WO2018090714A1 - 杀虫蛋白组合及其管理昆虫抗性的方法 - Google Patents

杀虫蛋白组合及其管理昆虫抗性的方法 Download PDF

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WO2018090714A1
WO2018090714A1 PCT/CN2017/102442 CN2017102442W WO2018090714A1 WO 2018090714 A1 WO2018090714 A1 WO 2018090714A1 CN 2017102442 W CN2017102442 W CN 2017102442W WO 2018090714 A1 WO2018090714 A1 WO 2018090714A1
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protein
cry2ab
corn borer
cry1ac
asian corn
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PCT/CN2017/102442
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English (en)
French (fr)
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陶青
杨旭
李建勇
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北京大北农科技集团股份有限公司
北京大北农生物技术有限公司
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Publication of WO2018090714A1 publication Critical patent/WO2018090714A1/zh

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    • 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
    • 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
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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

Definitions

  • the present invention relates to a pesticidal protein combination and a method for managing insect resistance, and in particular to a method for using the Cry1Ac protein and the Cry2Ab protein in combination to manage the resistance of Asian corn borer.
  • Ostracnia furnacalis commonly known as the borer, belongs to the family Lepidoptera, and is the main pest in corn production in China. It feeds on corn leaves and breaks into the main stem or ear of corn to reduce photosynthesis. Nutrient transportation can also lead to the production of various secondary diseases, resulting in reduced yield and quality of corn.
  • the damage of Asian corn borer has become more and more serious.
  • the object of the present invention is to provide a pesticidal protein combination and a method for managing the same, which not only firstly proposes a method for controlling resistant Asian corn borer through a combination of Cry2Ab protein and Cry1Ac protein, but also effectively delays the Asian corn borer to a single The protein produces resistance.
  • the present invention provides a method of managing insect resistance, the method comprising contacting the insect with at least a Cry2Ab protein and a Cry1Ac protein, wherein the insect comprises Asian corn borer.
  • the Cry2Ab protein and the Cry1Ac protein are present in a bacterium or a plant which produces at least the Cry2Ab protein and the Cry1Ac protein, the Asian corn mash by at least feeding the bacterium or the plant or a part thereof at least with the Cry2Ab protein Contact with the Cry1Ac protein, the Asian corn borer growth is inhibited and/or caused to death after exposure to achieve resistance to Asian corn borer.
  • the Asian corn borer is an Asian corn borer resistant to Cry1Ac protein
  • the Asian corn borer resistant to the Cry1Ac protein is at least contacted with the Cry2Ab protein and the Cry1Ac protein, the Cry2Ab protein and the Cry1Ac protein.
  • the Asian corn borer resistant to the Cry1Ac protein is at least associated with the Cry2Ab protein and the Cry1Ac protein by ingesting the bacterium or the plant or a part thereof Upon contact, the Asian corn borer resistant to the Cry1Ac protein is inhibited from growth and/or causes death after exposure to achieve resistance to the Asian corn borer which is resistant to the Cry1Ac protein.
  • the Asian corn borer is an Asian corn borer resistant to the Cry2Ab protein, and the Asian corn borer resistant to the Cry2Ab protein is at least contacted with the Cry2Ab protein and the Cry1Ac protein, the Cry2Ab protein and the Cry1Ac protein.
  • the Asian corn borer resistant to the Cry2Ab protein by ingesting the bacterium or the plant or a part thereof at least with the Cry2Ab protein and the Cry1Ac protein Upon contact, the Asian corn borer resistant to the Cry2Ab protein is inhibited from growth and/or causes death following exposure to achieve resistance to the Asian corn borer which is resistant to the Cry2Ab protein.
  • the plant is a transgenic plant and is in any growth period.
  • the plant part is a root, a leaf, a stem, a fruit, a tassel, an ear, an anther or a filament.
  • the management of resistance to Asian corn borer does not change due to changes in planting locations and/or planting times.
  • the plant is corn, wheat, sorghum, millet, rice, sunflower or soybean.
  • the Cry2Ab protein (1) has the amino acid sequence of SEQ ID NO: 1; (2) has one or several amino acid substitutions compared to the amino acid sequence shown by SEQ ID NO: Deletion, addition or insertion; or (3) having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 1.
  • polynucleotide encoding the Cry2Ab protein (1) has the nucleotide sequence shown in SEQ ID NO: 3; or (2) has the nucleotide sequence shown in SEQ ID NO: 3 At least 80% homology.
  • the Cry1Ac protein (1) has the amino acid sequence shown in SEQ ID NO: 2; (2) has one or several amino acid substitutions, deletions, and additions compared to the amino acid sequence shown in SEQ ID NO: Or insertion; or (3) having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 2.
