method of controlling a cartridgeworm insect

专利摘要:
Use of cry1da in combination with cry1be for resistant insect management The present invention includes methods and plants for the control of cartridgeworm insects, said plants comprising a cry1da insecticide protein and a cry1be insecticide protein and various combinations of other proteins comprising this pair of proteins to retard or prevent the development of resistance by insects.
公开号:BR112012014727B1
申请号:R112012014727
申请日:2010-12-16
公开日:2019-09-03
发明作者:T Woosley Aaron；J Sheets Joel；Narva Kenneth；P Storer Nicholas；L Burton Stephaine；Meade Thomas
申请人:Dow Agrosciences Llc；
IPC主号:

专利说明:

Descriptive Report of the Patent of Invention for METHOD OF CONTROL OF AN INSECT Caterpillar-DOCARTUCHO.
Background to the invention [001] Human beings grow corn for food and energy applications. Human beings also grow many other crops, including soybeans and cotton. Insects eat and damage plants and thus undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost due to the damage they cause. Synthetic organic chemical insecticides have been the primary tools used to control insect pests, but biological insecticides, such as insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect resistant plants through transformation with Bt insecticidal protein genes has revolutionized modern agriculture and highlighted the importance and value of insecticidal proteins and their genes.
[002] Several Bt proteins have been used to create the insect resistant transgenic plants that have been successfully registered and commercialized so far. These include Cry1Ab, Cry1Ac, Cry1F and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton and Cry3A in potato.
[003] Commercial products that express these proteins express a single protein, except in cases where the combined insecticidal spectrum of 2 proteins is desired (for example, Cry1Ab and Cry3Bb in maize combined to provide resistance to lepidopterans and chrysomelids, respectively) or where the independent action of proteins makes them useful as a tool to delay the
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2/54 development of resistance in susceptible insect populations (for example, Cry1Ac and Cry2Ab in cotton combined to confer resistance management to tobacco caterpillar). See also U.S. Patent Application Publication No. 2009/0313717, which refers to a Cry2 protein plus Vip3 Aa, Cry1F or Cry1A for the control of Helicoverpa zea or armigerain. WO 2009/1 32850 refers to Cry1F or Cry1A and Vip3Aa for the control of Spodoptera frugiperda. U.S. Patent Application Publication No. 2008/031 1096 relates, in part, to Cry1Ab for the control of Cry1F-resistant ECB.
[004] That is, some of the qualities of transgenic insect resistant plants that led to the rapid and widespread adoption of this technology have also given rise to concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested to preserve the utility of St-based insect resistance traits, which include using high-dose proteins in combination with a refuse and alternating with or co-employing different toxins (McGaughey et al. (1998) , Bt Resistance management, Nature Biotechnol. 16: 144-146).
[005] The proteins selected for use in an insect resistance management (MRI) stack need to exert their insecticidal effect independently, so that resistance developed to one protein does not confer resistance to the second protein (that is, there is no cross-resistance to proteins). If, for example, a selected pest population is resistant to Protein A it is sensitive to Protein B, it can be concluded that there is no cross-resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A only.
[006] In the absence of resistant insect populations, assess
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3/54 tions can be made based on other characteristics that are presumed to be related to the mechanism of action and cross-resistance potential. The usefulness of receptor-mediated binding in identifying insecticidal proteins that are unlikely to exhibit cross-resistance has been suggested (van Mellaert et al. 1999). The key predictor of the lack of cross-resistance inherent in this approach is that insecticidal proteins do not compete for receptors in a sensitive insect species.
[007] In the case where two Bt toxins compete for at least receptor, then, if that receptor mutates, so that one of the toxins no longer binds to that receptor and thus is no longer an insecticide against the insect, it could be the case where the insect would also be resistant to the second toxin (which binds competitively to the same receptor). That is, the insect is said to have cross-resistance to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to these two toxins.
[008] For example, Cry1Fa protein is useful in controlling many pests of lepidopteran species, including the European corn borer (ECB; Ostrinia nubilalis (Hübner)) and FAW and is active against the sugarcane borer ( SCB; Diatraea saccharalis). The Cry1Fa protein, as produced in transgenic corn plants containing the TC I 507 event, is responsible for an insect resistance trait unparalleled in the industry for FAW control. Cry1Fa is also used in Herculex®, SmartStax® and WideStrike® products.
[009] The ability to conduct receptor binding studies (competitive or homologous) using Cry1Fa protein is limited because the most common technique available for protein labeling for detection in receptor binding assays inactivates the insecticidal activity of the Cry1Fa protein.
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4/54 [0010] Additional Cry toxins are listed on the Official B.t. Nomenclature Committee (Crickmore et al .; lifesci.sussex.ac.uk/home/Neil_Crickmore/St/). There are currently almost 60 major groups of Cry toxins (Cry1-Cry59), with additional similar Cyt toxins and VIP toxins. Many of each numerical group have subgroups in uppercase and subgroups in uppercase have subgroups in lowercase. (Cry1 has A-L and Cry1A has a-i, for example).
Brief Summary of the Invention [0011] The present invention relates, in part, to the surprising discovery that Cry1Da and Cry1Be do not compete for binding sites in gut cell membrane preparations in caterpillar-cartridge (FAW; Spodoptera frugiperda). As those skilled in the field will recognize with the benefit of the present description, plants that produce both of these proteins (including insecticidal portions of the full-length proteins) can delay or prevent the development of resistance to either of these insecticidal proteins in isolation. Corn and soy are some favorite plants.
[0012] Thus, the present invention relates, in part, to the use of a Cry1Da protein in combination with a Cry1Be protein. Plants (and acres planted with such plants) that produce both of these proteins are included within the scope of the present invention.
[0013] The present invention also relates, in part, to triple cells or pyramids of three (or more) toxins, with Cry1Da and Cry1Be being the base pair. In some preferred pyramid modalities, the combination of selected toxins provides three sites of action against FAW. Some pyramid combinations with three preferred action sites include the base pair of proteins in question plus Cry1Fa, Vip3Ab or Cry1E as the third objective protein
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5/54 tion of FAW. Such particular triple cells would provide, advantageously and surprisingly, according to the present invention, three sites of action against FAW. This can help to reduce or eliminate the requirement for acres of protection.
[0014] Additional toxins / genes can also be added according to the present invention. For example, if Cry1Fa is stacked with the pair of proteins in question (Cry1Fa and Cry1Be are both active against FAW and European corn borer (ECB)), adding an extra protein to that triple cell, where the fourth protein added is objective ECB, would provide three action sites against FAW and three action sites against ECB. This added protein (the fourth protein) could be selected from the group consisting of Cry2A, Cry1I, D1G-3 and Cry1Ab. This would result in a pile of four proteins having three sites of action against two insects (ECB and FAW).
DETAILED DESCRIPTION OF THE INVENTION [0015] The present invention relates, in part, to the surprising discovery that Cry1Da and Cry1Be do not compete for binding to each other in the intestine of cartridge caterpillars (FAW; Spodoptera frugiperda). Thus, a Cry1Da protein can be used in combination with a Cry1Be protein in transgenic corn (and other plants; for example, cotton and soy, for example) to delay or prevent FAW from developing resistance to any of these proteins alone. The pair of proteins in question can be effective in protecting plants (such as corn plants and / or soybean plants) from damage by Cry-resistant cartridge caterpillar. That is, one use of the present invention is to protect maize and other economically important plant species from damage and loss of yield caused by populations of cartridge caterpillar that could develop resistance to Cry1Da or Cry1Be.
[0016] The present invention thus teaches a management stack
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6/54 Insect Resistant Management (IRM) comprising Cry1Da and Cry1Be to prevent or alleviate the development of resistance by FAW to either or both of these proteins.
[0017] The present invention provides compositions for the control of pest lepidopterans comprising cells that produce a Cry1Da insecticidal protein and a Cry1Be insecticidal protein.
[0018] The invention further comprises a host transformed to produce an insecticidal protein Cry1Da and an insecticidal protein Cry1Be, wherein said host is a microorganism or a plant cell. The polynucleotide (s) in question is preferably in a genetic construct under the control of (one) promoters that is not (are) of Bacillus thuringiensis. The polynucleotides in question may include the use of codons for enhanced expression in a plant.
[0019] Additionally, it is intended that the invention provides a method of controlling pest lepidopterans comprising contacting said pests or the environment of said pests with an effective amount of a composition containing a protein containing central toxin Cry1Da and still containing a protein containing central toxin Cry1Be.
[0020] One embodiment of the invention comprises a maize plant comprising a plant-capable gene encoding a Cry1Be insecticidal protein and a plant-capable gene encoding a Cry1Da insecticidal protein and cultivation of such a plant.
[0021] Another embodiment of the invention comprises a corn plant in which a gene capable of expression in a plant that encodes an insecticidal protein Cry1Be and a gene capable of expression in a plant that encodes an insecticidal protein Cry1Da were inspected 870180072826, of 20/08 / 2018, p. 16/73
7/54 trogredidos in said corn plant and cultivation of such plant.
[0022] As described in the Examples, competitive receptor binding studies using radiolabeled Cry1Da and Cry1Be proteins show that the Cry1Be protein does not compete for binding in FAW tissues to which Cry1Da binds. These results also indicate that the combination of proteins Cry1Da and Cry1Be can be an effective means to alleviate the development of resistance in FAW populations to any of these proteins. Thus, based, in part, on the data described here, it is believed that the coproduction (stacking) of the Cry1Be and Cry1Da proteins can be used to produce a high-dose MRI stack for FAW.
[0023] Other proteins can be added to this pair. For example, the present invention also relates, in part, to triple cells or pyramids of three (or more) toxins, with Cry1Da and Cry1Be being the base pair. In some preferred pyramid modalities, selected toxins have three distinct sites of action against FAW. Some pyramid combinations with three preferred action sites include the base pair of proteins in question plus Cry1Fa, Vip3Ab or Cry1E as the third protein for objectifying FAW. Such particular triple cells would provide, advantageously and surprisingly, according to the present invention, three sites of action against FAW. This can help to reduce or eliminate the requirement for acres of protection. By different sites of action, it is understood that any of the given proteins do not cause cross-resistance against each other.
