompassed by the invention are encoding other polypeptides, including those of other Bacillus thuringiensis insecticidal polypeptides or insecticidal proteins from other sources. For example, c/ylB genes can be maize optimized, and then stably introduced into plants, particularly maize. The sequence of a maize optimized crylB gene constructed in accordance with the present invention is set forth in SEQ ID NO:7. The present invention encompasses a unique chimeric insecticidal protein which is created by replacing all or part of the native protoxin segment with an alternate protoxin segment. In a preferred embodiment, the chimeric insecticidal protein comprises a CrylA(b) C-terminal protoxin portion and a CrylB core N-terminal toxin portion. A particularly preferred embodiment comprises a synthetic maize optimized c/ylB gene (SFLIB) at the 3' end of the full-length synthetic maize optimized cr lA(b) gene to generate a full-length hybrid cry\B gene (hyFLIB). This unique chimeric insecticidal protein has the full toxin portion of CrylB (amino acids 1-845) and a portion of the CrylA(b) protoxin portion (amino acids 846-1227). When the chimeric insecticidal protein gene is expressed in transgenic maize from both PEPC and pith promoters, insecticidal activity is observed in transgenic maize tissue against European corn borer (Ostrinia nubilalis). An additional aspect of the invention is recombinant, biologically pure microbial strains transformed with the hyFLIB gene which can be used in entomocidal formulations for the control of Lepidopteran insects. Yet another aspect of the invention are plants transformed with the toxin gene or active fragments thereof, particularly where the transforming sequences have been optimized for expression in maize. - 8 - It should also be recognized that the isolated novel toxin gene of the present invention can be transferred into any microbial host and confer its insecticidal properties upon that host. Alternate hosts for the novel toxin gene of the present invention can be selected as suitable for cloning purposes, for purposes of characterizing the form and function of the gene or encoded protein, for use as a fermentation host to increase production of the toxin protein, for purposes of delivering the toxin protein more effectively to the target insect pest, or introduction of the novel toxin gene into insect pathogens such as baculovirus to improve their effectiveness. The novel toxin gene or recombinant forms thereof can be transformed into such alternate hosts using a variety of art recognized methods. One such preferred method is electroporation of microbial cells, as described, for example, by the method of Dower (U.S. Patent No. 5,186,800). Another preferred method is that of Schurter et al. (Mol. Gen. Genet. 218: 177-181 (1989)), which is also disclosed in U.S. Serial No. 07/353,565 which is incorporated herein in its entirety. It is envisioned that said alternate host would be applied to the environment or plants or animals for insect control. Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation. Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., Bacillus, Caulobacter, Agmenellum, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Bacillus spp., Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium meliloti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir, and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. - 9 - pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms. The present invention further provides an entomocidai composition comprising a recombinant Bacillus thuringiensis strain containing the novel toxin gene in recombinant form, or a derivative or mutant thereof, together with an agricultural adjuvant such as a carrier, diluent, surfactant or application-promoting adjuvant. The composition may also contain a further biologically active compound selected from fertilizers, micronutrient donors, plant growth preparations, herbicides, insecticides, fungicides, bactericides, nematicides and molluscicides and mixtures thereof. The composition may comprise from 0.1 to 99% by weight of the recombinant Bacillus thuringiensis strain containing the novel gene in recombinant form, or the derivative or mutant thereof, from 1 to 99.9% by weight of a solid or liquid adjuvant, and from 0 to 25% by weight of a surfactant. The recombinant Bacillus thuringiensis strain containing the novel gene in recombinant form, or the composition containing it, may be administered to the plants or crops to be protected together with certain other insecticides or chemicals (1993 Crop Protection Chemicals Reference, Chemical and Pharmaceutical Press, Canada) without loss of potency. It is compatible with most other commonly used agricultural spray materials but should not be used in extremely alkaline spray solutions. It may be administered as a dust, a suspension, a wettable powder or in any other material form suitable for agricultural application. Target crops to be protected within the scope of the present invention comprise, e.g., the following species of plants: cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beet (sugar beet and fodder beet), forage grasses (orchardgrass, fescue, and the like), drupes, pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries and blackberries), leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons) fiber plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other Brassicaceae, onions, tomatoes, potatoes, paprika), lauraceae (avocados, carrots, cinnamon, camphor), deciduous trees and conifers (e.g. linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches, pines), or plants such as maize, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as well as ornamentals (including composites). - 10 - The recombinant Bacillus thuringiensis strain containing the novel gene in recombinant form is normally applied in the form of entomocidal compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with further biologically active compounds. These compounds may be both fertilizers or micronutrient donors or other preparations that influence plant growth. They may also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. The formulations, i.e. the entomocidal compositions, preparations or mixtures containing the recombinant Bacillus thuringiensis strain containing the novel gene in recombinant form as an active ingredient or combinations thereof with other active ingredients, and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, e.g., by homogeneously mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some cases surface-active compounds (surfactants). Suitable solvents are: aromatic hydrocarbons, preferably the fractions containing 8 to 12 carbon atoms, e.g. xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide, as well as vegetable oils or epoxidised vegetable oils such as epoxidised coconut oil or soybean oil; or water. The solid carriers used, e.g., for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues. - 11 - Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds. Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (C sub 10 -C sub 22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained, e.g. from coconut oil or tallow oil. Further suitable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids. More frequently, however, so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazoie derivatives or alkylarylsulfonates. The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally contain a C sub 8 -C sub 22 alkyl radical which also includes the alkyl moiety of acyl radicals, e.g. the sodium or calcium salt of lignosulfonic acid, of dodecylsulfate, or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazoie derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide. Non-ionic surfactant are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkyiphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkyiphenols. Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediaminopolypropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit. Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, - 12 - castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan, such as poiyoxyethylene sorbitan trioleate, are also suitable non-ionic surfactants. Cationic surfactants are preferably quaternary ammonium salts which contain, as N- substituent, at least one C sub 8 -C sub 22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or hydroxyl-lower alkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride or benzyldi-(2-chloroethyl)ethylammonium bromide. The surfactants customarily employed in the art of formulation are described, e.g., in "McCutcheon's Detergents and Emulsifiers Annual", MC Publishing Corp. Ridgewood, N.J., 1979; Dr. Helmut Stache, "Tensid Taschenbuch" (Handbook of Surfactants), Carl Hanser Verlag, Munich/Vienna. Another particularly preferred characteristic of an entomocidal composition of the present invention is the persistence of the active ingredient when applied to plants and soil. Possible causes for loss of activity include inactivation by ultra-violet light, heat, leaf exudates and pH. For example, at high pH, particularly in the presence of reductant, δ- endotoxin crystals are solubilized and thus become more accessible to proteolytic inactivation. High leaf pH might also be important, particularly where the leaf surface can be in the range of pH 8-10. Formulation of an entomocidal composition of the present invention can address these problems by either including additives to help prevent loss of the active ingredient or encapsulating the material in such a way that the active ingredient is protected from inactivation. Encapsulation can be accomplished chemically (McGuire and Shasha, 1992) or biologically (Barnes and Cummings, 1986). Chemical encapsulation involves a process in which the active ingredient is coated with a polymer while biological encapsulation involves the expression of the δ-endotoxin genes in a microbe. For biological encapsulation, the intact microbe containing the δ-endotoxin protein is used as the active ingredient in the formulation. The addition of UV protectants might effectively reduce irradiation damage. Inactivation due to heat could also be controlled by including an appropriate additive. The entomocidal compositions usually contain 0.1 to 99%, preferably 0.1 to 95%, of the recombinant Bacillus thuringiensis strain containing the novel gene in recombinant form, or combination thereof with other active ingredients, 1 to 99.9% of a solid or liquid adjuvant, - 13 - and 0 to 25%, preferably 0.1 to 20%, of a surfactant. Whereas commercial products are preferably formulated as concentrates, the end user will normally employ dilute formulations of substantially lower concentration. The entomocidal compositions may also contain further ingredients, such as stabilizers, antifoams, viscosity regulators, binders, tackifiers as well as fertilizers or other active ingredients in order to obtain special effects. A host plant expressing at least one of the novel toxin genes of the invention will have enhanced resistance to insect attack and will be thus better equipped to withstand crop losses