MXPA99010882A - Proteins having insecticidal activities and method of use - Google Patents
Proteins having insecticidal activities and method of useInfo
- Publication number
- MXPA99010882A MXPA99010882A MXPA/A/1999/010882A MX9910882A MXPA99010882A MX PA99010882 A MXPA99010882 A MX PA99010882A MX 9910882 A MX9910882 A MX 9910882A MX PA99010882 A MXPA99010882 A MX PA99010882A
- Authority
- MX
- Mexico
- Prior art keywords
- sequence
- ident
- amino acid
- leu
- sec
- Prior art date
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 130
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 130
- 230000000749 insecticidal Effects 0.000 title claims description 33
- 241000196324 Embryophyta Species 0.000 claims abstract description 98
- 229920001850 Nucleic acid sequence Polymers 0.000 claims abstract description 60
- 230000001131 transforming Effects 0.000 claims abstract description 18
- 241001343007 Pentaclethra Species 0.000 claims abstract description 8
- 230000001276 controlling effect Effects 0.000 claims abstract description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 49
- 230000014509 gene expression Effects 0.000 claims description 46
- 235000005822 corn Nutrition 0.000 claims description 45
- 235000005824 corn Nutrition 0.000 claims description 45
- 150000001413 amino acids Chemical class 0.000 claims description 41
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 35
- 229920001184 polypeptide Polymers 0.000 claims description 29
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 28
- 239000011780 sodium chloride Substances 0.000 claims description 24
- 230000000694 effects Effects 0.000 claims description 22
- 150000007523 nucleic acids Chemical group 0.000 claims description 20
- 239000002773 nucleotide Substances 0.000 claims description 16
- 125000003729 nucleotide group Chemical group 0.000 claims description 16
- 238000006467 substitution reaction Methods 0.000 claims description 16
- 230000002068 genetic Effects 0.000 claims description 10
- 238000003780 insertion Methods 0.000 claims description 10
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 9
- 241000209149 Zea Species 0.000 claims description 9
- 241000209510 Liliopsida Species 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 239000011778 trisodium citrate Substances 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 2
- 230000004059 degradation Effects 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 claims description 2
- 239000002699 waste material Substances 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims 2
- 102000035365 modified proteins Human genes 0.000 claims 1
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- 241000238631 Hexapoda Species 0.000 abstract description 54
- 239000000203 mixture Substances 0.000 abstract description 32
- 241000607479 Yersinia pestis Species 0.000 abstract description 27
- 235000018102 proteins Nutrition 0.000 description 112
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- 235000001014 amino acid Nutrition 0.000 description 37
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- 239000000523 sample Substances 0.000 description 24
- 238000009396 hybridization Methods 0.000 description 21
- ATIPDCIQTUXABX-UWVGGRQHSA-N Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ATIPDCIQTUXABX-UWVGGRQHSA-N 0.000 description 20
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- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 9
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- 150000001875 compounds Chemical class 0.000 description 9
- 108010089804 glycyl-threonine Proteins 0.000 description 9
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- IOUPEELXVYPCPG-UHFFFAOYSA-N val-gly Chemical compound CC(C)C(N)C(=O)NCC(O)=O IOUPEELXVYPCPG-UHFFFAOYSA-N 0.000 description 9
- HXWUJJADFMXNKA-UHFFFAOYSA-N Asparaginyl-Leucine Chemical compound CC(C)CC(C(O)=O)NC(=O)C(N)CC(N)=O HXWUJJADFMXNKA-UHFFFAOYSA-N 0.000 description 8
- UKGGPJNBONZZCM-WDSKDSINSA-N Aspartyl-L-proline Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O UKGGPJNBONZZCM-WDSKDSINSA-N 0.000 description 8
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 8
- DKEXFJVMVGETOO-LURJTMIESA-N Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CN DKEXFJVMVGETOO-LURJTMIESA-N 0.000 description 8
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- RNKSNIBMTUYWSH-YFKPBYRVSA-N L-prolylglycine Chemical compound [O-]C(=O)CNC(=O)[C@@H]1CCC[NH2+]1 RNKSNIBMTUYWSH-YFKPBYRVSA-N 0.000 description 8
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- KNPVDQMEHSCAGX-UHFFFAOYSA-N Phenylalanyl-Cysteine Chemical compound SCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 KNPVDQMEHSCAGX-UHFFFAOYSA-N 0.000 description 8
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Abstract
Compositions and methods for controlling pests, particularly insect pests, are provided. The compositions comprise proteins isolated from plants of the genus Pentaclethra. Nucleotide sequences encoding the proteins are also provided. Such sequences find use in transforming organisms for control of pests.
Description
PROTEINS CONTAINING INSECTICIDE ACTIVITIES AND METHOD OF USE DESCRIPTION OF THE INVENTION The invention relates to compositions and methods for controlling insect species. Additionally, the invention relates to plants and other organisms that have been genetically transformed with the compositions of the invention. Numerous insect species are problematic pests for common agricultural crops such as corn, soybeans, peas, cotton, and similar food and fiber crops. The main method to control these pests has been through the application of synthetic chemical compounds. However, the widespread use of chemical compounds has many problems with respect to the environment since there is no selectivity of the compounds and the development of insects resistant to chemicals. Other approaches to pest control have been attempted including the use of biological organisms that are normally "natural predators" of the species that are to be controlled. Said predators may include other insects, fungi, and bacteria such as Bacillus thuringiensis. Alternatively, in captivity insect pests have originated, sterilized and released into the environment in the hope that mating between sterile insects and fecund wild insects
the insect population will decrease. While these approaches have had some success, they involve considerable expense and severe current major difficulties. For example, it is difficult to both apply biological organisms to large areas and cause living organisms to remain in the treated area or in the treated plant species for an extended time. Predator insects can migrate and can wash fungi or bacteria from a plant or be removed from an area treated by rain. Consequently, while the use of such biological controls has convenient characteristics and has met with some success, these methods seem severely limited in practice. Advances in biotechnology in the last two decades have not presented new opportunities for pest control through genetic engineering. In particular, advances in plant genetics coupled with the identification of the development factors of insects and defensive compounds of plants present in nature or agents that offer the opportunity to create plants of transgenic crops capable of producing said defense agents and So protect the plants against the attack of insects. Transgenic plants that are resistant to specific insect pests have been produced using genes
which encode Bacill us thuringiensis (Bt) endotoxins or plant protease inhibitors (PIs). Transgenic plants that contain Bt endotoxin genes have been shown to be effective for the control of some insects. The protection of effective plants using genetic material from transgeneically inserted Pl has not yet been demonstrated in the field. While cultivators expressing Bt genes may currently exhibit resistance to some insect pests, resistance based on the expression of a single gene could eventually be lost due to the evolution of Bt resistance in insects. Therefore, the search for additional genes that can be inserted into plants to provide protection from insect pests is necessary. Scientists have identified some components of specific plants or compounds that act as defense agents to protect a plant from attack by pests and insect pathogens. While such components are usually present only at low levels in various plant tissues, some of them are also capable of being induced at higher levels by the attack of an insect pest or a pathogen. Examples of said defense compounds include alkaloids, terpenes, and various proteins such as enzymes, inhibitors of enzymes and lectins. Of particular interest are derived compounds
of plants that can block or alter normal biomolecular activity and therefore inhibit the growth of insects or kill the insect. The corn rootworm complex (CRW) in the United States consists of three species, Diabrotica barberi Smith and Lawrence (Northern), D. undecimpuncta ta howardi Barber (Southern) and D. virgifera virgifera LeConte (Western). Western and northern species contribute most of the economic damage to corn. The economic damage and control costs are estimated to exceed one billion dollars per year. As noted above, the main concerns of the use of pesticides to control damage by CRW are its negative effect on the environment and the development of resistance by the insect. Corn rotation is becoming less effective than a CRW control method due to the extended delay period in CRW western and to the modified behavior of laying eggs in CRW western. The generation of transgenic plants with resistance to CRW could have a greater economic impact. Unfortunately, there are relatively few, if any, genes available that can control CRW in transgenic plants. Therefore, there is a need for additional major insecticides, particularly those active against CRW. Compositions and methods for the control of insects and other pests is provided. The compositions
they comprise proteins that have pesticidal activities that can be isolated from plants of the genus Pentaclethra. The purified protein, as well as amino acid information and DNA sequence is provided for proteins that have rootworm activity. The DNA sequences encoding the pesticidal proteins can be used to transform plants, bacteria, fungi, yeasts, and other organisms for the control of pests. The compositions and methods of the invention can be used in a variety of systems to control pests that are plant and non-plant pests. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides the amino acid and nucleotide sequence of the corn root worm active ingredient cDNA sequence, pentin-1 from Pentaclethra SEC. of ID NOS. 1 and 2. Figure 2 provides the amino acid and nucleotide sequence of the Pentina-1 cDNA sequence, optimized for enhanced expression of SEC. of ID NOS: 3 and 4. Figure 3 provides the amino acid sequence of the Pentina-1 protein with an underlined portion representing the putative signal sequence. The AFS residues immediately after the signal sequence are the first three residues of the mature protein. The ASK waste starting at five residues from the start of AFS of the
Mature protein is designated to the apparent mature amino terminus region of pentin-1 expressed as the full-length protein and proteolyzed in corn roots. Figure 4 provides the expression cassette for the expression of Pentina-1 sequences. Compositions and methods are provided for controlling pests, particularly plant pests. In particular, novel pesticidal proteins are provided. The proteins are purified from members of the legume family, particularly the genus Pentaclethra de Leguminosa, more particularly the species P. macrophylla and P. macroloba. According to the invention, the pesticidal proteins produced by the members of the genus Pentaclethra can be isolated by methods known in the art. Methods for protein isolation include conventional chromatography, including ion exchange gel filtration and immunoaffinity chromatography by high performance liquid chromatography, such as reverse phase high performance liquid chromatography, ion exchange high performance liquid chromatography. , size exclusion high performance liquid chromatography, high performance chromatographic focus and hydrophobic interaction chromatography etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. See
for example Current Protocols in Molecular Biology, Vols. 1 and 2, Ausubel et al. (eds.), John Wiley & Sons, NY (1988), incorporated herein by reference. Once the purified protein is isolated, the protein or the polypeptides thereof comprised can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypeptides of which it is comprised, can be fragmented as with cyanogen bromide or proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike et al (1982) J. Biol Chem 257: 9751-9758 '; Liu et al (1983) Int. J. Pept. Protein Res. 21: 209-215). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electrospinning on PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automatic sequencers. It is recognized that the N-terminal, C-terminal or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the pesticide protein gene coding. In the same way, antibodies raised against partially purified or purified peptides can
be used to determine the spatial and temporal distribution of the protein of interest. Therefore, tissue in which the protein is more abundant, and possibly more highly expressed can be determined and expression banks can be constructed. Methods for the production of antibodies are known in the art. See for example Antibodies, A Labora tory Manual, Harlow and Lane (eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1988), and references cited therein. See also Radka et al (1983) J. Immunol. 128; 2804; and Radka et al (1984) Immunogenetics 19: 63. Such antibodies can be used to isolate proteins with similar binding domains and proteins tested for activity against insect pests of interest. It is recognized that any combination of methods can be used to purify proteins that have pesticidal properties. As an isolation protocol has been determined, the pesticidal activity can be tested for each fraction of material obtained after each purification step. Said purification protocols will result in a substantially purified protein fraction. By "substantially purified" or "substantially pure" it is intended that the protein be substantially free of any associated compound
normally with the protein in its natural state. "Substantially" protein preparations can be evaluated by the absence of other detectable protein bands after SDS-PAGE as determined visually - or by densitometry scanning. Alternatively, the absence of other amino terminal sequences or N-terminal residues in a purified preparation may indicate the level of purity. The purity can be verified by the rechromatography of the "pure" preparations that show the absence of other peaks by ion exchange, reverse phase or capillary electrophoresis. The terms "substantially pure" or "substantially purified" does not mean that artificial or synthetic mixtures of the proteins with other compounds are excluded. The terms also do not mean that they exclude the presence of minor impurities which do not interfere with the biological activity of the protein and which may be present, for example due to incomplete purification. From the fragments of the protein, the entire nucleotide sequence encoding the protein can be determined by PCR experiments. Thus, fragments obtained from PCR experiments can be used to isolate cDNA sequences from expression banks. See for example, Molecular Cloning, A Laboratory Manual, Second Edition, Vols. 1-3, Sambrook et al (eds.) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor NY (1989), and the references cited therein. In this way, the proteins and nucleotide sequences encoding the proteins can be isolated, which are inhibitory or toxic to the particular insect species. Said proteins and nucleotide sequences of the invention can be used to protect pest plants including insects, fungi, nematode bacteria, viruses or viroids and the like, particularly insect pests. In particular, sequences of proteins and nucleotides that are inhibitory or toxic to insects of the order of Coleoptera can be obtained. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, 'Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera. Pesticidal insects of the invention for the main crop include: Corn: Ostrinia nubilalis, European corn snap; Agrotis ipsilon, nocturnal caterpillar; Helicoverpa zea, corn scythe; Spodoptera frugiperda, devastating; Dia traea grandiosella, southwestern corn tick; Elasmopalpus lignosellus, tiny wheat straw tick; Dia traea saccharalis, sugarcane tick; Diabrotica virgifera, western corn rootworm; Diabrotica