  • polynucleotide encoding the Cry1Ac protein (1) has the nucleotide sequence shown in SEQ ID NO: 4; or (2) has the nucleotide sequence shown in SEQ ID NO: 4 At least 80% homology.
  • the present invention also provides a method of controlling Asian corn borer, which comprises contacting Asian corn borer with at least Cry2Ab protein and Cry1Ac protein to achieve control of Asian corn borer.
  • the Cry2Ab protein and the Cry1Ac protein are present in a bacterium or a plant which produces at least the Cry2Ab protein and the Cry1Ac protein, the Asian corn mash by at least feeding the bacterium or the plant or a part thereof at least with the Cry2Ab protein In contact with the Cry1Ac protein, the growth of the Asian corn borer is inhibited and/or caused to death after exposure to achieve control of the Asian corn borer hazard plant.
  • the Asian corn borer is an Asian corn borer resistant to Cry1Ac protein
  • the Asian corn borer resistant to the Cry1Ac protein is at least contacted with the Cry2Ab protein and the Cry1Ac protein, the Cry2Ab protein and the Cry1Ac protein.
  • the Asian corn borer resistant to the Cry1Ac protein is at least associated with the Cry2Ab protein and the Cry1Ac protein by ingesting the bacterium or the plant or a part thereof Upon contact, the growth of the Asian corn borer resistant to the Cry1Ac protein is inhibited and/or causes death after exposure to achieve control of the Asian corn borer-harming plant resistant to the Cry1Ac protein.
  • the Asian corn borer is an Asian corn borer resistant to the Cry2Ab protein, and the Asian corn borer resistant to the Cry2Ab protein is at least contacted with the Cry2Ab protein and the Cry1Ac protein, the Cry2Ab protein and the Cry1Ac protein.
  • the Asian corn borer resistant to the Cry2Ab protein by ingesting the bacterium or the plant or a part thereof at least with the Cry2Ab protein and the Cry1Ac protein Upon contact, the Asian corn borer resistant to the Cry2Ab protein is inhibited from growth and/or causes death after exposure to achieve control of the Asian corn borer hazard plant resistant to the Cry2Ab protein.
  • the plant is a transgenic plant and is in any growth period.
  • the plant part is a root, a leaf, a stem, a fruit, a tassel, an ear, an anther or a filament.
  • control of the Asian corn borer hazard plant does not change due to changes in plant planting locations and/or planting times.
  • the plant is corn, wheat, sorghum, millet, rice, sunflower or soybean.
  • the Cry2Ab protein (1) has the amino acid sequence of SEQ ID NO: 1; (2) has one or several amino acid substitutions compared to the amino acid sequence shown by SEQ ID NO: Deletion, addition or insertion; or (3) having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 1.
  • polynucleotide encoding the Cry2Ab protein (1) has the nucleotide sequence shown in SEQ ID NO: 3; or (2) has the nucleotide sequence shown in SEQ ID NO: 3 At least 80% homology.
  • the Cry1Ac protein (1) has the amino acid sequence shown in SEQ ID NO: 2; (2) has one or several amino acid substitutions, deletions, and additions compared to the amino acid sequence shown in SEQ ID NO: Or insertion; or (3) having at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 2.
  • polynucleotide encoding the Cry1Ac protein (1) has the nucleotide sequence shown in SEQ ID NO: 4; or (2) has the nucleotide sequence shown in SEQ ID NO: 4 At least 80% homology.
  • the present invention provides a use of a combination of a Cry2Ab protein and a Cry1Ac protein to prevent or delay the development of resistance of the Asian corn borer population to the Cry1Ac protein or the Cry2Ab protein.
  • the present invention also provides the use of a combination of a Cry2Ab protein and a CrylAc protein to control a population of Asian corn borer which is resistant to a Cry1Ac protein or a Cry2Ab protein.
  • the present invention also provides a use of a Cry2Ab protein in combination with a Cry1Ac protein for producing an insect-resistant transgenic plant.
  • Contact means that insects and/or pests touch, stay and/or ingest plants, plant organs, plant tissues or plant cells, and the plants, plant organs, plant tissues or plant cells can It is a pesticidal protein expressed in the body, and may also be a microorganism having a pesticidal protein on the surface of the plant, plant organ, plant tissue or plant cell and/or having a pesticidal protein.
  • Manage insect resistance” or “insect resistance management” as used in the present invention means to manage plants by using at least two toxins, preferably double toxins, in order to reduce the resistance of pests to insecticides (for example, Bt proteins).
  • the plant is preferably a transgenic Plants, more preferably transgenic crops, most preferably, the plants are insect-resistant transgenic crops or insecticidal transgenic crops.