associated with such attack. By plant is meant any plant species which can be genetically transformed by methods known in the art. Methods known in the art for plant transformation are discussed below. Host plants include, but are not limited to, those species previously listed as target crops. It has been discovered that the codon usage of a native Bacillus thuringiensis toxin gene is significantly different from that which is typical of a plant gene. In particular, the codon usage of a native Bacillus thuringiensis gene is very different from that of a maize gene. As a result, the mRNA from this gene may not be efficiently utilized. Codon usage might influence the expression of genes at the level of translation or transcription or mRNA processing. To optimize a toxin gene for expression in plants, for example in maize, the codon usage is optimized by using the codons which are most preferred in maize (maize preferred codons) in the synthesis of a synthetic gene which encodes the same protein as found for the native toxin gene sequence. The optimized maize preferred codon usage is effective for expression of high levels of the Bt insecticidal protein. Further details for constructing maize-optimized synthetic toxin genes can be found in U.S. Patent 5,625,136, herein incorporated by reference in its entirety. Toxin genes derived from microorganisms may also differ from plant genes. Plant genes differ from genes found in microorganisms in that their transcribed RNA does not possess defined ribosome binding site sequence adjacent to the initiating methionine. Consequently, microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210) has suggested the sequence GTCGACCATGGTC (SEQ ID NO: 9) as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (Nucl Acids Res 15: 6643- 6653,1987) has compared many plant sequences adjacent to the ATG and suggests the consensus TAAACAATGGCT (SEQ ID NO: 10). In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these - 14 - sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. By surveying the sequence of maize genes present in the GenBank/EMBL database it can be discerned which nucleotides adjacent to the ATG should be modified to enhance translation of the toxin gene introduced into maize. In addition, it has been shown that removal of illegitimate splice sites can enhance expression and stability of introduced genes. Genes cloned from non-plant sources and not optimized for expression in plants may contain motifs which can be recognized in plants as 5' or 3' splice sites. Consequently, the transcription process can be prematurely terminated, generating truncated or deleted mRNA. The toxin genes can be engineered to remove these illegitimate splice sites using techniques known in the art. It is within the scope of the present invention that genes encoding either the full-length protoxin form or the truncated toxic core fragment of the novel toxin proteins can be used in plant transformation vectors to confer insecticidal properties upon the host plant. The recombinant DNA molecules can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et al, Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986), /4grofcacter/ ' u/77-mediated transformation (Hinchee et al., Biotechnology 6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988); see also Weissinger et al., Annual Rev. Genet. 22:421 -477 (1988); Sanford et al., Particulate Science and Technology 5:27-37 91987)(onion); Christou et al., Plant Physiol. 87:671 -674 (1988)(soybean); McCabe etal., Bio/Technology 6:923-926 (1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rice); Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize); Klein et al., Bio Technology 6:559-563 (1988)(maize); Klein et al., Plant Physiol. 91 :440-444 (1988)(maize); Fromm etal., Bio/Technology 8:833-839 (1990); and Gordon-Kamm etal., Plant Cell 2:603-618 (1990)(maize); Svab et al. Proc. Natl. Acad. Sci. USA 87: 8526-8530 - 15 - (1990) (tobacco chloroplast); Koziel et al. (Biotechnology 11 : 194-200 (1993)) (maize); Shimamoto et al. Nature 338: 274-277 (1989) (rice); Christou et al. Biotechnology 9: 957- 962 (1991 ) (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (Biotechnology 11 : 1553-1558 (1993) (wheat); Weeks et al. (Plant Physiol. 102: 1077-1084 (1993) (wheat); Wan etal. (Plant Physiol. 104: 37-48 (1994)(barley)); Umbeck etal., (Bio/Technology 5: 263-266 (1987)(cotton). One particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment. An additional preferred embodiment is the protoplast transformation method for maize as disclosed in U.S. Patent No. 5,350,689, hereby incorporated by reference in its entirety. Transformation of plants can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with the novel toxin gene of the present invention. Methods using either a form of direct gene transfer or Agrobacterium-med ' aieό transfer usually, but not necessarily, are undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (phosphinothricin). The choice of selectable marker for plant transformation is not, however, critical to the invention. Numerous transformation vectors are available for plant transformation, and the genes of this invention can be used in conjunction with any such vectors. The selection of vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Vieira & Messing, Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res 18: 1062 (1990), Spencer et al. Theor AppI Genet 79: 625-631 (1990)), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger & Diggelmann, Mol Cell Biol 4: 2929- 2931), the dhfr gene, which confers resistance to methotrexate (Bourouis etal., EMBO J. 2: 1099-1104 (1983), and the mannose phosphate isomerase (PMI) gene, which allows selection on mannose as a carbon source (EP 530 129, WO 94/20627) Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 - 16 - (Bevan, Nucl. Acids Res. (1984)). In one preferred embodiment, the novel toxin gene of the present invention may be inserted into either of the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium. These vector cassettes for Agrobacterium-meόi ' aled transformation can be constructed in the following manner. pTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J Bacteriol. 