barber), western corn rootworm; Diabrotica undecimpuncta ta howardi, Moor cucumber beetle, Melanotus spp. weevil Cyclocephala borealis, western masked beetle (white larva); Cyclocephala immacula ta, southern masked beetle (white larva); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, bird bug maize; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus, chinche; Melanopl us femurrubrum, red-footed grasshopper; Melanoplus sanguinípes, migratory grasshopper; Delia platura, cresa of corn silk; Agromyza parvicornis, corn leaf miner; Anaphothrips obscrurus, trisanóptero; Solenopsis milesta, ant stealing; Tetranychus urticae, two-spotted spider tick; Busseola fusca, African corn stalk tick (AMB); Sesamia calamistis, African Rose tick (APB); Eldana sacchharina, African sugarcane tick (ASB); Chilo partell us, sorghum stem tick (SSB); Ostrinia furnacalis, Oriental corn tick (OCB); Sesamia nonagrioides, corn tick from Europe / N. Africa; Sorghum: Chilo partell us, sorghum tick; Spodoptera frugiperda, devastating autumn worm; Helicoverpa zea, corn scythe; Elasmopalpus Iignosell us, lower corn stem tick; Underground agrotis, granulated caterpillar; Phyllophaga crini ta, white larva; Eleodes,
Conoderus, and Aeol us spp. , one hundredpies; Oulema melanopus, cereal leaf caterpillar; Chaetocnema pulicaria, corn flying caterpillar; Sphenophorus maidis, corn bug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, sugar cane aphid, Blissus leucopterus, chinche; Contarinia sorghicola, sorghum mosquito; Tetranychus cinnabarinus, carmine spider tick; Tetranychus urticae, two-spotted spider tick; Schizophis graminum, green bug (aphid); Wheat: Pseudia unipuncta ta, devastating worm; Spodoptera frugiperda, autumn moth worm; Elasmopalpus l ignosellus, small wheat straw tick; Agrotis orthogonia, larva western plae; Oul ema melanopus, cereal leaf beetle; Hypera puncta ta, gorgoro sheet of clove leaf; Diabroti ca uncl ecimpuncta ta howardi, cucumber moro beetle; Russian wheat aphid; Schizophis graminum, green insect; Si tobion avenae, English grain aphid; Melanopl us femurrubrum, redlegged grasshopper; Melanoplus differen tialis, differential grasshopper;
Melanoplus sanguinipes, migratory grasshopper; Maye tiola destructor, Mosca Hessian; If todiplosis mosellana, wheat mosquito; Meromyza americana, wheat stem worm; Hylemya coarcta ta, wheat bulba fly; Frankliniella fusca, tobáceo thrips; Cephus cinctus, wheat trunk sawfly; Eriophyes tulipae, curly wheat tick; Sunflower: Suleima helian thana, head moth ofsunflower; Homeosoma ellectellum, sunflower head moth; Zygoramma exclaims thyesis, sunflower beetle; Bo thyrus gibbosus, carrot beetle; Neolasioptera murtpeldtiana, sunflower seed mosquito; Cochylis hospes, sunflower moth in bands; Rachipl usia nu, agentina linker; Smicronyx fulvu. s, gorgoro of sowing of red sunflower; Cylindrocopturus ad. spers. s, gorgoro of sowing of Moorish sunflower; Cotton: Heliothis virescens, tobacco budworm; tielioverpa zea, baga worm; Spodoptera exigua, beet moth worm; Pectinophora gossypiella, pink baga worm; An thonomus grandis, gorgoro de baga; Aphis gossypii, cotton aphid; Pseuda tomoscel is serious, your cotton flea; Trial eurodes abutilone, white fly with wings in bands; Lygus lineolaris, insect of spotted plants; Melanoplus femurrubrum, red-footed grasshopper; Melanoplus differen tial is, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella filsca, tobáceo thrips; Tetranychus cinnabarinus, carmine spider tick / Tetranychus urti falls, twospotted spider mite; Rice: Dia traea sacchuralis, cane sugar tick; Spodoptera frugiperda, autumn moth worm; Helicoverpa zea, corn land worm; Colaspis brzmnea, grape collapser; Lissorhoptrus oryzophilus, gorgoro of rice water; If tophil us oryzue, gorgoro of rice; Nephotettix nigropíctus, leaf jumper
of rice.; Blissus leucopterus, chinche; Acrosternum hilare, green wood insect; Soy: Pseuclopl usia incl uclens, soybean locker; Anticarsía gemma talis, caterpillar of alcatan; Pla thypena scabra, green clover worm; Os trinia nubilalis, European corn tick; Agrotis Ípsilon, black larva; Spodoptera exigua, beet moth worm; Heliothis virescens, cotton baga worm; Helicoverpa zea, cotton baga worm; Epilachna varivestis, Mexican bean beetle; Myzas persicae, green peach aphid; Empoasca fabae, potato leaf springer; Acrosternum hilare, green fato insect; Melanoplus femurrubrum, red-legged grasshopper; Melanoplus differen tialis, differential grasshopper; Delia platura, sowing wheat worm; Sericothrips variablis, soybean thrips; Thrips ta aci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, spider mite with two spots; Barley: Ostrinia nubilalis, European corn tick; Agrotis Ípsilon, black larva; Schizophis graminum, green insect; Blissus leucopterus, chinche; Acros ternum hilare, insect green fato; Euschistus serrus, insect fato coffee; Delia platura, sowing wheat worm; Mayetiola destructor, Hessian fly; Petrobia la tens, k brown wheat; Oilseed rape: Brevicoryne brassicae, cabbage aphid; Flea beetle, Phyllotreta spp.; Bertha moth worm; Mamestra confgura ta; Back-shaped moth
Diamond; Pl utella xylostella; Alfalfa: alfalfa harvester, Autographa californios; alfalfa beetle, Otiorhynchus ligusticií; lucerne caterpillar, Colias eurytheme; alfalfa stain climber, Agronyza fron tella; gorgoro of Egyptian lucerne, Hypera brunneipeonis; prairie grass insect, Philaerius spumarius; aphid of Moro alfalfa, Theriophis mecula ta; gorgoro of trevol leaf, Hypera puncta ta; pea aphid, Acyrthosiphon pisum; blue alfalfa aphid, Acyrthosiphor kondoi; green clover worm, Pla thypena scabia; clover root curculio, Si tona hispidulus; chalcid of alfalfa seed, Brachophagus roddi; spotted plant insect, Lygus lineolaris; insect fato, Chlorochroa sayi; caterpillar of velvety bean, Anticarsia friegiperda, gorgoro - of alfalfa, Hypera postica; autumn moth worm, Spodoptera; potato climber, Empoasca fabae; soybean crusher, Psuedol usia incl udens; Alfalfa tepador with three angles surrounded, Spissistil us festinus; See, for example, Manya B. Stoetzel (1989) Common Ñames of Insects & Related Organisms, Enlomological Society of America, incorporated herein by reference. The nucleotide sequences of the invention can be used to isolate other homologous sequences in other plant species, particularly other legume species. The methods are available in the art for the hybridization of nucleic acid sequences. The
Coding sequences from other plants can be isolated according to well-known techniques based on their sequence homology to the coding sequences shown herein. In these techniques all or part of the coding sequence is used as a probe that selectively hybridizes to other coding sequences of pesticides present in a population of cloned genomic DNA fragments or cDNA fragments (ie genomic cDNA libraries) of an elected body. For example, the entire Pentina-1 sequence or portions thereof can be used as probes capable of "specifically hybridizing to the corresponding coding sequences and messenger RNAs." To achieve specific hybridization under a variety of conditions said probes include sequences which are unique and preferably are less than about 10 nucleotides in length and more preferably at least 20 nucleotides in length.These probes can be used to amplify pentin-1 coding sequences of an organism chosen by the well-known process of the reaction Polymerase chain (PCR) This technique can be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of? entin-1 coding sequences in an organism.
Such techniques include sieving by hybridization of plaque-plated DNA banks (any plaques or colonies, see for example Sambrook et al., Molecular Cloning, eds., Cold Spring Harbor Laboratory Press (1989)) and amplification using PCR of oligonucleotide primers. which correspond to domains of conserved sequences between the amino acid sequences (see, for example Innis et al., PCR Protocols, to Guide to Methods and Applications, eds., Academic Press (1990)). For example, hybridization of the sequences can be carried out under reduced restriction conditions, medium restriction or restriction conditions (for example, conditions represented by a wash restriction of 35-40% formamide with 5x Denhardt's solution, 0.5 % SDS and lx SSPE at 37 ° C, conditions represented by a wash restriction of 40-45% formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSP at 42 ° CM and conditions represented by a restriction of 50% formamide wash with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42 ° C, respectively), to DNA encoding insecticide genes described herein in a normal hybridization analysis. See J. Sambrook et al., Molecular Cloning A Labora tory Manual 2d Ed. (1989) Cold Spring Harbor Laboratory.
The terms "stringent conditions" or "stringent hybridization conditions" include the reference for conditions under which a probe can be hybridized to its target sequence to a detectably greater extent than other sequences (eg, at least 2 folds over the background) . Strict conditions depend on the sequence and will be different in different circumstances. By controlling the restriction under the conditions of hybridization and / or washing, the white sequences can be identified which are 100% complementary to the probe (homologous probe). Alternatively, the restriction conditions can be adjusted to allow some sequence inequality so that lower degrees of similarity are detected (heterologous sounding). In general, a probe is less than about 1,000 nucleotides in length, preferably less than about 500 nucleotides in length, typically about 50 about 300 nucleotides in length. Normally, the stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, normally about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature at least is 30 for short probes (for example, 10 to 50 nucleotides) and at least around 60 ° C for long probes (for
example, greater than 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low restriction conditions include hybridization with a pH buffer of 30 to 35 percent formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C and a wash at IX to 2X SSC (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) from 50 ° C to 55 ° C. Illustrative moderate restriction conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 ° C, and a 0.5X to IX SSC wash at 55 ° C to 60 ° C. Illustrative high restriction conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 ° C, and a 0.1X SSC wash at 60 ° C to 65 ° C. The specificity is usually the function of washings after hybridization, the critical factors being the ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem 138: 267-284 (1984): Tm = 81.5C + 16.6 (log M) + 0.41 (% GC) -0.61 (form%) - 500 / L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in DNA,% is the percentage of formamide in guanosine nucleotides and cytosine in DNA,% is the percentage of formamide in the solution of
Hybridization and L is the length of the hybrid in base pairs. The Tm is the temperature (under the defined ionic strength and pH) at which 50% of a complementary white sequence is preferably hybridized to a matched probe. Tm is reduced by approximately 1 ° C for every 1% inequality; therefore, Tm, hybridization and / or washing conditions can be adjusted to hybridize sequences of the desired identity. For example, if the sequences with > 90% identity is sought, the Tm can decrease 10 ° C. Generally, the restriction conditions selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence and its complement to a defined ionic strength and pH. However, severely stringent conditions can utilize hybridization and / or washing at 1, 2, 3 or 4 ° C lower than the thermal melting point (Tm); moderately stringent conditions may utilize a hybridization and / or wash at 6, 7, 8 or 9 or less than the thermal melting point (Tm); the low restriction conditions can use a hybridization and / or washing at 11, 12, 13, 14, 15 or 20 lower than the thermal melting point (Tm) using the equation, hybridization and washing compositions and desired Tm to those of experience ordinary will understand that variations in the hybridization restriction and / or wash solutions are inherently described. If the degree
The desired inequality results in a Tm less than 45 ° C (aqueous solution) or 32 ° C (formamide solution) it is preferred to increase the concentration of SSC so that a higher temperature can be used. An extensive guide to nucleic acid hybridization is found in Tijssen, Labora tory Techniques in Biochemistry and Molecular Biology - Hybridization, Nucleic Acid Probes, Part I, Chapter 2"Overview of Principles of Hybridization and the Strategy of Nucleic Acid Test assays ", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). In general, the sequences encoding Pentina-1 and other insecticidal peptins of the invention and hybridizing to the gene described herein will be at least approximately 50% homologous, approximately 70% homologous, up to about 85% homologous or homologous up to about 90% to about 95% with the sequence described. That is, the sequence similarity of the sequence can vary by sharing at least about 50%, about 70% and about 85% up to about 90% to 95% sequence similarity. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "window"
for comparison ", (c)" sequence identity ", (d)" percentage of sequence identity "and (e)" substantial identity. "(a) As used herein," reference sequence "is a sequence defined as a basis for the comparison of sequences A reference sequence can be a subgroup or the entirety of a specific sequence, for example as a segment of a full-length cDNA or gene sequence, or the entire cDNA or sequence of gene (b) As used herein, "comparison window" means that it includes reference to a contiguous and specific segment of a polynucleotide sequence, wherein the polynucleotide sequence can be compared to a reference sequence and in wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (ie, spaces) compared to the reference sequence (which does not comprise additions or deletions for optimal alignment). ma of the two sequences. In general, the comparison window is at least 20 contiguous nucleotides in length, and optionally may be 30, 40, 50, 100 or longer. Those experts in the material will understand that to avoid a high similarity to a reference sequence due to the inclusion of spaces in the sequence of
polynucleotides, a space of penalty is usually introduced and subtracted from the number of equalities. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be carried out by the local homology algorithm of Smithy aterman, Adv. Appl. Ma th 2: 482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 45: 443 (1970); by the investigation of the similarity method of Pearson and Lipman, Proc. Na ti. Acad. Sci. 85: 2444 (1988); for computerized implementations of these algorithms including, but not limited to, CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Groups (GCG) , 575 Science Drive, Madison, Wisconsin, USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet et al., Nucleic Acids Research 15: 10881-90 (1988); Huang et al., Compu ter Applications in the Biosciences S: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 301-331 (1994). The BLAST family of programs that can be used for database similarity investigations includes: BLASTN for nucleotide screening sequences against nucleotide sequences; BLASTX for sequences of
research of nucleotides against protein database sequences; BLASTP for protein research sequences against protein database sequences; TBLASTN for research sequences of proteins against nucleotide base sequences; and TBLASTX for nucleotide research sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds. Greene Publishing and Wiley-Interscience, New York (1995). As those skilled in the art will understand, investigations in BLAST assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences that may be homopolymer tracts, short period repeats or regions enriched in one or more amino acids. Said regions of low complexity can be aligned between unrelated proteins although other regions of proteins are completely different. A number of low complexity filter programs can be used to reduce such low complexity alignments. For example, the low complexity filters SEG (Wooten and Federhen, Comput.Chem, 17: 149-163 (1993) and XNU (Claverie and States, Comput.Chem., 17: 191-201 (1993)) that can be used alone or in combination.
