CN114680127A - Control of noctuid, snout moth's larva and snout moth's larva harmful organism - Google Patents

Control of noctuid, snout moth's larva and snout moth's larva harmful organism Download PDF

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CN114680127A
CN114680127A CN202011586496.7A CN202011586496A CN114680127A CN 114680127 A CN114680127 A CN 114680127A CN 202011586496 A CN202011586496 A CN 202011586496A CN 114680127 A CN114680127 A CN 114680127A
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曹光宇
段倩倩
龙颖
陆天聪
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Syngenta Crop Protection AG Switzerland
Syngenta Biotechnology China Co Ltd
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Syngenta Biotechnology China Co Ltd
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    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance

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Abstract

The present invention provides methods for controlling Lepidoptera (Lepidoptera), such as Noctuidae (Noctuidae), Pyralidae (Crambidae) and Pyralidae (Pyralidae), and protecting crops, in particular maize, from economic damage caused by Lepidoptera, such as Noctuidae, Pyralidae and Pyralidae. The invention further relates to the use of plants stably transformed with nucleic acid molecules encoding the Cry proteins of the invention (alone or in combination with other insecticidal proteins) for controlling or combating lepidoptera (e.g., noctuidae, chilo-suppressalis and snout moth's family).

Description

Control of noctuid, snout moth's larva and borer moth pests
Technical Field
The present invention relates to compositions and methods for using pesticidal proteins and nucleic acid molecules encoding them to control or combat Noctuidae (Noctuidae), Pyralidae (Crambidae) and Pyralidae (Pyralidae) pests.
Background
Bacillus thuringiensis (Bt) is a gram-positive, spore-forming soil bacterium characterized by its ability to produce crystalline inclusions that are specifically toxic to certain orders and species of plant pests, including insects, but not harmful to plants and other non-target organisms. Thus, compositions comprising a bacillus thuringiensis strain or insecticidal proteins thereof can be used as environmentally acceptable insecticides for controlling agricultural insect pests or insect vectors of a wide variety of human or animal diseases.
Crystal (Cry) proteins from bacillus thuringiensis have potent insecticidal activity against predominantly lepidopteran, dipteran, and coleopteran pest insects. These proteins also show activity against pests in the orders Hymenoptera (Hymenoptera), Homoptera (Homoptera), pediculoptera (phirapera), Mallophaga (Mallophaga) and acarina (Acari) and other invertebrate orders, such as the phylum lineates (Nemathelminthes), phylum Platyhelminthes and phylum sarcophaga (sarcophaga) (Feitelson, j.1993.the Bacillus thunringiensis family tree in Advanced Engineered pestides Marcel, inc. These proteins were originally classified as CryI to CryVI, primarily based on their insecticidal activity classification. The main classes are lepidopteran-specific (I), lepidopteran-and dipteran-specific (II), coleopteran-specific (III), dipteran-specific (IV) and nematode-specific (V) and (VI). The proteins are further classified into subfamilies; within each family, more highly related proteins are assigned grouping letters, such as CryIA, CryIB, CryIC, and the like. Within each packet, the more closely related proteins are given names such as cryic (a), cryic (b), and the like. The terms "Cry toxin" and "delta-endotoxin" have been used interchangeably with the term "Cry protein". Current nomenclature for Cry proteins and genes is based on amino acid sequence homology, not insect target specificity (Crickmore et al, (1998) Microbiol. mol. biol. Rev.62: 807-813). In this more accepted classification, each toxin is assigned a unique name that incorporates a first rank (arabic numeral), a second rank (upper case), a third rank (lower case), and a fourth rank (another arabic numeral). In the current taxonomy, the roman numerals have been transposed to arabic numerals in the first level. For example, "CryIA (a)" under the old nomenclature is now "Cry 1 Aa" under the current nomenclature. According to Ibrahim et al (2010, bioenng. bugs,1:31-50), Cry toxins can still be classified into six major classes according to their insect host specificity, and include: group 1 — lepidoptera (e.g., Cry1, Cry9, and Cry 15); group 2-lepidoptera and diptera (e.g., Cry 2); group 3 — coleoptera (Cry3, Cry7, and Cry 8); group 4-twin wings (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19 and Cry 20); group 5-lepidopteran and coleopteran species (Cry 1I); and group 6-nematodes (Cry 6). The Cry1I, Cry2, Cry3, Cry10 and Cry11 toxins (73-82kDa) are unique in that they appear as natural truncations of the larger Cry1 and Cry4 proteins (130-140 kDa).
Cry proteins are globular protein molecules that accumulate as protoxins in crystal form during the sporulation phase of Bt. Upon ingestion by pests, the crystals typically dissolve to release protoxins, ranging in size, for example, from 130-140kDa for many lepidopteran active Cry proteins (e.g., Cry1 and Cry9), and from 60-80kDa for coleopteran active Cry3 proteins and lepidopteran/dipteran active Cry2 proteins. After the crystals are solubilized by susceptible insects, the released protoxin is processed in the insect gut by proteases (e.g., trypsin and chymotrypsin) to produce protease-resistant core Cry protein toxins. This proteolytic processing involves the removal of amino acids from different regions of various Cry protoxins. For example, the 130-plus 140kDa Cry protoxin is typically activated by: proteolytically removes the 25-30 amino acid N-terminal peptide and about half of the remaining protein from the C-terminus, resulting in a mature Cry toxin of approximately 60-70 kDa. Protoxins of 60-80kDa (e.g., Cry2 and Cry3) are also processed, but to a different extent than the larger protoxins. Smaller protoxins typically remove the same or more amino acids from the N-terminus, but less amino acids from the C-terminus than larger protoxins. For example, proteolytic activation of members of the Cry2 family typically involves removal of approximately 40-50N-terminal amino acids. Many of the Cry proteins are quite toxic to specific target insects, but many have a narrow activity spectrum.
Cry proteins typically have five conserved sequence domains and three conserved domains (see, e.g., de Maagd et al, (2001) Trends Genetics 17: 193-199). The first conserved domain (called domain I) typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three β -sheets arranged in a Greek-key configuration, and domain III typically consists of two antiparallel β -sheets constructed in a "pancake-type" configuration (de Maagd et al, 2001, supra). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.
Many commercially valuable plants, including common crops, are vulnerable to attack by plant pests, including insect and nematode pests, resulting in a significant reduction in crop yield and quality. For example, plant pests are a major cause of loss of important crops of the world. Approximately 15-20% of the harvestable grain is lost annually in china due to insect pests and diseases. In addition, in the united states alone, there are approximately 80 billion dollars lost annually due to infestation by invertebrate pests (including insects). Insect pests are also a burden for vegetable and fruit growers, ornamental flower producers, and home gardeners.
Insect pests are controlled primarily by intensive application of chemical pesticides that exert activity by inhibiting insect growth, preventing insect feeding or reproduction, or causing death. Biological pest control agents, such as bacillus thuringiensis strains expressing pesticidal toxins (e.g., Cry proteins), have also been applied to crop plants with satisfactory results, providing alternatives or supplements to chemical pesticides. Genes encoding some of these Cry proteins have been isolated, and their expression in heterologous hosts (e.g., transgenic plants) has been shown to provide another tool for controlling economically important insect pests. Most Cry proteins are active against a very limited spectrum of insect pests. And typically, activity against one insect species cannot be predicted against a different insect species.
Lepidopteran pests, including Noctuid (Noctuid), Pyralid (Crambid), and borer (Pyralid) pests of the present disclosure, remain a problem in china and other countries where such pests are present. In addition, the threat of resistance to existing insecticidal proteins means that it is important to introduce new insecticidal proteins. Thus, there remains a need to identify insecticidal proteins capable of controlling lepidopteran pests (such as noctuid, snout moth and snout moth pests of the present disclosure).