[0024] Additional toxins / genes can also be added according to the present invention. For example, if Cry1Fa is stacked with the pair of proteins in question (Cry1Fa and Cry1Be are both active against FAW and European corn borer (ECB)), adding an extra protein to that triple cell, where the fourth protein is added
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8/54 objective target ECB, would provide three sites of action against FAW and three sites of action against ECB. These added proteins (the fourth protein) could be selected from the group consisting of Cry2A, Cry1I, DIG-3 (see US Patent Application Serial No. 61 / 284,278 (filed December 16, 2009) and US 2010 00269223) and Cry1Ab (US 2008 031 1096). This would result in a pile of four proteins having three sites of action against two insects (ECB and FAW).
[0025] Thus, one option of use is to use the pair of proteins in question in combination with a third toxin / gene and use that triple cell to alleviate the development of resistance, in FAW, to any of these toxins. Therefore, the present invention also relates, in part, to triple cells or pyramids of three (or more) toxins. In some preferred pyramid modalities, selected toxins have three distinct sites of action against FAW.
[0026] Included among the options of use of the present invention would be the use of two, three or more proteins of the proteins in question in growing regions of crops where FAW can develop resistant populations. For example, for use of Cry1Fa plus Cry1D, see U.S. Patent Application Serial No. 61 / 284,252 (filed December 16, 2009), which shows that Cry1D is active against Cry1F-resistant FAW. This order shows that Cry1D does not compete with Cry1F for binding to FAW membrane preparations. For guidance regarding the use of Cry1Fa and Cry1Be, see U.S. Patent Application Serial No. 61 / 284,294 and with Vip3Ab, see concurrently filed application COMBINED USE OF Vip3Ab AND Cry1Fa FOR MANAGEMENT OF RESISTANT INSECTS. With Cry1Fa being active against FAW (and European corn borer (ECB)), Cry1Da plus Cry1Be plus Cry1Fa would provide, advantageously and surprisingly, according to the present invention,
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9/54 three places of action against FAW. This can help to reduce or eliminate the requirement for acres of protection.
[0027] Cry1Fa is used in Herculex®, SmartStax® and WidesStrike® products. The pair of genes in question (Cry1 Da and Cry1 Be) could be combined, for example, in a Cry1Fa product, such as Herculex®, SmartStax® and WideStrike®. Therefore, the pair of proteins in question could be significant in reducing the selection pressure on these and other commercialized proteins. The pair of proteins in question could thus be used as in the three gene combinations for corn and other plants (cotton and soy, for example).
[0028] As discussed above, additional toxins / genes can also be added according to the present invention. For objectification of ECB, Cry2A, Cry1I and / or DIG-3 can be used. See U.S. Patent Application Serial No. 61 / 284,278 (filed December 16, 2009). For the addition of Cry1Ab (for ECB control), see US Patent Application Publication No. 2008/031 1096. For the use of Cry1E for FAW control, see US Patent Application Serial No. 61 / 284,278 (deposited on December 16, 2009).
[0029] Plants (and acres planted with such plants) that produce any of the protein combinations in question are included within the scope of the present invention. Additional toxins / genes can also be added, but the particular cells discussed above provide, advantageously and surprisingly, multiple sites of action against FAW and / or ECB. This can help to reduce or eliminate the requirement for acres of protection. A field thus planted over ten acres is thus included within the present invention.
[0030] GENBANK can also be used to obtain information
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10/54 quenches for any of the genes and proteins discussed here. See Appendix A below. Patents also describe relevant sequences. For example, U.S. Patent No. 5,188,960 and U.S. Patent No. 5,827,514 describe proteins containing central Cry1Fa toxin suitable for use in carrying out the present invention. U.S. Patent No. 6,218,188 describes plant-optimized DNA sequences encoding proteins containing central Cry1Fa toxin that are suitable for use in the present invention.
[0031] Protein combinations described here can be used to control pest lepidopterans. Adult lepidopterans, for example, butterflies and moths, feed primarily on flower nectar and are a significant pollinator. Almost all lepidopteran larvae, that is, caterpillars, feed on plants and many are serious pests. Caterpillars feed on or within the foliage or on the roots or stems of a plant, depriving the plant of nutrients and often destroying the plant's physical support structure. Additionally, caterpillars feed on stored fruits, fabrics and grains and flours, ruining these products for sale or seriously reducing their value. As used here, reference to pest lepidoptera refers to various stages of the pest's life, including larval stages.
[0032] Some chimeric toxins of the present invention comprise a complete N-terminal central toxin portion of a Bt toxin and, at some point beyond the end of the central toxin portion, the protein has a transition to a heterologous pro-toxin sequence. The N-terminal insecticide-active toxin portion of a Bt toxin is referred to as the central toxin. The transition from the central toxin segment to the heterologous pro-toxin segment can occur approximately at the toxin / pro-toxin junction or, alternatively, a portion of the native pro-toxin (which extends beyond the toxin portion
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11/54 central) can be retained, with the transition to the heterologous pro-toxin portion occurring downstream.
[0033] As an example, a chimeric toxin of the present invention is a portion of the complete central toxin of Cry1 Da (amino acids 1 to 601) and / or a heterologous pro-toxin (amino acids 602 to the C-terminus). In a preferred embodiment, the portion of a chimeric toxin comprising the pro-toxin is derived from a Cry1Ab protein toxin. In a preferred embodiment, the portion of a chimeric toxin comprising the pro-toxin is derived from a Cry1Ab protein toxin.
[0034] Those skilled in the field will appreciate that Bt toxins, even within a certain class, such as Cry1Be, will vary to some extent in the length and precise location of the transition from the central toxin portion to the pro-toxin portion . Typically, Cry1Be toxins are about 1150 to about 1200 amino acids in length. The transition from the central toxin portion to the pro-toxin portion will typically occur between about 50% to about 60% of the full-length toxin. The chimeric toxin of the present invention will include the full extent of that portion of the central N-terminal toxin. Thus, the chimeric toxin will comprise at least about 50% of the total length of the Cry1Be protein. This will typically have at least about 590 amino acids. With respect to the pro-toxin portion, the total extension of the Cry1Ab pro-toxin portion extends from the end of the central toxin portion to the C-terminus of the molecule.
[0035] Genes and Toxins. The genes and toxins useful in accordance with the present invention include not only the full-length sequences disclosed, but also fragments of those sequences, variants, mutants and fusion proteins which retain the pesticidal activity characteristic of the toxins specifically exemplified
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12/54 here. As used herein, the terms variants or variations of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term equivalent toxins refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.
[0036] As used here, the limits represent approximately 95% (Cry1Da's and Cry1Be's), 78% (Cry1D's and Cry1B's) and 45% (Cry1's) of sequence identity, as per Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins, N. Crickmore, DR Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum and DH Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. These cuts can also be applied to central toxins only.
[0037] It will be evident to those skilled in the field that genes encoding active toxins can be identified and obtained through various means. The specific genes or gene portions exemplified here can be obtained from the isolates deposited in a culture depository. These genes, or portions or variants thereof, can also be constructed synthetically, for example, using a gene synthesizer. Gene variations can also be readily constructed using standard techniques to make point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut nucleotides from the ends of these genes. Genes that encode active fragments can also be obtained using a variety of restriction enzymes. Proteases can be used to directly obtain active fragments of these toxins from
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13/54 protein.
[0038] Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the present invention. Also, due to the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed here. It is well within the ability of those skilled in the field to create these alternative DNA sequences that encode the same or essentially the same toxins. Such variant DNA sequences are within the scope of the present invention. As used here, reference to essentially the same sequence refers to sequences which have amino acid substitutions, deletions, additions or insertions which do not materially affect pesticidal activity. Fragments of genes that encode proteins that retain pesticidal activity are also included in this definition.
[0039] Another method for identifying the genes encoding toxins and gene portions useful in accordance with the present invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. Such sequences can be detectable by virtue of an appropriate label or can be made inherently fluorescent, as described in International Application No. WO93 / 16094. As is well known in the art, if the probe molecule and the nucleic acid sample hybridize by forming a strong bond between the two molecules, it can reasonably be assumed that the probe and the sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by methods well known in the art as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pages 169-170. Some examples of combinations of salt and temperatu concentrations
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14/54 ra are as follows (in ascending order of stringency): 2X SSPE or SSC at room temperature; I X SSPE or SSC at 42 ° C; 0.1 X SSPE or SSC at 42 ° C; 0.1 X SSPE or SSC at 65 ° C. Probe detection is a means of determining, in a known way, whether hybridization has occurred. Such probe analysis provides a quick method for identifying genes that encode toxin of the present invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. Such nucleotide sequences can also be used as PCR primers to amplify the genes of the present invention.