(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid sequences or polypeptides includes references to the residues in the two sequences which are the same when aligned for maximum correspondence on a specific comparison window. When the percentage of sequence identity is used in reference to proteins it is recognized that positions in the residues that are identical often differ by conservative amino acid substitutions, where the amino acid residues are substituted for other amino acid residues with properties similar chemicals (eg, charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When the sequences differ and in conservative substitutions, the percent sequence identity can be adjusted upwardly to correct the conservative nature of the substitution. The sequences that differ by said conservative substitutions are those that have "sequence similarity" or "similarity". The means for making this adjustment are well known to those skilled in the art. Normally this involves classifying a conservative substitution as a partial inequality instead of a complete inequality, thus increasing the percent sequence identity. Therefore, for example, where even an identical amino acid is given a classification of 1 and a
Non-conservative substitution is given a zero classification, a conservative substitution is given a classification between zero and 1. The classification of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4: 11-11 (19878) for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, California, USA). (d) As used herein, "percent sequence identity" means the value determined to compare two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison sale may comprise additions or deletions (ie, spaces) compared to the reference sequence (which does not comprise additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the nucleic acid base or identical amino acid residues are presented in both sequences to give the number of different positions, dividing the number of equal positions by the total number of positions in the advantage of comparison and multiplying the result by 100 to give the percentage of sequence identity. (e) (i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide
it comprises a sequence having at least 70% sequence identity, preferably at least 80 ', more preferably at least 90% and even more preferably at least 95%, compared to a reference sequence using only one of the alignment programs described using normal parameters. One with experience will recognize that these values can be adjusted approximately to determine the corresponding identity of proteins encoded by two nucleotide sequences taking into account codon degeneracy, amino acid similarity, reading frame placement and the like. The substantial identity of the amino acid sequences for this purpose usually means the sequence identity of at least 60%, more preferably at least 70%, 80%, 90% and more preferably at least 95%. Another indication that the nucleotide sequences are substantially identical as if two molecules hybridized to one another under normal conditions. However, nucleic acids that do not hybridize to one another under stringent conditions are substantially identical and the polypeptides they encode are substantially identical. This can occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code. An indication that two sequences of
Nucleic acids are substantially identical in that the polypeptide which is the first nucleic acid coding is of immunologically cross-reactive reaction with the polypeptide encoded with the second nucleic acid sequence. (e) (ii) The terms "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, even more preferably at least 90% or 95% sequence identity for the reference sequence over a specific comparison window. Preferably, the optimal alignment is carried out using the homology alignment algorithm of Needleman and Wunschm J. Mol. Biol. 48: 443 (1970). An indication that two peptide sequences are substantially identical is that a peptide is immunologically reactive with antibodies raised against the second peptide. Therefore, a peptide is substantially identical to a second peptide, for example, wherein the two peptides differ only by a conservative substitution. Peptides that are "substantially similar" share sequences as noted above except that positions of residues that are not identical may differ by conservative amino acid changes.
It is recognized that pesticidal proteins can be oligomeric and that they will vary in molecular weight, number of promoters, component peptides, activity against particular pests and in other characteristics. However, by the methods set forth herein, proteins active against a variety of pests can be isolated and characterized. Of particular interest are proteins that are active against the corn rootworm (CRW). Therefore, the purified or partially purified proteins of the invention are tested for insecticidal activity against corn rootworm, including Diabrotica barberi
(Northern), D. undecimpuncta ta howardi (Southern), and D. virgifera vergifera (Western). In this way, a protein designated Pentina-1 has been isolated which has insecticidal activity for the corn rootworm. Pentina-1 is a glycosylated protein of about 45 about 50 kDal. The amino acid and nucleotide sequence of the Pentina-1 protein is given in Figure 1 and SEC. of ID. NOS 1 and 2- The highest concentration of Pentina-1 in the plant seems to be in the mature seeds. The protein is heat stable and has an LC50 of approximately 10 μg / ml of diet against the corn rootworm. Pentina-1 and other proteins of the invention can be altered in various ways including substitutions,
deletions, truncations and amino acid insertions. Methods for such manipulations are generally known in the art. For example, the amino acid sequence variants of the pesticidal proteins can be prepared by mutations in the DNA. Methods for mutagenesis and alterations in nucleotide sequence are well known in the art. See for example, Kunkel, T. (1985) Proc Na ti. Acad Sci, USA 82: 488-492; Kunkel et al (1987) Methods in Enzymol. 154: 361-382; U.S. Patent No. 4,873,192; Walker and Gaastra (eds.) Techniques in Molecular Biology, MacMillan Publishig Company, NY (1983) and the references cited therein. Therefore, the genes and nucleotide sequences of the invention include both the sequences present in nature and the mutant forms. Likewise, the proteins of the invention encompass both the proteins present in nature as well as the variations and modified forms thereof. Said variants will continue to have a desired pesticidal activity. Obviously, the mutations that will be made in the DNA encoding the variant should not place the sequence outside of the reading frame and preferably would not create complementary regions that could produce secondary RNA structures. See, EP Patent Application Publication No. 75,444.
In this way, the present invention encompasses the pesticidal proteins as well as components and fragments thereof. That is, it is recognized that the promoters of components, polypeptides or fragments of the proteins can be produced which retain pesticidal activity. These fragments include truncated sequences, as well as N-terminal, C-terminal internal and internally suppressed amino acid sequences of the proteins. Most deletions, insertions and substitutions of protein sequences are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of substitution, deletion, or insertion in their advancement, one skilled in the art will appreciate that the effect will be evaluated by routine screening analysis. In other words, the activity can be evaluated by insect toxicity analysis. The nucleotide sequences can be used intermixed DNA protocols. The intermixing of DNA is a process for recursive recombination and mutation, carried out by the random fragmentation of a coalition of related genes, by the reassembly of the fragments and PCR without primers. See, for example Stemmer, W.P.C. (1994) Proc. Na ti. Acad. Sci. USA 91: 101-1-10151; Stemmer, W.P.C. (1994) Na ture 370: 38 9-391; Zhang et al (1997)
Proc. Na ti. Acad. Sci. USA 94: 4504-4509; and PCT Publication No. 96/19256. An advantage of DNA intermixing of a rotational design is that intermixing can optimize gene function without first determining which gene product is regime-limiting. The present invention provides methods for intermixing sequenced using polypeptides of the invention and compositions resulting therefrom. Generally, sequenced intermixing provides a means to generate libraries of polynucleotides having a desired characteristic that can be selected or screened. Banks of recombinant polypeptides are generated from a population of polypeptides of related sequences that comprise sequenced regions that have substantial sequence identity and that can be homologously recombined in vitro or in vivo. The population of sequenced-recombined polynucleotides comprises a subpopulation of polynucleotides having desired or advantageous characteristics and which can be selected by a suitable selection or screening methods. The characteristics may be any property or attribute capable of being selected for, or detected in a screening system and may include properties of: an encoded protein, a transcriptional element, a sequence control transcript, RNA processing,
RNA stability, chromatin confirmation, translation or other expression property of a gene or transgene, an element of replication, a protein binding element or the like, such as any characteristic that confers a selectable or detectable property. In some embodiments, the selected feature will have one Km and / or Kcat increased over the wild type protein as tested herein. In other embodiments, a protein or polynucleotide generated from the sequenced entanglement will have a higher ligand binding affinity than the non-interspersed wild type polynucleotide. The increase in said properties may be at least 110%, 120%, 130%, 140% or at least 150% of the wild type value. Pentina-1 is a member of a wider gene family of esterases, and more specifically acyl hydrolases of lipids as determined by sequence similarity. The intermingling of genes is a method that can improve or alter a biological activity of a given gene product. The intermixing of the gene, together with a selection strategy, can be used to improve properties such as substrate specificity, solubility, temperature and optimal pH of a protein or enzyme by directed molecular evolution. In the case of Pentina-1 toxicity towards insects as determined by lethal concentrations is a more relevant parameter.
The intermingling of genes can be applied to a single gene that introduces mutations into the gene at a given frequency. The combinations of synergistic mutations that can be selected by subsequent generations of intermingling of genes from the primary mutant population. This approach can be applied to Pentina-1. Alternatively, different members of the gene families are encoded by divergent or related sequences can be used for the intermixing of genes. This may include but not be limited to Pentaclethra's Pentina-1 and an expressed corn sequence tag identified as 5C9 that encodes a cDNA that is approximately 57% identical to the pentin-1 level of nucleotides. See co-pending patent application 08 / 449,986 filed May 25, 1995, incorporated herein by reference. Concomitant mutations will also be introduced by intermixing genes further contributing to the diversity of genetics. Then synergistic combinations of fusions between members of the gene family and newly introduced mutations can be selected by directed molecular evolution strategies. Acyl lipid hydrolases comprise a family of multiple diverse genes that are conserved through
of many plant species. The enzymes exhibit hydrolyzing activity for many glycols and phospholipids. Substrates include monogalactosyldiacylglycerol, acylterglycoside, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, as well as many other lipid substrates. Similarly, the membrane composition of various insects as well as plants can vary from species to species and can be affected by diet or growth conditions. Consequently, the activity of a given lipid acyl hydrolase for a given substrate could affect both specificity and potency. The altered substrate specificity could be a parameter to select products of intermingling of genes. Protein solubility and stability can also be selected from intermixed gene products. Insecticidal proteins are active in the severe environment of the lumen of the insect's intestine. Their proteins are digested by proteases and are affected by reduction or oxidation conditions that vary according to the species of insects tested. The solubility and stability of acyl hydrolases of lipids in both the transgenic plant and in the intestine lumen of insects could affect biological activity and may be altered by the strategies of entanglement of genes.
Conditions for the reaction of enzymes such as optimum pH and temperature can also affect the insecticidal activity of Pentina-1. The intestinal pH of the corn rootworm is 5.5-6.0. The section of the products of genes of Pentina-1 intertwined for enzymatic activity towards lipid substrates in this pH scale is of another parameter that could affect the toxicity. Therefore, the pentin sequence of the present invention can be used in the entanglement experiments of genes with other lipid hydrolases such as patatins, and in particular with 5C9. The proteins or other component polypeptides described herein may be used alone or in combination with other proteins or agents to control different insect pests. Other insecticidal proteins include those of Basillus, including d-endotoxins and vegetative insecticidal proteins as well as protease inhibitors (both types of serine and cysteine), lectins, α-amylases, peroxidases, cholesterol oxidase, and the like. In one embodiment, the expression of the proteins of the invention in a transgenic plant is accompanied by the expression of one or more d-endotoxins of Bacillus thuringensis (Bt). This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically treating a plan to contain and express all the Necessary genes Alternatively, a plant, Parent 1, can be genetically treated for the expression of proteins of the invention. A second plant, Father 2, can be treated genetically for the expression of other principles such as Bt d-endotoxins. Crossing Father 1 with Father 2, progeny plants can be obtained that express all the genes present in both Parents 1 and 2. The present invention also encompasses nucleotide sequences from organisms other than Penta cle thra, where the proteins cross-react with antibodies raised against the proteins of the invention or wherein the nucleotide sequences are isolated by hybridization with the nucleotide sequences of the invention. Proteins isolated or encoded by the nucleotide sequences can be tested for pesticidal activity. The isolated proteins can be analyzed for pesticidal activity by the methods described herein or others well known in the art. In another embodiment, the proteins of the invention can be used in combination with seed coatings available in the art. In this way, the transformed seeds are coated with applications of available insecticide sprays or powders. Such insecticides are known in the art. See for example North American Patents Nos. 5,696,144; 5,695,763; 5,420,318;
,405,612; 4,596,206; 4,356,934; 4,886,541; etc., incorporated herein by reference. Once the nucleotide sequences encoding the pesticidal proteins of the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants. The proteins of the invention can be used to protect agricultural crops and pest products by introducing via a suitable vector into a microbial host and the host applied to the environment or plants. Guests of microorganisms can be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and / or rhizoplane) of one or more crops of interest. These organisms are selected so that they are able to compete successfully in the environment in particular with wild-type microorganisms, provide stable maintenance and expression of the polypeptide-expressing gene, and conveniently provide improved pesticide protection from the degradation and environmental inactivation. The proteins of the invention can be used in expression cassettes for expression in any host of interest. Said expression cassettes will comprise a transcriptional initiation region linked to the gene that
encodes the pesticide gene of interest. Said expression cassette is provided with a plurality of restriction sites for the insertion of the gene of interest that will be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes suitable for the particular host organism. The transcriptional initiation region, the promoter, may be natural or analogous or foreign or heterologous to the host. Additionally, the promoter may have a natural sequence or alternatively have a synthetic sequence. It is pretended that the transcriptional initiation region is not based on the wild-type host into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to the transcription initiation region that is heterologous to the coding sequence. While any promoter or promoter element capable of driving the expression of a coding sequence can be used, the root promoters are of particular interest for expression in plants (Bevan et al (1993) in Gene Conservation and Exploitation.) Proceedings of the 20th Stadler Genetics
Symposium, Gustafson et al (eds.) Plenum Press, New York pp. 109-129; Brears et al. (1991) Plan t J. 1: 235-244; Lorenz et
al (1993) Plant J. 4: 545-554; US Patent Nos. 5,459,252; 5,608,149; 5,599,670);; marrow (U.S. Patent Nos. 5,466,785; 5,451,514; 5,391,725); or other tissue-specific and constitutive promoters (See, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142, incorporated herein by reference.) The transcriptional cassette will include in the 5 'direction -3 'of transcription, a region of transcriptional initiation and translation, a DNA sequence of interest and a transcriptional termination and functional translation region in plants.The termination region may be natural with the transcriptional initiation region, may be native with the DNA sequence of interest or can be carried from another source Convenient termination regions are available from Ti plasmid and A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. al., (1991) Mol Gen. Genet 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes D Ev. 5: 141-149; Mogen et al. (1990) Plan t Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Bailas et al. (1989) Nucleic Acids Res. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.
The nucleotide sequences encoding the proteins or polypeptides of the invention are particularly useful in the genetic manipulation of plants. In this way, the genes of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5 'and 3' regulatory sequences operably linked to the gene of interest. The gene may additionally contain at least one additional gene that will be cotransformed or ligated and transformed in the organism. Alternatively, the genes of interest may be provided in another expression cassette. When appropriate, the genes can be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using preferred codons of plants for improved expression. The methods are available in the art to synthesize preferred plant genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference. Depending on where the DNA sequence of interest is to be expressed, it may be convenient to synthesize the sequence with preferred codons of plants or alternatively with preferred codons of chloroplasts. The preferred codons of plants can be determined from the codons of higher frequency in the proteins expressed in the highest amount in the plant species.
particular of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Na ti. Acad. Sci. USA 88: 3324-3328; and Murray et al. (1989). Nuclei c Acids Research 17: 477-498. In this way, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthetic. "That is, synthetic or partially optimized sequences can also be used.