Disclosure of Invention
The present disclosure provides compositions and methods for controlling Lepidopteran (Lepidopteran) pests (e.g., noctuid, snout moth and borer pests) and protecting crops, particularly corn and rice, from economic damage caused by such pests. The present disclosure further relates to the use of plants, especially monocotyledonous plants, in particular maize (corn, maize, Zea mays) and rice (rice, Oryza sativa) stably transformed with a nucleic acid molecule encoding a Cry protein of the disclosure (alone or in combination with other insecticidal proteins) for the control or combating of lepidoptera (e.g., noctuid, snout moth's larva and borer moth). The disclosure still further relates to the use of insecticidal formulations comprising the Cry proteins of the disclosure for protecting plants from lepidoptera (e.g., noctuid, snout moth's larva, and snout moth's larva). The disclosure also relates to plants, especially monocots, in particular corn plants or rice plants, which can be infested with lepidopteran pests (e.g., noctuids, snout moth's larva, and borer moths) and transformed with an expressible nucleic acid molecule encoding a Cry protein of the disclosure to combat or control lepidopteran pest populations (e.g., noctuids, snout moth's larva, and borer moths).
According to the present disclosure, a method is provided for combating and/or controlling insects of the order Lepidoptera (e.g. Spodoptera exigua, Cnaphalocrocis medinalis, and borer) and Helicoverpa armigera (Cotton bollworm/Old World bollworm), Mythimna orientalis (Mythimna sepenata, Oriental armyworm), Athetis lepigone (Two-spoted armyworm), Spodoptera litura (Common cutworm/Oriental leaf worm), cutworm (Agrotis ipsilon, Black cutworm), Cymbopogon punctatus (Conogetheus puttiferalis, Yollow pest), Chilo suppressalis (Chitosa punctifera), Chilo suppressalis (Ossorethes punctalis), Ostrea punctiferalis (Ostrea bombesi), Ostrea punctifera (Piercea punctifera), or Ostrinia japonica (Ostrinia japonica) species, comprising the sequence thereof, or the step of contacting these insects with Osmanthus insecta officinalis, Osmanthus insect (Ostrinia terrestris) or Osmanthus insect pest insect (Osmanthus insect pest), or Osmanthus insect pest 3, or Osmanthus insect species comprising the step thereof. In some embodiments, the black cutworm is from a population of black cutworms in china.
In some embodiments, the contacting step can be performed with an insecticidal composition comprising: a Cry protein of the disclosure, or an insecticidal fragment thereof, and an acceptable agricultural carrier. In some embodiments, the contacting of the insect can be with a plant (particularly a monocot plant, particularly a maize plant or a rice plant) stably transformed with an expressible nucleic acid molecule encoding a Cry protein of the disclosure, such that the transformed plant expresses the Cry protein of the disclosure or an insecticidal fragment thereof in an amount effective to control the insect.
Furthermore, plants, especially monocotyledonous plants, in particular maize plants or rice plants, which are infested with lepidoptera (e.g. noctuids, snout moth's larva and stem borers) are protected from continuing economic damage from the insect by stable transformation with a gene encoding the Cry protein of the disclosure.
Brief description of the sequences in the sequence listing
SEQ ID NO. 1 is the amino acid sequence of the BT-0032 protein.
SEQ ID NO. 2 is the amino acid sequence of CryET72 protein.
SEQ ID NO 3 is the amino acid sequence of the second BT-0032 protein.
Detailed Description
This description is not intended to be an exhaustive list of all the different ways in which the invention may be practiced or to add all the features of the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. In addition, numerous variations and additions to the various embodiments set forth herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention. Accordingly, the following description is intended to illustrate certain specific embodiments of the invention and is not intended to be exhaustive or to limit all permutations, combinations and variations thereof.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.
As used herein, the word "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").
The term "about" is used herein to mean approximately, or on the left or right. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by a 20%, preferably 10% (higher or lower) variation. With respect to temperature, the term "about" means ± 1 ℃, preferably ± 0.5 ℃. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., without "about") is preferred.
By "controlling" an insect is meant inhibiting the ability of an insect pest to survive, grow, feed or reproduce by toxic effects, or limiting insect-related damage or loss in crop plants, or protecting crop yield potential when the crop is grown in the presence of insect pests. "controlling" an insect may or may not mean killing the insect, although it preferably means killing the insect.
The terms "comprises" or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.
As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) means that the scope of the claims is to be interpreted as encompassing the explicitly recited materials or steps recited in the claims, as well as those materials or steps that do not materially alter the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of …" is not intended to be construed as equivalent to "comprising" when used in the claims of this invention.
As used herein, the term "Cry protein" means an insecticidal protein that can occur in crystal form in bacillus thuringiensis or related bacteria. The term "Cry protein" can refer to the protoxin form or any insecticidal fragment or toxin thereof.
By "delivering" a composition or toxic protein is meant that the composition or toxic protein comes into contact with the insect, which facilitates oral ingestion of the composition or toxic protein, resulting in toxic effects and control of the insect. The composition or toxic protein may be delivered in a number of recognized ways including, but not limited to, transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait matrices, or any other art recognized protein delivery system.
By "effective insect-controlling amount" is meant a concentration of a toxic protein that inhibits the ability of an insect to survive, grow, feed or reproduce by a toxic effect, or limits insect-related damage or loss in crop plants, or protects the yield potential of a crop when the crop is grown in the presence of an insect pest. An "effective insect-controlling amount" may or may not mean killing the insect, although it preferably means killing the insect.
A "gene" is defined herein as a genetic unit comprising one or more polynucleotides, which occupy a specific position on a chromosome or plasmid, and which comprise genetic instructions for a specific feature or trait in an organism.
As used herein, "pesticidal," "insecticidal," and the like, refer to the ability of a Cry protein of the disclosure to control a pest organism, or to the amount of a Cry protein that can control a pest organism as defined herein. Accordingly, pesticidal Cry proteins can kill or inhibit the ability of a pest organism (e.g., an insect pest) to survive, grow, feed, or reproduce.
Nucleotides are designated herein by the following standard abbreviations: adenine (a), cytosine (C), thymine (T) and guanine (G). Likewise, amino acids are designated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
The present invention is based on the results of a toxicity assay performed by feeding certain noctuid, snout moth and snout moth insects with an artificial diet comprising purified Cry toxins and surprisingly shows that certain Cry proteins are toxic to one or more of the insects tested (see example 1). Thus, these active Cry proteins can be used to provide maximum protection against such pests, and can prevent or reduce the development of insect resistance to Cry insecticidal formulations in the field.
The "Cry proteins" of the present disclosure can be naturally occurring or engineered and encompass full-length proteins (protoxins) having the amino acid sequences shown in any of SEQ ID NOs 1-3 of the sequence listing, as well as any insecticidally active fragments thereof.
The present disclosure also includes polynucleotides that are fragments of polynucleotides encoding Cry protein protoxins. By "fragment" is meant a portion of the nucleotide sequence encoding the Cry protein. A fragment of a nucleotide sequence may encode a biologically active portion of a Cry protein, a so-called "toxin fragment," or it may be a fragment that can be used as a hybridization probe or PCR primer by using the methods disclosed below. Nucleic acid molecules that are fragments of a nucleotide sequence encoding a Cry protein comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 consecutive nucleotides, or up to the number of nucleotides present in the nucleotide sequence encoding a full-length Cry protein disclosed herein, depending on the intended use. "contiguous" nucleotides means nucleotide residues that are immediately adjacent to each other. Some fragments of the nucleotide sequences of the present disclosure will encode toxin fragments that retain the biological activity of the Cry proteins, and thus, the insecticidal activity. By "retains insecticidal activity" is meant that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, and even more preferably at least about 80% of the insecticidal activity of the Cry protein. Methods for measuring insecticidal activity are well known in the art. See, e.g., Czapla and Lang (1990) J.Econ.Entomol.83: 2480-2485; andrews et al, (1988) biochem. J.252: 199-206; marron et al, (1985) J.of Economic Entomogy 78: 290-293; and U.S. patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.