[0040] Variant Toxins. Certain toxins of the present invention have been specifically exemplified here. Since these toxins are merely exemplary of the toxins of the present invention, it will be readily apparent that the present invention comprises variant or equivalent toxins (and nucleotide sequences encoding equivalent toxins) having the same or similar pesticidal activity as the exemplified toxin. Equivalent toxins will have amino acid homology with an exemplified toxin. Such an amino acid homology will typically be greater than 75%, preferably greater than 90% and, even more preferably, greater than 95%. The amino acid homology will be greater in critical regions of the toxin which account for biological activity or are involved in determining the three-dimensional configuration which, ultimately, is responsible for biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical for activity or are conservative amino acid substitutions which do not affect the molecule's three-dimensional configuration. For example, amino acids can be placed in the following classes: no
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15/54 polar, uncharged, basic and acidic polar. Conservative substitutions by which an amino acid of a class is replaced by another amino acid of the same type fall within the scope of the present invention, provided that the substitution does not materially alter the biological activity of the compound. Below is a list of examples of amino acids belonging to each class.
Class of Amino Acid Examples of Amino Acids Non-polar Wing, Val, Leu, Ile, Pro, Met, Phe, Trp Polar not charged Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acid Asp, Glu Basic Lys, Arg, His
[0041] In some cases, non-conservative substitutions can also be made. The critical factor is that these substitutions should not significantly impair the biological activity of the toxin.
[0042] Recombinant hosts. The genes encoding the toxins of the present invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the present invention. Other host organisms can also be transformed with one or both of the toxin genes, then used to obtain the synergistic effect. With suitable microbial hosts, for example, Pseudomonas, microbes can be applied to the pest site, where they will proliferate and be ingested. The result is pest control. Alternatively, the microbe that hosts the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the target pest environment.
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16/54 [0043] Where the Bt toxin gene is introduced via a suitable vector into a microbial host and said host is applied to the environment in a living state, it is essential that certain host microbes are used. Microbial hosts are selected which are known to occupy the phytosphere (phyloplane, phyllosphere, rhizosphere and / or rhizoplane) of one or more crops of interest. These microorganisms are selected in order to be able to successfully compete in the particular environment (crop and other insect habitats) with wild type microorganisms, they provide maintenance and stable expression of the gene that expresses the polypeptide pesticide and, desirably , provide enhanced protection to the pesticide against environmental degradation and inactivation.
[0044] A large number of microorganisms are known to inhabit the phylloplane (the surface of flat leaves) and / or the rhizosphere (the soil surrounding the roots of plants) in a wide variety of important crops. These microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, for example, the genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostat; fungi, particularly yeast, for example, the genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of particular interest are bacterial species of the phytosphere, such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus and Azotobacter vinii; and phytosphere yeast species, such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Sac
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17/54 charomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae and Aureobasidium pollulans. Of particular interest are pigmented microorganisms.
[0045] A wide variety of methods are available for introducing a Bt gene that encodes a toxin into a microbial host under conditions which allow for stable maintenance and expression of the gene. Such methods are well known to those skilled in the field and are described, for example, in U.S. Patent No. 5,135,867, which is incorporated herein by reference.
[0046] Cell Treatment. Bacillus thuringiensis or recombinant cells that express Bt toxins can be treated to prolong the activity of the toxin and stabilize the cell. The pesticidal microcapsule that is formed comprises the toxin or Bt toxins within a cell structure that has been stabilized and will protect the toxin when the microcapsule is applied to the target pest environment. Suitable host cells can include prokaryotes or eukaryotes, usually being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application low enough to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be prokaryotes and lower eukaryotes, such as fungi.
[0047] The cell will usually be intact and will be substantially in the proliferative form when treated, rather than in a spore form although, in some cases, spores may be employed.
[0048] Treatment of the microbial cell, for example, a microbe containing the B.t. toxin gene or genes, can be by means
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18/54 chemical or physical or through a combination of chemical and / or physical means, as long as the technique does not adversely affect the properties of the toxin, nor does it diminish the cellular protective capacity of the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic number 17-80. More particularly, iodine can be used under mild conditions and for a time sufficient to obtain the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; antiinfectives, such as zefiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl alcohol and ethanol; various histological fixators, such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative (see: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host's environment. Examples of physical media are short wavelength radiation, such as gamma radiation and X-rays, freezing, UV irradiation, lyophilization and the like. Methods for treating microbial cells are described in the Patents. U.S. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.
[0049] Cells generally have enhanced structural stability, which will enhance resistance to environmental conditions. Where the pesticide is in a proforma, the cell treatment method should be selected so as not to inhibit the processing of the proforma to the mature form of the pesticide by the target pathogenic pest. For example, formaldehyde will cross-link proteins and could inhibit the processing of a polypeptide pesticide proforma. The treatment method should retain at least a substantial portion of the toxin's bioavailability or bioactivity.
[0050] Characteristics of particular interest in the selection of a
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19/54 host cells for production purposes include ease of introduction of the B.t. in the host, availability of expression systems, expression efficiency, pesticide stability in the host and the presence of auxiliary genetic capabilities. Features of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation and intracellular engulfment or the formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness for ingestion by pests; ease of death and fixation without damage to the toxin; and the like. Other considerations include ease of formulation and handling, costs, storage stability and the like.
[0051] Growth of Cells. The cell host containing the B.t. it can be grown in any convenient nutrient medium, where the DNA construct confers a selective advantage, constituting a selective medium, so that substantially all cells retain the B.t. These cells can then be collected according to conventional ways. Alternatively, the cells can be treated before collection.
[0052] B.t. that produce the toxins of the invention can be grown using standard means in the field and fermentation techniques. At the end of the fermentation cycle, the bacteria can be collected first by separating the spores and crystals from B.t. fermentation broth by means well known in the art. B.t. spores and crystals. recovered can be formulated into a wet powder, liquid concentrate, granules or other formulations by adding surfactants, dispersants, inert vehicles and other components to facilitate handling and application to particular target pests. Such formulations and application procedures are all well known in the art.
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20/54 [0053] Formulations. Formulated bait granules containing an attractant and spores, crystals and toxins from B.t. or recombinant microbes comprising the genes obtainable from B.t. described here, can be applied to the soil. The formulated product can also be applied as a seed coating or root treatment or treatment for the whole plant in the later stages of the harvest cycle. Soil and plant treatments with B.t. can be used as moisturizing powders, granules or powders, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates and the like) or botanical materials (powdered corn cobs, rice husks, nut shells) and the like). The formulations can include dispersant-adherent adjuvants, stabilizing agents, other pesticidal or surfactant additives. Liquid formulations can be aqueous or non-aqueous and used as foams, gels, suspensions, emulsifiable concentrates or the like. The ingredients can include rheological agents, surfactants, emulsifiers, dispersants or polymers.
[0054] As will be appreciated by those skilled in the art, the concentration of pesticide will vary widely, depending on the nature of the particular formulation, particularly if it is a concentrate or has to be used directly. The pesticide will be present in at least 1% by weight and can be 100% by weight. Dry formulations will have about 1-95% by weight of the pesticide, whereas liquid formulations will generally have about 1-60% by weight of solids in the liquid phase. The formulations will generally have from about 10 ² to about 10 ⁴ cells / mg. These formulations will be administered at about 50 mg (liquid or dry) at 1 kg or more per hectare.
[0055] The formulations can be applied to the pest environment
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21/54 lepidóptera, for example, foliage or soil, through spraying, sprinkling, spraying or similar.
[0056] Transformation of Plant. A preferred recombinant host for producing the insecticidal proteins of the present invention is a transformed plant. Genes encoding Bt toxin proteins, as disclosed here, can be inserted into plant cells using a variety of techniques, which are well known in the art. For example, a large number of cloning vectors comprising a replication system in Escherichia coli and a marker that allows selection of transformed cells are available to prepare for the insertion of foreign genes in higher plants. The vectors comprise, for example, pBR322, the pUC series, M 13mp series, pACYC184, inter alia. Accordingly, the DNA fragment having the sequence encoding the Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. E. coli cells are grown in an appropriate nutrient medium, then collected and subjected to lysis. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis and other molecular-biochemical biological methods are, in general, performed as a method of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned into the same or other plasmids. Depending on the method of inserting the desired genes into the plant, other DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right edge, but often the right and left edges of the Ti or Ri T-DNA plasmids have to be joined together as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of
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22/54 plant has been extensively researched and sufficiently described in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et al., (1986) and An et al., (1985) and is well established in technical.