164: 446-455 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Vieira & Messing, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers were ligated to the EcoRV fragment of pCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the X ιo/-digested fragment was cloned into Sa//-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 which created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul. pCIB2001 , in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for /4gfro_}acfer/um-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the Or/Tand Or/Vfunctions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals. An additional vector useful for Agrobacterium-medlateό transformation is the binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives of pCIBIO have been constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717). Other transformation techniques which do not rely on Agrobacterium, the so-called direct gene transfer methods, are also useful for the introduction of the novel toxin gene of the present invention, including transformation by microprojectile bombardment, protoplast - 17 - uptake (e.g. PEG and electroporation) and microinjection. The choice of vector for these methods depends largely on the preferred selection for the species being transformed. One such vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is pCIB3064. This vector is based on the plasmid pCIB246, which comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278, herein incorporated by reference. The gene providing resistance to phosphinothricin is the bar gene from Streptomyces hygroscopicus (Thompson et al. EMBO J 6: 2519-2523 (1987)). This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals. An additional transformation vector is pSOG35 which utilizes the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker conferring resistance to methotrexate. PCR was used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp; see Dennis etal., Nucleic Acid Res. 12:3983-4000 (1984)) and 18 bp of the GUS untranslated leader sequence (see Jefferson etal., Proc. Nat. Acad. Sci. USA 83: 8447- 8451 (1986). A 250 bp fragment encoding the E. coli dihydrofolate reductase type II gene was also amplified by PCR and these two PCR fragments were assembled with a Sacl-PstI fragment from pBI221 (Clontech) which comprised the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generated pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Psf/ and EcoRI sites available for the cloning of foreign sequences. The coding sequence of the novel insecticidal proteins of the present invention can be operably fused to a variety of promoters for expression in plants including constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters to prepare recombinant DNA molecules, i.e., chimeric genes. Preferred constitutive promoters include the CaMV 35S and 19S promoters (Fraley etal., U.S. Patent No. 5,352,605, issued October 4, 1994). An additionally preferred promoter is derived from any one of several of the actin genes, which are known to be expressed in most cell types. The promoter expression cassettes described by McElroy et - 18 - al. (Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expression of the novel toxin gene and are particularly suitable for use in monocotyledonous hosts. Yet another preferred constitutive promoter is derived from ubiquitin, which is another gene product known to accumulate in many cell types. The ubiquitin promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991 ), maize - Christensen et al. Plant Molec. Biol. 12: 619-632 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of the novel toxin gene in transgenic plants, especially monocotyledons. Tissue-specific or tissue-preferential promoters useful for the expression of the novel toxin gene in plants, particularly maize, are those which direct expression in root, pith, leaf or pollen. Such promoters, e.g. those isolated from PEPC or trpk, are disclosed in U.S. Patent No. 5,625,136, or MTL, disclosed in U.S. Patent No. 5,466,785. Both U.S. patents are herein incorporated by reference in their entirety. Chemically inducible promoters useful for directing the expression of the novel toxin gene in plants are disclosed in U.S. Patent No. 5,614,395, herein incorporated by reference in its entirety. in addition to promoters, a variety of transcriptional terminators are also available for use in chimeric gene construction using the novel toxin gene of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator and others known in the art. These can be used in both monocotyledons and dicotyledons. Numerous sequences have also been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the novel toxin gene of this invention to increase their expression in transgenic plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adh1 gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Callis et al., Genes Develop. 1 : 1183-1200 (1987)). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader. - 19 - A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the "Ω-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15; 65-79 (1990)) EXAMPLES The following examples further describe the materials and methods used in carrying out the invention and the subsequent results. They are offered by way of illustration, and their recitation should not be considered as a limitation of the claimed invention. Example 1: Construction of a Maize Optimized crylB Full-length Hybrid Gene CrylB is a δ-endotoxin of 1207 amino acids and is approximately 130 Kd in size. To design a synthetic crylB gene, the deduced peptide sequence of CrylB (Brizzard & Whiteley, Nucl Acids Res. 16:2723, 1988) (Genbank accession number X06711 ) is backtranslated using the "Backtranslation" program found in the University of Wisconsin GCG group of programs using a maize preference codon table (Murray et al., Nucl Acids Res. 17:477-498, 1989). By inserting unique restriction sites to facilitate cloning, the "maize optimized " sequence is further modified resulting in a synthetic full-length crylB sequence (SFLIB). This gene is designed for cloning from 12 parts. Each fragment of the synthetic gene is constructed by hybridization of 5-7 pairs of oligomers 60-75 nucleotides (nt) in length representing both strands of the gene. A 15 nt overlap is designed between sequential oligonucleotide pairs for correct orientation and assembly. The ends of each fragment are designed to have ends cohesive with DNA digested with restriction enzyme EcoRI at the 5' end and Hindlll at the 3' ends. Oligos are synthesized by either IDT Inc. (Iowa ) or Oligos etc.(Oregon). Fragments of the gene are constructed either by hybridization and ligation of the oligonucleotides and cloning into pUC18 vector digested with EcoRI and Hindlll or by PCR amplification. Amplification is carried out using an equimolar mixture of oligomers as the template and primers with sequence corresponding to the ends of the fragment. Taq polymerase is used in the - 20 - reactions, following standard conditions from the supplier (Perkin-Elmer). The amplified DNA for each fragment is cloned into a T-vector made from pBSSK+ ® (Marchuk et al., Nucl Acids Res. 19:1154, 1991 ). Gene fragments with the correct sequence are joined using the overlapping restriction endonuclease sites. Approximately 2.5 Kb of the synthetic crylB gene (including the complete toxin region) is subcloned behind the Lac Z promoter derived from pUC18 resulting in pCIB5518. The protein from E. coli transformed with pCIB5518 is 100% active in bioassay against ECB (Ostrinia nubilalis). Eight of these fragments make up 2.54 Kb of the c/ylB gene, and end at a Clal restriction site. This position in the cryB gene corresponds to the analogous position (and restriction site) in the crylA(b) gene (at the beginning of the thermostable element described by Geiser et al., Gene 48:109-118, 1986). Homology at the protein and amino acid level is 80% in the region beyond amino acid residue 850 in CrylB (and corresponding to amino acid residue 804 in CrylA(b) and the end of both genes. The 2.54 Kb of the synthetic c/ylB gene is joined to the 3' end of the full-length synthetic crylA(b) described in U.S. Patent No. 5,625,136, herein incorporated by reference (SEQ ID NOS: 3, 5). This has been referred to as the full-length hybrid cry\B gene (hyFLIB). The nucleotide sequence of the coding region is shown in SEQ ID NO:1 , and the encoded protein sequence is set forth in SEQ ID NO:2. Example 2: Maize Transformation Vectors Comprising the hyFLIB Gene For maize transformation, the 2.54 kb BamHI -Clal fragment of the synthetic cry\B gene is cloned into pCIB6014 which contained the PEPC promoter and the full-length fully synthetic c/ylA(b) gene (construction of the full-length synthetic cry\A(b) gene is set forth in U.S. Patent No. 5,625,136, herein incorporated by reference), replacing the BamHI -Clal synthetic crylA(b) fragment with the synthetic cry\B gene fragment described above and creating a full-length c/ylB/crylA(b) hybrid gene called hyFLIB. This full-length hybrid gene containing the PEPC promoter is called pCIB5520 (SEQ ID NO: 1). The pCIB5520 clone was deposited under the terms of the Budapest Treaty on April 1 , 1998, with the Agricultural Research Service, Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 Northern University Street, Peoria, Illinois 61604, USA and assigned accession number NRRL B-21972. The full-length hybrid gene is subcloned into plant expression vector cassettes containing the pith promoter, the MTL promoter and the maize ubiquitin promoter to give pCIB4784 , pCIB9830 and pCIB9831 , respectively. - 21 - Example 3: Bioassav of Transformed Maize for Insecticidal Activity Transformed plants are assayed for insecticidal activity and the presence of a BT protein resulting from the expression of the maize-optimized coding sequence of a synthetic BT gene. The procedure is similar for any maize plant transformed with a BT gene but is described here using as an example the HyFLIB gene, its expressed product, and resistance to European corn borer. Insecticidal activity is determined by insect bioassay. One to four 4 cm sections are cut from an extended leaf of a transformed maize plant. Each leaf piece is placed on a moistened filter disc in a 50 X 9 mm Petri dish. Five neonate European corn borer larvae are placed on each leaf piece. Since each plant is sampled multiple times this makes a total of 5-20 larvae per plant. The Petri dishes are incubated at 29.5 °C and leaf feeding damage and mortality data are