Additional sequence modifications are known to improve the expression of genes in a cellular host. These include the elimination of sequences encoding false polyadenylation signals, exons-introns cleavage site signals, transposon-like repeats and other well-characterized sequences that may be detrimental to gene expression. The G-C content of the sequences can be adjusted to average levels for a given cell host, as calculated by reference in the known genes expressed in the host cell. When possible, the sequence can be modified to avoid the secondary mRNA structures provided by crochet. The expression cassettes can additionally contain 5 'leader sequences in the construction of expression cassettes. These leading sequences can act to improve translation. Translation leaders know each other
in the matter and include: leaders of picornaviruses, for example leader of EMCV (5 'non-coding region of Encephalomyocarditis) (Elroy-Stein, 0., Fuerst, TR, and Moss, B. (1989) PNAS USA, 86: 6126-6130); potivirus leaders, for example, leader of TEV (Tobacco etching virus) (Allison et al. (1986)); MDMV leader (Maize Dwarf Mosaic Virus) Virology, 154: 9-20); and human immunoglobulin thick chain binding protein (BIP), (Macejak, DG, and Sarnow, P. (1991) Na ture, 353: 90-94, untranslated leader of the mosaic virus mRNA coating protein of alfalfa (AMV RNA 4), (Jobling, SA, and Gehrke, L., (1987) Na ture, 325: 622-625), leader of tobacco mosaic virus (TMV), (Gallie, DR et al. (1989) Molecular Biology of RNA, pages 237-256) and leader of maize chlorotic vein virus (MCMV) (Lommel, SA et al. (1991) Virology, 81: 382-385) See also, Della-Cioppa et al. (1987) Plan t Physiology, 84: 965-968 Other known methods for improving translation can also be used, for example introns and the like The genes of the present invention can be directed to the chloroplast or amyloplast for expression. In this form, where the gene of interest is not directly inserted into the chloroplast or amyloplast, the expression cassette will additionally contain a gene encoding a peptide tem poral to direct the gene of interest to the chloroplast.
Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) Biol. Chem. 264: 17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481. The construct can also include any other necessary regulator such as nuclear localization signals (Kalderon et al (1984) Cell 39: 499-509; and Lassner et al. (1991) Plant Molecular Biology 17: 229-234); consensus sequences of translational translation of plants (Joshi),
C.P. (1987) Nucleic Acids Research 15: 6643-6653), introns
(Luehrsen and Walbot (1991) Mol, Gen. Genet, 225: 81-93) and the like, operably linked to the nucleotide sequence of interest. It is recognized that the protein can be expressed comprising the sequences of native signals. See Figure 3. Alternatively, other signal sequences in the art, for example the barley alpha amylase signal sequence can be used. To prepare the expression cassette, several DNA fragments can be manipulated so as to provide the DNA sequences in the proper orientation and as appropriate, in the appropriate reading frame. To this end, adapters or linkers can be used to join DNA fragments
or other manipulations may be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, repair in the primer, restriction, recosido, resecado, binding, PCR, or the like can be used, where insertions, deletions or substitutions may be involved, for example, transitions and transversions. The compositions of the present invention can be used to transform any plant. In this way, genetically modified plants, plants, plant tissues, seeds and the like can be obtained. Transformation protocols may vary from the type of plant or plant cell, i.e., monocotyledons or dicotyledons targeted for transformation. Suitable methods for transforming transformation plant cells include microinjection (Crossway et al (1986) Bio techni ques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Na ti. Acad. Sci. USA, 83: 5602-5606, Agrobacterium-mediated transformation (Hinchee et al. (1988) Biotechnology, 6: 915-921), direct gene transfer (Paszkowski et al.
(1984) EMBO J., 3: 2111-2122), and acceleration of leaf spring particles (see, for example, Sanford et al., Patent
North American No. 4,945,050; and, McCabe et al. (1988) Bio technology, 6: 923-926). Also see, Weissinger et al.
(1988) Annual Rev. Genet., 22: 421-477; Sanford et al. (1987)
Particulate Science and Technology, 5: 27-37 (onion);
Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean);
McCabe et al. (1988) Bio / Technology, 6: 923-926 (soybean); Datta et al. (1990) Biotechnology, 8: 736-740 (rice); Klein et al.
(1988) Proc. Nati Acad. Sci. USA, 85: 4305-4309 (corn); Klein et al. (1988) Biotechnology, 6: 559-563 (corn); Klein et al.
(1988) Plant Physiol., 91: 440-444 (corn); Fromm et al. (1990)
Biotechnology, 8: 833-839; Tomes et al. "Direct DNA transfer into intact plant cells via microprojectile bombardment, In:
Gamborg and Phillips (eds) Plant Cell, Tissue and Organ
Culture: Fundamental Methods, Springer-Verlag, Berlin, 1995
(corn); Hooydaas-Van Slogteren & Hooykaas (1984) Nature
(London), 311: 763-764; Bytebier et al. (1987) Proc. Nati Acad. Sci. USA, 84: 5345-5349 (Liliaceae); De Wet et al.
(1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., Pp. 197-209. Longman, NY (pollen); Kaeppler et al. (1990) Plant Cell Reports, 9: 415-418; and Kaeppler et al. (1992) Theor. Appl. Genet., 84: 560-566 (mediated transformation-brush); D = Halluin et al. (1992) Plant Cell, 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports, 12: 250-255 and Christou and Ford (1995) Annals of Botany, 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology, 14: 745-750 (corn via Agrobacterium
tumefaciens); all of which are incorporated here by reference. Where it is convenient, the plant plastid can be transformed directly. Stable transformation of plastids has been reported in higher plants, see for example SVAB et al. (1990) Proc. Na ti. Acad. Sci. USA 87: 8526-8530; SVAB & Maliga (1993) Proc. Na ti. Acad. Sci. USA 90: 913-917; Staub & Maliga (1993) Embo J. 12: 601-606. The method is based on the delivery with a DNA particle gun containing a selectable marker and a DNA direction to the plastid genome through homologous recombination. Additionally, the transformation of plastids can be achieved by the transactivation of a transgene with plastics at rest by the tissue-specific reaction of a nuclear-encoded RNA polymerase directed to the plastid. Ducho system has been reported in McBride et al. (1994) Proc. Na ti. Acad. Sci. USA 91: 7301-7305. The cells that have been transformed can be grown in plants according to conventional forms. See, for example, McCormick et al. (1986) Plant Cell Reports, 5: 81-84. These plants can be developed and polinated with the same transformed strain or different strains and the resulting offspring having the desired phenotypic characteristics identified. Two or more generations can be developed to ensure that the phenotypic characteristic of the subject is
maintain stably and be hereditary and then the amides are cultured to ensure the desired phenotype or other property or that another property has been achieved. The proteins will be expressed in transformed organisms in amounts that are toxic to the insects of interest or inhibitors for the development of insects. The following examples are offered by way of illustration and not by way of limitation. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention described. EXPERIMENTAL Purification of Pentina-1 Seeds of P. macroloba were collected from the humid lowland forest of Costa Rica and transported to the laboratories of the inventors where they were cut, lyophilized and stored at -20 ° C before use. The frozen seeds were cut into smaller pieces and homogenized using a Brinkman homogenizer. In a normal procedure, 10 g of seed material were homogenized with 1-2 g of insoluble polyvinylpyrrolidone and 50-100 ml of 10 mM phosphate buffer solution.
sodium, pH 7.5. The homogenate was then stirred at 4 ° C for 8 to 10 hours and centrifuged at 5,000 rpm for 15 minutes. The supernatant fluid was carefully decanted and poured through a single layer of Miracloth, and was collected in a manner that prevented the transfer of lipid-like materials in the extract that separated and solidified on the surface during centrifugation. The pellet was discarded and the recovered liquid that was still nebulized was centrifuged a second time at 18,000 rpm in a Sorval SS-34 rotor or its equivalent for 30 minutes. The slightly cloudy supernatant liquid, hereinafter called the crude extract, was recovered and the pellet was discarded. A sample of the crude extract was saved for testing and the remainder was dialyzed using cut membrane (MWCO) of molecular weight of 3,500 against five changes of 10 mM sodium phosphate pH buffer, pH 7.5 at 3 ° C to 4 ° C. The ratio of dialysis fluid to extract was at least 20: 1. Dialysis was continued for 8 to 16 hours by exchange of buffer solution. The extract became somewhat turbid during dialysis as a result of the precipitation of the protein. Therefore, the dialyzed extract was clarified by centrifugation at 18,000 rpm for 30 minutes to remove the denatured proteins. The resulting material, after the centrifugation hereafter, is called crude dialyzed extract. The extracts dialyzed and
Raw dialysates were analyzed for composition or protein content and found to contain a substance that was insecticidally active against the corn rootworm (CRW) in biological analyzes. The insecticide was found to be a protein or protein substance. A 100 ml sample of the dialyzed crude extract was heated to about 80 ° C using a water bath and kept at this temperature for about 5 minutes. The hot extract was then cooled to less than 25 ° C using an ice bath and then cooled, centrifuging for 15-30 minutes at 18,000 rpm using a Sorval SS-34 rotor. The clear supernatant fluid was removed, stored and designated from witch onwards as the heat-treated extract. The material formed in pellets was discarded. It was observed that sometimes the heat-treated extract receives a tendency to gel. The heat treated extract was protein analyzed using the Brandford method with BSA as normal and was found to have insecticidal activity against CRW in biological analysis. A sample of the hot extract was fractionated and concentrated using ammonium sulfate. The sample was cooled using an ice bath and ammonium sulfate powder, 0.6 g / ml sample, added slowly with stirring. Once the addition was complete, ammonium sulfate, the sample was maintained at ice bath temperatures during
approximately 30 minutes. The sample was then centrifuged at 4 ° C for 20 minutes at 18,000 rpm using a Sorval SS-34 rotor. The supernatant liquid and the material formed into pellets were separated and the material formed into pellets was re-solubilized in a minimum amount of 10 mm sodium phosphate buffer, pH 7.5 and extensively dialyzed against its sodium phosphate buffering action and 10 mM, pH 7.5. The supernatant liquid and the material formed in resolubilized pellets were analyzed for protein content by the Bradfort method using BSA as the standard and tested for biological activity against CRW. The majority of the Pentina-1 was found in the material formed in pellets and was insecticide against CRW. Alternatively, the volume of the hot extract was reduced by centrifugal concentration using Contricon ™ or similar concentration devices according to the manufacturer's instructions. Proteins were also fractionated by size exclusion chromatography on a Pharmacia Sephacryl S-200 column or a Pharmacia Superego column 12. Different sizes of columns were used depending on the amount of protein in the sample that was chromatographed. Generally, the sample volume was not greater than 0.5-1% of the column volume. The column was equilibrated with at least two or three column volumes of 10 mM sodium phosphate buffer, pH 7.5, before the sample was collected.
will apply to the column. Proteins were eluted from the column with sodium phosphate buffer and 10 mM pH 7.5. Fractions were analyzed to contain protein by the Bradford method using BSA as the standard and bioanalyzed using corn rootworm larvae. Crude or dialyzed extracts, hot extracts, fractions resolved after the precipitation of ammonium sulfate, and extracts of concentrated fractions by other methods can be chromatographed by this method. The biologically active material was eluted just after the void volume. Suggesting that the active material is of moderately high molecular weight. This result is consistent with the size calculations obtained using Centricon ™ filter devices with different molecular weight scales. The latter indicated that the active material has a natural molecular weight greater than 100 kFa, the likely result of combining a plurality of subunits of molecular weight 40-55 + 5 Kda. The fraction purity was calculated after molecular weight termination using SDS-PAGE as described below. These fractions were essentially pure with a primary band detected with a subunit molecular weight estimated at the scale of 40-55 + 5 kDa. Heat treated samples or samples that were subjected to size exclusion chromatography were fractionated by anion exchange chromatography using
a column of Pharmacia Q Sepharose or a column of Pharmacia Q. Before placing the sample in the column, the column was first washed with 25 mM Tris-HCl or suitable buffer solution containing 1 M NaCl, and then equilibrated with the same solution Regulator without NaCl. The pH of the buffer used in the chromatography varied between pH 4 and pH 10. In order to illustrate the methods, chromatography, using buffer solution of Tris-HCl of 25 mM, pH 9.0 is described herein. Before injecting the sample into the column, the sample was dialyzed using a MWCO membrane of 3,500 through 2-3 exchanges of 25 mM HCl buffer solution without 1 M NaCl. After placement of the column, a step flow was collected and the column washed with 25 mM Tris HCl, pH 9.0. the wash was also collected. The column was then eluted with a gradient varying from 25 mM Tris HCl, pH 9.0, without NaCl to 25 mM Tris HCl, pH 9.0, 1 M NaCl. All collected fractions were dialyzed with a minimum of two exchanges of buffer solution against 10 mM pH 7.5 sodium phosphate. Fractions through flow, wash and eluted with salt were analyzed by protein by the Bradford method using BSA as the standard and bioanalyzed using CRW. The active material was found in the flow and in the fractions that were eluted between 0.2 and 0.5 M NaCl. To determine if the capacity of the column was exceeded, resulting in
Additional materials passing through the non-union column, the active material in the flow of the step was reapplied to the column after rebalancing. The majority of the 280 nm UV absorbent material passed through the column. These observations suggest that this active material has different properties than the material that binds to the column and was eluted with increases in NaCl. The other buffer solutions used were also suitable for anion exchange chromatography as shown by those familiar with the subject. The active material can also be purified by cation exchange chromatography. The Pentina-1 material was purified to near homogeneity by size exclusion chromatography or anion exchange chromatography. The minor protein bands were removed by high pressure liquid chromatography (HPLC) using a reverse phase column before amino acid analysis and amino acid sequence determination. The purity of the samples and the subunit molecular weight were determined by SDS-PAGE using 12% polyacrylamide gels and generally following the method of Laemmli, Na ture 227: 680-685 (1970). The gels were stained with Coomassie Blue R250 using standard protocols or stained with silver (Hammer et al., Phytochemistry 28: 3019-3026
(1989)). For SDS-PAG, the subunit molecular weight of the active substance was found to be in the range of 40-55,000 + 5,000 Daltones. In addition to the methods described above, other methods can be used to separate the active substance from a raw seed extract. For example, an extract can be subjected to isoelectric focusing (IEF) using a Rotofor system (Bio Rad). The Rotofor separates the molecules on the basis of its pl or isoelectric point. Each molecule will have a specific charge, either positive or negative at a specific pH. The Rotofor, using an electric current, moves the molecules through a pH gradient until they reach their pl; that is, the pH which has a net charge of zero. The seals of molecules that migrate in their pl because they are no longer affected by the electric current. The Rotofor focusing chamber is separated into 20 smaller chambers by permeable membranes. These twenty samples are removed simultaneously to ensure as little mixing as possible. Normally, a sample is placed in the focusing medium, a pH-regulating solution (see manufacturer's instructions) that includes 12.5% (v / v) glycerol and 2.5% pH 3-10 Amfolitos (Bio Rad). After the focus, the fractions are collected, the pH of each is determined and each fraction is performed against 1 M of NaCl using a MWCO membrane of
3,500 to remove the Amfolitos. The samples are then dialyzed against deionized water to remove the NaCl. Each fraction was lyophilized and resuspended in 0.4 ml of 10 mM NaCl. Rotofor fractions containing active material can be determined by protein analysis and bioanalysis with insect larvae. The Rotofor fractions can then be subjected to further treatment or separation as described above. Biological Analysis The bioassay was carried out using CRW neonate larvae placed in artificial diets containing Pentina-1 obtained from P. macroloba as described herein. Pentina-1 can be from a crude extract or purified as taught herein. Pentina-1 was applied topically to the diet surface or incorporated into the diet as taught by Czapla and Lang, J. Econo. In to 83 (6): 2480-2485 (1990). The cultivation tray used in the bionalysis was divided into treatment groups. One of a plurality of Pentina-1 preparations or fractions of the various separation were screened on each tray; each preparation or fraction being applied to a plurality of cells; each cell is infested with one or more larvae of neonates. A laminar film with ventilation holes was fixed on the top of each tray to prevent leakage and allow air exchange.