[0057] Once the inserted DNA has been integrated into the plant's genome, it is relatively stable. The transformation vector normally contains a selectable marker that gives the transformed plant cells resistance to a biocide or an antibiotic, such as Bialaphos, anamycin, G418, Bleomycin or Hygromycin, inter alia. The individually employed marker should therefore allow the selection of transformed cells, rather than cells that do not contain the inserted DNA.
[0058] A large number of techniques are available for inserting DNA into a host plant cell. These techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation, fusion, injection, biolistic (microparticle bombardment) or electroporation agent, as well as other possible methods. If Agrobacteria are used for transformation, the DNA to be inserted has to be cloned into special plasmids, that is, an intermediate vector or a binary vector. The intermediate vector can be integrated into the Ti or Ri plasmid through homologous recombination by virtue of sequences that are homologous to the sequences in T-DNA. The Ti or Ri plasmid also comprises the region necessary for the transfer of TDNA.
[0059] Intermediate vectors cannot be reproduced in Agrobacteria. The intermediate vector can be transferred in Agrobacterium tumefaciens by means of an auxiliary plasmid (conjugation). Binary vectors can reproduce in E. coli and in Agrobacteria. They comprise a selection marker gene and a ligand or polylinker which is networked across the right T-DNA border regions
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23/54 and Left. They can be transformed directly into Agrobacteria (Holsters et.al., 1978). Agrobacteria used as a host cell must comprise a plasmid bringing a region to come. The vir region is necessary for the transfer of T-DNA in the plant cell. Additional T-DNA may be contained. The bacterium thus transformed is used for the transformation of plant cells. Plant explants can advantageously be grown with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of DNA in the plant cell. Whole plants can then be regenerated from the infected plant material (for example, leaf pieces, stem segments, roots, but also protoplasts or cells grown in suspension) in a suitable medium, which may contain antibiotics or biocides. for selection. The plants thus obtained can then be tested for the presence of the inserted DNA. No special demands are made with respect to plasmids in the case of injection and electroporation. It is possible to use common plasmids such as, for example, pUC derivatives.
[0060] The transformed cells grow inside the plants in the usual way. They can form germ cells and transmit the transformed trait (s) to the offspring. Such plants can be grown in the normal way and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
[0061] In a preferred embodiment of the present invention, plants will be transformed with genes in which the use of codons has been optimized for plants. See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference. Although some truncated toxins are exemplified here, it is well known in the Bt technique that 130 kDa type toxins (full length) have a goal
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24/54 N-terminal which is the central toxin and a C-terminal half which is a pro-toxin tail. Thus, appropriate tails can be used with truncated / central toxins of the present invention. See, for example, U.S. Patent No. 6,218, 188 and U.S. Patent No. 6,673,990. In addition, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). A non-limiting example of a preferred transformed plant is a fertile maize plant comprising an expressionable plant gene encoding a Cry1Da protein and further comprising a second plant expressible gene encoding a Cry1Be protein.
[0062] Transfer (or introgression) of the trait (s) determined by Cry1Da and Cry1Be in endogenous maize lines can be obtained through improvement by recurrent selection, for example, by means of crossbreeding. In this case, a desired recurring parent is first mated with an endogenous donor (the non-recurring parent) that brings the appropriate gene (s) to the trait (s) determined by Cry1Da and Cry1Be. The offspring of that crossing is then crossed again with the recurring parent, followed by selection in the resulting offspring so that the desired trait (s) is (are) transferred from the non-recurring parent. After three, preferably four, more preferably five or more generations of recruitments with the recurrent parent with selection by the desired trait (s), the offspring will be heterozygous for loci that control the trait (s) that is (are) being transferred, but will be similar to the parental applicant in most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th ^to Ed., 172175; Fehr (1987) Principles of Cultivar Development, Vol. 1: Theory and Technique, 360-376).
[0063] Insect Resistance Management Strategies
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25/54 (IRM). Roush et al., For example, describes two toxin strategies, also called pyramidation or stacking for the management of transgenic crop insecticides. (The Royal Society. Phil. Trans. R. Soc. Lond. B (1998) 353, 1777-1786).
[0064] On its website, the United States Environmental Protection Agency (epa.gov/oppbppdl/biopesticides/pips/Bt_corn_refuge_2006.htm) publishes the following requirements for providing non-GM (ie non-Bt) waste (a crop section) / non-Bt corn) for use with transgenic crops that produce a single Bt protein active against target pests.
[0065] The specific structured requirements for corn borerprotected Bt (Cry1Ab or Cry IF) corn products are as follows: Structured wastes: 20% non-lepidopteran Bt corn waste in the corn belt;
50% non-lepidopteran Bt waste in the cotton belt
Blocks
Internal (that is, within the Bt field) [0066] External (that is, distinct fields within a mile (¼ mile if possible) of the Bt field to maximize random crossover)
In-field strips [0067] Strips should be at least 4 rows wide (preferably 6 rows) to reduce the effects of larval movement [0068] In addition, the National Corn Growers Association, on its website:
(ncga.com/insect-resistance-management-fact-sheet-Btcorn) also provides similar guidance regarding requirements 870180072826, 8/20/2018, p. 35/73
26/54 waste. For example:
[0069] IRM requirements for corn borer:
- Plant at least 20% of your corn acres for refuse hybrids
- In regions that produce cotton, the waste must be 50%
- It should be planted within ^1/2 mile from scrap hybrids
- Refuse can be planted as strips within the field.
Bt; the refuge the strips must be at least 4 rows wide
- The refuse can be treated with conventional pesticides only if economic thresholds are reached for the target insect
- Sprayable insecticides based on Bt cannot be used on waste corn
- Proper waste must be planted on each farm with Bt 'corn [0070] As stated by Roush et al. (on pages 1780 and 1784, right column, for example), stacking or forming a pyramid of two different proteins, each effective against target pests and with little or no cross resistance, may allow the use of a smaller refuse. Roush suggests that, for a successful stack, a protection size of less than 10% scrap, can provide resistance management comparable to about 50% scrap for a single trace (not in a pyramid). For pyramid Bt corn products currently available, the U.S. Environmental Protection Agency requires significantly less (usually 5%) structured non-Bt corn waste to be planted than for products with a single dash (generally 20%).
[0071] There are several ways to check the MRI effects of a protection, including various geometric planting patterns in the fields (as mentioned above) and seed mixtures in bags, depending on
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27/54 was still discussed by Roush et al. (supra) and U.S. Patent No. 6,551,962.
[0072] The above percentages or similar scrap ratios can be used for the double or triple piles or pyramids in question. For triple piles with three sites of action against a single target pest, a target would be zero refuse (or less than 5% refuse, for example). This is particularly true for commercial acres - over 10 acres, for example.
[0073] All patents, patent applications, provisional applications and publications mentioned or cited here are incorporated by reference in full to the extent that they are not inconsistent with the explicit teachings of this specification.
[0074] Unless specifically indicated or implied, the terms one, one, o and a mean at least one (a), as used here.
[0075] The following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture ratios are by volume, unless otherwise noted. All temperatures are in degrees Celsius.
EXAMPLES
Example 1 - ¹²⁵ I Labeling of Cry Proteins [0076] Iodination of Cry toxins. Purified truncated Cry toxins were iodinated using Iodine-Beads or Iodine-gen (Pierce). Briefly, two Iodine Beads were washed twice with 500 pl of phosphate buffered saline, PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.5) and placed in a centrifuge tube.
1.5 ml behind the prototype protection. To these were added 100 µl of PBS. In a hood and using appropriate
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28/54 radioactive manipulation, 0.5 μθΐ Na ¹²⁵ I (17.4 Ci / mg, Lot 0114, Amersham) were added to the PBS solution with Iodine-Bead. The components were allowed to react for 5 minutes at room temperature, then 2-25 pg of highly pure truncated Cry protein was added to the solution and allowed to react for an additional 3-5 minutes. The reaction was terminated by removing the solution from the iodine beads and applying it to a 0.5 ml Zeba desalination centrifugation column (Invitrogen) balanced in PBS. The sludge-bead was washed twice with 10 pl of PBS each and the washing solution also applied to the desalination column. The radioactive solution was eluted through the desalination column by centrifugation at 1,000 xg for 2 min. In the case of Cry1Da, the sludge-gen method was used to conduct the radioactive staining procedure. Using this procedure, the Cry toxin in 100 mM phosphate buffer (pH 8) was first cleared of lipopolysaccharides (LPS) by passing it through a small 0.5 ml polymyxin column multiple times. To the sludge-gen tube (Pierce Chem. Co.) was added 20 pg of the Cry1Da toxin without LPS, then 0.5 l of Na ¹²⁵ l. The reaction mixture was stirred for 15 min at 25 ° C. The solution was removed from the tube and 50 µl of 0.2M unmarked Nal added to dissipate the reaction. The protein was subjected to dialysis vs PBS with 3 buffer changes to remove any unbound ¹²⁵ l.
[0077] The radiopurity of iodinated Cry proteins was determined using SDS-PAGE, Phosphor-lmaging and gamma counting. Briefly, 2 pl of the radioactive protein was separated by SDS-PAGE. After separation, the gels were dried using a BioRad gel drying apparatus following the manufacturer's instructions. The dry gels were formed by rolling them in Mylar film (12 pm thickness) and exposing them to a Molecular Dynamics Storage Phosphor Screen (35 cm x 43 cm) for 1 hour. At
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29/54 slides were developed using a Molecular Dynamics Storm 820 Phosphor-Imager and the image analyzed using ImageQuant® software. The radioactive band, along with areas immediately above and below the band, were cut using a scalpel and counted on a gamma counter. Radioactivity was detected only in the Cry protein band and in areas below the band. No radioactivity was detected above the band, indicating that all radioactive contaminants consisted of smaller protein components than the truncated Cry protein. These components most likely represent degradation products.