scored at 48 hours. Example 4: Transformation of maize with novel toxin gene Transformation of maize with at least one of the novel toxin genes prepared according to any of the above methods is achieved by microprojectile bombardment of either immature zygotic embryos or serially-propagatable Type I embryogenic callus. Type I embryogenic maize callus cultures (Green et al, Miami Winter Symposium 20,1983) are initiated from immature embryos, 1.5 - 2.5 mm in length, from greenhouse grown material. Embryos are aseptically excised from surface-sterilized ears approximately 14 days after pollination. Embryos may be placed on D callus initiation media with 2% sucrose and 5 mg/L chloramben (Duncan et al, Planta 165: 322-332,1985) or onto KM callus initiation media with 3% sucrose and 0.75mg/L 2,4-d (Kao and Michayluk, Planta 126:105- 110, 1975). Embryos and embryogenic cultures are subsequently cultured in the dark. Embryogenic responses are removed from the explants after -14 days. Embryogenic responses from D callus initiation media are placed onto D callus maintenance media with 2% sucrose and 0.5mg/L 2,4-d while those from KM callus initiation media are placed onto KM callus maintenance media with 2% sucrose and 5mg/L Dicamba. After 3 to 8 weeks of weekly selective subculture to fresh maintenance media, high quality compact embryogenic cultures are established. Actively growing embryogenic callus pieces are selected as target tissue for gene delivery. The callus pieces are plated onto target plates containing maintenance medium with 12% sucrose approximately 4 hours prior to gene delivery. The - 22 - callus pieces are arranged in circles, with radii of 8 and 10 mm from the center of the target plate. Plasmid DNA is precipitated onto gold microcarriers as described in the DuPont Biolistics manual. Two to three μg of each plasmid is used in each 6 shot microcarrier preparation. Genes are delivered to the target tissue cells using the PDS-1000He Biolistics device. The settings on the Biolistics device are as follows: 8 mm between the rupture disc and the macrocarrier, 10 mm between the macrocarrier and the stopping screen and 7 cm between the stopping screen and the target. Each target plate is shot twice using 650psi rupture discs. A 200 X 200 stainless steel mesh (McMaster-Carr, New Brunswick, NJ) is placed between the stopping screen and the target tissue. Seven days after gene delivery, target tissue pieces are transferred from the high osmotic medium to selection media. For selection using the BAR gene, target tissue pieces are placed onto maintenance medium containing 100 mg/L glufosinate ammonium (Basta®) or 20 mg/L bialaphos (Herbiace®). All amino acids are removed from the selection media. After 5 to 8 weeks on these high level selection media, any growing callus is subcultured to media containing 3-20mg/L Basta®. For selection using the Mannose Phosphate Isomerase gene, target tissues are placed on their respective maintenance media containing no sucrose and 1 % mannose. The amino acids are not removed from these media. After 5 to 8 weeks, growing callus is either subcultured to D callus maintenance medium containing no sucrose and 1.5% mannose or KM callus maintenance medium containing 1% sucrose and 0.5% mannose. Embryogenic callus growing on selection media is subcultured every 2 weeks for 4 to 8 weeks until enough callus is produced to generate 10-20 plants. Tissue surviving selection from an original target tissue piece is subcultured as a single colony and designated as an independent transformation event. At that point, colonies selected on Basta® are transferred to a modified MS medium (Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) containing 3% sucrose (MS3S) with no selection agent and placed in the light. Either 0.25mg/L ancymidol and 0.5mg/L kinetin are added to this medium to induce embryo germination or 2mg/L benzyl adenine is added. Colonies selected using mannose are transferred onto a modified MS medium containing 2% sucrose and 1 % mannose (MS2S + 1 M) with the ancymidol and kinetin additions described above or a modified MS medium containing 2% sucrose and 0.5% mannose (MS2S + 0.5M) with the benzyl adenine addition described above. - 23 - Regenerating colonies from Basta® selection are transferred to MS3S media without ancymidol and kinetin or benzyl adenine after 2 weeks. Regenerating colonies from mannose selection are transferred to MS2S + 1 M and MS2S + 0.5M media respectively without hormones after 2 weeks. Regenerating shoots with or without roots from all colonies are transferred to Magenta boxes containing MS3S medium and small plants with roots are eventually recovered and transferred to soil in the greenhouse. Plants are tested for expression of the PMI gene using a modified 48-well chlorophenol red assay where the media contains no sucrose and 0.5% mannose. Leaf samples (~5mm x 5mm) are placed on this assay media and grown in the dark for -72 hours. If the plant is expressing the PMI gene, it can metabolize the mannose and the media will turn yellow. If not, the media will remain red. Transformation events have also been created using Type I callus obtained from immature zygotic embryos using standard culture techniques. For gene delivery, approximately 300 mg of the Type I callus is prepared by subculturing to fresh media 1 to 2 days prior to gene delivery, selecting target tissue pieces and placing them in a ring pattern 10 mm from the center of the target plate on medium again containing 12% sucrose. After approximately 4 hours, the tissue is bombarded using the PDS-1000/He