  • control and/or “control” means that the Asian corn borer pest is in contact with at least the Cry2Ab protein and the Cry1Ac protein, and the growth of the Asian corn borer pest is inhibited and/or causes death after contact. Further, the Asian corn borer pest is at least contacted with the Cry2Ab protein and the Cry1Ac protein by feeding the plant tissue, and all or part of the Asian corn borer pest growth is inhibited and/or causes death after the contact.
  • “Inhibition” refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation.
  • plants and/or plant seeds controlling Asian corn borer pests containing a polynucleotide encoding a Cry2Ab protein and a Cry1Ac protein are subjected to artificial inoculation of Asian corn borer pests and/or Asian corn borer pests.
  • Transgenic wild-type plants have reduced plant damage compared to, but are not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like.
  • control and / or “control” effects of Cry2Ab and Cry1Ac on Asian corn borer can exist independently and are not attenuated by other substances that can "control” and / or “control” Asian corn borer pests. / or disappear.
  • any tissue of a transgenic plant (containing a polynucleotide encoding a Cry2Ab protein and a Cry1Ac protein) is present and/or asynchronously, present and/or produced, a Cry2Ab protein and a Cry1Ac protein, and optionally a controllable Asian corn borer
  • Another substance of the pest the presence of the other substance does not affect the "control” and / or “control” effects of the Cry2Ab protein and the Cry1Ac protein on Asian corn borer, nor does it lead to the "control” and / Or “control” is achieved completely and / or partially by the other substance, regardless of the Cry2Ab protein and the Cry1Ac protein.
  • Any tissue of a plant (containing a polynucleotide sequence encoding a Cry2Ab protein and a Cry1Ac protein) is present in dead Asian corn borer pests, and/or Asian corn borer pests on which growth inhibition is inhibited, and/or with non-transgenic wild
  • the plant has reduced plant damage compared to the method and/or use of the present invention, that is, the method and/or use of controlling the Asian corn borer pest by contacting the Asian corn borer pest with at least the Cry2Ab protein and the Cry1Ac protein.
  • plant includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps, and intact plants in plants or plant parts.
  • Cells such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like.
  • the genome of a plant, plant tissue or plant cell as referred to in the present invention refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • Recombination as used in the present invention means that it is usually not found in nature and is therefore produced by human intervention.
  • Such manual intervention can produce recombinant DNA molecules and/or recombinant plants.
  • the "recombinant DNA molecule” is obtained by artificially combining two sequence segments which are otherwise isolated, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulation are well known.
  • protoxin refers to the initial translation product of a full-length gene encoding a pesticidal protein prior to any cleavage in the midgut.
  • toxin or "insecticidal toxin” or “minimum toxic fragment” is understood to mean a part of a pesticidal protein, such as a Cry2Ab or Cry1Ac protein, which can be digested by trypsin or by (target insects such as Asian corn borer). It is obtained by hydrolysis in the midgut fluid and still has insecticidal activity.
  • the Cry1 toxin has a molecular weight of about 60-65 kD and the Cry2A toxin has a molecular weight of about 50-58 kD.
  • the Cry2Ab protein and the Cry1Ac protein are expressed in a transgenic plant. Co-expression of such more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant (the first parent) can express the Cry2Ab protein by genetic engineering
  • the second plant (second parent) can express the Cry1Ac protein by genetic engineering. Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • the transgenic plants producing the Cry1Ac protein include, but are not limited to, TT51 transgenic rice events and/or plant material comprising TT51 transgenic rice events (as described in CN100582223C and CN101302520B), 223F-S21 transgenic rice lines and / or plant material containing 223F-S21 transgenic rice lines (as described in CN103773759A), Mon15985 transgenic cotton events and/or plant material containing Mon15985 transgenic cotton events (as described in CN101413028B), Mon531 transgenic cotton events and / or plant material containing the Mon531 transgenic cotton event (as described in USDA APHIS Unregulated Status Application 00-342-01p), COT67B transgenic cotton event and/or plant material containing COT67B transgenic cotton event (eg in USDA APHIS non- Regulatory Status Application 07-108-01p) or 3006-210-23 GM cotton events and/or plant material containing 3006-210-23 GM cotton events (
  • the transgenic plants producing the Cry2Ab protein include, but are not limited to, the Mon89034 transgenic maize event and/or plant material comprising the Mon89034 transgenic maize event (as described in CN101495635A), the Mon87751 transgenic soybean event, and/or comprise Mon87751 Plant material for the transgenic soybean event (as described in USDA APHIS Unregulated Status Application 13-337-01p), or Mon15985 transgenic cotton event and/or plant material containing the Mon15985 transgenic cotton event (as described in CN101413028B).