Example 2 - BBMV Preparation Protocol [0078] Preparation and fractionation of solubilized BBMV's. Larvae of Spodoptera frugiperda, Ostrinia nubilalis or Heleothis zea in the last stage were fasted overnight and then dissected in the morning after cooling on ice for 15 minutes. The midgut tissue was removed from the body cavity, leaving the rear intestine attached to the integument behind. The median intestine was placed in 9X volume of cold homogenization buffer (300 mM mannitol, 5 mM EGTA, 17 mM tris base, pH 7.5), supplemented with Protease Inhibitor Cocktail ¹ (Sigma P-2714 ) diluted as recommended by the supplier. The tissue was homogenized with 15 pressures from a glass tissue homogenizer. BBMV's were prepared using Wolfersberger's (1993) MgCb precipitation method. Briefly, an equal volume of a 24 mM MgCl2 solution in 300 mM mannitol was mixed with the midgut homogenate, stirred for 5 minutes and left to rest on ice for 15 min. The solution was centrifuged at 2,500 xg for 15 min at 4 ° C. The supernatant was stored and the pellet suspended in the original volume of 0.5 X diluted homogenization buffer and centrifuged again. The two supernatants went with
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30/54 binated, centrifuged at 27,000 x g for 30 min at 4 ° C to form the BBMV fraction. The pellet was suspended in 10 ml of homogenization buffer and supplemented with protease inhibitors and centrifuged again at 27,000 x g for 30 min at 4 ° C to wash the BBMV's. The resulting pellet was suspended in BBMV Storage Buffer (10 mM HEPES, 130 mM KCl, 10% glycerol, pH 7.4) to a concentration of about 3 mg / ml protein. The protein concentration was determined using the method of Bradford (1976) with bovine serum albumin (Bovine Serum Albumin - BSA) as the standard. Alkaline phosphatase determination was made before freezing the samples using the Sigma assay, following the manufacturer's instructions. The specific activity of this marker enzyme in the BBMV fraction increased, typically, 7 times compared to that found in the midgut homogenate fraction. BBMV's were aliquoted in 250 pl samples, quickly frozen in liquid N2 and stored at -80 ° C.
[0079] ¹ The final concentration of cocktail components (in μΜ) are AEBSF (500), EDTA (250 mM), Bestatin (32), E-64 (0.35), Leupeptin (0.25) and Aprotinin ( 0.075).
Example 3 - Method for Measuring the Binding of ¹²⁵ I Cry Proteins to BBMV Proteins [0080] Binding of ¹²⁵ I Cry Proteins to BBMV's. To determine the optimal amount of BBMV protein to be used in binding assays, a saturation curve was generated. ¹²⁵ I radiolabeled Cry protein (0.5 nM) was incubated for 1 hour at 28 ° C with varying amounts of BBMV protein, ranging from 0-500 pg / pl in binding buffer (8 mM NaHPO4, KbPO4 at 2 mM, 150 mM NaCl, 0.1% bovine serum albumin, pH 7.4). The total volume was 0.5 ml. Bound ¹²⁵ I Cry protein was separated from unbound protein by collecting 150 pl of the triplicate reaction mixture from a centrifuge tube
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31/54 1.5 ml in a 500 μΙ centrifuge tube and centrifuging the samples at 14,000 x g for 6 minutes at room temperature. The supernatant was gently removed and the pellet was gently washed three times with cold binding buffer. The bottom of the centrifuge containing the pellet was cut and placed in a 13 x 75 mm glass culture tube. The samples were counted for 5 minutes each in the gamma counter. The counts contained in the sample were subtracted from the base counts (reaction with any protein) and were plotted against the BBMV protein concentration. The optimal amount of protein to be used was determined to be 0.15 mg / ml BBMV protein.
[0081] To determine the binding kinetics, a saturation curve was generated. Briefly, BBMV's (150 μg / ml) were incubated for 1 hour at 28 ° C with increasing concentrations of ¹²⁵ I Cry toxin, ranging from 0.01 to 10 nM. The total binding was determined by collecting 150 μl of each concentration in triplicate, centrifuging the sample and counting as described above. Non-specific binding was determined in the same way, with the addition of 1,000 nM of homologous trypsinized non-radioactive Cry toxin added to the reaction mixture to saturate all non-specific receptor binding sites. The specific connection was calculated as the difference between the total connection and the non-specific connection.
[0082] Homologous and heterologous competitive binding assays were conducted using 150 μg / mL of BBMV protein and 0.5 nM of ¹²⁵ I radiolabeled Cry protein. The concentration of competitive non-radiolabeled Cry toxin added to the reaction mixture fluctuated from 0.045 to 1,000 nM and was added at the same time as the radioactive ligand to ensure competition for true binding. Incubations were performed for 1 hour at 28 ° C and the amount of ¹²⁵ I Cry protein bound to its receptor, me
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32/54 taken as described above, with subtraction of the non-specific connection. One hundred percent total binding was determined in the absence of any competing ligands. The results were plotted on a semi-logarithmic plot as a specific percentage total bond versus the concentration of competitive ligand added.
Example 4 - Summary of Results [0083] Figure 1 shows the specific percentage binding of ¹²⁵ I Cry1Be (0.5 nM) in BBMV's from FAW versus competition for homologous Cry1Be unlabeled (o) and heterologous Cry1Da (·). The displacement curve for homologous competition by Cry1Be results in a sigmoidal shape curve showing a 50% displacement of the radioligand to about 2 nM of Cry1Be. Cry1Da, at a concentration of 1,000 nM (2,000 times more than 125I Cry1Be being displaced) results in less than 50% displacement. Error bars represent the range of values obtained from triplicate determinations.
[0084] Figure 2 shows the specific percentage binding of ¹²⁵ I Cry1Da (0.5 nM) in FAW BBMV's versus competition for unlabeled homologous Cry1Da (o) and heterologous Cry1Be (·). The displacement curve for homologous competition by Cry1Da results in a sigmoidal shape curve showing 50% displacement of the radioligand at about 1.5 nM of Cry1 Da. Cry1Be does not prevent the specific binding of ¹²⁵ I Cry1 Da at any concentration tested, up to 1,000 nM or 2,000 times the concentration of ^l25 I Cry1Da used in the assay.
Bibliography
Heckel, D.G., Gahan, L.J., Baxter, S.W., Zhao, J.Z., Shelton, A.M., Gould, F. and Tabashnik, B.E. (2007). The diversity of Bt resistance genes in species of Lepidoptera. J Invertebr Pathol 95, 192197.
Luo, K., Banks, D. and Adang, M.J. (1999). Toxicity, binding, and permeability analyzes of four Bacillus thuringiensis cryl delta
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33/54 endotoxins using brush border membrane vesicles of spodoptera exigua and spodoptera frugiperda. Appl. Environ. Microbiol. 65, 457-464.
Palmer, M., Buchkremer, M., Valeva, A. and Bhakdi, S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Analytical Biochemistry 253, 175-179. 1997. Ref Type: Journal (Full)
Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory).
Schlenz, M. L., Babcock, J. M. and Storer, N. P. Response of CrylF-resistant and Susceptible European Corn Borer and Fall Armyworm Colonies to Cry1A.105 and Cryl2Ab2. DAI 0830, 2008. Indianapolis, Dow AgroSciences. Derbi Report.
Sheets, J. J. and Storer, N.P. Analysis of CrylAc Binding to Proteins in Brush Border Membrane Vesicles of Corn Earworm Larvae (Heleothis zea). Interactions with Cry1F Proteins and Its Implication for Resistance in the Field. DAI-0417, 1-26. 2001. Indianapolis, Dow AgroSciences.
Tabashnik, B.E., Liu, Y.B., Finson, N., Masson, L. and Heckel, D.G. (1997). One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. Proc. Natl. Acad. Sci. U. S.A., 94, 16401644.
Tabashnik, B.E., Malvar, T., Liu, Y.B., Finson, N., Borthakur, D., Shin, B.S., Park, S.H., Masson, L., by Maagd, R.A. and Bosch, D. (1996). Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 62, 2839-2844.
Tabashnik, B.E., Roush, R.T., Earle, E.D. and Shelton, A.M. (2000). Resistance to Bt toxins. Science 287, 42.
Wolfersberger, M.G. (1993). Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the gypsy moth (Lymantria dispar). Arch. InPetição 870180072826, of 08/20/2018, p. 43/73
34/54 sect Biochem. Physiol 24, 139-147.
Xu, X., Yu, L. and Wu, Y. (2005). Disruption of a cadherin gene associated with resistance to CrylAc {deltaj-endotoxin of Bacillus thuringiensis in Helicoverpa armigera. Appl Environ Microbiol 71, 948954.
APPENDIX A
List of delta-endotoxins - from the website of Crickmore et al. (quoted in the request)
Accession number is NCBI entry
Name No. Ac. Authors Year Source strain Comment Cry1Aa1 AAA22353 Schnepf et al. 1985 Bt kurstaki HD1Crv1Aa2 AAA22552 Shibano et al. 1985 Bt sottoCrv1Aa3 BAA00257 Shimizu et al. 1988 Bt aizawai ILP7Crv1Aa4 CAA31886 Masson et al. 1989 Bt entomocidusCrv1Aa5 BAA04468 Udayasuriyan et al 1994 Bt Fu-2-7Crv1Aa6 AAA86265 Masson et al. 1994 Bt kurstakiNRD-12Crv1Aa7 AAD46139 Osman et al. 1999 BtC12Crv1Aa8 126149 Liu 1996Sequence ofDNA only Crv1Aa9 BAA77213 Nagamatsu et al. 1999 Bt dendrolimusT84A1Crv1Aa10 AAD55382 Hou and Chen 1999 Bt kurstaki HD-1-02Crv1Aa11 CAA70856 Tounsi et al. 1999 Bt kurstakiCrv1Aa12 AAP80146 Yao et al. 2001 Bt Ly30Crv1Aa13 AAM44305 Zhong et al. 2002 Bt sottoCrv1Aa14 AAP40639 Ren et al. 2002 Not publishedCrv1Aa15 AAY66993 Sauka et al. 2005 Bt INTA Mol-12Crv1Ab1 AAA22330 Wabiko et al. 1986 Bt berliner1715Crv1Ab2 AAA22613 Thorne et al. 1986 Bt kurstakiCrv1Ab3 AAA22561 Geiser et al. 1986 Bt kurstaki HD1Crv1Ab4 BAA00071 Kondo et al. 1987 Bt kurstaki HD1Crv1Ab5 CAA28405 Hofte et al. 1986 Bt berliner1715Crv1Ab6 AAA22420 Hefford et al. 1987 Bt kurstakiNRD-12
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Crv1Ab7 CAA31620 Haider & Ellar 1988 Bt aizawai IC1 Crv1Ab8 AAA22551 Oeda et al.1987 Bt aizawai IPL7Crv1Ab9 CAA38701 Chak & Jen1993 Bt aizawaiHD133Crv1Ab7 CAA31620 Haider & Ellar1988 Bt aizawai IC1Crv1Ab8 AAA22551 Oeda et al.1987 Bt aizawai ILP7Crv1Ab9 CAA38701 Chak & Jen1993 Bt aizawaiHD133Crv1Ab10 A29125 Fischhoff et al.1987 Bt kurstaki HD1Crv1Ab11 112419 Ely & Tippett1995 Bt A20 Sequence ofDNA only Crv1Ab12 AAC64003 Silva-Werneck et 1998 Bt kurstaki S93Crv1Ab13 AAN76494 Tan et al.2002 Bt c005Crv1Ab14 AAG16877 Meza-Basso & 2000 Bt Chilean nati- Theoduloz goCry1Ab15 AA013302 Li et al.2001 BtB-Hm-16Crv1Ab16 AAK55546 Yu et al.2002 BtAC-11Crv1Ab17 AAT46415 Huang et al.2004 BtWB9Crv1Ab18 AAQ88259 Stobdan et al.2004 BtCrv1Ab19 AAW31761 Zhong et al.2005 BtX-2Crv1Ab20 ABB72460 Liu et al.2006 BtC008Crv1Ab21 ABS18384 Swiecicka et al.2007 BtlS5056Crv1Ab22 ABW87320 Wu and Feng2008 BtS2491AbCrvIAb-like AAK14336 Nagarathinam et 2001 Bt kunthala Sequence al. RX24 indeterminate CrvIAb- AAK14337 Nagarathinam et 2001 Bt kunthala Sequence likeal. RX28 indeterminate CrvIAb- AAK14338 Nagarathinam et 2001 Bt kunthala Sequence likeal. RX27 indeterminate Crv 1Ab- ABG88858 Lin et al.2006 Bt Iy4a3 Sequence likeinsufficient Crv1Ac1 AAA22331 Adang et al.1985 Bt kurstakiHD73Crv1Ac2 AAA22338 Von Tersch et al. 1991 Bt kenyaeCrv1Ac3 CAA38098 Dardenne et al.nineteen ninety Bt BTS89ACrv1Ac4 AAA73077 Feitelson1991 Bt kurstakiPS85A1Crv1Ac5 AAA22339 Feitelson1992 Bt kurstakiPS81GGCry1Ac6 AAA86266 Masson et al.1994 Bt kurstaki
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NRD-12
Crv1Ac7 AAB46989 Herrera et al. 1994 Bt kurstakiHD73 Crv1Ac8 AAC44841 Omolo et al. 1997 Bt kurstakiHD73Crv1Ac9 AAB49768 Gleave et al. 1992 Bt DSIR732CrvIAc 10 CAA05505 Sun 1997 Bt kurstakiYBT-1520Crv1Ac11 CAA10270 Makhdoom &Riazuddin 1998 Crv1Ac12 112418 Ely & Tippett 1995 BtA20 Sequence ofDNA only Crv1Ac13 AAD38701 Qiao et al. 1999 Bt kurstaki HD1Crv1Ac14 AA006607 Yao et al. 2002 Bt Ly30Crv1Ac15 AAN07788 Tzeng et al. 2001 Taiwan BTCrv1Ac16 AAU87037 Zhao et al. 2005 Bt H3Crv1Ac17 AAX18704 Hire et al. 2005 Bt kenyaeHD549Crv1Ac18 AAY88347 Kaur & Aliam 2005 Bt SK-729Crv1Ac19 ABD37053 Gao et al. 2005 Bt C-33Crv1Ac20 ABB89046 Tan et al. 2005 Crv1Ac21 AAY66992 Sauka et al. 2005 TNTA Mol-12Crv1Ac22 ABZ01836 Zhang & Fang 2008 BÍW015-1Crv1Ac23 CAO30431 Kashyap et al. 2008 BtCrv1Ac24 ABL01535 Arango et al. 2008 Bt 146-158-01Cry1Ac25 FJ513324 Guan Peng et al. 2008 Bt Tm37-6 No link onNCBIJuly 9 Cry1Ac26 FJ617446 Guan Peng et al. 2009 Bt Tm41-4 No link onNCBIJuly 9 Cry1Ac27 FJ617447 Guan Peng et al. 2009 Bt TM44-1B No link onNCBIJuly 9 Cry1Ac28 ACM90319 Li et al. 2009 Bt Q-12Cry1Ad1 AAA22340 Feitelson 1993 Bt aizawaiPS81ICrv1Ad2 CAA01880 Anonymous 1995 Bt PS81RR1Crv1Ae1 AAA22410 Lee & Aronson 1991 Bt alestiCrvAfl AAB82749 Kang et al. 1997 Bt NT0423Crv1Aa1 AAD46137 Mustafa 1999