Toxin fragments of the Cry proteins of the disclosure will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, and 450 consecutive amino acids, or up to the total number of amino acids present in the full-length Cry proteins of the disclosure.
As used herein, a Cry protein that is "toxic" to an insect pest means that the Cry protein functions as an orally active insect control agent to kill the insect pest, or that the Cry protein is capable of disrupting or arresting insect feeding or causing growth inhibition to the insect pest, both of which may or may not cause death of the insect. When a Cry protein of the present disclosure is delivered to an insect or an insect comes into oral contact with said Cry protein, the result is typically death of said insect, or growth of said insect is slowed, or said insect stops feeding so that said insect can obtain a source of said toxic Cry protein.
In some embodiments, the present disclosure provides a method of inhibiting the growth of or killing a pest, the method comprising contacting the pest with a Cry protein comprising an amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of cotton bollworm, oriental armyworm, athetis lepigone, spodoptera litura, black cutworm, peach borer, chilo suppressalis, spodoptera littoralis, and ostrinia furnacalis. In some embodiments, the black cutworm is from a population of black cutworms in china.
In some embodiments, the present disclosure provides methods for controlling a pest population comprising contacting the pest population with an insecticidally effective amount of a Cry protein comprising an amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of cotton bollworm, oriental armyworm, athetis lepigone, spodoptera litura, black cutworm, dichocrocis punctifera, chilo suppressalis, noctuid rice and ostrinia nubilalis. In some embodiments, the black cutworm is from a population of black cutworms in china.
In a further embodiment of the present disclosure, the pest or pest population is further contacted with a second insecticidal protein that is different from the Cry protein comprising the amino acid sequence of any of SEQ ID NOs 1-3. In still other embodiments, the second insecticidal protein is selected from the group consisting of Bacillus thuringiensis (Bt) insecticidal proteins, Xenorhabdus (Xenorhabdus) insecticidal proteins, Photorhabdus (phorhabdus) insecticidal proteins, Brevibacillus laterosporus (Brevibacillus laterosporus) insecticidal proteins, Bacillus sphaericus (Bacillus sphaericus) insecticidal proteins, perforins, protease inhibitors (both serine and cysteine types), lectins, alpha-amylases, peroxidases, cholesterol oxidases, and double stranded rna (dsrna) molecules.
In other embodiments of the present disclosure, the contacting step (wherein the Cry protein of the present disclosure is contacted with a pest) is performed with a microorganism or plant that expresses the protein or insecticidal fragment thereof. In other embodiments, the plants are stably transformed with a nucleic acid molecule encoding a Cry protein of the disclosure or an insecticidal fragment thereof. In yet other embodiments, the plant is a monocot or a dicot. In other embodiments, the monocot plant is a maize plant or a rice plant, or the dicot plant is a soybean plant.
In some embodiments, the present disclosure provides methods for protecting a plant from a pest, comprising expressing in the plant or cell thereof an insecticidally effective amount of a Cry protein comprising an amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of cotton bollworm, oriental armyworm, athetis lepigone, prodenia litura, black cutworm, peach borer, chilo suppressalis, stem borer, and asian corn borer. In some embodiments, the black cutworm is from a population of black cutworms in china.
To be effective against the insect pests of the present disclosure, the Cry proteins are first taken orally by the insects. However, the Cry proteins can be delivered to the insects in a number of recognized ways. Means for oral delivery of a protein to an insect include, but are not limited to, (1) providing the protein in a transgenic plant, wherein the insect eats (ingests) one or more parts of the transgenic plant, thereby ingesting a polypeptide expressed in the transgenic plant; (2) providing the protein in a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) providing the protein in a protein composition that can be applied to a surface, e.g., sprayed onto the surface of plant parts, and then ingested by the insect as the insect eats one or more of the sprayed plant parts; (4) a bait matrix; or (5) any other art-recognized protein delivery system. Thus, any method of oral delivery to insects can be used in the methods of the present disclosure to deliver the toxic Cry proteins of the present disclosure. In some particular embodiments, the Cry proteins of the disclosure are delivered orally to insects, wherein the insects ingest one or more portions of a transgenic plant.
In other embodiments, the Cry proteins of the disclosure are delivered orally to insects, wherein said insects ingest one or more portions of a plant sprayed with a composition comprising the Cry proteins of the disclosure. Delivery of the compositions of the present disclosure to a plant surface can be carried out by using any method known to those skilled in the art for applying compounds, compositions, formulations, etc. to a plant surface. Some non-limiting examples of delivery to or contact with a plant or portion thereof include spraying, dusting, spraying, spreading, misting, atomizing, broadcasting, soaking, soil drenching, soil incorporation, drenching (e.g., root, soil treatment), maceration, irrigation, cladding, leaf or stem infiltration, side application, or seed treatment, and the like, as well as combinations thereof. These and other procedures for contacting plants or parts thereof with compounds, compositions or formulations are well known to those skilled in the art.
In some embodiments of the present disclosure, the insecticidal Cry proteins of the present disclosure are expressed in higher organisms (e.g., plants). In this case, transgenic plants expressing an effective amount of an insecticidal protein protect themselves from plant pests, such as insect pests. When an insect pest larva begins to feed on such a transgenic plant, it will take up the expressed insecticidal Cry protein. This may prevent the insect from further biting into the plant tissue, or may even injure or kill the insect. The polynucleotides encoding the Cry proteins of the disclosure are inserted into an expression cassette, which is then stably integrated into the genome of the plant. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus. Plants transformed according to the present disclosure may be monocotyledonous or dicotyledonous plants, and include, but are not limited to, corn (maize), soybean, rice, wheat, barley, rye, oat, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanut, vegetables (including sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melon, chili, celery, squash, pumpkin, honey pumpkin), fruits (including apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana), and specialty plants (e.g., Arabidopsis (Arabidopsis)), as well as woody plants (e.g., coniferous trees and deciduous trees). Preferably, the plants of the present disclosure are crop plants, such as maize, soybean, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, sugar beet, sugarcane, tobacco, barley, oilseed rape and the like. Once the desired polynucleotide has been transformed into a particular plant species, it can be propagated in that species or moved to other varieties of the same species (including particularly commercial varieties) by using conventional breeding techniques.
Polynucleotides encoding the Cry proteins of the disclosure are expressed in transgenic plants, thereby causing biosynthesis of the encoded Cry protein (in protoxin or toxin form) in the transgenic plants. In this way, transgenic plants with enhanced yield protection in the presence of insect pest population pressure are produced. In order to express them in transgenic plants, the nucleotide sequences encoding the Cry proteins may need to be modified and optimized. Although in many cases genes from microorganisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that living organisms have a particular preference for codon usage, and that the codons of the nucleotide sequences described in the present disclosure can be changed to conform to plant preferences while retaining the amino acids encoded thereby. Furthermore, high expression in plants (e.g., maize plants) is optimally achieved from coding sequences having at least about 35% GC content, or at least about 45% GC content, or at least about 50% GC content, or at least about 60% GC content. Microbial nucleotide sequences with low GC content may be poorly expressed in plants due to the presence of ATTTA motifs which may destabilize the information and AATAAA motifs which may cause inappropriate polyadenylation. Although certain gene sequences can be expressed adequately in both monocot and dicot species, the sequences can be modified to address specific codon preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al, Nucl. acids Res.17:477-498 (1989)). In addition, nucleotide sequences are screened for the presence of an irregular splice site that may result in truncation of the message. All changes that need to be made within the nucleotide sequence (e.g., those described above) are made by techniques using well known site-directed mutagenesis, PCR, and synthetic gene construction (using methods such as those described in U.S. patent nos. 5,625,136, 5,500,365, and 6,013,523).