  • the Mon89034 transgenic maize event and/or plant material comprising the Mon89034 transgenic maize event as described in CN101495635A
  • the Mon87751 transgenic soybean event and/or comprise Mon87751 Plant material for the transgenic soybean event (as described in USDA APHIS Unregulated Status Application 13-337-01p)
  • Mon15985 transgenic cotton event and/or plant material containing the Mon15985 transgenic cotton event as described in CN101413028
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • To express a protein in vivo one strand of DNA is typically transcribed into a complementary strand of mRNA that is used as a template to translate the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “coding” strand consists of a series of codons (codons are three nucleotides, three reads at a time to produce a specific amino acid), which can be read as an open reading frame (ORF) to form the protein of interest. Or peptide.
  • the invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • nucleic acid hybridization or amplification method can be used to identify the presence of the pesticidal gene of the present invention.
  • a nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances.
  • two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes.
  • the stringent conditions of the present invention may be specific hybridization with SEQ ID NO: 3 and SEQ ID NO: 4 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1 %SDS and 1 ⁇ SSC, 0.1% SDS each wash film 1 time.
  • sequences having insect resistance and hybridizing to SEQ ID NO: 3 or SEQ ID NO: 4 of the present invention under stringent conditions is included in the present invention.
  • These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93.
  • nucleotide sequence of the polynucleotide encoding the Cry2Ab protein is as shown in SEQ ID NO: 3
  • nucleotide sequence of the polynucleotide encoding the Cry1Ac protein is shown in SEQ ID NO: 4.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or end ratios compared to full length proteins). Deletions), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • the "variant” or “mutant” refers to a nucleotide sequence encoding the same protein or an equivalent protein encoding insecticidal activity.
  • the "equivalent protein” refers to a biologically active protein of the same or substantially the same anti-Lepidopteran insect pest (such as Asian corn borer) as the Cry2Ab protein and the Cry1Ac protein.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide sequence or amino acid sequence) involved or an artificially engineered form thereof (eg, suitable for plant expression)
  • the sequence of the foregoing sequence may vary in length, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A gene encoding an active fragment can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
  • the present invention may derive equivalent proteins and/or genes encoding these equivalent proteins from Bacillus thuringiensis (B.t.) isolates and/or DNA libraries.
  • Bacillus thuringiensis (B.t.) isolates and/or DNA libraries There are various methods for obtaining the pesticidal protein of the present invention.
  • antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from protein mixtures.
  • antibodies may be caused by protein portions that are most constant in protein and most different from other Bt proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies that specifically bind to the proteins disclosed in the present invention or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
  • substantially identical sequence refers to a sequence which has an amino acid substitution, deletion, addition or insertion but does not substantially affect the insecticidal activity, and also includes a fragment which retains insecticidal activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R. L.
  • amino acid residues necessary for their activity and thus selected for unsubstituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique introduces a mutation at each positively charged residue in the molecule, and detects the insecticidal activity of the resulting mutant molecule, thereby determining an amino acid residue important for the activity of the molecule.
  • the substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, eg, de Vos et al., 1992, Science 255). : 306-312; Smith et al, 1992, J. Mol. Biol 224: 899-904; Wlodaver et al, 1992, FEBS Letters 309: 59-64).
  • the amino acid sequence of the Cry2Ab protein of the present invention is shown in SEQ ID NO: 1
  • the amino acid sequence of the Cry1Ac protein of the present invention is shown in SEQ ID NO: 2.
  • an amino acid sequence having a certain homology with the amino acid sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 is also included in the present invention. These sequences are typically greater than 78%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99%, similar to the sequences of the present invention.
  • the invention may also be defined in terms of a more specific range of identity and/or similarity. Preferred polynucleotides and proteins.
  • sequences of the examples of the present invention are 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity.
  • cross-resistance means that a strain of an insect is also resistant to an agent or a class of agents that have never been used other than the selected agent due to the same resistance mechanism or a similar mechanism of action or a similar chemical structure.
  • the phenomenon in particular, “interaction resistance” in the present invention refers to a phenomenon in which resistance to an insecticidal protein or a class of insecticidal proteins which have never been used other than the selected insecticidal protein. Proteins selected for insect resistance management need to exert their insecticidal effects independently, so that resistance to one protein does not confer resistance to the second protein (ie no cross-resistance to the second protein) Or have lower cross resistance).
  • a population of insects resistant to "Protein A” is sensitive to "Protein B", one can conclude that Protein A and Protein B are not cross-resistant and that their combination is effective in delaying resistance to a single Protein A.
  • a population of insect species "produces resistance”, “has developed resistance” or “has been resistant” to insecticidal proteins and/or plants expressing insecticidal proteins that previously controlled or killed the insect population.
  • “Resistant” means that the control effect of the same insect species and/or the yield loss of the plant caused by the same insect species is compared to the level of loss of the same insect species when the insecticidal protein and/or the plant is first introduced, A dull and significant yield loss was observed to be mitigated by the insect population control effect and/or caused.