For topical analyzes, a solution containing 2% Pentina-1 was prepared in 0.1 M phosphate buffered saline (PBS), pH 7.8. Seventy-five microliters of Pentina-1 buffer was pipetted into Stonville diet medium in each cell. The culture tray was rotated to ensure equal distribution to the pentin solution in the diet medium. The cells were infected and sealed as described above. The control was 75 μl of 0.1 M PBS (only) per cell. For the analysis of diet incorporation, the Stoneville medium was prepared in standard form, but only with 90% of the prescribed water. Pentina-1 was added so that the amount in the diet was on the scale of 1-5 μg / g. The control treatment consisted of 0.9 ml of buffer added to 8.1 g of the medium. The medium was poured into the cells and the cells were infested and covered as described above. The weights of the insects (Weight or Average Weight) were determined on day 7 and are given in the tables. Table A. Effect of Peptin-1 on Southern Corn Rootworm Sample Concentration (diet μg / ml) of Mortality
Control 0 Crude 1,000 100 Crude 400 15
AS75 400 60 Size Fraction 17 8 14 Size Fraction 18 8 45 Size Fraction 19 8 29 Notes: AS75 = Ammonium sulfate 75% population. Once the Pentina-1 was purified and its insecticidal activity was established, planning efforts were carried out. The first step of the process was to determine by western blot analysis the temporal and spatial distribution of Pentina-1 in order to identify the plant or seed tissue or tissues that most likely express this protein. Since Pentina-1 was not isolated in amounts that allow the production of antibodies, the protein was sequenced in order to allow the design of peptides by synthesis. The amino acid sequence data for Pentina-1 are shown below in Figure 1. The carboxy terminus and internal sequence of approximately 40% of the Pentina-1 peptides were collected from 15 peptides purified from LysC and CNBr digestion. purified pentin protein. The NH2-terminal sequence was not identified during this process.
Antibodies were raised against five of the peptides. The synthetic peptides used to produce antibodies are listed below. Synthetic peptide No. 1 (SEQ ID NO: 5) Met Ser Thr Ser Ala Ala Pro lie Val Phe Pro Pro Tyr Tyr Phe Lys Note: Corresponds to amino acid numbers 213-228 of the
Figure 1. Synthetic peptide No. 2 (SEQ ID NO: 6) Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Gln Glu Try Asp Leu Asp Note: Corresponds to amino acid numbers 344-360 of Figure 1. Synthetic Peptide No. 3 (SEQ ID NO: 7) Pro Asp Trp Val Val lie Arg Ser Glu Ser Val Gly Lys Note: Does not correspond to the amino acids of Figure 1. Synthetic Peptide No. 4 (SEQ ID. NO: 8) Lys Wing Phe Val Asn Gly Val Tyr Phe lie Asn Thr Tyr Asp Ser Wing Note: Does not correspond to the amino acids in Figure 1. Synthetic peptide KS (SEQ ID NO: 9) Asn Asn Tyr Leu Arg lie Gln Glu Tyr Asp Leu Pro Pro Ala Leu Note: Corresponds to the amino acid numbers 349-363 of Figure 1. Western blots of Pentina-1, each of the synthetic peptides and an experimental protein designated 5C9 incubated with each of the antibodies. The incubation results indicated that the antibody
originated against the synthetic peptide KS (anti-KS antibody) and the antibody raised against the synthetic peptide (anti-2 antibody) recognized Pentina-1. Western blot analyzes of tissue extracts of Pentaclethra macroloba treated with anti KS antibodies indicated that the largest recognition was with mature seeds 30-40 mm in diameter or larger. The total RNA was isolated from these seeds. Genomic DNA was isolated, the degenerate oligonucleotides in the codons based on the peptides were used to amplify genomic fragments by PCR. The sequence of exons of the resulting clones was used to perform RT-PCR with specific oligos, then RT-PCR experiments were carried out to obtain at least a partial Pentina-1 cDNA for probing the expression bank. The information obtained from the sequencing of random cDNA clones from immature seed bank of P. macroloba was used to generate a table of use of nascent codons. The data obtained indicated that the P. macroloba tree did not have a strong codon inclination and that the content of GC is moderate. A matrix for degenerating the forward and reverse primers corresponding to the Pentina-1 peptides were selected for use. The initiator sequence below was VVKRLAGYFDV (amino acids of Pentina-1 Nos. 76-86: Val Val Lys Arg Leu Ala
Gly Tyr Phe Asp Val) (SEQ ID NO: 10) and the reverse primer sequence was ENMENLEK, (amino acids of Pentina-1 Nos. 372-379: Glu Asn Met Glu Asn Leu Glu Lys) (SEQ ID. NO: 11). Due to the small amount of tissue available, the initial primer test was carried out using genomic DNA derived from P macroloba leaves. One of the sixty-four possible primer combinations gave a 3.0 kb fragment that encoded the Pentina-1 peptide sequence. The pair of forward and backward primers were used to amplify a 0.8 kB cDNA fragment of the total DNA isolated from mature seeds (30-40 mm). Subsequent screening of the mature seed expression bank with this 0.8 kb cDNA probe yielded several related clones, one of which is a 1.4 kb clone encoding twelve of the fifteen Pentina-1 peptide sequences (SEQ. IDENT. NO: l). Western blots were carried out with Pentina-1, synthetic Pentina-1 peptides, 5C9 proteins and BSA after exposure to selected antibodies. Stain analyzes were treated with a dilution of 1 / 10,000 of antibodies raised against each of the peptides and 5C9. Each antibody recognized its antigen without detectable cross-reactivity to BSA, the negative control. All antibodies, except those raised against the number of synthetic peptides 1, recognized 1.0 micrograms of 5C9.
Although the synthetic peptides KS and number 2 were 74% identical, the anti-2 antibody did not recognize KS, but detected Pentina-1 and 5C9. The nucleic acid sequence of the Pentina-1 clone was determined by standard procedures known to those skilled in the art. The cDNA sequence and the predicted pentin-1 protein sequence were provided in Figure 1 and SEQ. FROM IDENT. NO: 1. Bioanalysis of Cloned Material Western blight worm (WCR) bioassay was performed using sound treated E. coli that was transformed with one of several listed plasmids (Table 1). Transformed cells were grown in approximately 25-35 ml of TB broth. The cells were harvested after 24 hours by centrifugation. The pellet was resuspended in approximately 1 ml of PBS buffer and treated with sound. The resulting mixture was then loaded on the upper surface of the diet, then infested with neonatal WCR larvae. Mortality was recorded after 4 days. A positive result indicated 100% mortality. A negative result indicated mortality less than 10%. A similar experiment involved the use of transformed cells grown on an agar plate. The cells were scraped after sufficient growth, suspended in a small amount of buffer
of PBS and then the solution was incorporated into the insect diet. A 4-day bionalysis was also carried out with recorded mortality. Table 1 shows the results of two replicated bioassays. All cells transformed with putative negative plasmids (non-lethal WCR genes) did not cause larval mortality in any test. These plasmids are P7725, P88126, and P11426. The two plasmids containing the coding sequence for Pentina-1 but not promoters to produce the current protein, PGEM and P11394 did not exhibit WCR activity. However, all plasmids containing the coding region for Pentina-1 (SEQ ID NO: 1) and a functional expression cassette exhibited excellent activity against WCR larvae. All these treatments had 100% mortality. Preliminary Western spot analysis indicated that a protein similar in size to Pentina-1 was present in these cell extracts, but not in negative control samples. The activity was observed in both types of cell preparation and bionalysis.
TABLE 1 Plasmid # Treatment Content Type of Bioanalysis Construction Result bioanalysis control
P7725 UBI-Bt Broth plate Negative top load Neg. Negative microincorporation Neg.
P8812 Bt Broth plate Top load of Negative Neg. Negative microincorporation Neg.
MoPentina / PGEM Broth plate Negative top load Neg. Negative microincorporation Neg.
P11184 Pentin-1 cDNA Broth plate Top loading of Positive N / A complete of UBI of microincorporation Positive N / A Clon-Pinll bank P11335 UBI-Pentina Mod. (ATG- Broth plate Top load of Positive N / A TGA ) -PinII (modified microincorporation Positive N / A of the complete clone, but not putative mature P11361 Pentina-1 of cDNA in broth plate Top loading of Positive N / A pBK-CMV (promoter lacZ) microincorporation N / A
P11394 Pentina-1 (protein Broth plate Negative top load Neg. Mature) without promoter or microincorporation Negative Neg. start codon) P11426 UBI-moPAT-CAMV35s Broth plate Top load of Negative Neg. Negative microincorporation Neg.
P11443 Ubi-moPentina-1-Pin Broth plate Top load of Positive N / A II / CAMV35S-Pat-CAMV35S microincorporación Positivo N / A
Transformation of Isolated Plotoplasts of Maize Suspension Cells with Three Gene Constructions of Pentin for Gene Expression and for CRW I Bioanalysis. Protoplast Transformation Protocol The established Hill (GS3) suspension cells were used to form protoplasts. Cells were recovered 3-4 days after subculture. Digestion of cells: The cells were directed in enzyme solution at 27 ° C for 3-5 hours with stirring speed of 50-60 RPM. The cell wall was digested with cellulase and pectolyase to release the protoplasts. Protoplast recovery: the digested material was passed through the 30 mm filter and the protoplasts were recovered by centrifugation of the filtrate at 1, 000 RPM for 10 minutes. The protoplast pellet was resuspended in 20 ml or 40 ml of KMC solution. The protoplast density and total protoplast yield was determined by counting the protoplast number with a hemacytometer. The suspension was centrifuged to form protoplast pellets. The protoplast pellet was suspended in the MaMg transformation solution at a concentration of 2 million protoplasts per ml. The solution in quantities of 2 ml (approximately 4 million protoplasts) was dispersed in bottom tubes
round of 15 ml. Each tube was a replication. At least three replicates were used for each gene construction of Pentina-1. The constructs included native Pentina-1 and the optimized Pentina-1 sequence. See SEC. FROM IDENT. NOS: 1 and 3, respectively. Plasmid DNA was added to the suspension of protoplasts in the tubes (15 mg of plasmid DNA / million protoplasts) and mixed. After incubation for 5 minutes, 2 ml 40% polyethylene glycol (PEG-8000, Sigma) was added to the protoplast / DNA mixture (the final PEG concentration is about 20%) and mixed by inverting tubes several times and incubated at room temperature for 20-30 minutes. About 3 ml of W5 saline was added to each tube. The tubes were covered and inverted gently. This was repeated twice until the final volume was 13-14 ml. The suspension was centrifuged 8 minutes at 1,000 RPM. 3 ml of FW medium was used to resuspend the protoplasts. (See right away). Using a compressible plastic Pasteur pipette, a treatment (3 ml) was dispensed into two wells of a 6 well culture plate, sealed with parafilm and incubated 24-48 hours in the dark at 28 ° C. After the culture, the protoplasts were transferred in a 15 ml tube using a Pasteur pipette
compressible plastic and centrifuged 8 minutes at 500 RPM to form the pellet of the protoplasts. Protein analysis and bioanalysis: From one-fifth to one-fourth of the protoplast pellet in each replication for each transformation treatment was sampled for analysis of Peptina-1 expression by Western analysis. The rest of the protoplast pellet was used for bioassay. All replication samples from the same transformation treatment, which is transformed with the same Peptina-1 construct, were combined and incorporated into the diet for CRW bioassay. The results of bioanalysis are given in Table 2.
Table 2. Effect of Petina-1 when it was expressed temporarily in corn protoplasts against WCR larvae. The data is a combination of 6 replicated experiments over time. The protoplasts were treated with sound and the entire mixture was incorporated into the client.
Plasmid # Construction Content Analysis of Bioanalysis Mortality Staining for Western Control Pyrin P111841 UBI-Pentina 1 Complete Positive cDNA 32- 'N / A Clone-PinlI Bank P11335 UBI-Mod Pentina (ATG-TGA) - Positive 05 N / A Pinll N / A weak (modified from the full clone but not putative) P11443 Ubi-mo Pentin-1-Pin-II / Positive 54% N / A Strong P8126 UBI-Bt Negative 0% Negative Control
P3953 UBI-Gus Negative 0% Negative Control
1P11184 was only used in the last four experiments.