For efficient translation initiation, the sequence adjacent to the initiating methionine may need to be modified. For example, they may be modified by including sequences known to be effective in plants. Suitable consensus sequences for plants have been proposed by Joshi (NAR 15:6643-6653 (1987)). These consensus sequences are suitable for use with the nucleotide sequences of the present disclosure. The sequence is incorporated into a construct comprising the nucleotide sequence up to and including the ATG (with the second amino acid being unmodified), or alternatively up to and including the GTC following the ATG (with the possibility of modifying the second amino acid of the transgene).
Polynucleotide sequences encoding the Cry proteins of the disclosure can be operably fused to a wide variety of promoters for expression in plants (including constitutive promoters, inducible promoters, temporally regulated promoters, developmentally regulated promoters, chemically regulated promoters, tissue-preferred promoters, and tissue-specific promoters) to produce recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements for expression and also depending on the target species. Thus, expression of the nucleotide sequences of the present disclosure in leaves, in stalks or stems, in ears, in inflorescences (e.g., panicles, cobs, etc.), in roots, or in seedlings is preferred. However, in many cases, it is desirable to seek protection against more than one type of insect pest, and thus expression in a variety of tissues. While many promoters from dicots have been shown to function in monocots, and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots. However, there is no limitation on the origin of the promoter selected; they are sufficient to be operable in driving expression of the nucleotide sequence in the desired cell.
Suitable constitutive promoters include, for example, the CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); the Arabidopsis thaliana At6669 promoter (see PCT publication WO04081173A 2); maize Ubi 1(Christensen et al, Plant mol.biol.18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2:163-171, 1990); pEMU (Last et al, the or. appl. Gene.81: 581-588, 1991); CaMV 19S (Nilsson et al, Physiol. plant 100:456-462, 1997); GOS2(de Pater et al, Plant J November,2(6): 837-; ubiquitin (Christensen et al, Plant mol.biol.18:675-689, 1992); rice cyclophilins (Bucholz et al, Plant Mol biol.25(5):837-43, 1994); maize H3 histone (Lepetit et al, mol.Gen.Genet.231:276-285, 1992); actin 2(An et al, Plant J.10(1),107-121, 1996); the constitutive root tip CT2 promoter (PCT application No. IL/2005/000627); and Synthetic Super MAS (Ni et al, The Plant Journal 7:661- "76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463 and 5,608,142.
Tissue-specific or tissue-preferential promoters useful for expressing Cry protein coding sequences of the disclosure in plants (particularly maize) are those that direct expression in roots, pith, leaves, or pollen. Suitable tissue-specific promoters include, but are not limited to: leaf-specific promoters [ e.g., from Yamamoto et al, Plant J.12:255-265, 1997; kwon et al, Plant Physiol.105:357-67, 1994; yamamoto et al, Plant Cell physiol.35:773-778, 1994; gotor et al, Plant J.3:509-18, 1993; orozco et al, Plant mol.biol.23:1129-1138, 1993; and Matsuoka et al, Proc. Natl. Acad. Sci. USA 90:9586-9590,1993, seed-preferred promoters [ e.g.from seed-specific genes (Simon et al, Plant Mol. Biol.5.191, 1985; Scofield et al, J.biol. chem.262:12202,1987; Baszczynski et al, Plant Mol. Biol.14:633,1990), Brazilian nut albumin (Pearson' et al, Plant Mol. Biol.18:235-245,1992), legumin (Ellis et al, Plant Mol. biol.10:203-214,1988), gluten (rice) (Takaiwa et al, Mol. Genet.208:15-22,1986; Takaiwa et al, FEke.221: 43-47,1987), gluten (rice) (Takaiwa et al, wheat gluten; wheat gluten et al, wheat gluten: 8732; wheat gluten, wheat gluten 121; wheat gluten, 2: 32; wheat gluten, 2-32; wheat gluten, 1988; wheat gluten et al, wheat gluten, 15-32; Skinson, 1988; S1, wheat gluten, 15-32; Skinson, Skoff; Skohlia, Skohlia et al, Skohlia, Skohli et al, Skohli, Skohlia, wheat a, B and g gliadins (EMB03:1409-, sorghum gamma-sorghum prolamin (Plant mol.biol.32: 1029-35,1996) ], embryo-specific promoters [ e.g.rice OSH1(Sato et al, Proc. Natl.Acad.Sci.USA,93:8117-8122), KNOX (Postma-Haarsma et al, Plant mol.biol.39:257-71,1999), rice oleosins (Wu et al, J.biochem.,123:386,1998) ], flower-specific promoters [ e.g.AtPRP 4, chalcone synthase (chsA) (Van der Meer et al, Plant mol.biol.15,95-109,1990), LAT52(Twell et al, mol.Gen.217: 240-245,1989) ], apetala-3], Plant reproductive tissue [ e.g.OsMADS promoter (U.S. Pat. No. 2007/0006344.
The nucleotide sequence may also be expressed under the control of a chemically regulated promoter. This allows crop plants to synthesize the Cry proteins of the disclosure only when the crop plants are treated with an induction chemical. Examples of such techniques for chemical induction of gene expression are described in detail in published application EP 0332104 and U.S. patent No. 5,614,395. In one embodiment, the chemically regulated promoter is a tobacco PR-1a promoter.
Another class of promoters useful in the present disclosure are wound-inducible promoters. Numerous promoters have been described that express at the site of a wound and also at the site of a plant pathogen infection. Ideally, such promoters should be active only locally at the insect infestation site, and in this way, the insecticidal proteins accumulate only in cells that need to synthesize the insecticidal proteins to kill the invading insect pest. Examples of promoters of this class include those described by: stanford et al, mol.Gen.Genet.215:200-208 (1989); xu et al, Plant mol.biol.22: 573-588 (1993); logemann et al, Plant Cell 1:151-158 (1989); rohrmeier & Lehle, Plant Molec.biol.22: 783. Bucking792 (1993); firek et al, Plant mol.biol.22: 129-142 (1993); and Warner et al, Plant J.3:191-201 (1993).
Non-limiting examples of promoters useful in the present disclosure that cause tissue-specific expression patterns include green tissue-specific promoters, root-specific promoters, stem-specific promoters, or flower-specific promoters. Promoters suitable for expression in green tissues include a number of promoters regulating genes involved in photosynthesis, and many of them have been cloned from monocotyledons and dicotyledons. One such promoter is the maize PEPC promoter from the phosphoenolcarboxylase gene (Hudspeth & Grula, Plant molecular. biol.12:579-589 (1989)). Another promoter for root-specific expression is that described by de Framond (FEBS 290:103-106(1991) or U.S. Pat. No. 5,466,785). Another promoter useful in the present disclosure is the stem-specific promoter described in U.S. patent No. 5,625,136, which naturally drives expression of the maize trpA gene.
In addition to selecting an appropriate promoter, constructs for expressing insecticidal toxins in plants also require an appropriate transcription terminator to be operably linked downstream of the Cry protein coding sequence of the disclosure. Several such terminators are available and known in the art (e.g., tml from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of the present disclosure.
Numerous other sequences can be incorporated into the expression cassettes described in this disclosure. These include sequences that have been shown to enhance expression, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).