  • Asian corn borer (Ostrinia furnacalis) and cotton bollworm (HelicovercrumigeraHubner) belong to the same order Lepidoptera
  • Asian corn borer and cotton bollworm are biologically distinct and distinct species. At least the following main differences: 1) Asian corn The genus Aphididae, the cotton bollworm is a genus Noctuidae. 2) Different eating habits. Asian corn borer most commonly harms corn, which can damage various parts of the corn plant, including leaves, tassels, stalks, stalks, and cobs.
  • the morphological characteristics of eggs, larvae, pupa and adults of Asian corn borer and cotton bollworm are quite different. 5) Growth habits and occurrence patterns are different.
  • the occurrence algebra of corn borer varies significantly with latitude: In China, north latitude 45° north, 1st generation, 45°-40°2 generation, 40°-30°3 generation, 30°-25° 4 generation, 25°-20° 5-6 generation. The higher the altitude, the less algebra occurs. In Sichuan province, 2-4 generations occur in one year. The temperature is high, the altitude is low, and algebras are more common. Usually, the mature larvae are in corn stalks, cobs or sorghum, sunflower stalks.
  • a female moth can lay eggs 500-1000 in a lifetime. Up to 2,700 capsules, the eggs are mostly produced on the back of the leaves, and are also produced on the front, top core, petiole, tender stems or other plants such as weeds; weeds have hatch habits after hatching, and newly hatched larvae have The group has a restriction on eating habits. Two or three heads, three or five heads are on the front or back of the leaves, and the heads are arranged in the leaf margins and feed inward from the leaf margins. As a result, the leaves are eaten, leaving only the main veins and petioles, or netted withered.
  • the age of the insects is not neat; the most suitable temperature for the occurrence of cotton bollworm is 25-28 ° C, the relative humidity is 70-90%; the second and third generations are the most serious, serious insects
  • the density is 98 heads/louvers, the rate of insects is 60-70%, the individual plots are 100%, the damaged leaves are more than 1/3, the leaf yield is 20%, the quality is reduced by at least 1 grade, and the growth of seedlings has great influence.
  • Asian corn borer and cotton bollworm are of the same order Lepidoptera, there are many differences in morphological characteristics and harmful habits, and the relationship between them is far away, and they cannot be mated to produce offspring. Therefore, the receptors that bind to Bt toxin on the upper surface of the intestinal epithelial cell membrane are also different.
  • the resistance mechanism of insects to Bt protein is not single. Heckel (1994) analyzed the potential mechanism of insect resistance to Bt protein. It is believed that the production of resistance is mainly related to the following factors: 1) Solubility of Bt insecticidal protein : protoxin cannot dissolve or decrease solubility; 2) proteolyticity of Bt protoxin: insufficient hydrolysis or excessive hydrolysis; 3) Bt Binding of a protein to a receptor on a cell membrane: binding of a Bt protein to a receptor is blocked by competitive inhibition, and the modification of the primary structure or secondary structure of the receptor is altered, resulting in a decrease in the binding site of the Bt protein to the receptor.
  • the formation of cavities on the cell membrane is the formation or obstruction of the cavity; 5) the repair of the midgut epithelium; 6) the behavioral mechanism.
  • the change in the ability of the toxin to bind to receptors on the cell membrane is the primary resistance mechanism.
  • the Cry2Ab protein may have the amino acid sequence shown by SEQ ID NO: 1 in the Sequence Listing; the Cry1Ac protein may have the amino acid sequence shown by SEQ ID NO: 2 in the Sequence Listing.
  • other elements may be included, such as a coding region encoding a transit peptide or a protein encoding a selectable marker, and the nucleotide sequences of the Cry2Ab protein and the Cry1Ac protein provided by the present invention may be conventional. Means form a construct.
  • a construct comprising a Cry2Ab protein and a Cry1Ac protein of the invention may also be expressed in a plant together with at least one protein encoding a herbicide resistance gene including, but not limited to, glyphosate resistant Sex genes (such as bar gene, pat gene), benzoin resistance genes (such as pmph gene), glyphosate resistance genes (such as EPSPS gene), bromoxynil resistance gene, sulfonylurea resistance Gene, resistance gene to herbicide tortoise, resistance gene to cyanamide or glutamine synthetase inhibitor (such as PPT), thereby obtaining high insecticidal activity and herbicide resistance Sexual transgenic plants.
  • glyphosate resistant Sex genes such as bar gene, pat gene
  • benzoin resistance genes such as pmph gene
  • glyphosate resistance genes such as EPSPS gene
  • bromoxynil resistance gene sulfonylurea resistance Gene
  • resistance gene to herbicide tortoise resistance gene to cyanamide
  • the regulatory sequences of the invention include, but are not limited to, a promoter, a transit peptide, a terminator, an enhancer, a leader sequence, an intron, and other nucleotides operably linked to the Cry2Ab protein or the Cry1Ac protein.