Biolistic device from DuPont. The plasmids pCIB5520 (SEQ ID NO: 1 ) and pUBIAc (SEQ ID NO:12) are precipitated onto 1 μm gold particles using the standard protocol from DuPont. Genes are delivered using two shots per target plate at 650psi. Approximately 16 hours after gene delivery the callus is transferred to standard culture medium containing 2% sucrose with no selection agent. At 12 or 13 days after gene delivery, target tissue pieces are transferred to selection media containing 40mg/l phosphinothricin as either Basta or bialaphos. The callus is subcultured on selection for 12 to 16 weeks, after which surviving and growing callus is transferred to standard regeneration medium containing 3mg/l phosphinothricin as Basta for the production of plants. Example 5: Transformation of soybean with novel toxin gene Protoplasts of Glycine max axe prepared by the methods as described by Tricoli et al., 1986 (Plant Cell Rep. 5: 334-337), or Chowhury and Widholm, 1985 (Plant Cell Rep. 4: 289-292), or Klein et al., 1981 (Planta 152: 105-114). The protoplast suspension is distributed as 1 ml aliquots into plastic disposable cuvettes. For transformation, 10 μg of DNA is added in 10 μl sterile distilled water and sterilized as described by Paszkowski et al., 1984 (EMBO J. 3: 2717-2722). The solution is mixed gently and then subjected at room temperature (24 to - 24 - 28°C) to a pulse of 400 Vcm "1 with an exponential decay constant of 10 ms from a BTX- Transfector 300 electroporation apparatus using the 471 electrode assembly. The above is repeated with one or more of the following modifications: (1) The voltage used is 200 Vcm "1 , or between 100 Vcm '1 and 800 Vcm "1 (2) The exponential decay constant is 5 ms, 15 ms or 20 ms (3) 50 μg of sheared calf thymus DNA in 25 μl sterile water is added together with the plasmid DNA (4) The plasmid DNA is linearized before use by treatment with an appropriate restriction enzyme (e.g. BamHI) The protoplasts are cultured as described in Klein et al., 1981 (Planta 152: 105-114), Chowhury and Widholm, 1985 (Plant Cell Rep. 4: 289-292), or Tricoli et al., 1986 (Plant Cell Rep. 5: 334-337), without the addition of alginate to solidify the medium. Example 6: Transformation of cotton with novel toxin gene Agrobacterium strains containing the binary vectors for transformation that are constructed by standard methods are grown 18 to 24 hours in glutamate salts media adjusted to pH 5.6 and supplemented with 0.15% mannitol, 50 μg/ml kanamycin, 50 μg/ml spectinomycin and 1 mg/ml streptomycin before they are diluted to an OD600 of 0.2 in the same media without the antibiotics. The bacteria are then grown for three to five hours before dilution to an OD600 of 0.2 to 0.4 and then used for inoculation of discs cut from surface sterilized cotton seeds. The cotton seeds are soaked 20 min in 10% chlorox and rinsed with sterile water. The seeds are germinated on 0.7% water agar in the dark. The seedlings are grown for one week before inoculation of the bacteria onto the cotyledon surface. The inoculated cotyledons are allowed to form callus before they are cut and placed on 0.7% agar containing MS salts, 3% sucrose, 100 μg/ml carbenicillin, and 100 μg/ml mefoxim. The callus is transferred to fresh media every three weeks until sufficient quantity is obtained for 4 plates. Half of the callus growing from the virulent Agrobacterium strains is transferred to media without hormones containing 50 μg/ml kanamycin. - 25 - Insecticidal Activity of Transgenic Maize Containing hyFLIB Event No. Transforming Plasmid Activity against Activity Against European Corn Borer Corn Ear Worm 601 PCIB5520, pUBIAc + - 602 pCIB5520, pUBIAc + Not tested 605 PCIB5520, pCIB4784 + Not tested pUBIAc Brief description of the sequences in the sequence listing: SEQ ID NO: 1 Sequence of pCIB5520 containing coding sequence for HyFLIB protein SEQ ID NO: 2 Amino acid sequence of HyFLIB protein encoded by coding sequence of SEQ ID NO:1 SEQ ID NO: 3 Full-length pure maize optimized synthetic CrylA(b) SEQ ID NO: 4 Amino acid sequence of full-length CrylA(b) protein SEQ ID NO: 5 Full length synthetic maize optimized CrylA(b) SEQ ID NO: 6 Amino acid sequence of full-length CrylA(b) protein encoded by coding sequence of SEQ ID NO:5 SEQ ID NO: 7 Full length pure maize optimized CrylB SEQ ID NO: 8 Amino acid sequence of full-length CrylB protein SEQ ID NO: 9 Clontech sequence SEQ ID NO:10 Joshi sequence SEQ ID NO:11 DNA sequence of pCIB9818 Ubi/PMI selectable marker plasmid SEQ ID NO:12 DNA sequence of pUBIAc Ubi/PAT selectable marker plasmid Deposits Deposited material Accession number Name and address of Date of deposit depository pCIB5520 NRRL B-21972 Northern Regional Research April 1 , 1998 Center, 1815 Northern University Street, Peoria, Illinois 61604, USA -26- BUDAPEST TREAT". ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURES INTERNATIONAL FORM τo RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Novart s Corp issued purβuant to Rule 7 1 by the c/o Novartiβ AO INTERNATIONAL DEPOSITARY AUTHORITY P. O. Box 12257 identified at the bottom of this page Research Triangle Park, NC 27709 NAME AND ADDRESS OF DEPOSITOR IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession numbe-r given by the DEPOSITOR INTERNATIONAL DEPOSITARY AUTHORITY Bacteria ep PCIB5520 NRRL B-21972 II SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOKIC DES CGNATIQN The microorganism identified under I. above was accompanied by R3 a scientific description j [ a propoβed taxonomic designation (Mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE This International Depoaitary Authority accepts the microorganiβm identified under I above, which was received by it on April 1, 1998 (date of the original deposit) IV RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I. above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) . V INTERNATIONAL DEPOSITARY AUTHORITY Name: Agricultural Research Culture Signature (s) cf person (s) having the power Collection (NRRL) to represent the International Depositary International Depositary Authority Authority or of authorized official ( B ) Address. 