II. Constructions of Pentina-1 gene for Transformation
P11184 - Ubi promoter :: Pentina-1 (original full-length clone) P11335 - Ubi promoter :: Pentina-1 (partially modified gene) P11443 - Ubi promoter:: moPentin-l (optimized gene) Solutions and Medium Used for Transformation Solution KMC - 1,000 ml KCI 8.65 g MgC12-6H20 16.47 g CaC12-2H20 12.50 g MES 0.5% 5.0 g PH 5.8 with KOH It is sterilized with filter MaMg transmition solution - 1,000 ml M manitol 108.1 g 15 mM MgC12-6H20 3.05 g 10 mM MONTH 1.95 g PH 5.7 Sterilized with filter 40% PEG - 100 ml 40 g of PEG are added to 60 ml of MaMg transformation solution. The microwaves are placed briefly to dissolve PEG. More MaMg solution is added to a
final volume of 100 ml. It adjusts to pH 7.0. It is sterilized with filter. Enzyme solution to digest the cell suspension (Enzyme Solution) The enzyme solution contains 3% RS cellulose and
0. 3% of pectoliase Y23 in the protoplast solution. Protoplast Solution - 1,000 ml M manitol 108.1 g 10 mM MES 1.95 g 1 mM CaC12-2H20 147 mg 1 mM MgC12-6H20 203 mg 1% BSA (optional) 1 g PH 5.7 Sterilize with filter Salt solution W5 - 1,000 ml 154 mM NaCl 9.0 g 125 mM CaC12-2H20 18.56 g 5 mM KCI 0.373 g 5 M Glucose 0.901 g PH 5.5 with KOH Is sterilized with filter Medium FW - 1,000 ml MS salts (Sigma M5519) 4.3 g sucrose 30.0 g mannitol 54.0 g
Proline 1.5 g 2, 4-D 3.0 mg l, 000x B5 Vitamins 1 ml PH 5.8 Sterilized with filter Transformation and Regeneration of Corn Callus The immature maize embryos of greenhouse donor plants were bombarded with a plasmid containing all three constructions of Pentina-1 plus a plasmid containing the selectable marker gene, PAT, (Wohlleden, W., Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Puehler, A. "Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tue494 and its expression in Mcotiana tabacum "Gene 70: 25-37 (1988) which confers resistance to the herbicidal Bialophos by the following method: Note: All medium formulas are in the Appendix. of white tissue: The ears were superficially sterilized in 30% Clorox bleach plus 0.55 Micro detergent for 20 minutes and rinsed twice with sterile water.The immature embryos were removed and placed it's embryos upside down (at the top is the scutellum), 25 embryos per plate. These were cultivated in 560L medium four days before the bombardment in the dark. The day of the bombing,
the embryos were transferred to the 560Y medium for 4 hours, they were placed within the 2.5 cm white zone. DNA preparation: 100 μl of tungsten particles prepared in water 10 μl of DNA (1 μg) in TrisEDTA buffer (lμg in total) 100 μl 2.5 M CaC12 10 μl of 0.1 spermidine Each reagent was added sequentially to the suspension of tungsten particles while kept in the rotator of multiple tubes. The plasmids were adjusted for a final ratio of 1: 1 per size. The final mixture was briefly treated and allowed to incubate under constant revolution for 10 minutes. After the precipitation period, the tubes were centrifuged briefly, the liquid was removed, washed with 500 ml of 100% ethanol and centrifuged 30 seconds. The new liquid was removed and 105 μl of 100% ethanol was added to the pellet of final tungsten particles. For particle gun bombardment, the tungsten / DNA particles were briefly treated with sound and 10 μl was placed in the center of each macrocarrier and allowed to dry approximately 2 minutes before bombardment. Particle Gun Treatment: The sample plates were bombarded at level # 4 on the gun
particles # HE34-1 or # HE34-2. All samples received a single shot at 650 PSI, with a total of ten aliquots taken from each prepared particle / DNA tube. Subsequent treatment: After bombardment, embryos were maintained in 560Y medium for 2 days then transferred to selection medium of 560R containing 3 mg / liter Bialphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, resistant callus selection clones were sampled for PCR and for TLC activity analysis of fumonisin esterase. Positive lines were transferred to medium 288J to initiate plant regeneration. After the maturation of somatic embryos (2-4 weeks), well-developed somatic embryos were transferred to the medium for germination and transferred to the enlightened culture environment. Approximately 7-10 days later, the developing seedlings were transferred to medium in tubes for 7-10 days until the seedlings were well established. The plants were transferred to flat inserts (equivalent to pots of 6.35 cm (2.5")) containing potting soil and developed 1 week in a growth chamber, subsequently developed for 1-2 weeks in the greenhouse, then transferred to 600 Classic pots (1.6 gallons) and developed to maturity.
APPENDIX Ingredient Quantity Unit
D-I H20 900,000 ml
Basic Sales CHU (N6) (SIGMA C-1416) 1,600 g
N6 Macronutrients 10X Stock ## 60,000 ml
Potassium Nitrate 1,680 Lower salts B5H 1000X ### 0.600 ml
B5H Fe Na EDTA IOOX #### 6.000 ml
Mixture of Vitamin Eriksson (lOOOX SIGMA-1511) 0.400 ml
S & H Mixture of vitamin lOOx Stock (S3766) 6 000 m?
Thiamine .HCL 0.4mg / ml 0.500 ml
L-Proline 1.980 g
Casein Hydrolyzate (acid) 0.300 g
Sucrose 20,000 g
Glucose O.600 g
2,4-D 0.5 mg / ml 1,600 ml
Gelrita @ 2,000 g
Dicamba 1 mg / ml # 1,200 ml
Silver Nitrate 2 mg / ml # 1700 ml
Directions: @ = Add after reaching the indicated volume # = Add after sterilizing and cooling to temperature
Dissolve ingredients in D-I H2O polished in sequence Adjust to pH 5.8 Reach volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 ° C. ## = Dissolve 1,660 g of calcium chloride dihydrate in
950. 000 ml of D-I H20 polished. Then dissolve 4,629 of ammonium sulfate; 4,000 g of potassium phosphate Monobasic
KH2P04; 1850 g of Magnesium Sulphate 7-H20, MgSO4, 7H20; Y
28. 300 g of Potassium Nitrate in sequence. Get a volume with D-I H20 polished. ### = Dissolve 3,000 g of Boric Acid; 10,000 g of Sulphate
Manganese Monohydrate; 0.250 g of Molybdate Dihydrate
Sodium; and 0.750 g of Potassium Iodide in D-I H20 polished in sequence. Reach a volume with polished D-I H20. #### = Dissolve 3,700 g of Disodium Dihydrate EDTA and 2,790 g of Ferrous Sulphate 7-Hydrate in D-I H20. Reach a volume with D-I H20. Total Volume (L) = 1.00
604 A Ingredient Quantity Unit
D-I H? 0 900,000 ml
Basic Sales CHU (N6) (SIGMA C-1416) 1,600 g
N6 Macronutrients 10X Stock ## 60,000 ml
Potassium Nitrate 1,680 g
Lower salts B5H ÍOOOX ### 0.600 ml
B5H Fe Na EDTA lOOx #### 6.000 ml
Mixture of Vitamin Eriksson (1000X SIGMA-1511) 0.400 ml
I S & H Mixture of vitamin lOOx Stock (S3766) 6 000 ml
1 Thiamine .HCL 0.4mg / ml 0.500 ml
L-Proline 1.980 g
Casein Hydrolyzate (acid) 0.300 g
Sucrose 20,000 g
Glucose O.600 g
2,4-D 0.5 mg / ml 1,600 ml
Gelrita @ 2,000 g
Dicamba 1 mg / ml # 1,200 ml
Directions: @ = Add after reaching the indicated volume # = Add after sterilizing and cooling to temperature
Dissolve ingredients in D-I H20 polished in sequence
Adjust to pH 5.8 Reach volume with polished D-I H20 then adjust pH
Sterilize and cool to 60 ° C. ## = Dissolve 1,660 g of calcium chloride dihydrate in
950. 000 ml of D-I H20 polished. Then dissolve 4,629 of ammonium sulfate; 4,000 g of potassium phosphate Monobasic
KH2P04; 1850 g of Magnesium Sulphate 7-H20, MgSO4, 7H20; Y
28. 300 g of Potassium Nitrate in sequence. Get a volume with D-I H20 polished. ### = Dissolve 3,000 g of Boric Acid; 10,000 g of Sulphate
Manganic Monohydrate; 0.250 g of Sodium Molybdate Dihydrate; and 0.750 g of Potassium iodide in D-I H20 polished in sequence. Reach a volume with polished D-I H20. #### = Dissolve 3,700 g of Disodium Dihydrate EDTA and 2,790 g of Ferrous Sulphate 7-Hydrate in D-I H20. Reach a volume with D-I H20. Total Volume (L) = 1.00
605 J
Directions: @ = Add after reaching the indicated volume # = Add after sterilizing and cooling to temperature
Dissolve ingredients in D-I H20 polished in sequence
Adjust to pH 5.8 Reach volume with polished D-I H20 then adjust pH
Sterilize and cool to 60 ° C. ## = Dissolve 1,660 g of calcium chloride dihydrate in
950. 000 ml of D-I H20 polished. Then dissolve 4,629 of ammonium sulfate; 4,000 g of potassium phosphate Monobasic
KH2P04; 1850 g of Magnesium Sulphate 7-H20, MgSO4, 7H20; Y
28. 300 g of Potassium Nitrate in sequence. Get a volume with D-I H20 polished. ### = Dissolve 3,000 g of Boric Acid; 10,000 g of Sulphate
Manganese Monohydrate; 0.250 g of Molybdate Dihydrate
Sodium; and 0.750 g of Potassium Iodide in D-I H20 polished in sequence. Reach a volume with polished D-I H20. #### = Dissolve 3,700 g of Disodium Dihydrate EDTA and 2,790 g of Ferrous Sulphate 7-Hydrate in D-I H20. Reach a volume with D-I H20. Total Volume (L) = 1.00
604 S
Directions: @ = Add after reaching the indicated volume # = Add after sterilizing and cooling to temperature
Dissolve ingredients in D-I H20 polished in sequence Adjust to pH 5.8 Reach volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 ° C. ## = Dissolve 1,660 g of calcium chloride dihydrate in
950. 000 ml of D-I H20 polished. Then dissolve 4,629 of ammonium sulfate; 4,000 g of potassium phosphate Monobasic
KH2P04; 1850 g of Magnesium Sulphate 7-H20, MgSO4, 7H20; Y
28. 300 g of Potassium Nitrate in sequence. Get a volume with D-I H20 polished. ### = Dissolve 3,000 g of Boric Acid; 10,000 g of Sulphate
Manganese Monohydrate; 0.250 g of Molybdate Dihydrate
Sodium; and 0.750 g of Potassium Iodide in D-I H20 polished in sequence. Reach a volume with polished D-I H20. #### = Dissolve 3,700 g of Disodium Dihydrate EDTA and 2,790 g of Ferrous Sulphate 7-Hydrate in D-I H20. Reach a volume with D-I H20. Total Volume (L) = 1.00
272 V Ingredient Quantity Unit
D-I H20 900,000 ml
Sales MS (GIBCO 11117-074) 4,300 g
Myo-Inositol 0.100 g
Vitamins MS Solution Stock ## 5.000 ml
Sucrose 40,000 g
Bacto-Agar 6,000 g
Directions: @ = Add after reaching the indicated volume Dissolve ingredients in D-I H20 polished in sequence Adjust to pH 5.8 Reach volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 ° C. ## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.
HCL; 0.100 g of pyridoxine. HCL; and 0.400 g of glycine in
875. 00 ml of D-I H20 polished in sequence. Reach a volume with polished D-I H20. Made in 400 ml portions. Thiamine.
HCL & Pyridoxine HCL. They are in Dark Descicator. Store for one month, unless contamination or precipitation occurs, then form a fresh raw material. Total Volume (L) = 1.00
288 J Ingredient Quantity Unit
D-I H20 950,000 ml
Sales MS 4,300 g
Myo-Inositol 0.100 g
Vitamins MS Solution Stock ## 5.000 ml
Zeatin .5 mg / ml 1.00 ml
Sucrose 60,000 g
Gelrita @ 3,000 g
Acetic Acid Indole 0.5 mg / ml # 2,000 ml
. lmM Absinthic Acid 1,000 ml
Bialasfos lmg / ml # 3,000 ml
Directions: @ = Add after reaching the indicated volume Dissolve ingredients in D-I H20 polished in sequence Adjust to pH 5.8 Reach volume with polished D-I H20 after adjusting pH
Sterilize and cool to 60 ° C. Add 3.5 g / L of Gelrite for cell biology ## = Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Tíamina.
HCL; 0.100 g of pyridoxine. HCL; and 0.400 g of glycine in
875. 00 ml of D-I H20 polished in sequence. Reach a volume with polished D-I H20. Made in 400 ml portions. Thiamine.