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Crv1Ah1 AAQ14326 Tan et al. 2000 Crv1Ah2 ABB76664 Qi et al.2005 Bt alestiCrv1Ai1 AA039719 Wang et al.2002 Crv1A-like AAK14339 Nagarathinam et 2001 Bt Kunthala Sequence al. nags3 indeterminate Crv1Ba1 CAA29898 Brizzard & White- 1988 Bt thuringiensis ley HD2Crv1Ba2 CAA65003 Soetaert1996 Bt entomocidusHD110Crv1Ba3 AAK63251 Zhang et al.2001 Crv1Ba4 AAK51084 Nathan et al.2001 Bt entomocidusHD9Crv1Ba5 ABO20894 Song et al.2007 Bt sfw-12Crv1Ba6 ABL60921 Martins et al.2006 Bt S601Crv1Bb1 AAA22344 Donovan et al.1994 Bt EG5847Crv1Bc1 CAA86568 Bishop et al.1994 Bt morrisoniCrv1Bd1 AAD10292 Kuo et al.2000 Bt wuhanensisHD525Crv1Bd2 AAM93496 Isakova et al.2002 Bt 834Crv1Be1 AAC32850 Payne et al.1998 Bt PS158C2Crv1Be2 AAQ52387 Baum et al.2003 Cry1Be3 FJ716102 Xiaodong Sun et 2009 Bt No link on al. NCBI
July
Cry1Bf1 CAC50778 Arnaut et al. 2011 Cry1Bf1 AAQ52380 Baum et al. 2003Cry1Bg1 AA039720 Wang et al. 2002Cry1Ca1 CAA30396 Honee et al. 1988 Bt entomocidus60.5 Cry1Ca2 CAA31951 Sanchis et al. 1989 Bt aizawai 7.29 Cry1Ca3 AAA22343 Feitelson 1993 Bt aizawaiPS81I Cry1Ca4 CAA01886 Van Mellaert al. et 1990 Bt entomocidusHD110 Cry1Ca5 CAA65457 Strizhov 1996 Bt aizawai 7.29 Cry1Ca6 AAF37224 Yu et al. 2000 Bt AF-2 Cry1Ca7 AAG50438 Aixing et al. 2000 Bt J8 Cry1Ca8 AAM00264 Chen et al. 2001 Bt c002 Cry1Ca9 AAL79362 Kao et al. 2003 BtG10-01A Cry1Ca10 AAN16462 Lin et al. 2003 Bt E05-20a
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Cry1Ca11 AAX53094 Cai et al. 2005 Bt C-33 Cry1Cb1 M97880 Kalman et al. 1993 Bt galleriae Sequence of HD29 DNA only Cry1Cb2 AAG35409 Song et al. 2000 Bt c001Cry1Cb3 ACD50894 Huang et al. 2008 Bt 087CrylCb- AAX63901 Thammasittirong 2005 BtTA476-1 Sequence likeet al. insufficient Cry1Da1 CAA38099 Hofte et al. nineteen ninety Bt aizawaiHD68Cry1Da2 176415 Payne & Sick 1997Sequence ofDNA only Cry1Db1 CAA80234 Lambert 1993 Bt BTS00349ACry1Db2 AAK48937 Li et al. 2001 Bt B-Pr-88Cry1Dc1 ABK35074 Lertwiriyawong et g | 2006 Bt JC291Cry1Ea1 CAA37933 Visser et al. nineteen ninety Bt kenyae 4F1Cry1Ea2 CAA39609 Bosse et al. nineteen ninety Bt kenyaeCry1Ea3 AAA22345 Feitelson 1991 Bt kenyaePS81FCry1Ea4 AAD04732 Barboza-Corona 1998 Bt kenyae et al.LBIT-147Cry1Ea5 A15535 Botterman et al. 1994 Bt YBT-032 Sequence ofDNA only Cry1Ea6 AAL50330 Sun et al. 1999 Bt JC190Cry1Ea7 AAW72936 Huehne et al. 2005 Bt HZM2Cry1Ea8 ABC11258 Huang et al. 2007 Bt aizawaiCry1Eb1 AAA22346 Feitelson 1993 PS81A2Cry1Fa1 AAA22348 Chambers et al. 1991 Bt aizawaiCry1Fa2 AAA22347 Feitelson 1993 EG6346Cry1Fb1 CAA80235 Lambert 1993 Bt aizawaiPS81ICry1Fb2 BAA25298 Masuda & Asano 1998 Bt morrisoniINA67Cry1Fb3 AAF21767 Song et al. 1998 Bt morrisoniCry1Fb4 AAC10641 Payne et al. 1997 Cry1Fb5 AAO13295 Li et al. 2001 Bt B-Pr-88Cry1Fb6 ACD50892 Huang et al. 2008 Bt012Cry1Fb7 ACD50893 Huang et al. 2008 Bt 087Cry1Ga1 CAA80233 Lambert 1993 Bt BTS0349ACry1Ga2 CAA70506 Shevelev et al. 1997 Bt wuhanensis
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Cry1Gb1 AAD10291 Kuo & Chak 1999 Bt wuhanensisHD525 Cry1Gb2 AAQ13756 Li et al. 2000 Bt B-Pr-88CrylGc AAQ52381 Baum et al. 2003 Cry1Ha1 CAA80236 Lambert 1993 BtBTS02069AACry1Hb1 AAA79694 Koo et al. 1995 Bt morrisoniBF190Cry1H- AAF01213 Srifah et al. 1999 Bt JC291 Sequence like insufficient Cryllal CAA44633 Tailor et al. 1992 Bt kurstakiCry1la2 AAA22354 Gleave et al. 1993 Bt kurstakiCry1la3 AAC36999 Shin et al. 1995 Bt kurstaki HD1Cry1la4 AAB00958 Kostichka et al. 1996 Bt AB88Cry1la5 CAA70124 Selvapandiyan 1996 Bt61Cry1la6 AAC26910 Zhong et al. 1998 Bt kurstakiS101Cry1la7 AAM73516 Porcar et al. 2000 BtCry1la8 AAK66742 Song et al. 2001 Cry1la9 AAQ08616 Yao et al. 2002 Bt Ly30Cry1la10 AAP86782 Espíndola et al. 2003 Bt thuringiensisCry1la11 CAC85964 Tounsi et al. 2003 Bt kurstakiBNS3Cry1la12 AAV53390 Grossi de Sa et 2005 BtCry1la13 ABF83202 Martins et al. 2006 BtCry1la14 ACG63871 Liu & Guo 2008 Bt11Cry1la15 FJ617445 Guan Peng et al. 2009 Bt E-1B No link onNCBIJuly 9 Cry1la16 FJ617448 Guan Peng et al. 2009 Bt E-1A No link on
July NCBI
Cryllbl AAA82114 Shin et al. 1995 Bt entomocidusBP465 Cry1lb2 ABW88019 Guan et al. 2007 Bt PP61 Cry1lb3 ACD75515 Liu & Guo 2008 Bt GS8 Cryllcl AAC62933 Osman et al. 1998 BtC18 Cry1lc2 AAE71691 Osman et al. 2001Crylldl AAD44266 Choi 2000
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Cryllel AAG43526 Song et al. 2000 Bt BTC007 Cryllfl AAQ52382 Baum et al. 2003 Cry1 l-like AAC31094 Payne et al. 1998Insufficient sequence Cry1-like ABG88859 Lin & Fang 2006 Bt Iy4a3 Insufficient sequence Cry1Ja1 AAA22341 Donovan 1994 Bt EG5847Cry1Jb1 AAA98959 Von Tersch &Gonzalez 1994 Bt EG5092Cry1Jc1 AAC31092 Payne et al. 1998 Cry1Jc2 AAQ52372 Baum et al. 2003 Cry1Jd1 CAC50779 Arnaut et al. 2001 BtCry1Ka1 AAB00376 Koo et al. 1995 Bt morrisoniBF190Crv1La1 AAS60191 Je et al. 2004 Bt kurstaki KlCrv1-like AAC31091 Payne et al. 1998Insufficient sequence Crv2Aal AAA22335 Donovan et al. 1989 Bt kurstakiCrv2Aa2 AAA83516 Widner & Whiteley 1989 Btkurstaki HDICrv2Aa3 D86064 Sasaki et al. 1997 Bt sotto Sequence ofDNA only Crv2Aa4 AAC04867 Misra et al. 1998 Bt kenyaeHD549Crv2Aa5 CAA10671 Yu & Pang 1999 Bt SL39Crv2Aa6 CAA10672 Yu & Pang 1999 BÍYZ71Crv2Aa7 CAA Yu & Pang 1999 Bt CY29 10670 Crv2Aa8 AAO13734 Wei et al. 2000 Bt Dongbei 66Crv2Aa9 AA013750 Zhang et al. 2000 Crv2Aa10 AAO04263 Yao et al. 2001 Crv2Aa11 AAQ52384 Baum et al. 2003 Crv2Aa12 ABI83671 Tan et al. 2006 Bt Rpp39Crv2Aa13 ABL01536 Arango et al. 2008 Bt146-158-01Crv2Aa14 ACF04939 Hire et al. 2008 Bt HD-550Crv2Ab1 AAA22342 Widner & Whiteley 1989 Btkurstaki HD1Crv2Ab2 CAA39075 Dankocsik et al. nineteen ninety Btkurstaki HD1Crv2Ab3 AAG36762 Chen et al. 1999 Bt BTC002Crv2Ab4 AAO13296 Li et al. 2001 Bt B-Pr-88
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Crv2Ab5 AAQ04609 Yao et al. 2001 Bt ly30 Crv2Ab6 AAP59457 Wang et al. 2003 Bt WZ-7 Crv2Ab7 AAZ66347 Udayasuriyan g | et 2005 Bt 14-1 Crv2Ab8 ABC95996 Huang et al. 2006 BtWB2 Crv2Ab9 ABC74968 Zhang et al. 2005 Bt LLB6 Crv2Ab10 EF157306 Lin et al. 2006 BtLyD Crv2Ab11 CAM84575 Saleem et al. 2007 Bt CMBL-BT1 Crv2Ab12 ABM21764 Lin et al. 2007 BtLyD Crv2Ab13 ACG76120 Zhu et al. 2008 Bt ywc5-4 Crv2Ab14 ACG76121 Zhu et al. 2008 BTS Crv2Ac1 CAA40536 Aronson 1991 Bt shanghai S1 Crv2Ac2 AAG35410 Song et al. 2000Crv2Ac3 AAQ52385 Baum et al. 2003Crv2Ac4 ABC95997 Huang et al. 2006 BtWB9 Crv2Ac5 ABC74969 Zhang et al. 2005Crv2Ac6 ABC74793 Xia et al. 2006 Bt wuhanensis Crv2Ac7 CAL18690 Saleem et al. 2008 BtSBSBT-1 Crv2Ac8 CAM09325 Saleem et al. 2007 Bt CMBL-BT1 Crv2Ac9 CAM09326 Saleem et al. 2007 Bt CMBL-BT2 Crv2Ac10 ABN15104 Bai et al. 2007 BtQCL-1 Cry2Ac11 CAM83895 Saleem et al. 2007 Bt HD29 Crv2Ac12 CAM83896 Saleem et al. 2007 Bt CMBL-BT3 Crv2Ad1 AAF09583 Choi et al. 1999 Bt BR30 Crv2Ad2 ABC86927 Huang et al. 2006 BtWB10 Crv2Ad3 CAK29504 Saleem et al. 2006 Bt5_2AcT (1) Crv2Ad4 CAM32331 Saleem et al. 2007 Bt CMBL-BT2 Crv2Ad5 CA078739 Saleem et al. 2007 Bt HD29 Crv2Ae1 AAQ52362 Baum et al. 2003Crv2Af1 ABO30519 Beard et al. 2007 BtC81 Crv2Ae ACH91610 Zhu et al. 2008 BOF19-2 Cry2Ah EU939453 Zhang et al. 2008 Bt
July NCBI
ACL80665 Zhang et al. 2009 Bt BRC-ZQL3 ry2Ah2Cry2Ai FJ788388 Udayasuriyan et 2009 Bt No link on al. NCBIJuly 9 Crv3Aa1 AAA22336 Herrnstadt et al. 1987 BT San Diego
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Crv3Aa2 AAA22541 Sekar et al. 1987 Bt tenebrionis Crv3Aa3 CAA68482 Hofte et al. 1987 Crv3Aa4 AAA22542 McPherson et al. 1988 Bt tenebrionisCry3Aa5 AAA50255 Donovan et al. 1988 Bt morrisoniEG2158Crv3Aa6 AAC43266 Adams et al. 1994 Bt tenebrionisCrv3Aa7 CAB41411 Zhang et al. 1999 Bt22Crv3Aa8 AAS79487 Gao & Cai 2004 Bt YM-03Crv3Aa9 AAW05659 Bulla and Candas 2004 Bt UTD-001Crv3Aa10 AAU29411 Chen et al. 2004 Bt 886Crv3Aa11 AAW82872 Kurt et al. 2005 Bt tenebrionisMm2Crv3Aa12 ABY49136 Sezen et al. 2008 Bt tenebrionisCrv3Ba1 CAA34983 Sick et al. nineteen ninety Bt tolworthi 43 FCrv3Ba2 CAA00645 Peferoen et al. nineteen ninety Bt PGSI208Crv3Bb1 AAA22334 Donovan et al. 1992 Bt EG4961Crv3Bb2 AAA74198 Donovan et al. 1995 Bt EG5144Crv3Bb3 115475 Peferoen et al. 1995Sequence ofDNA only Crv3Ca1 CAA42469 Lambert et al. 1992 Bt kurstakiBtl109PCrv4Aa1 CAA68485 Ward & Ellar 1987 Bt israelensisCrv4Aa2 BAA00179 Sen et al. 1988 Bt israelensisHD522Crv4Aa3 CAD30148 Berry et al. 2002 Bt israelensisCrv4A-like AAY96321 Mahalakshmi et al. 2005 Bt LDC-9 Insufficient sequence Crv4Ba1 CAA30312 Chungjatpornchai et al. 1988 Bt israelensis4Q2-72Cry4Ba2 CAA30114 Tungpradubkul et al 1988 Bt israelensisCry4Ba3 AAA22337 Yamamoto et al. 1988 Bt israelensisCry4Ba4 BAA00178 Sen et al. 1988 Bt israelensisHD522Cry4Ba5 CAD30095 Berry et al. 2002 Bt israelensisCry4Balike ABC47686 Mahalakshimi et al. 2005 Bt LDC-9 Insufficient sequence Cry4Ca1 EY646202 Shu et al. 2008No link onNCBI
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July 9 Cry4Cb1 FJ403208 Jun & Furong 2008 Bt HS18-1 No link onNCBIJuly 9 Cry4Cb2 FJ597622 Jun & Furong 2008 Bt Ywc2-8 No link on
July NCBI
Cry4Cc1 FJ403207 Jun & Furong 2008 Bt MC28 No link onNCBIJuly 9 Crv5Aa1 AAA67694 Narva et al. 1994 Bt darmstadiensis PS17Crv5Ab1 AAA67693 Narva et al. 1991 Bt darmstadiensis PS17Crv5Ac1 134543 Narva et al. 1997Sequence of
DNA only
Crv5Ad1 ABQ82087 Lenane et al. 2007 Bt L366 Crv5Ba1 AAA68598 FoncerradaNarva & 1997 Bt PS86Q3 Crv5Ba2 ABW88932 Guo et al. 2008 YBT 1518 Crv6Aa1 AAA22357 Narva et al. 1993 Bt PS52A1 Crv6Aa2 AAM46849 Bai et al. 2001 YBT 1518 Crv6Aa3 ABH03377 Jia et al. 2006 Bt96418 Crv6Ba1 AAA22358 Narva et al. 1991 Bt PS69DI Crv7Aa1 AAA22351 Lambert et al. 1992 Bt galleriaePGSI245 Crv7Ab1 AAA21120 Narva & Fu 1994 Bt dakotaHD511 Crv7Ab2 AAA21121 Narva & Fu 1994 Bt kumamoto-ensis 867 Crv7Ab3 ABX24522 Song et al. 2008 Bt WZ-9 Cry7Ab4 EU380678 Shu et al. 2008 Bt No link in
July NCBI
Crv7Ab5 ABX79555 Aguirre-Arzola al. et 2008 Bt monterreyGM-33 Crv7Ab6 ACI44005 Deng et al. 2008 Bt HQ122 Cry7Ab7 FJ940776 Wang et al. 2009Without link noNCBI July 9 Cry7Ab8 GU145299 Feng Jing 2009Without link no
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44/54
November NCBI
Crv7Ba1 ABB70817 Zhang et al. Crv7Ca1 ABR67863 Gao et al. Crv7Da1 ACQ99547 Yi et al. Cry8Aa1 AAA21117 Narva & Fu Cry8Ab1 EU044830 Cheng et al. Cry8Ba1 AAA21118 Narva & Fu Crv8Bb1 CAD57542 Abad et al. Crv8Bc1 CAD57543 Abad et al. Crv8Ca1 AAA21119 Sato et al. Crv8Ca2 AAR98783 Shu et al. Cry8Ca3 EU625349 Du et al. Crv8Da1 BAC07226 Asano et al. Crv8Da2 BD133574 Asano et al. Crv8Da3 BD133575 Asano et al. Crv8Db1 BAF93483 Yamaguchi et al Crv8Ea1 AAQ73470 Fuping et al. Cry8Ea2 EU047597 Liu et al. Crv8Fa1 AAT48690 Shu et al. Crv8Ga1 AAT46073 Shu et al. Crv8Ga2 ABC42043 Yan et al. Cry8Ga3 FJ198072 Xiaodong et al. Cry8Ha1 EF465532 Fuping et al.
2006 Bt huazhogen-
sis2007 Bt BTH-132009 Bt LH-21992 Bt kumamotoensi2007 Bt B-JJX No link onNCBIJuly 9 1993 Bt kumamotoensis2002 2002 1995 Bt japonensis Buibui2004 Bt HBF-12008 Bt FTL-23 No link onNCBIJuly 9 2002 Bt galleriae2002 Bt Sequence ofDNA only 2002 Bt Sequence ofDNA only 2007 Bt BBT2-52003 Bt 1852007 Bt B-DLL No link onNCBIJuly 9 2004 Bt 185 alsoAAW81032 2004 Bt HBF-182008 Bt 1452008 BtFCD114 No link onNCBIJuly 9 2006 Bt 185 No link onNCBI
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45/54 July