It may be preferred to target expression of the nucleotide sequences of the present disclosure to different cellular locations in a plant. In some cases, localization in the cytosol may be desirable, while in other cases, localization in some subcellular organelles may be preferred. Any mechanism for targeting gene products (e.g., in plants) can be used in the practice of the present invention, and such mechanisms are known to exist in plants and the sequences that control the functioning of these mechanisms have been characterized in greater detail. Sequences have been characterized that cause targeting of gene products to other cellular compartments. The amino-terminal sequence may be responsible for targeting the protein of interest to any cellular compartment, such as the vacuole, mitochondria, peroxisomes, proplastids, endoplasmic reticulum, chloroplasts, amyloid granules, amyloplasts, apoplast, or cell wall of a Plant (e.g., Unger et al, Plant mol. biol.13: 411. sup. 418 (1989); Rogers et al, (1985) Proc. Natl. Acad. Sci. USA 82: 6512. sup. 651; U.S. Pat. No. 7,102,057; WO 2005/096704, all of which are incorporated herein by reference). Optionally, the signal sequence may be an N-terminal signal sequence from waxy, an N-terminal signal sequence from gamma-zein, a starch binding domain, a C-terminal starch binding domain, a chloroplast targeting sequence which imports the mature protein into the chloroplast (Comai et al, (1988) J.biol.Chem.263: 15104-minus 15109; van den Broeck et al, (1985) Nature 313: 358-minus 363; U.S. Pat. No. 5,639,949) or a secretory signal sequence from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). In addition, the amino-terminal sequence together with the carboxy-terminal sequence is responsible for vacuolar targeting of the gene product (Shinshi et al, (1990) Plant mol. biol.14: 357-368). In one embodiment, the selected signal sequence includes a known cleavage site, and the fusion constructed takes into account any amino acids (which are required for cleavage) following the cleavage site. In some cases, this requirement can be met by adding a small number of amino acids between the cleavage site and the transgenic ATG or alternatively replacing some amino acids within the transgenic sequence. These construction techniques are well known in the art and apply equally to any cellular compartment.
It will be appreciated that the mechanisms described above for cell targeting may be used not only in conjunction with their homologous promoters, but also in conjunction with heterologous promoters, in order to achieve specific cell targeting goals under the transcriptional regulation of promoters with expression patterns different from those of the promoter from which the targeting signal is derived.
Procedures for transforming plants are well known in the art and are described throughout the literature. Non-limiting examples of methods for plant transformation include transformation by: bacteria-mediated nucleic acid delivery (e.g., via Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microprojectile bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), or biological mechanism that results in the introduction of nucleic acid into a plant cell, including any combination thereof. General guidelines for various Plant transformation Methods known in the art include Miki et al ("Procedures for Introducing DNA for insect Plants", in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E., eds. (CRC Press, Inc., Boca Raton,1993), pp.67-88) and Rakowczy-Trojanowska (cell. mol. biol. Lett.7: 849-.
For Agrobacterium-mediated transformation, one or more binary vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer (e.g., particle bombardment, etc.), any vector is suitable and linear DNA containing only the construct of interest may be used. In the case of direct gene transfer, transformation or co-transformation with a single DNA species may be used (Schocher et al, Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and agrobacterium-mediated transfer, transformation is typically (but not necessarily) performed with a selectable marker, which may be positive selection (phosphomannose isomerase), providing resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (glyphosate or glufosinate). However, the choice of selectable marker is not critical to the present invention.
Agrobacterium-mediated transformation is a commonly used method for transforming plants because of its high transformation efficiency and because of its wide availability with many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying the foreign DNA of interest to a suitable Agrobacterium strain, which may depend on the complement of the vir gene carried by the host Agrobacterium strain either on the co-resident Ti plasmid or on the chromosome (Uknes et al, (1993) Plant Cell 5: 159-169). Transfer of the recombinant binary vector to agrobacterium may be accomplished by a triparental mating procedure using Escherichia coli (Escherichia coli) carrying the recombinant binary vector, a helper Escherichia coli strain carrying a plasmid capable of mobilizing the recombinant binary vector to the target agrobacterium strain. Alternatively, the recombinant binary vector may be transferred to Agrobacterium by nucleic acid transformation (A)
Figure BDA0002867281840000191
&Willmitzer(1988)Nucleic Acids Res.16:9877)。
Both dicotyledonous and monocotyledonous plants can be transformed by using Agrobacterium. Methods for agrobacterium-mediated transformation of rice include well-known methods for rice transformation, such as those described in any of the following: european patent application EP 1198985a 1; aldemita and Hodges (Planta 199: 612. sup. -. 617. sup.,1996); chan et al (Plant Mol Biol 22(3):491-506, 1993); hiei et al (Plant J6 (2):271-282,1994), the disclosure of which is incorporated herein by reference. In the case of maize transformation, preferred methods are described in Ishida et al (nat. Biotechnol 14(6):745-50,1996) or Frame et al (Plant Physiol 129(1):13-22,2002), the disclosures of which are incorporated herein by reference. As an example, the method is further described in the following documents: jenes et al, Techniques for Gene Transfer, in Transgenic Plants, Vol.1, Engineering and validation, edited by S.D. Kung and R.Wu, Academic Press (1993) 128-143; and Potrykus Annu. Rev. plant Physiol. plant mol. biol.42(1991) 205-225. The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transformation of Agrobacterium tumefaciens (Agrobacterium tumefaciens), for example pBin19(Bevan et al, nucleic acids Res.12(1984) 8711). The agrobacterium transformed with such a vector can then be used in a known manner for the transformation of plants, for example plants used as models (e.g. arabidopsis thaliana) or crop plants (e.g. tobacco plants), for example by immersing the comminuted leaves or the minced leaves in an agrobacterium solution and then cultivating them in a suitable medium. Plant transformation with the aid of Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl.acid Res. (1988)16,9877 or is known, inter alia, from F.F.white, Vectors for Gene Transfer in Higher Plants, in Transgenic Plants, Vol.1, Engineering and Ultilization, edited by S.D.Kung and R.Wu, Academic Press,1993, pages 15 to 38.
Plant transformation by recombinant agrobacterium typically involves co-cultivation of agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissues, which carry antibiotic or herbicide resistance markers between the binary plasmid T-DNA borders, are regenerated on selection medium.
As previously discussed, another method for transforming plants, plant parts, and plant cells involves the advancement of inert or biologically active particles to plant tissues and cells. See, for example, U.S. Pat. nos. 4,945,050, 5,036,006, and 5,100,792. Generally, such methods involve propelling inert or biologically active particles into plant cells under conditions effective to penetrate the outer surface of the cell and provide for incorporation into the interior of the cell. When inert particles are used, the vector may be introduced into the cell by coating the particles with a vector comprising the nucleic acid of interest. Alternatively, the cell may be surrounded by the carrier such that the carrier is carried into the cell following the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria, or phage, each containing one or more nucleic acids sought to be introduced) can also be propelled into plant tissue.
In other embodiments, polynucleotides encoding the Cry proteins of the disclosure can be transformed directly into the plastid genome. The main advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without major modification, and plastids are capable of expressing multiple open reading frames under the control of a single promoter. Plastid transformation techniques are described extensively in U.S. Pat. Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT application No. WO 95/16783, and in McBride et al, (1994) Proc. Nati. Acad. Sci. USA 91, 7301-. The basic technique for chloroplast transformation involves introducing a region of cloned plastid DNA flanked by a selectable marker along with the gene of interest into a suitable target tissue, for example by using biolistic or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation). The 1 to 1.5kb flanking regions, called targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastid group (plastome). Initially, point mutations in the chloroplast 16SrRNA and rps12 genes conferring resistance to spectinomycin or streptomycin could be used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-. The presence of cloning sites between these markers allows the creation of plastid targeting vectors for the introduction of foreign genes (Staub, J.M. and Maliga, P. (1993) EMBO J.12, 601-606). A significant increase in transformation frequency can be obtained by replacing the recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker (bacterial aadA gene, which encodes the spectinomycin detoxification enzyme, aminoglycoside-3' -adenylyl transferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90,913 + 917). Previously, this marker has been successfully used for the high frequency transformation of the plastidic genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. acids Res.19: 4083-. Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present disclosure. Typically, about 15-20 cell division cycles are required after transformation to achieve a homogeneous state. Plastid expression, in which the gene is inserted by homologous recombination into the entire thousands of copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number of genes over nuclear expression, allowing expression levels that can easily exceed 10% of the total soluble plant protein. In one embodiment, a polynucleotide of the present disclosure may be inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Thus, plants of the same type as for the plastid genome comprising the nucleotide sequences of the present disclosure can be obtained, which are capable of high expression of the polynucleotides.