  • the regulatory sequence of the sequence include, but are not limited to, a promoter, a transit peptide, a terminator, an enhancer, a leader sequence, an intron, and other nucleotides operably linked to the Cry2Ab protein or the Cry1Ac protein.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S derived from cauliflower mosaic virus Promoter, maize Ubi promoter, promoter of rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • Effectively linked or “operably linked” in the context of the invention denotes a linkage of a nucleic acid sequence which results in a A strip sequence provides the functionality needed for a connected sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • Insecticide or “insect-resistant” as used in the present invention means toxic to crop pests, thereby achieving "control” and/or “control” of crop pests.
  • said "insecticide” or “insect-resistant” means killing crop pests.
  • the target insect is an Asian corn borer pest.
  • the plant of the present invention contains exogenous DNA in its genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1Ac protein and a Cry2Ab protein, and the Asian corn borer pest by feeding plant tissue with Cry1Ac protein and Cry2Ab Protein contact, post-contact Asian corn borer pest growth is inhibited and / or cause death, while Asian corn borer will delay the production of resistance to Cry1Ac protein. Inhibition refers to death or sub-lethal death.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the plant substantially eliminates the need for chemical or biological insecticides.
  • a foreign DNA is introduced into a plant by a transformation method, such as a gene or an expression cassette or a recombinant vector encoding the Cry2Ab protein and/or the Cry1Ac protein, which is introduced into a plant cell.
  • a transformation method such as a gene or an expression cassette or a recombinant vector encoding the Cry2Ab protein and/or the Cry1Ac protein, which is introduced into a plant cell.
  • Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microprojection bombardment, direct uptake of DNA into protoplasts, electroporation or whisker silicon mediated DNA introduction.
  • the present invention provides a pesticidal protein combination and a method for managing insect resistance, which have the following advantages:
  • the invention enables the expression of Cry1Ac protein and Cry2Ab protein in plants, and only needs to be able to grow the transgenic plants without using other measures, thereby saving a lot of manpower, material resources and financial resources, and the effect is stable and thorough. .
  • Figure 1 is a schematic view showing the structure of a recombinant expression vector Cry2Ab-pET30 containing a Cry2Ab nucleotide sequence according to the insecticidal protein combination of the present invention and a method for managing insect resistance;
  • Figure 2 is a schematic diagram showing the corrected mortality of larvae of ACB-BtS and ACB-AcR fed Cry1Ac protein-treated artificial diet of the insecticidal protein combination of the present invention and its method for managing insect resistance;
  • Figure 3 is a schematic diagram showing the corrected mortality of larvae of ACB-BtS and ACB-AcR fed with Cry2Ab protein-treated artificial diet of the insecticidal protein combination of the present invention and its method for managing insect resistance;
  • Figure 4 is a schematic diagram showing the growth inhibition rate of larvae of ACB-BtS and ACB-AcR fed with Cry1Ac protein-treated artificial diet according to the insecticidal protein combination of the present invention and the method for managing insect resistance;
  • Figure 5 is a schematic diagram showing the growth inhibition rate of larvae of ACB-BtS and ACB-AcR fed with Cry2Ab protein-treated artificial diet of the insecticidal protein combination of the present invention and its method for managing insect resistance.
  • the amino acid sequence of the Cry2Ab insecticidal protein (634 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; the nucleotide sequence of the Cry2Ab insecticidal protein is optimized according to the preference of the E. coli codon, and the coding is obtained corresponding to The Cry2Ab nucleotide sequence (1905 nucleotides) of the amino acid sequence of the Cry2Ab insecticidal protein is shown as SEQ ID NO: 3 in the Sequence Listing.
  • the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 3 in the sequence listing) is from Nanjing Kingsray Biotechnology
  • the structure of the recombinant expression vector Cry2Ab-pET30 synthesized by the company is shown in Figure 1 (wherein, Kan represents the kanamycin resistance gene; f1 represents the origin of replication of phage f1; Cry2Ab is the Cry2Ab nucleotide) Sequence (SEQ ID NO: 3); Lac I represents an operon; ori represents an origin of replication).
  • the recombinant expression vector Cry2Ab-pET30 was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l of E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant) Expression vector Cry2Ab-pET30), water bath at 42 ° C for 30 seconds; shake culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl) 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH, and grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ l of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose).
  • the TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was dissolved and precipitated; the RNA was digested in a water bath at 37 ° C for 30 min; and stored at -20 ° C until use.
  • the Cry2Ab nucleotide sequence inserted into the recombinant expression vector Cry2Ab-pET30 was the nucleus represented by SEQ ID NO: 3 in the sequence listing.
  • the nucleotide sequence, the Cry2Ab nucleotide sequence, was correctly inserted.