Cry8la1 EU381044 Yan et al. 2008 Bt su4 No link onNCBIJuly 9 Cry8Ja1 EU625348 Du et al. 2008 Bt FPT-2 No link onNCBIJuly 9 Cry8Ka1 FJ422558 Quezado et al. 2008No link onNCBIJuly 9 Cry8Ka2 ACN87262 Noguera & Ibarra 2009 Bt kenyaeCrv8-like FJ770571 Noguera & Ibarra 2009 Bt canadensis Sequence ofDNA only Crv8-like ABS53003 Mangena et al. 2007 BtCrv9Aa1 CAA41122 Shevelev et al. 1991 Bt galleriaeCrv9Aa2 CAA41425 Gleave et al. 1992 Bt DSIR517Cry9Aa3 GQ249293 Su et al. 2009 Bt SC5 (D2) No link onNCBIJuly 9 Cry9Aa4 GQ249294 Su et al. 2009 Bt T03C001 No link onNCBIJuly 9 Cry9Aa- AAQ52376 Baum et al. 2003Sequence like incomplete Crv9Ba1 CAA52927 Shevelev et al. 1993 Bt galleriaeCry9Bb1 AAV28716 Silva-Werneck et al 2004 Bt japonensisCrv9Ca1 CAA85764 Lambert et al. 1996 Bt tolworthiCrv9Ca2 AAQ52375 Baum et al. 2003 Crv9Da1 BAA Asano 1997 Bt japonensis 19948 N141Crv9Da2 AAB97923 Wasano & Ohba 1998 Bt japonensisCry9Da3 GQ249295 Su et al. 2009 Bt T03B001 No link onNCBIJuly 9 Cry9Da4 GQ249297 Su et al. 2009 Bt T03B001 No link onNCBIJuly 9 Cry9Db1 AAX78439 Flannagan & 2005 Bt kurstaki AbadDP1019Crv9Ea1 BAA34908 Midoh & Oyama 1998 Bt aizawai
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46/54
SSK-10
Crv9Ea2 AA012908 Li et al. 2001 Bt B-Hm-16 Crv9Ea3 ABM21765 Lin et al.2006 Bt lyACrv9Ea4 ACE88267 Zhu et al.2008 Bt ywc5-4Crv9Ea5 ACF04743 Zhu et al.2008 BtCrv9Ea6 ACG63872 Liu & Guo2008 Bt 11Cry9Ea7 FJ380927 Sun et al.2008No link onNCBIJuly 9 Cry9Ea8 GQ249292 Su et al.2009 GQ249292 No link onNCBIJuly 9 Cry9Eb1 CAC50780 Arnaut et al.2001 Cry9Eb2 GQ249298 Su et al.2009 Bt T03B001 No link onNCBIJuly 9 Cry9Ec1 AAC63366 Wasano et al.2003 Bt galleriaeCrv9Ed1 AAX78440 Flannagan & 2005 Bt kurstaki Abad DP1019Cry9Ee1 GQ249296 Su et al.2009 Bt T03B001 No link onNCBIAugust 9 Cry9-like AAC63366 Wasano et al.1998 Bt galleriae Insufficient sequence Crv10Aa1 AAA22614 Thorne et al.1986 Bt israelensisCrv10Aa2 E00614 Aran & Toomasu 1996 Bt israelensis Sequence ofONR-60A DNA only Crv10Aa3 CAD30098 Berry et al.2002 Bt israelensisCrvIOA- DQ167578 Mahalakshmi et 2006 Bt LDC-9 Sequence likeal. incomplete Crv11Aa1 AAA22352 Donovan et al.1988 Bt israelensisCrv11Aa2 AAA22611 Adams et al.1989 Bt israelensisCrv11Aa3 CAD30081 Berry et al.2002 Bt israelensisCrv11Aa- DQ166531 Mahalakshmi et 2007 Bt LDC-9 Sequence likeal. incomplete Crv11Ba1 CAA60504 Delecluse et al.1995 Bt jegathesan367Cry11Bb1 AAC97162 Orduz et al.1998 Bt medellinCrv12Aa1 AAA22355 Narva et al.1991 BtPS33F2Crv13Aa1 AAA22356 Narva et al.1992 Bt PS63BCrv14Aa1 AAA21516 Narva et al.1994 Bt sotto
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47/54
PS80JJ1
Crv15Aa1 AAA22333 Brown & Whiteley 1992 Bt thompsoni Crvl6Aa1 CAA63860 Barloy et al. 1996 Cb malaysiaCH18Crv17Aa1 CAA67841 Barloy et al. 1998 Cb malaysiaCH18Crv18Aa1 CAA67506 Zhang et al. 1997 Paenibacillus popilliaeCrv18Ba1 AAF89667 Patel et al. 1999 Paenibacillus popilliaeCrv19Ca1 AAF89668 Patel et al. 1999 Paenibacillus popilliaeCrv19Aa1 CAA68875 Rosso & Delecluse 1996 Bt jegathesan367Crv19Ba1 BAA32397 Hwang et al. 1998 Bt higoCrv20Aa1 AAB93476 Lee & Gill 1997 Bt fukuokaensisCrv20Ba1 ACS93601 Noguera & Ibarra 2009 Bt higo LBIT-976Crv20-like GO144333 Yi et al. 2009 Bt Y-5 Sequence ofDNA only Crv21Aa1 132932 Payne et al. 1996Sequence ofDNA only Crv21Aa2 I66477 Feitelson 1997Sequence ofDNA only Crv21Ba1 BAC06484 Sato & Asano 2002 Bt roskildiensisCrv22Aa1 134547 Payne et al. 1997Sequence ofDNA only Crv22Aa2 CAD43579 Isaac et al. 2002 BtCrv22Aa3 ACD93211 Du et al. 2008 Bt FZ-4Crv22Ab1 AAK50456 Baum et al. 2000 Bt EG4140Crv22Ab2 CAD43577 Isaac et al. 2002 BtCry22Ba1 CAD43578 Isaac et al 2002 BtCrv23Aa1 AAF76375 Donovan et al. 2000 Bt Binary withCry37Aa1 Crv24Aa1 AAC61891 Kawalek & Gill 1998 Bt jegathesanCrv24Ba1 BAD32657 Ohgushi et al. 2004 Bt sottoCrv24Ca1 CAJ43600 Beron & Salerno 2005 Bt FCC-41Crv25Aa1 AAC61892 Kawalek & Gill 1998 Bt jegathesanCrv26Aa1 AAD25075 Wojciechowska 1999 Bt finitimus B-
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Crv27Aa1 BAA82796 et al.Saitoh 1999 1166Bt higo Crv28Aa1 AAD24189 Wojciechowska et al. 1999 Bt finitimus B-1161Crv28Aa2 AAG00235 Moore & Debro 2000 Bt finitimusCrv29Aa1 CAC80985 Delecluse et al. 2000 Bt medellinCrv30Aa1 CAC80986 Delecluse et al. 2000 Bt medellinCrv30Ba1 BAD00052 Ito et al. 2003 Bt entomocidusCrv30Ca1 BAD67157 Ohgushi et al. 2004 Bt sottoCrv30Ca2 ACU24781 Sun & Park 2009 Bt jegathesan367Cry30Da1 EF095955 Shu et al. 2006 Bt Y41 No link onNCBIJuly 9 Crv30Db1 BAE80088 Kishida et al. 2006 Bt aizawaiBUN1-14Crv30Ea1 ACC95445 Fang et al. 2007 Bt S2160-1Cry30Ea2 FJ499389 Jun et al. 2008 Bt Ywc2-8 No link onNCBIJuly 9 Crv30Fa1 ACI22625 Tan et al. 2008 Bt MC28Crv30Ga1 ACG60020 Zhu et al. 2008 Bt HS18-1Crv31Aa1 BAB11757 Saitoh & Mizuki 2000 Bt 84-HS-1-11Crv31Aa2 AAL87458 Jung & Cote 2000 BtM15Crv31Aa3 BAE79808 Uemori et al. 2006 Bt B0195Crv31Aa4 BAF32571 Yasutake et al. 2006 Bt 79-25Crv31Aa5 BAF32572 Yasutake et al. 2006 Bt 92-10Crv31Ab1 BAE79809 Uemori et al. 2006 Bt B0195Crv31Ab2 BAF32570 Yasutake et al. 2006 Bt31-5Crv31Ac1 BAF34368 Yasutake et al. 2006 Bt 87-29Crv32Aa1 AAG36711 Balasubramanian et al. 2001 Bt yunnanensisCrv32Ba1 BAB78601 Takebe et al. 2001 BtCrv32Ca1 BAB78602 Takebe et al. 2001 BtCrv32Da1 BAB78603 Tabeke et al. 2001 BtCrv33Aa1 AAL26871 Kim et al. 2001 Bt dakotaCrv34Aa1 AAG50341 Ellis et al. 2001 Bt PS80JJ1 Binary withCry35Aa1 Crv34Aa2 AAK64560 Rupar et al. 2001 Bt EG5899 Binary with
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Crv34Aa3 AAT29032 Schnepf et al Crv34Aa4 AAT29030 Schnepf et al Crv34Ab1 AAG41671 Moellenbeck al. Crv34Ac1 AAG50118 Ellis et al. Crv34Ac2 AAK64562 Rupar et al. Crv34Ac3 AAT29029 Shnepf et al. Crv34Ba1 AAK64565 Rupar et al. Crv34Ba2 AAT29033 Schnepf et al Crv34Ba3 AAT29026 Schnepf et al Crv35Aa1 AAG50342 Ellis et al. Crv35Aa2 AAK64561 Rupar et al. Crv35Aa3 AAT29028 Schnepf et al Crv35Aa4 AAT29025 Schnepf et al Crv35Ab1 AAG41672 Moellenbeck al. Crv35Ab2 AAK64563 Rupar et al. Crv35Ab3 AY536891 AAT29024 Crv35Ac1 AAG50117 Ellis et al. Crv35Ba1 AAK64566 Rupar et al. Crv35Ba2 AAT29027 Schnepf et al Crv35Ba3 AAT29026 Schnepf et al Crv36Aa1 AAK64558 Rupar et al.
Cry35Aa2 2004 Bt PS69Q BinaryCry35Aa3 with2004 Bt PS185GG BinaryCry35Aa4 with et 2001 Bt PS149BI BinaryCry35Ab1 with2001 Bt PS167H2 BinaryCry35Ac1 with2001 Bt EG9444 BinaryCry35Ab2 with2004 Bt KR1369 BinaryCry35Ab3 with2001 Bt EG4851 BinaryCry35Ba1 with2004 Bt PS201L3 Binary with Cry35ABa22004 Bt PS201HH2 BinaryCry35Ba3 with2001 Bt PS80JJ1 BinaryCry34Aa1 with2001 Bt EG5899 BinaryCry34Aa2 with2004 Bt PS69Q BinaryCry34Aa3 with2004 Bt PS185GG BinaryCry34Aa4 with et 2001 Bt PS149B1 BinaryCry34Ab1 with2001 Bt EG9444 BinaryCry34Ac2 with2004 Bt KR1369 BinaryCry34Ab3 with2001 Bt PS167H2 BinaryCry34Ac1 with2001 Bt EG4851 BinaryCry34Ba1 with2004 Bt PS201L3 BinaryCry34Ba2 with2004 Bt PS201HH2 BinaryCry34Ba3 with2001 Bt
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Crv37Aa1 AAF76376 Donovan et al. 2000 Bt Binary withCry23Aa Crv38Aa1 AAK64559 Rupar et al. 2000 BtCrv39Aa1 BAB72016 Ito et al. 2001 Bt aizawaiCrv40Aa1 BAB72018 Ito et al. 2001 Bt aizawaiCrv40Ba1 BAC77648 Ito et al. 2003 Bun 1-14Cry40Ca1 EU381045 Shu et al. 2008 Bt Y41 No link on
July NCBI
Crv40Da1 ACF15199 Zhang et al. 2008 Bt S2096-2 Crv41Aa1 BAD35157 Yamashita et al. 2003 Bt A1462 Crv41Ab1 BAD35163 Yamashita et al. 2003 Bt A1462 Crv42aa1 BAD35166 Yamashita et al. 2003 Bt A1462 Crv43Aa1 BAD15301 Yokoyama and Ta- 2003 P. lentimorbus nakasemadara Crv43Aa2 BAD95474 Nozawa 2004 P. popilliaepopilliae Crv43Ba1 BAD Yokoyama and Ta- 2003 P. lentimorbus 15303 nakasemadara Crv43-like BAD Yokoyama and Ta- 2003 P. lentimorbus 15305 nakasemadara Crv44Aa BAD08532 Ito et al. 2004 Bt entomocidusINA288 Crv45Aa BAD22577 Okumura et al. 2004 Bt 89-T-34-22 Crv46Aa BAC79010 Ito et al. 2004 Bt dakota Crv46Aa2 BAG68906 Ishikawa et al. 2008 Bt A1470 Crv46Ab BAD35170 Yamagiwa et al. 2004 Bt Crv47Aa AAY24695 Kongsuwan et al. 2005 Bt CAA890 Crv48Aa CAJ18351 Jones and Berry 2005 Bs IAB59 Binary49Aa with Crv48Aa2 CAJ86545 Jones and Berry 2006 Bs 47-6B Binary49Aa2 with Crv48Aa3 CAJ86546 Jones and Berry 2006 Bs NHA15b Binary49Aa3 with Crv48Ab CAJ86548 Jones and Berry 2006 Bs LP1H Binary49Ab1 with Crv48Ab2 CAJ86549 Jones and Berry 2006 Bs 2173 Binary49Aa4 with Crv49Aa CAH56541 Jones and Berry 2005 Bs IAB59 Binary48Aa with Crv49Aa2 CAJ86541 Jones and Berry 2006 Bs 47-6B Binary with
Petition 870180072826, of 8/20/2018, p. 60/73
51/54
48Aa2
Crv49Aa3 CAJ86543 Jones and Berry 2006 BsNHA15b Binary with48Aa3 Crv49Aa4 CAJ86544 Jones and Berry 2006 Bs 2173 Binary with48Ab2 Crv49Ab1 CAJ86542 Jones and Berry 2006 Bs LP1G Binary with48Ab1 Crv50Aa1 BAE86999 Ohgushi et al. 2006 Bs sottoCrv51Aa1 ABII14444 Meng et al. 2006 Bt F14-1Cry52Aa1 EF613489 Song et al. 2007 Bt Y41 No link onNCBIJuly 9 Cry52Ba1 FJ361760 Jun et al. 2008 Bt BM59-2 No link onNCBIJuly 9 Cry53Aa1 EF633476 Song et al. 2007 Bt Y41 No link onNCBIJuly 9 Cry53Ab1 FJ361759 Jun et al. 2008 Bt MC28 No link onNCBIJuly 9 Crv54Aa1 ACA52194 Tan et al. 2009 Bt MC28Crv55Aa1 ABW88931 Guo et al. 2008 YBT 1518Crv55Aa2 AAE33526 Bradfisch et al. 2000 Bt Y41Cry56Aa1 FJ597621 Jun & Furong 2008 Bt Ywc2-8 No link onNCBIJuly 9 Cry56Aa2 GQ483512 Guan Peng et al. 2009 Bt G7-1 No link onNCBIAugust 9 Crv57Aa1 ANC87261 Noguera & Ibarra 2009 Bt kimCrv58Aa1 ANC87260 Noguera & Ibarra 2009 Bt entomocidusCrv59Aa1 ACR43758 Noguera & Ibarra 2009 Bt kim LBIT-
980
Vip3Aa1 Vip3Aa AAC37036 Estruch et al. 1996 PNAS 93.5389-5394 AB88Vip3Aa2 Vip3Ab AAC37037 Estruch et al. 1996 PNAS 93,5389-5394 AB424
Petition 870180072826, of 8/20/2018, p. 61/73
52/54
Vip3Aa3 Vip3AcEstruch et al. 2000 US 6137033Dec 2000 Vip3Aa4 PS36ASup AAR81079 Feitelson et al. 1998 US 6656908Dec 2003 Bt PS36A WO9818932 (A2, A3)May 7, 1998 Vip3Aa5 PS81FSup AAR81080 Feitelson et al. 1998 US 6656908Dec 2003 Bt PS81F WO9818932 (A2, A3)May 7, 1998 Vip3Aa6 Jav90Sup AAR81081 Feitelson et al. 2003 US 6656908Dec 2003 Bt WO9818932 (A2, A3)May 7, 1998 Vip3Aa7 Vip83 AAK95326 Cai et al. 2001 Not published Bt YBT-833Vip3Aa8 Vip3A AAK97481 Loguercio et al. 2001 Not published Bt HD125Vip3Aa9 VipS CAA76665 Selvapandiyan et al. 2001 Not published Bt A13Vip3Aa10 Vip3V AAN60738 Doss et al. 2002 Protein Expr. Purif 26, 82-88 BtVip3Aa11 Vip3A AAR36859 Liu et al. 2003 Not published Bt C9Vip3Aa12 Vip3A-WB5 AAM22456 Wu and Guan 2003 Not published BtVip3Aa13 Vip3A AAL69542 Chen et al. 2002 Sheng Wu,Gong ChengXue Bao 18, 687-692 Bt S184Vip3Aa14 VIP AAQ12340 Polumetla et al. 2003 Not published Bt tolworthiVip3Aa15 Vip3A AAP51131 Wu et al. 2004 Not published Bt WB50Vip3Aa16 Vip3LB AAW65132 Mesrati et al. 2005 FEMS MicroLett 244, 353-358 BtVip3Aa17 Jav90Feitelson et al. 1999 US 6603063August 2003 Javelinnineteen ninety WO9957282 (A2, A3)11 Nov 1999 Vip3Aa18AAX49395 Cai and Xiao 2005 Not published Bt 9816CVip3Aa19 Vip3ALD DQ241674 Liu et al. 2006 Not published Bt ALVip3Aa19 Vip3A-1 DQ539887 Hart et al. 2006 Not published Vip3Aa20 Vip3A-2 DQ539888 Hart et al. 2006 Not published Vip3Aa21 VIP ABD84410 Panbangred 2006 Not published Bt aizawaiVip3Aa22 Vip3A- AAY41427 Lu et al. 2005 Not published Bt LS1
Petition 870180072826, of 8/20/2018, p. 62/73
53/54
LS1 Vip3Aa23 Vip3A-LS8 AAY41428 Lu et al. 2005 Not published Bt LS8Vip3Aa24BI880913 Song et al. 2007 Not published Bt WZ-7Vip3Aa25EF608501 Hsieh et al 2007 Not published Vip3Aa26EU294496 Shen and Guo 2007 Not published Bt TF9Vip3Aa27EU332167 Shen and Guo 2007 Not published Bt 16Vip3Aa28FJ494817 Xiumei Yu 2008 Not published Bt JF23-8Vip3Aa29FJ626674 Xieumei et al 2009 Not published Bt JF21-1Vip3Aa30FJ626675 Xieumei et al 2009 Not published MD2-1Vip3Aa31FJ626676 Xieumei et al 2009 Not published JF21-1Vip3Aa32FJ626677 Xieumei et al 2009 Not published MD2-1Vip3Ab1 Vip3B AAR40284 Feitelson et al. 1999 US 6603063August 2003 BtKB59A4-6 WO9957282 (A2, A3)11 Nov 1999 Vip3Ab2 Vip3D AAY88247 Feng and Shen 2006 Not published BtVip3Ac1 PS49CNarva et al.US Order20040128716 Vip3Ad1 PS158C2Narva et al.US Order20040128716 Vip3Ad2 ISP3B CAI43276 Van Rie et al. 2005 Not published BtVip3Ae1 ISP3C CAI43277 Van Rie et al. 2005 Not published BtVip3Af1 ISP3A CAI43275 Van Rie et al. 2005 Not published BtVip3Af2 Vip3C ADN08753 SyngentaWO03/075655 Vip3Ag1 Vip3B ADN08758 SyngentaWO02/078437 Vip3Ag2FJ556803 Audtho et al. 2008BtVip3Ah1 Vip3S DQ832323 Li and Shen 2006 Not published BtVip3Ba1AAV70653 Rang et al. 2004 Not published
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54/54
Vip3Bb1 Vip3Z ADN08760 SyngentaWO03/075655 Vip3Bb2EF439819 Akhurst et al. 2007
Petition 870180072826, of 8/20/2018, p. 64/73