HCL & Pyridoxine HCL. They are in Dark Descicator. Store for one month, unless contamination or precipitation occurs, then form a fresh raw material. Total Volume (L) = 1.00
560 L Ingredient Quantity Unit
D-I Water, filtered 950,000 ml
Basic Sales CHU (N6) (SIGMA C -1416) 4.000 g
Mixture of Eriksson Vitamin (1000X SIGMA-1511 0.400 ml Thiamine .HCL 0.4 mg / ml 1.250 ml
Sucrose 20,000 g
2,4-D 0.5 mg / ml 2,000 ml
L-Proline 2.880 g
Gelrita @ 2,000 g
Silver Nitrate 2 mg / ml # 4.250 ml
Directions: @ = Add after reaching the indicated volume # = Add after sterilization and cooling to temperature. Dissolve ingredients in D-I H20 in sequence Adjust to pH 5.8 with KOH Reach volume with D-I H20 Sterilize and cool to room temperature. Total Volume (L) = 1.00
560 R Ingredient Quantity Unit
D-I Water, filtered 950,000 ml
Basic Sales CHU (N6) (SIGMA C -1416) 4.000 g
Mixture of Erik's Vitamin (1000X SIGMA-1511 1,000 ml Thiamine .HCL 0.4 mg / ml 1,250 ml
Sucrose 30,000 g
2,4-D 0.5 mg / ml 4,000 ml
Gelrita @ 3,000 g
Silver Nitrate 2 mg / ml # 0.450 ml
Bialofos lmg / ml # 3,000 ml
Directions: @ = Add after reaching the indicated volume # = Add after sterilization and cooling to temperature. Dissolve ingredients in D-I H20 in sequence Adjust to pH 5.8 with KOH Reach volume with D-I H20 Sterilize and cool to room temperature. Total Volume (L) = 1.00
560 Y Ingredient Quantity Unit
D-I Water, filtered 950,000 ml
CHU (N6) Basic Salts (SIGMA C-1416) 4.000 Eriksson Vitamin Mix (1000X SIGMA-1511 1.000 ml Thiamine .HCL 0.4 mg / ml 1.250 ml
Sucrose 120,000 2, 4-D 0.5 mg / ml 2,000 ml
L-Proline 2.880 g
| Gelrita @ 2,000 g
Silver Nitrate 2 mg / ml # 4.250 ml
Directions: @ = Add after reaching the indicated volume # = Add after sterilization and cooling to temperature. Dissolve ingredients in D-I H20 in sequence Adjust to pH 5.8 with KOH Reach volume with D-I H20 Sterilize and cool to room temperature. ** Minor autoclave time because there is a content of
Sfcarose increased ** Total Volume (L) = 1.00
Plasmids PHP11511 were deposited with American type Cultere collection, Bethesda, Maryland, and are given Access numbers 209026 and 209025, respectively. PHP11361 comprises the nucleotide sequence of natural Pentiná-1 sequence. PHP11511 comprises the optimized Pentina-1 sequence. All publications and patent applications mentioned in the specification indicate the level of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each publication or individual patent application will be specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: CIGAN, AMY L CZAPLA, THOMAS H FALLÍS, LYNN MEYER, TERRY E MUNDELL, SCOTT TO SABUS, BRIAN SCHUBERT, KAREL (ii) TITLE OF THE INVENTION: PROTEINS THAT HAVE INSECTICIDE ACTIVITIES AND METHOD OF USE (iii) SEQUENCE NUMBERS: 11 (iv) DIRECT CORRESPONDENCE: (A) RECIPIENT: W. MURRAY SPRUILL (ALSTON &BIRD, LLP) (B) STREET: 3605 GLENWOOD AVE. (C) CITY: RALEIGH (D) STATE: NC (E) COUNTRY: USA (F) ZIP: 27622 (v) METHOD OF READING ON THE COMPUTER (A) TYPE OF MEDIA: Soft disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30
(vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: SPRUILL, W. MURRAY (B) REGISTRATION NUMBER: 32,943 (C) REFERENCE / FILE NUMBER: 5718-9 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 919 420 2202 (B) TELEFAX: 919 881 3175 FORMATION FOR SEC. FROM IDENT. NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1469 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANIZATION: Pentaclethra macroloba (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 31..1257
(xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT . : 1: CGGCACGAGC TCGTACAGAT TCTATCCATT ATG AAG TCG AAA ATG GCC ATG CTC 54 Met Lys Ser Lys Met Wing Met Leu 1 5 CTT TTG TTA TTT TGT GTG TTA TCT AAT CAG CTA GTG GCA GCA TTT TCC 102 Leu Leu Leu Phe Cys Val Leu Ser Asn Gln Leu Val Wing Wing Phe Ser 10 15 20 AC CAA GCG AAA GCT TCT AAA GAT GGA AAC TTA GTC ACA GTT CTT GCC 150 Thr Gln Wing Lys Wing Ser Lys Asp Gly Asn Leu Val Thr Val Leu Wing 25 30 35 40 ATT GAT GGA GGT GGT ATC AGA GGA ATT ATC CCC GGA GTT ATT CTC AAA 198 He Asp Gly Gly Gly He Arg Gly He He Pro Gly Val He Leu Lys 45 50 55 CAA CTA GAA GCT ACT CTT CAG AGA TGG GAC TCA AGT GCA AGA CTA GCA 2 6 Gln Leu Glu Wing Thr Leu Gln Arg Trp Asp Ser Wing Wing Arg Leu Wing 60 65 70 GAG TAT TTT GAT GTG GTT GCC GGG ACG AGC ACT GGA GGG ATT ATA ACT 294 Glu Tyr Phe Asp Val Val Wing Gly Thr Ser Thr Gly Gly He He Thr 75 80 85 GCC ATT CTA ACT GCC CCG GAC CCA CAA AAC AAG GAC CGT CCT TTG TAT 342 Wing He Leu Thr Wing Pro Asp Pro Gln Asn Lys Asp Arg Pro Leu Tyr 90 95 100 GCT GCC GAA GAA ATT ATC GAC TTC T AC ATA GAG CAT GGT CCT TCC ATT 390 Wing Wing Glu Glu He He Asp Phe Tyr He Glu His Gly Pro Ser He 105 - "110 115 120
TTT AAT AAA TCC ACC GCC TGC TCG TTG CCT GGT ATC TTT TGT CCA AAG 438 Phe Asn Lys Ser Thr Ala Cys Ser Leu Pro Gly He Phe Cys Pro Lys 125 130 135 TAT GAT GGG AAG TAT TTA CA GAA ATA ATA AGC CAG AAA TTG AAT GAA 486
Tyr Asp Gly Lys Tyr Leu Gln Glu He He Ser Gln Lys Leu Asn Glu 140 145 150 ACÁ CTA CTA GAC CAG ACÁ ACÁ ACÁ AAT GTT GTT ATC CCT TCC TTC GAC 534
Thr Leu Leu Asp Gln Thr Thr Thr Asn Val Val Pro Pro Ser Phe Asp 155 160 165 ATC AAG CTT CTT CGT CCA ACC ATA TTC TCA ACT TTC AAG TTA GAG GAA 582
He Lys Leu Leu Arg Pro Thr He Phe Ser Thr Phe Lys Leu Glu Glu 170 175 180 GTT CCT GAG TTA AAT GTC AAA CTC TCC GAT GTA TGC ATG GGA ACT TCA 630
Val Pro Glu Leu Asn Val Lys Leu Ser Asp Val Cys Met Gly Thr Ser 185 190 195 200 GCA GCA CCA ATC GTA TTT CCT CCC TAT TAT TTC AAG CAT GGA GAT ACT 678
Wing Wing Pro He Val Phe Pro Pro Tyr Tyr Phe Lys His Gly Asp Thr 205 210 215 GAA TTC AAT CTC GTT GAT GGT GCA ATC ATC GCT ATT CCG GCC CCG 726 Glu Phe Asn Leu Val Asp Gly Wing He He Wing Asp He Pro Wing Pro 220 225 230 GTT GCT CTC AGC GAG GTG CTC CAG CAA GAA AAA TAC AAG AAA GAA 774
Val Ala Leu Ser Glu Val Leu Gln Gln Glu Lys Tyr Lys Asn Lys Glu 235 240 245 ATC CTT TTG CTG TCT ATA GGA ACT GGA GTT GTA AAA CCT GGT GAG GGT 822
He Leu Leu Leu Be He Gly Thr Gly Val Val Lys Pro Gly Glu Gly 250 255 260 TAT TCT GCT AAT CGT ACT TGG ACT ATT TTC GAT TGG AGT AGT GAA ACT 870
Tyr Ser Wing Asn Arg Thr Trp Thr He Phe Asp Trp Ser Ser Glu Thr 265 270 275 280 TTA ATC GGG CTT ATG GGT CAT GGA ACG AGA GCC ATG TCT GAT TAT TAC 918
Leu He Gly Leu Met Gly His Gly Thr Arg Wing Met Ser Asp Tyr Tyr 285 290 295 GTT GGC TCA CAT TTC AAA GCC CTT CAA CCC CAG AAT AAC TAC CTC CGA 966
Val Gly Ser His Phe Lys Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg 300 305 310 ATT CAG GAA TAC GAT TTA GAT CCG GCA CTG GAA AGC ATT GAT GAT GCT 1014
He Gln Glu Tyr Asp Leu Asp Pro Wing Leu Glu Ser He Asp Asp Wing 315 320 325 TCA ACG GAA AAC ATG GAG AAT CTG GAA AAG GTA GGA CAG AGT TTG TTG 1062
Be Thr Glu Asn Met Glu Asn Leu Glu Lys Val Gly Gln Ser Leu Leu 330 335 340 AAC GAA CCA GTT AAA AGG ATG AAT CTG AAT ACT TTT GTC GTT GAA GAA 1110
Asn Glu Pro Val Lys Arg Met Asn Leu Asn Thr Phe Val Val Glu Glu 345 350 355 360 ACA GGT GAA GGT ACC AAT GCA GAA GCT TTA GAC AGG CTG GCT CAG ATT 1158
Thr Gly Glu Gly Thr Asn Wing Glu Wing Leu Asp Arg Leu Wing Gln He 365 370 375 CTT TAT GAA GAA AAG ATT ACT CGT GGT CTC GGA AAG ATA TCT TTG GAA 1206
Leu Tyr Glu Glu Lys He Thr Arg Gly Leu Gly Lys He Ser Leu Glu 38Q 385 390 GTG GAT AAC ATT GAT CCA TAT ACT GAA GTT AGG AAA CTG CTA TTC 1254 Val Asp Asn As Asp Pro Tyr Thr Glu Arg Val Arg Lys Leu Leu Phe 395 400 405 TGA TACGAATTGA AGTTGTTTCC TCCTTGCCAT ATAGCCTCAC TTTGTTTGGC 1307
AATAAATAAA TAAATAAATG TAATCGTTTG GTTTGATGTC CTTGACTTTG TCATATATGC 1367 TGGCTCTATA AGAAGCACCA GCAGATAAAT AAAGGTTAAT GTTTGAGGTA TWAARWAAAA 1427 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAA AAAAAAACTC GA 1469
(2) INFORMATION FOR SEC. FROM IDENT. NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 409 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO.:2: Met Lys Ser Lys Met Met Wing Leu Leu Leu Leu Phe Cys Val Leu Ser 1 5 10 15 Asn Gln Leu Val Wing Wing Phe Ser Thr Gln Wing Lys Wing Ser Lys Asp 20 25 30 Gly Asn Leu Val Thr Val Leu Ala He Asp Gly Gly Gly He Arg Gly 35 40 45 He He Pro Gly Val He Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg 50 55 60
Trp Asp Ser Wing Arg Leu Wing Glu Tyr Phe Asp Val Val Wing Gly
65 70 75 80
Thr Ser Thr Gly Gly He He Thr Wing He Leu Thr Wing Pro Asp Pro 85 90 95
Gln Asn Lys Asp Arg Pro Leu Tyr Ala Wing Glu Glu He He Asp Phe
100 105 110 Tyr He Glu His Gly Pro Ser He Phe Asn Lys Ser Thr Ala Cys Ser
115 120 125 Leu Pro Gly He Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu
130 135 140 He He Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr 145 150 155 160
Asn Val Val He Pro Ser Phe Asp He Lys Leu Leu Arg Pro Thr He 165 170 175
Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu
180 185 190 Ser Asp Val Cys Met Gly Thr Ser Wing Ala Pro He Val Phe Pro Pro
195 200 205 Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Ala
210 215 220 He He Wing Asp He Pro Wing Pro Val Wing Leu Ser Glu Val Leu Gln 225 230 235 240
Gln Glu Lys Tyr Lys Asn Lys Glu He Leu Leu Leu Ser He Gly Thr 245 250 255
Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Wing Asn Arg Thr Trp Thr
260 265 270 He Phe Asp Trp Ser Ser Glu Thr Leu He Gly Leu Met Gly His Gly 275 280 285 Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu 290 295 300 Gln Pro Gln Asn Asn Tyr Leu Arg He Gln Glu Tyr Asp Leu Asp Pro 305 310 315 320
Ala Leu Glu Ser He Asp Asp Ala Ser Thr Glu Asn Met Glu Asn Leu 325 330 335
Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn
340 345 350 Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn Ala Glu 355 360 365 Wing Leu Asp Arg Leu Wing Gln He Leu Tyr Glu Glu Lys He Thr Arg 370 375 380 Gly Leu Gly Lys He Ser Leu Glu Val Asp Asn He Asp Pro Tyr Thr 385 390 395 400
Glu Arg Val Arg Lys Leu Leu Phe * 405 (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1227 base pairs (B) TYPE: nucleic acid (C) CHAIN FORM: simple
(D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "cDNA of Pentin-1 optimized for enhanced expression" (vi) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (ix) ) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1..1227 (xi) SEQUENCE DESCRIPTION: SEC. FROM IDENT. NO.:3: ATG AAG TCC AAG ATG GCC ATG CTC CTC CTC CTC TTC TGC GTG CTC TCC 48 Met Lys Ser Lys Met Met Wing Leu Leu Leu Leu Phe Cys Val Leu Ser 410 415 420 425 AAC CAG CTC GTG GCC GCG TTC TCC ACC CAG GCC AAG GCC TCC AAG GAC 96 Asn Gln Leu Val Wing Wing Phe Ser Thr Gln Wing Lys Wing Ser Lys Asp 430 435 440 GGC AAC CTC GTG ACC GTG CTC GCC ATC GAC GGC GGC GGC ATC CGC GGC 1 4 Gly Asn Leu Val Thr Val Leu Wing He Asp Gly Gly Gly He Arg Gly 445 450 455 ATC ATC CCG GGC GTG ATC CTC AAG CAG CTC GAG GCG ACC CTC CAG AGG 192 He He Pro Gly Val He Leu Lys Gln Leu Glu Wing Thr Leu Gln Arg 460 465 470 TGG GAC TCC AGC GCC AGG CTC GCG GAG TAC TTC GAC GTG GTG GCC GGC 240 Trp Asp Ser Ser Wing Arg Leu Wing Glu Tyr Phe Asp Val Val Wing Ala Gly 475 480 485
ACC TCC ACC GGC GGC ATC ATC ACC GCC ATC CTC ACC GCC CCG GAC CCG 288
Thr Ser Thr Gly Gly He He Thr Wing He Leu Thr Wing Pro Asp Pro 490 495 500 505 CAG AAC AAG GAC CGC CCG CTC TAC GCC GCC GAG GAG ATC ATC GAC TTC 336
Gln Asn Lys Asp Arg Pro Leu Tyr Wing Wing Glu Glu He He Asp Phe 510 515 520 TAC ATC GAG CAC GGC CCG TCC ATC TTC AAC AAG TCC ACC GCC TGC TCC 384
Tyr He Glu His Gly Pro Ser He Phe Asn Lys Ser Thr Ala Cys Ser 525 530 535 CTC CCG GGC ATC TTC TGC CCG AAG TAC GAC GGC AAG TAC CTC CAG GAG 432
Leu Pro Gly He Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu 540 545 550 ATC ATC TCC CAG AAG CTC AAC GAG ACC CTC CTC GAC CAG ACC ACC ACC 480
He He Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr 555 560 565 AAC GTG GTG ATC CCG TCC TTC GAC ATC AAG CTC CTC CGC CCG ACC ATC 528
Asn Val Val He Pro Ser Phe Asp He Lys Leu Leu Arg Pro Thr He 570 575 580 585 TTC TCC ACC TTC AAG CTC GAG GAG GTG CCG GAG CTC AAC GTG AAG CTC 576
Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu 590 595 600 TCC GAC GTG TGC ATG GGC ACC TCC GCC GCC CCG ATC GTG TTC CCG CCG 624
Being Asp Val Cys Met Gly Thr Ser Wing Ala Pro He Val Phe Pro Pro 605 610 615 TAC TAC TTC AAG CAC GGC GAC ACC GAG TTC AAC CTC GTC GAC GGC GCG 672
Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Wing 620 625 630 ATC ATC GCG GAC ATC CCA GCC CCG GTG GCC CTC TCC GAG GTG CTC CAG 720
He He Wing Asp He Pro Wing Pro Val Wing Leu Ser Glu Val Leu Gln 635 640 645 CAG GAG AAG TAC AAG AAC AAG GAG ATC CTC CTC CTG AGC ATC GGC ACC 768 Gln Glu Lys Tyr Lys Asn Lys Glu He Leu Leu Leu Ser He Gly Thr 650 655 660 665 GGC GTG GTG AAG CCG GGC GAG GGC TAC TCC GCC AAC CGC ACC TGG ACC 816 Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Wing Asn Arg Thr Trp Thr 670 675 680 ATC TTC GAC TGG TCC TCC GAG ACC CTC ATC GGC CTC ATG GGG CAC GGC 864 He Phe Asp Trp Ser Ser Glu Thr Leu He Gly Leu Met Gly His Gly 685 690 695 ACC CGC GCC ATG TCC GAC TAC TAC GTG GGC TCC CAC TTC AAG GCC CTC 912
Thr Arg Wing Met As Asp Tyr Tyr Val Gly Ser His Phe Lys Wing Leu 700 705 710 CAG CCG CAG AAC AAC TAC CTC CGC ATC CAG GAG TAC GAC CTC GAC CCG 960
Gln Pro Gln Asn Asn Tyr Leu Arg He Gln Glu Tyr Asp Leu Asp Pro 715 720 725 GCC CTC GAG TCC ATC GAC GAC GCC TCC ACC GAG AAC ATG GAG AAC CTC 1008