  • the correctly sequenced recombinant expression vector Cry2Ab-pET30 was transformed into E. coli expression host BL21 (DE3) (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).
  • the specific transformation method was as follows: 50 ⁇ L of melted BL21(DE3) competent state on ice Cells, add plasmid DNA (recombinant expression vector Cry2Ab-pET30) and gently mix, let stand on ice for 25min; water bath at temperature 42 ° C for 90s, then quickly put back on ice and let stand for 2min; add to the centrifuge tube 800 ⁇ L of antibiotic-free LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, adjusted to pH 7.5 with NaOH), mixed and resuscitated at 37 ° C, 150 rpm for 60 min.
  • antibiotic-free LB liquid medium tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/
  • the cells were collected, and about 100 ⁇ L of the supernatant was gently pipetted, resuspended, and coated on an LB plate containing 50 ⁇ g/mL kanamycin (trypeptin 10 g/L, yeast extract 5 g/L). It was grown overnight on NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH. After obtaining the expression strain, the expression was induced according to the following procedure:
  • Step 121 Pick a positive monoclonal containing the recombinant expression vector Cry2Ab-pET30 in 5 mL of LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, kanamycin 50 ⁇ g/ mL, adjusted to pH 7.5 with NaOH, shake culture at 37 ° C, so that the OD 600 value reaches 0.5-0.6;
  • Step 122 taking 0.5 mL of the bacterial liquid of the above step 121 and centrifuging at a rotational speed of 12000 rpm for 10 min, respectively taking the supernatant and the precipitate as negative controls for uninduced expression;
  • Step 123 adding isopropyl thiogalactoside (IPTG) to the remaining 4.5 mL of the bacterial solution of the above step 121 to a final concentration of 1 mM, and continuing to induce culture for 20 hours at a rotation speed of 150 rpm and a temperature of 16 ° C;
  • IPTG isopropyl thiogalactoside
  • Step 124 after the induced bacterial solution was centrifuged at a rotational speed of 12000 rpm for 10 min, the cells were collected and suspended by adding 10 mM Tris (pH 8.0);
  • Step 125 ultrasonically disrupt the suspended cells, centrifuge at a rotational speed of 12000 rpm for 10 min, separately collect the supernatant and precipitate and perform SDS-PAGE protein electrophoresis detection (refer to "Protein Electrophoresis Experimental Technology (Second Edition)", edited by Guo Yujun, Science Press, 2005)
  • SDS-PAGE protein electrophoresis detection Refer to "Protein Electrophoresis Experimental Technology (Second Edition)", edited by Guo Yujun, Science Press, 2005
  • the expression of Cry2Ab protein showed that Cry2Ab protein expression (72KDa) was found in both supernatant and pellet.
  • the Cry2Ab protein was expressed in a large amount according to the above steps 121-125, and the collected crude protein was purified by a nickel column (His-Trap, FFGE Healthcare).
  • the specific steps are as follows:
  • Step 131 the cells collected in the above step 124 are dissolved in binding buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 20 mM imidazole, pH 8.0), and then ultrasonically disrupted on ice, and centrifuged at a rotational speed of 12000 rpm for 30 min. After collecting the supernatant, the supernatant was 0.45uM filter to remove impurities;
  • binding buffer 50 mM NaH 2 PO 4 , 500 mM NaCl, 20 mM imidazole, pH 8.0
  • Step 132 equilibrating the nickel column with the binding buffer, eluting 5 column volumes, the flow rate is 2 mL / min;
  • Step 133 the filtered supernatant is loaded, the flow rate is 0.5 mL / min;
  • Step 134 re-eluting 5 column volumes with the binding buffer, the flow rate is 2 mL / min, so that the Cry2Ab protein is fully hung on the column;
  • Step 135, continue to rinse with the binding buffer, wash away the protein, the flow rate is 2mL / min;
  • Step 136 using a elution buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 500 mM imidazole, pH 8.0) for gradient elution (in order: 10% concentration of the elution buffer eluted 3 column volumes)
  • the elution buffer with a concentration percentage of 20% eluted 3 column volumes, 40% of the elution buffer eluted with 3 column volumes, and the concentration percentage was 100% of the elution buffer.
  • the liquid was eluted with 5 column volumes, the flow rate was 2 mL/min, and the elution peak and eluate were collected separately. After purification by SDS-PAGE protein electrophoresis, the purified Cry2Ab protein was obtained.
  • the purified Cry2Ab protein was dissolved in 50 mM sodium carbonate buffer (pH 10.0); trypsin was formulated into an aqueous solution having a concentration of 1 mg/mL, and the ratio of 100:1 (Cry2Ab protein: trypsin) was used to Cry2Ab.