权利要求:
Claims (1)
[1]
CLAIM
1. Method of control of a cartridge caterpillar insect, characterized by the fact that it comprises contacting said insect, or the environment of said insect, with an effective amount of an insecticidal protein Cry1Be and an insecticidal protein Cry1Da, in which the said Cry1Be insecticidal protein comprises SEQ ID NO: 1, and said Cry1Da insecticidal protein comprises SEQ ID NO: 2.

类似技术:

---

公开号 | 公开日 | 专利标题

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EP2513317B1|2018-01-24|Use of cry1da in combination with cry1be for management of resistant insects

---

EP2513318B1|2018-01-24|Use of cry1ab in combination with cry1be for management of resistant insects

---

EP2513314B2|2019-09-11|Combined use of cry1ca and cry1ab proteins for insect resistance management

---

ES2700458T3|2019-02-15|Combined use of Vip3Ab with Cry1Ca to suppress resistant insects

---

ES2704652T3|2019-03-19|Combined use of CRY1Da and CRY1Fa proteins to manage the resistance of insects

---

EP2512225B1|2017-08-23|Use of vip3ab in combination with cry1ca for management of resistant insects

---

EP2513316B1|2018-11-28|Use of cry1da in combination with cry1ca for management of resistant insects

---

RU2624031C2|2017-06-30|Application of insecticidic crystalline protein dig3 in combination with cry1ab for regulation of stability to corn borer

---

BR102012019662A2|2015-05-05|Use of dig3 crystal insecticide protein in combination with cry1ab for European corn borer resistance control

同族专利:

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公开号 | 公开日

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JP2016154536A|2016-09-01|

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MX2012007126A|2012-11-12|

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US20120331590A1|2012-12-27|

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KR101841296B1|2018-03-22|

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EP2513317A4|2013-10-02|

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MX350002B|2017-08-22|

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KR20120115980A|2012-10-19|

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IL220332D0|2012-08-30|

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CA2782565A1|2011-07-14|

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AR079506A1|2012-02-01|

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US9499835B2|2016-11-22|

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CO6561806A2|2012-11-15|

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RU2012129910A|2014-01-27|

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RU2590592C2|2016-07-10|

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WO2011084630A1|2011-07-14|

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NZ601102A|2014-10-31|

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AU2010339919B2|2016-01-21|

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JP5969921B2|2016-08-17|

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CN102753696A|2012-10-24|

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ZA201204925B|2013-03-27|

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JP2013514774A|2013-05-02|

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EP2513317B1|2018-01-24|

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BR112012014727A2|2015-08-25|

---

IL220332A|2017-12-31|

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AU2010339919A1|2012-07-12|

---

CL2012001623A1|2013-01-25|

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EP2513317A1|2012-10-24|

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法律状态:
2018-05-22| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2018-12-26| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

---

2019-05-28| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2019-09-03| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/12/2010, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/12/2010, OBSERVADAS AS CONDICOES LEGAIS |

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2021-01-12| B21F| Lapse acc. art. 78, item iv - on non-payment of the annual fees in time|Free format text: REFERENTE A 10A ANUIDADE. |

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2021-05-04| B24J| Lapse because of non-payment of annual fees (definitively: art 78 iv lpi, resolution 113/2013 art. 12)|Free format text: EM VIRTUDE DA EXTINCAO PUBLICADA NA RPI 2610 DE 12-01-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDA A EXTINCAO DA PATENTE E SEUS CERTIFICADOS, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

优先权:

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申请号 | 申请日 | 专利标题

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US28425209P| true| 2009-12-16|2009-12-16|

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US28429009P| true| 2009-12-16|2009-12-16|

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PCT/US2010/060829|WO2011084630A1|2009-12-16|2010-12-16|Use of cry1da in combination with cry1be for management of resistant insects|

---