1B15 N University Street / Peoria, Illiπolo 61604 U.S A Date S-N -ir ' Where Rule €.4(d) appliee, such date is the αate on which the status of international depositary authority was acquired -27- BUDAPEST TREATY ON THE INTERITATICNAL R__OGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PROCEDURES INTERNATIONAL FORM TO raBiLiTY gτaτ-M-wτ Novar ia Corp. c/o Novartis AG issued pursuant to Rule 10.2 by the P. O. Box 122S7 INTERNATIONAL DEPOSITARY AUTHORITY Research Triangle Park, NC 27709 identified at the bottom of this page NAME AND ADDRESS OF THE PARTY TO WHOM THE VIABILITY STATEMENT IS ISSUED DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM Name: Novartis Corp Depositor's taxonomic designation and c/o Novartis AG accession number given by the Address: P. 0. Box 122S7 INTERNATIONAL DEPOSITARY AUTHORITY: Research Triangle Park, Bacteria sp . NRRL B-21972 NC 27709 Date of:April 1, 199β I2SJ ' Original Deposit L_J * New Deposit L_J ' Repropagation of Original Deposit III . ( a) VIABILITY STATEMENT Deposit was found: Viable D Nonviable on April 2, 1398 (Date) International Depositary Authority's preparation was found viable on April 10, 1998 (Date)' III. (b) DEPOSITOR'S EQUIVALENCY DECLARATION Depositor determined the International Depositary Authority's preparation was L_J • Equivalent I—I ' Not equivalent to deposit on (Date) Signature of Depositor IV. CONDITIONS UNDER WHICH THE VIABILITY TEST WAS PERFORMED (Depositors /Depositary) ' INTERNATIONAL DEPOSITARY AUTHORITY Name: Agricultural Research Culture Signature (β) of person (B) having the power Collection (NRRL) to represent the International Depositary International Depositary Authority Authority or of authorized official (e) : Address: 1815 N. University Street Peoria, Illinois 61604 U.S.A. Date: f- .M -1* ' Imlirβcr πie date ot the original deposit o r w en t ι. w -let-cmlt hi* been m».Jβ . * mr- witn * cross the. .lppll C iiMft r > r > x . * in urn cam .o xiβe] io in Rule 1C . 2 ( a ) l li l ■<"" I ' 1 ' ■ i "t* c Co the maat c»cβnt vl.ιr.1. * Kill if- It e ? . ' r.inru *. tot* lie* tiamn requeeceo .

Claims: -28- What is claimed is: 1. A DNA molecule comprising a nucleotide sequence encoding a chimeric insecticidal protein of 1200 to 1300 amino acids, wherein said insecticidal protein comprises a crylB core N-terminal toxin portion of 600 to 1000 amino acids and a c/ylA(b) C-terminal protoxin portion of 300 to 600 amino acids. 2. A DNA molecule according to claim 1 , wherein said insecticidal protein comprises a crylB core N-terminal toxin portion comprising about two thirds of the amino acids of the protein and a crylA(b) C-terminal protoxin portion comprising about one third of the amino acids of the protein. 3. A DNA molecule according to claim 1 , wherein said nucleotide sequence encodes a chimeric insecticidal protein of 1227 amino acids, wherein said insecticidal protein comprises a crylB core N-terminal toxin portion of 845 amino acids and a crylA(b) C- terminal protoxin portion of 382 amino acids, as set forth in SEQ ID NO:1. 4. A DNA molecule according to any one of claims 1 to 3 comprising additionally a promoter operably linked to the nucleotide sequence encoding the chimeric insecticidal protein. 5. The DNA molecule of claim 4 wherein said promoter is selected from the group consisting of a PEPC promoter, pith promoter, MTL promoter, and ubiquitin promoter. 6. A plant stably transformed with the DNA molecule of claim 4. 7. A plant according to claim 6 which is a maize plant. 8. A recombinant host transformed to express a chimeric insecticidal protein comprising a nucleotide sequence according to any one of claims 1 to 3. 9. The recombinant host according to claim 8 which is selected from bacteria, algae, and fungi. -29- 10. The recombinant host of claim 9 which is selected from bacteria known to occupy the phytosphere. 11. The recombinant host of claim 10 which is Bacillus thuringiensis. 12. The insecticidal protein set forth in SEQ ID NO:2. 13. A method of protecting plants against insect pests comprising expressing in said plant a nucleotide sequence according to any one of claims 1 to 3 to produce an anti-insect protein. 14. A method of protecting plants against insect pests comprising applying to said plant the insecticidal protein of claim 12. 15. The method according to any one of claims 13 or 14, wherein the insect pest is caused by the European corn borer. 16. An entomocidal composition for the control of Lepidopteran insects comprising a recombinant host according to any one of claims 8 to 11 or the insecticidal protein of claim 12 or a combination thereof in an insecticidally effective amount together with an agriculturally acceptable carrier and, optionally, any other agricultural adjuvant.

Inventors: DESAI NALINI MANOJ, DESAI, NALINI, MANOJ
Applicants: DESAI NALINI MANOJ, NOVARTIS AG, NOVARTIS ERFIND VERWALT GMBH, DESAI, NALINI, MANOJ, NOVARTIS-ERFINDUNGEN VERWALTUNGSGESELLSCHAFT M.B.H.
Application year: 1999
Main classification: C07K14
IPC codes: A01H5, A01N63, C07K14, C12N1, C12N15, C12R1