Methods of selecting transformed transgenic plants, plant cells, or plant tissue cultures are conventional in the art and may be used among the methods of the present disclosure provided herein. For example, the recombinant vectors of the present disclosure may also include an expression cassette comprising a nucleotide sequence for a selectable marker that can be used to select for transformed plants, plant parts, or plant cells. As used herein, a "selectable marker" means a nucleotide sequence which, when expressed, confers a different phenotype on a plant, plant part or plant cell expressing the marker and thus allows such transformed plants, plant parts or plant cells to be distinguished from those plants, plant parts or plant cells not having the marker. Such nucleotide sequences may encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, e.g., through the use of a selection agent (e.g., an antibiotic, herbicide, etc.), or whether the marker is simply a trait that can be identified by observation or testing, e.g., by screening (e.g., an R-locus trait). Of course, many examples of suitable selectable markers are known in the art and may be used among the expression cassettes described herein.
Examples of selectable markers include, but are not limited to: a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, etc. (Potrykus et al, (1985) mol.Gen.Genet.199: 183-188); a nucleotide sequence encoding bar which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase which confers resistance to glyphosate (Hinche et al, (1988) Biotech.6: 915-922); nucleotide sequences encoding nitrilases (e.g., bxn from Klebsiella odoriphaga (Klebsiella ozaenae)), which confer resistance to bromoxynil (Stalker et al, (1988) Science 242: 419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinones, sulfonylureas, or other ALS-inhibiting chemicals (european patent application No. 154204); a nucleotide sequence encoding dihydrofolate reductase (DHFR) resistant to methotrexate (Thillet et al (1988) J.biol.chem.263: 12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)) which confers the ability to metabolize mannose (U.S. Pat. nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph, which confers resistance to hygromycin. One skilled in the art will be able to select suitable selectable markers for use in the expression cassettes of the present disclosure.
Additional selectable markers include, but are not limited to: a nucleotide sequence encoding a β -glucuronidase, or uida (gus), which encodes an enzyme for which various chromogenic substrates are known; nucleotide sequences of the R-locus which encode products which modulate the production of anthocyanidin pigment (red) in plant tissue (Dellaporta et al, "Molecular cloning of the mail R-nj insulator by transloson-tagging with Ac" 263-282, in Chromosome Structure and Function: Impact of New Concepts, 18th Stacker Genetics Symposium (edited by Gustafson & applications, Plenum Press 1988)); nucleotide sequences encoding beta-lactamase, an enzyme for which various chromogenic substrates (e.g., PADAC, chromogenic cephalosporin) are known (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75: 3737-; a nucleotide sequence encoding xylE, which encodes catechol dioxygenase (Zukowsky et al, (1983) Proc. Natl. Acad. Sci. USA 80: 1101-1105); nucleotide sequences encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone (which then condenses to form melanin) (Katz et al, (1983) J.Gen.Microbiol.129: 2703-2714); a nucleotide sequence encoding beta-galactosidase, an enzyme for which a chromogenic substrate is present; nucleotide sequences encoding a luciferase (lux) that allows bioluminescent detection (Ow et al, (1986) Science 234: 856-859); nucleotide sequences encoding aequorin, which can be used in a calcium-sensitive bioluminescence assay (Prasher et al, (1985) biochem. Biophys. Res. Comm.126: 1259-1268); or a nucleotide sequence encoding a green fluorescent protein (Niedz et al, (1995) Plant Cell Reports 14: 403-. One skilled in the art will be able to select suitable selectable markers for use in the expression cassettes of the present disclosure.
In addition, as is well known in the art, whole transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts by using any of a variety of known techniques. Plant regeneration starting from Plant cells, Plant tissue Cultures or cultured protoplasts is described, for example, in Evans et al (Handbook of Plant Cell Cultures, Vol.1, MacMilan Publishing Co.New York (1983)); and Vasil I.R (eds.) (Cell Culture and social Cell Genetics of Plants, Acad.Press, Orlando, Vol.I (1984) and Vol.II (1986)).
In addition, the genetic traits described above that are engineered into the transgenic seeds and plants, plant parts, or plant cells of the present disclosure can be transmitted by sexual reproduction or vegetative growth, and thus can be maintained and propagated in progeny plants. Generally, maintenance and propagation utilize known agricultural methods developed to meet specific objectives (e.g., harvesting, sowing, or farming).
Thus, the polynucleotide may be introduced into the plant, plant part, or plant cell in any number of ways well known in the art (as described above). Thus, independent of the particular method used to introduce the polynucleotide or polynucleotides into the plant, any method that allows for stable integration of the polynucleotide or polynucleotides into the plant genome may be used. When more than one polynucleotide is to be introduced, the respective polynucleotides may be assembled as part of a single nucleic acid molecule, or as separate nucleic acid molecules, and may be located on the same or different nucleic acid molecules. Thus, the polynucleotides may be introduced into the target cells in a single transformation event, in separate transformation events, or as part of a breeding scheme, e.g., in a plant.
In some embodiments, the present disclosure provides a method of controlling a pest, the method comprising contacting the pest with a composition comprising a first insecticidal protein and a second pest control agent different from the first insecticidal protein, wherein the first insecticidal protein is a Cry protein comprising the amino acid sequence of any of SEQ ID NOs 1-3, wherein the pest is selected from the group consisting of cotton bollworm, oriental armyworm, athetis lepigone, spodoptera litura, black cutworm, peach borer, chilo suppressalis, spodoptera worm and asian corn borer. In other embodiments, the composition is a formulation for topical application to a plant. In yet other embodiments, the composition is a transgenic plant. In a further embodiment, the composition is a combination of agents topically applied to the transgenic plant. In some embodiments, when the transgenic plant comprises a second pest control agent, the formulation comprises a first Cry protein of the disclosure. In other embodiments, when the transgenic plant comprises the first Cry protein of the disclosure, the formulation comprises a second pest control agent.
In some embodiments, the second pest control agent may be an agent selected from the group consisting of: chemical pesticides (e.g., insecticides), bacillus thuringiensis (Bt) insecticidal proteins, xenorhabdus insecticidal proteins, photorhabdus insecticidal proteins, brevibacillus laterosporus insecticidal proteins, bacillus sphaericus insecticidal proteins, perforins, protease inhibitors (both serine and cysteine types), lectins, alpha-amylases, peroxidases, cholesterol oxidases, and double stranded rna (dsrna) molecules.