Wing Leu Glu Ser He Asp Asp Wing Ser Thr Glu Asn Met Glu Asn Leu 730 735 740 745 GAG AAG GTG GGC CAG TCC CTC CTC AAC GAG CCG GTG AAG CGC ATG AAC 1056
Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn 750 755 760 CTC AAC ACG TTC GTC GTG GAG GAG ACC GGC GAG GGG ACC AAC GCC GAG 1104 Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn Wing Glu 765 770 775 GCG CTC GAC CGC CTC GCC CAG ATC CTC TAC GAG GAG AAG ATC ACC CGC 1152 Wing Leu Asp Arg Leu Wing Gln He Leu Tyr Glu Glu Lys He Thr Arg 780 785 790 GGC CTC GGC AAG ATC TCC CTC GAG GTG GAC AAC ATC GAC CCG TAC ACC 1200 Gly Leu Gly Lys He Ser Leu Glu Val Asp Asn As Asp Pro Tyr Thr 795 800 805 GAG CGC GTG CGC AAG CTC CTC TTC TGA 1227 Glu Arg Val Arg Lys Leu Leu Phe * 810 815 (2 ) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 409 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT. Met Lys Ser Lys Met Met Wing Leu Leu Leu Leu Phe Cys Val Leu Ser 1 5 10 15 Asn Gln Leu Val Wing Wing Phe Ser Thr Gln Wing Lys Wing Ser Lys Asp 20 25 30
Gly Asn Leu Val Thr Val Leu Ala He Asp Gly Gly Gly He Arg Gly
40 45 He He Pro Gly Val He Leu Lys Gln Leu Glu Wing Thr Leu Gln Arg 50 55 60 Trp Asp Ser Wing Arg Leu Wing Glu Tyr Phe Asp Val Val Wing Ala Gly 65 70 75 80
Thr Ser Thr Gly Gly He He Thr Wing He Leu Thr Wing Pro Asp Pro 85 90 95
Gln Asn Lys Asp Arg Pro Leu Tyr Wing Wing Glu Glu He He Asp Phe 100 105 110 Tyr He Glu His Gly Pro Be He Phe Asn Lys Ser Thr Wing Cys Ser
115 120 125 Leu Pro Gly He Phe Cys Pro Lys Tyr Asp Gly Lys Tyr Leu Gln Glu
130 135 140 He He Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp Gln Thr Thr Thr
145 150 155 160
Asn Val Val He Pro Ser Phe Asp He Lys Leu Leu Arg Pro Thr He 165 170 175
Phe Ser Thr Phe Lys Leu Glu Glu Val Pro Glu Leu Asn Val Lys Leu 180 185 190 Ser Asp Val Cys Met Gly Thr Ser Ala Ala Pro He Val Phe Pro Pro
195 200 205 Tyr Tyr Phe Lys His Gly Asp Thr Glu Phe Asn Leu Val Asp Gly Wing 210 215 220
He He Wing Asp He Pro Wing Pro Val Wing Leu Ser Glu Val Leu Gln 225 230 235 240
Gln Glu Lys Tyr Lys Asn Lys Glu He Leu Leu Leu Ser He Gly Thr 245 250 255
Gly Val Val Lys Pro Gly Glu Gly Tyr Ser Wing Asn Arg Thr Trp Thr
260 265 270 He Phe Asp Trp Ser Ser Glu Thr Leu He Gly Leu Met Gly His Gly
275 280 285 Thr Arg Ala Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu
290 295 300 Gln Pro Gln Asn Asn Tyr Leu Arg He Gln Glu Tyr Asp Leu Asp Pro 305 310 315 320
Ala Leu Glu Ser He Asp Asp Ala Ser Thr Glu Asn Met Glu Asn Leu 325 330 335
Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn
340 345 350 Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn Ala Glu
355 360 365 Wing Leu Asp Arg Leu Wing Gln He Leu Tyr Glu Glu Lys He Thr Arg
370 375 380 Gly Leu Gly Lys He Ser Leu Glu Val Asp Asn He Asp Pro Tyr Thr 385 390 395 400
Glu Arg Val Arg Lys Leu Leu Phe * 405
(2) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) FORM OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT . : 5
Met Ser Thr Ser Wing Wing Pro He Val Phe Pro Pro Tyr Tyr Phe Lys 1 5 10 15 (2) INFORMATION FOR SEC. FROM IDENT. NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) FORM OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT . : 6: Ala Leu Gln Pro Gln Asn Asn Tyr Leu Arg Gln Glu Tyr Asp Leu Asp 1 5 10 15 (2) INFORMATION FOR SEC. FROM IDENT. NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) FORM OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT . : 7 Pro Asp Trp Val Val He Arg Ser Gln Ser Val Gly Lys 1 5 10 (2) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) CHAIN FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT . : 8:
Lys Wing Phe Val Asn Gly Val Tyr Phe He Asn Thr Tyr Asp Ser Wing 1 5 10 15 (2) INFORMATION FOR SEC. FROM IDENT. NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STPANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO.:9: Asn Asn Tyr Leu Arg He Gln Glu Tyr Asp Leu Pro Pro Ala Leu 1 5 10 15 (2) INFORMATION FOR SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) FORM OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO.:10; Val Val Lys Arg Leu Wing Gly Tyr Phe Asp Val 1 5 10 (2) INFORMATION FOR SEC. FROM IDENT. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) FORM OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGINAL SOURCE: (A) ORGANISM: Pentaclethra macroloba ( xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO.:ll Glu Asn Met Glu Asn Leu Glu Lys 1 5
Claims (32)
- CLAIMS 1. A substantially purified protein isolated from the genus Pentaclethra, characterized because it has insecticidal properties.
- 2. A substantially purified polypeptide characterized in that it comprises an amino acid sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ. FROM IDENT. DO NOT. : 2; b) residues 22-409 of the sequence established in SEC. FROM IDENT. NO .: 2; c) residues 29-409 of the amino acid sequence set forth in SEQ. FROM IDENT. NO .: 2; and d) a pesticidically active fragment of the amino acid sequence set forth in SEQ. FROM IDENT. DO NOT. : 2. 3. A polypeptide having insecticidal activity against the corn rootworm wherein the polypeptide comprises a variation of an amino acid sequence selected from the group characterized in that it consists of: a) the amino acid sequence set forth in SEQ. FROM IDENT. DO NOT. : 2; b) residues 22-409 of the sequence established in SEC. FROM IDENT. NO .: 2; Y c) residues 29-409 of the amino acid sequence set forth in SEQ. DE IDENT.NO.:2, wherein the variation comprises at least one substitution, truncation, deletion or internal insertion of amino acids. 4. The protein according to claim 1, characterized in that the protein has an amino acid sequence as set forth in Figure 1 and SEQ. FROM IDENT. US: 1 and 2. 5. A substantially modified protein having insecticidal activity, characterized in that the protein comprises the amino acid sequence set forth in Figure 1 and SEC. FROM IDENT. NOS: 1 and 2. 6. A DNA sequence encoding the protein according to claim 5. 7. A vector characterized in that it comprises the DNA sequence according to claim 6. 8. An isolated nucleotide sequence encoding a polypeptide having insecticidal activity for corn rootworm, characterized in that the nucleotide sequence encodes a polypeptide selected from the group consisting of: a) the amino acid sequence set forth in SEQ. FROM IDENT. DO NOT . : 2; b) residues 22-409 of the sequence established in SEC. FROM IDENT. NO .: 2; c) residues 22-409 of the amino acid sequence set forth in SEQ. FROM IDENT. DO NOT . : 2; d) a polypeptide comprising a variation of (a), (b) or (c), wherein the variation comprises at least one substitution, truncation, deletion or internal insertion of amino acids. 9. An isolated nucleotide molecule that encodes a polypeptide having insecticidal activity, the molecule having a sequence selected from the group consisting of: (a) the sequence set forth in Figure 1 and SEC. FROM IDENT. NO: 1; (b) nucleotide sequences that encode a polypeptide having insecticidal activity and that hybridize to the sequences of (a) above under strict conditions defined by a wash restriction of 0.3 M NaCl, 0.03 M sodium citrate, 0.1% SDS at 70 ° C; (c) nucleotide sequences which encode a polypeptide having insecticidal activity and which differ from the sequences of (a) and (b) due to the degeneracy of the genetic code. 10. An organism characterized in that it has been transformed with the vector according to claim 7. 11. An isolated nucleotide sequence characterized in that it encodes the protein established in the Figure 1. 12. The nucleotide sequence according to claim 11, characterized in that the nucleotide sequence is the sequence established in Figure and SEC. FROM IDENT. NO: l. 13. The DNA sequence according to claim 11, characterized in that the sequence is a synthetic sequence. 1 . The DNA sequence according to claim 13, characterized in that the sequence has been optimized for expression in corn. 15. A plant that has been stably transformed with an expression cassette characterized in that it comprises a promoter that drives expression in a plant cell operably linked to a nucleotide sequence encoding an insecticidal protein wherein the protein is selected from the group that consists of: a) the amino acid sequence established in SEC. FROM IDENT. DO NOT. :2; b) residues 22-409 of the sequence established in SEC. FROM IDENT. DO NOT. : 2 c) waste 29-409. of the amino acid sequence established in SEC. FROM IDENT. NO .: 2; d) a polypeptide comprising a variation of (a), (b) or (c) wherein the variation comprises at least one substitution, truncation, deletion or internal insertion of amino acids. 16. The plant according to claim 15, characterized in that the plant is corn. 17. The seed of the plant according to claims 15 or 16. 18. The plant according to claim 15, characterized in that the nucleotide sequence encodes the amino acid sequence established in Figure 1 and SEQ. FROM IDENT. NOS: 1 and 2. 19. The plant according to claim 15, characterized by the nucleotide sequence is the sequence established in Figure 1 and SEC. FROM IDENT. NO .: 1. The plant according to claim 15, characterized in that the nucleotide sequence is operably linked to a preferential root promoter. 21. The plant according to claim 18, characterized in that the nucleotide sequence is operably linked to a preferential root promoter. 22. The plant according to claim 19, characterized in that the nucleotide sequence is operably linked to a preferential root promoter. 23. A method for controlling the corn rootworm, the method is characterized in that it comprises: transforming a plant cell with an expression cassette comprising a promoter that drives an expression in a plant cell operably linked to a nucleotide sequence which encodes an insecticidal protein wherein the protein is selected from the group consisting of: a) the amino acid sequence set forth in SEC. FROM IDENT. NO .: '2; b) residues 22-409 of the sequence established in SEC. FROM IDENT. DO NOT . : 2; c) residues 29-409 of the amino acid sequence set forth in SEC. FROM IDENT. NO.:2; d) a polypeptide comprising a variation of (a), (b) or (c) wherein the variation comprises at least one replacement, truncation, deletion or internal insertion of amino acids. 24. The method according to claim 23, characterized in that the promoter is a preferential root promoter. 25. The method according to claim 24, wherein the nucleotide sequence has a sequence that is selected from the group consisting of: (a) the sequence set forth in Figure 1 and 1 SEC. FROM IDENT. N0: 1; (b) nucleotide sequences which encode a polypeptide having insecticidal activity and which hybridizes to the sequences of (a) above under stringent conditions comprising; and, (c) nucleic acid sequences that encode a polypeptide having insecticidal activity and which differ from the sequences of (a) and (b) due to the degradation of the genetic code. 26. The method of compliance with the claim 25, characterized in that the plant cell is monocotyledonous. 27. The method of compliance with the claim 26, characterized in that the monocotyledone is corn. 28. A plant cell that has been stably transformed with an expression cassette comprising a promoter that drives expression in a plant cell operably linked to a nucleotide sequence that encodes an insecticidal protein wherein the protein has the activity of corn rootworm and is selected from the group consisting of: a) the amino acid sequence set forth in SEC. FROM IDENT. NO .: 2; b) residues 22-409 of the sequence established in SEC. FROM IDENT.NO.:2; c) residues 29-409 of the amino acid sequence set forth in SEC. FROM IDENT. NO .: 2; d) a polypeptide comprising a variation of (a), (b) or (c) wherein the variation comprises at least one replacement, truncation, deletion or internal insertion of amino acids. 29. The plant cell according to claim 28, characterized in that the promoter is a preferential promoter for roots. 30. The plant cell according to claim 29, characterized in that the nucleotide sequence has a sequence that is selected from the group consisting of: (a) the sequence set forth in Figure 1 and SEQ. FROM IDENT. DO NOT. : 1; (b) nucleotide sequences that encode a polypeptide having insecticidal activity and that hybridizes the sequences of (a) above under strict conditions defined by a wash restriction of 0.
- 3 M NaCl, 0.03 M sodium citrate, 0.01% SDS at 70 ° C; and (c) nucleotide sequences that encode a polypeptide having insecticidal activity and which differ from the sequences of (a) and (b) due to the degeneracy of the genetic code. 31. The plant cell according to claim 30, characterized in that the plant cell is monocotyledonous. 32. The plant according to claim 31, characterized in that the monocot is corn.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/047864 | 1997-05-29 | ||
US047864 | 1997-05-29 | ||
US074912 | 1998-05-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99010882A true MXPA99010882A (en) | 2000-05-01 |
Family
ID=
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