In other embodiments, the second pest control agent is a chemical pesticide selected from the group consisting of: pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrolides, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics. In other embodiments, the chemical pesticide is selected from the group consisting of: abamectin (abamectin), acephate (acephate), acetamiprid (acetamiprid), sulfadimidine (amidoflumethet) (S-1955), avermectin (avermectins), azadirachtin (azadirachtin), bayanphos (azinphos-methyl), bifenthrin (bifenthrin), bifenazate (bifenazate), buprofezin (buprofefazine), carbofuran (carbofuran), chlorfenapyr (chlorfenapyr), chlorfluazuron (chlorfluazuron), chlorpyrifos (chlorpyrifos), chlorpyrifos-methyl, chromafenozide (chlorofenthiflufen), cyhalothrin (cyhalothrin), cyfluthrin (cyhalothrin), cyhalothrin (lambda-cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin, cyhalothrin, Endosulfan (endosulfan), fenpropathrin (fenproparate), ethiprole (ethiprole), fenoxycarb (fenoxycarb), fenpropathrin (fenpropathrin), fenpyroximate (fenproximate), fenvalerate (fenvallate), fipronil (fipronil), flonicamid (flonicamid), flufenvalerate (fluythrinate), flucyanthrin (tau-fluvalinate), pyrimethanil (UR-50701), flufenoxuron (flufenoxuron), phosmet (fenofos), chlorfenapyr (halofenozide), flufenoxuron (hexuron), imidacloprid (imidacloprid), indoxacarb (indoxacarb), isofenphos (isophos), lufenuron (fenuron), fenthion (fenpyrazone), pyriproxyfen (imidacloprid), thion (fenpyrazofenozide (fenpyrazone), methoxyfenozide (metafenozide (XD) (methoxyfenozide), methoxyfenozide (methoate) (metafenozide), methoxyfenozide (methoxyfenozide) (XD) (methoxyfenozide), methoxyfenozide (methoxyfenozide), methoxyfenozide (methoxyfenozide), chlorpyrifos (methoxyfenozide), methoxyfenozide (methofenozide), methoxyfenozide (methoprene), chlorpyrifos (methofenozide), chlorpyrifos (methoprene), thiuron (methoprene), chlorpyrifos (methofenozide), thiuron (methoprene), thiuron (methyl-ethyl (methyl-ethyl), thiuron (methyl-ethyl), thiuron (methyl-ethyl (methyl-ethyl), thiuron (methyl-, Oxamyl, parathion-methyl, permethrin, phorate, phosmet, pyridalyl, pyriproxyfen, prothromofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spirotetramine, BSN 2060, thioprofenofos, tebufenozide, flubenzuron, tefluthrin, terbufos, tebufenthrin, thiacloprid, fenthiuron, fenproxyfen, thiacloprid, chlorpyrifos, thiacloprid, chlorpyrifos, thiacloprid, Cyhexatin (cyhexatin), dicofol (dicofol), dichloflufen (dienochlor), etoxazole (etoxazole), fenazaquin (fenazaquin), fenbutatin oxide (fenbutatin oxide), fenpropathrin, fenpyroximate, hexythiazox (hexythiazox), propargite (prodigite), pyridaben (pyridaben), and tebufenpyrad (tebufenpyrad). In yet other embodiments, the chemical pesticidal agent is selected from the group consisting of: cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tetrabromthrin, fenoxycarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, emamectin benzoate, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, bendiofen, pyriproxyfen, and amitraz.
In additional embodiments, the second pest control agent may be one or more of any number of bacillus thuringiensis insecticidal proteins, including but not limited to Cry proteins, Vegetative Insecticidal Proteins (VIPs), and insecticidal chimeras of any of the foregoing insecticidal proteins. In other embodiments, the second pest control agent is a Cry protein selected from the group consisting of: cry1, Cry2, Cry7, Cry8, Cry7, Cry8, Cry1, Cry2, Cry1, Cry2, Cry1, Cry2, Cry4, Cry5, Cry7, Cry2, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, and Cry73 Aa.
In further embodiments, the second pest control agent is a Vip3 vegetative insecticidal protein selected from the group consisting of: vip3Aa, Vip3Ab, Vip3Aa, Vip3Ab, Vip3 Bp 3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip3Ab, Vip 3.
In a still further embodiment, the first Cry protein of the disclosure and the second pest control agent are co-expressed in a transgenic plant. This co-expression of more than one pesticidally active ingredient in the same transgenic plant can be achieved by genetically engineering the plant to contain and express all the necessary genes. Alternatively, a plant "parental 1" can be genetically engineered to express a Cry protein of the disclosure. A second plant "parent 2" may be genetically engineered to express a second pest control agent. By crossing "parent 1" with "parent 2", progeny plants expressing all the genes introduced into "parent 1" and "parent 2" are obtained.
In a further embodiment, the present disclosure provides a method of producing a pest-resistant (e.g., insect-resistant) transgenic plant, the method comprising introducing into a plant a polynucleotide, chimeric gene, recombinant vector, expression cassette or nucleic acid molecule comprising a nucleotide sequence encoding a Cry protein of the disclosure, wherein said nucleotide sequence is expressed in said plant, thereby conferring resistance to said plant to a pest, and producing an insect-resistant transgenic plant, wherein said pest is selected from the group consisting of: cotton bollworm, oriental armyworm, athetis lepigone, prodenia litura, black cutworm, dichocrocis punctiferalis, chilo suppressalis, rice stem borer and Asiatic corn borer. In some embodiments, the black cutworm is from a population of black cutworms in china. In some embodiments, said introducing is effected by transforming said plant. In other embodiments, the introduction is achieved by crossing a first plant comprising a chimeric gene, recombinant vector, expression cassette, or nucleic acid molecule of the disclosure with a second, different plant.
In some embodiments, the present disclosure includes methods of providing a farmer with a means for controlling pests, the method comprising supplying or selling to the farmer plant material (e.g., seeds) comprising a polynucleotide, chimeric gene, expression cassette or recombinant vector capable of expressing a Cry protein of the invention in plants grown from the seeds, as described above, wherein the pests are selected from the group consisting of: cotton bollworm, oriental armyworm, athetis lepigone, prodenia litura, black cutworm, dichocrocis punctiferalis, chilo suppressalis, rice stem borer and Asiatic corn borer. In some embodiments, the black cutworm is from a population of black cutworms in china.
Embodiments of the invention may be better understood by reference to the following examples. The foregoing and following description of embodiments of the invention, as well as the various embodiments, is not intended to limit the claims, but is merely illustrative thereof. Therefore, it is to be understood that the claims are not to be limited to the specific details of these embodiments. Those skilled in the art will recognize that other embodiments of the invention can be practiced without departing from the spirit and scope of the present disclosure, which is defined by the appended claims.
Examples example 1 activity of Cry proteins against noctuidae, sodalidadae and snout moth's pests
BT-0032 and CryET72 proteins of table 1 were tested in an artificial diet bioassay against chinese populations of each of cotton bollworm, oriental armyworm, athetis lepigone, prodenia litura, black cutworm, dichocrocis punctiferalis, chilo suppressalis, rice stem moth, and asian corn borer. As shown in table 1, these Cry proteins have been previously documented.
TABLE 1 references to Cry protein disclosures
Cry proteins SEQ ID NO: Publication number
BT-0032 protein 1 WO2017007679
CryET72 protein 2 WO2001019859
Mutant BT-0032 proteins 3 WO2017007679
Essentially, equal amounts of protein in solution were applied to the surface of the artificial insect diet in the multi-well plate. After the diet surface was dried, 24 larvae were added to each well. The plates were sealed and maintained at ambient laboratory conditions (with respect to temperature, light and relative humidity). The positive control group consisted of larvae exposed to known active Cry proteins. The negative control group consisted of larvae exposed to insect diet treated with buffer solution only. Mortality was assessed after approximately 3-4 days. The experiment was repeated at least twice for each protein.
The results of the bioassay are shown in table 2, where "-" indicates 0% mortality, "+/-" indicates 1-9% mortality (this category also includes 0% mortality with strong inhibition of larval growth), "+" indicates 10-24% mortality, "+ +" indicates 25-74% mortality, and "+++" indicates 75-100% mortality. Also shown in table 2 are indications of the activity of the Cry proteins against four north american pest insect species in the family noctuidae, including black cutworm (Agrotis ipsilon), Spodoptera frugiperda (fall armyworm), corn earworm (Helicoverpa zea) and European corn borer (Ostrinia nubilalis). For these four insect species, the activity is simply indicated as "+" or "-", and no percent mortality is indicated based on published data. The cell labeled "nt" indicates that the protein has not been tested against the pest species or that no published information indicates that the Cry protein has been tested against the pest species.
TABLE 2 results of bioassays with Cry proteins
Figure BDA0002867281840000311
Example 2 expression and Activity of Cry proteins in maize plants
Transgenic maize plants are prepared using the Cry proteins of the disclosure. Transformation of immature maize embryos is performed essentially as described in Negrotto et al, 2000, Plant Cell Reports 19: 798-. Briefly, agrobacterium strain LBA4404(pSB1) was transformed with an expression vector comprising two expression cassettes, wherein the first expression cassette comprises a plant-expressible promoter operably linked to a Cry protein coding sequence operably linked to a terminator, and the second expression cassette comprises a plant-expressible promoter operably linked to a selectable marker operably linked to a terminator. Expression of the selectable marker allows transgenic plants to be identified on selective media. Both expression cassettes were cloned into appropriate vectors for agrobacterium-mediated maize transformation. The transformed Agrobacterium strain was grown for 2-4 days at 28 ℃ on YEP (yeast extract (5g/L), peptone (10g/L), NaCl (5g/L), 15g/L agar, pH 6.8) solid medium. Will be about 0.8 x 109The individual Agrobacterium cells were suspended in LS-inf medium supplemented with 100. mu.M As. The bacteria were pre-induced in this medium for approximately 30-60 minutes.
Immature embryos from inbred maize lines were excised from 8-12 day old ears into liquid LS-inf +100 μ M As. Embryos were rinsed once with fresh infection medium. Then, the agrobacterium solution was added and the embryos vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. Then, the embryos were transferred scutellum side up to LSAs medium and cultured in the dark for 2 to 3 days. Subsequently, approximately 20 to 25 embryos per culture dish were transferred to LSDc medium supplemented with cefotaxime (250mg/l) and silver nitrate (1.6mg/l) and cultured in the dark at approximately 28 ℃ for 10 days.
Immature embryos producing embryogenic callus were transferred to lsd1m0.5s medium. The cultures were selected on this medium for about 6 weeks with a subculture step performed at about 3 weeks. Surviving calli were transferred to Reg1 medium supplemented with mannose. After culturing under light (16 hour light/8 hour dark split), the green tissue was then transferred to Reg2 medium without growth regulators and incubated for approximately 1-2 weeks. The plantlets were transferred to a Magenta GA-7 box (Magenta Corp, Chicago Ill.) containing Reg3 medium and allowed to grow under light. After about 2-3 weeks, plants were tested by PCR for the presence of the selectable marker gene and the Bt cry gene. Positive plants from the PCR assay were transferred to the greenhouse for further evaluation.
Transgenic plants were evaluated for copy number (as determined by Taqman analysis), protein expression level (as determined by ELISA), and efficacy against insect species (in the bioassay of example 1). Specifically, plant tissue (leaves or silks) were excised from the single copy event (V3-V4 stage) and infested with neonatal larvae of the pest in example 1, followed by incubation at room temperature for 5 days.
Example 3 expression and Activity of Cry proteins in Rice plants
Transgenic rice plants are prepared using the Cry proteins of the disclosure. Methods for rice transformation are known in the art, for example, the protocols described by Hiei et al (Plant Journal 1994,6(2):271-282) and Zaidi et al (Mol Biotechnol 2009,43: 232-242).
Transgenic plants were evaluated for copy number (as determined by Taqman analysis), protein expression level (as determined by ELISA), and efficacy against insect species (in the bioassay of example 1). Specifically, plant tissue (leaves or tillers) were excised from the single copy event and infested with newborn larvae of the pest in example 1, followed by incubation at room temperature for 4 days.
The results of the transgenic plant tissue bioassays from example 2 and example 3 would confirm that the Cry proteins of the disclosure are toxic to the pests in example 1 when expressed in transgenic plants.
Example 4 expression and Activity of Cry proteins in Soybean plants
Transgenic soybean plants are made using the Cry proteins of the disclosure. Methods of soybean transformation are known in the art, for example, the experimental protocol set forth in U.S. patent publication No. US 2004034889. Binary vectors for soybean transformation were constructed with soybean suitable promoters that drive expression of the Cry proteins of the disclosure. Genes encoding the Cry proteins of the disclosure can be codon optimized for soybean expression based on the predicted amino acid sequences of their coding regions. Agrobacterium binary transformation vectors containing expression cassettes comprising Cry protein coding sequences are also constructed by adding transformation selectable marker genes. The selectable marker coding sequence may also be codon optimized for expression in soybean.
Transgenic plants were evaluated for copy number (as determined by Taqman analysis), protein expression level (as determined by ELISA), and efficacy against insect species (in the bioassay of example 1). Specifically, plant tissue (leaves) was excised from single copy events and infested with the neonatal larvae of example 1, followed by 4 days of incubation at room temperature.
The results of the transgenic plant tissue bioassay from example 4 will confirm that the Cry proteins of the disclosure are toxic to the pest in example 1 when expressed in the transgenic plant.
Sequence listing
<110> Xianzhengda Biotechnology (China) Co., Ltd
Syngenta Crop Protection JSC
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Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr
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Arg Asp Tyr Leu Arg Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys Ile
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Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe
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Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile
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Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn
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Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr
545 550 555 560
Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr Asn
565 570 575
Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile
580 585 590
Gly Asn Leu Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile Asn
595 600 605
Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Ile Met Asn Ile Met Leu
610 615 620
Val Pro Thr Asn Ile Ser Pro Leu Tyr
625 630

Claims (11)

1. A method of inhibiting the growth or killing a pest, comprising contacting the pest with a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Oriental armyworm (Mythimna separata, Oriental armyworm), Spodoptera litura (Common cutword/Oriental leaf), Chilo supress (Striped stee), Trichoplusia oryzae (Sesamia incerens, Pink stem borer), and Asian corn borer (Ostrinia furnacalis, Asian corn borer).
2. A method for controlling a pest population, said method comprising contacting said pest population with an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein said pest is selected from the group consisting of oriental armyworm, spodoptera litura, chilo suppressalis, spodoptera oryza incertulas, and ostrinia furnacalis.
3.The method of claim 1 or 2, wherein said pest or pest population is further contacted with a second insecticidal protein different from a Cry protein.
4. The method of claim 3 wherein said second insecticidal protein is selected from the group consisting of a second Cry protein, a Vip protein, a protease inhibitor, a lectin, an alpha-amylase, and a peroxidase.
5. The method of any one of claims 1 to 4, wherein the contacting step is performed with a microorganism or plant expressing the protein or insecticidal fragment thereof.
6. The method of claim 5, wherein said plant is stably transformed with a DNA sequence encoding said protein or insecticidal fragment thereof.
7. The method of claim 6, wherein the plant is a monocot or a dicot.
8. The method of claim 7, wherein the monocot plant is a maize plant or a rice plant.
9. The method of claim 7, wherein the dicot is a soybean plant.
10. A method for protecting a plant from a pest, said method comprising expressing in said plant or cell thereof an insecticidally effective amount of a Cry protein comprising an amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein said pest is selected from the group consisting of oriental armyworm, spodoptera litura, chilo suppressalis, phoma oryza incertulas, and ostrinia nubilalis.
11. A method for controlling a pest population, said method comprising contacting said pest population with an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs 1-3, or an insecticidal fragment thereof, wherein said pest is selected from the group consisting of oriental armyworm, spodoptera litura, chilo suppressalis, spodoptera oryza incertulas, and ostrinia furnacalis.
CN202011586496.7A 2020-12-29 2020-12-29 Control of noctuid, snout moth's larva and snout moth's larva harmful organism Pending CN114680127A (en)

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