  • the trypsin solution was added to the protein; after mixing, it was digested at a temperature of 37 ° C for 1-3 hours. SDS-PAGE protein electrophoresis was performed on the activated Cry2Ab protein, and the results showed that the Cry2Ab protein was obtained after digestion.
  • the Cry1Ac protein used in the following experiment was purchased from Beijing Le Shining Technology Co., Ltd.
  • the test insects were Bt-sensitive strains of Asian corn borer (Ostrinia furnacalis) (hereinafter, ACB-BtS represents Asian corn borer Bt-sensitive strain) and Asian corn borer (Ostrinia furnacalis) Bt-resistant strain (hereinafter as ACB- AcR represents the Asian corn borer Bt resistant strain), and the insects of the above two strains are all from the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences.
  • ACB-BtS represents Asian corn borer Bt-sensitive strain
  • ACB- AcR represents the Asian corn borer Bt resistant strain
  • Bioassays were performed using different concentrations for each Bt protein, and the concentration of the Cry1Ac protein was determined to range from 0 ⁇ g/g to 50 ⁇ g/g, and the concentration of the Cry2Ab protein was determined to range from 0 ⁇ g/g to 100 ⁇ g/g.
  • the Bt protein solution is prepared by mixing Cry1Ac protein with distilled water (or 20 mM Tris-HCl buffer, pH 8.0) in a certain ratio to a concentration of 0 ⁇ g/g, 0.01 ⁇ g/g, 0.05 ⁇ g/g, 0.1, respectively.
  • Cry1Ac protein solution of ⁇ g/g, 0.5 ⁇ g/g, 1 ⁇ g/g, 5 ⁇ g/g, 10 ⁇ g/g, and 50 ⁇ g/g.
  • the Cry2Ab protein was mixed with distilled water (or 20 mM Tris-HCl buffer, pH 8.0) in a certain ratio to prepare concentrations of 0 ⁇ g/g, 0.01 ⁇ g/g, 0.05 ⁇ g/g, 0.1 ⁇ g/g, and 0.5 ⁇ g/g, respectively. 1 ⁇ g/g, 5 ⁇ g/g, 10 ⁇ g/g, 50 ⁇ g/g, and 100 ⁇ g/g of Cry2Ab protein solution.
  • Bt protein solution Cry1Ac protein solution or Cry2Ab protein solution
  • artificial feed artificial feed from the Institute of Plant Protection of Chinese Academy of Agricultural Sciences
  • Larval mortality is manifested by "actual mortality” (hereinafter referred to as mortality), and mortality is calculated taking into account actual dead larvae and surviving larvae that do not show a significant increase in body weight ( ⁇ 0.1 mg/larva).
  • the LC 50 value calculated based on ACB-BtS-based larval mortality was 0.21 ⁇ g/g; the LC 50 value calculated based on ACB-AcR-based larval mortality was greater than 1000 ⁇ g/g, thus ACB-BtS
  • the difference in LC 50 value from ACB-AcR is extremely significant, and the difference in resistance to Cry1Ac protein is also extremely significant, that is, Cry1Ac protein has extremely weak insecticidal activity against ACB-AcR.
  • the larval mortality of ACB-BtS and ACB-AcR fed the Cry2Ab protein-treated mixed diet increased with increasing Cry2Ab protein concentration, and the larval mortality of ACB-BtS was higher than ACB-AcR at the same concentration.
  • Table 1 and Figure 3 the LC 50 value calculated based on ACB-BtS-based larval mortality was 1.23 ⁇ g/g; the LC 50 value calculated based on ACB-AcR larval mortality was 6.71 ⁇ g/g, thus ACB-BtS 50 and the difference value LC ACB-AcR is significant, but for the ACB-BtS Cry2Ab protein and ACB-AcR have pesticidal activity.
  • the abscissa is the logarithm of the concentration of Bt protein used ( ⁇ g/g), the ordinate is the growth inhibition rate of the corresponding Asian corn borer, and the ACB-BtS and ACB-AcR fed the Cry1Ac protein-treated mixed feed.
  • Larval growth The inhibition rate increased with the increase of Cry1Ac protein concentration, and the growth inhibition rate of ACB-BtS larvae was significantly higher than that of ACB-AcR at the same concentration; the concentration of Cry1Ac protein required for ACB-BtS reached 70% growth inhibition rate.
  • the concentration of Cry1Ac protein required for ACB-AcR was about 500 ⁇ g/g, indicating that the Cry1Ac protein has weak inhibitory activity against ACB-AcR.
  • the larval growth inhibition rates of ACB-BtS and ACB-AcR fed Cry2Ab protein-treated mixed feed increased with increasing Cry2Ab protein concentration, and larval growth inhibition of ACB-BtS at the same concentration.
  • the rate is higher than ACB-AcR; when the 100% growth inhibition rate is reached, the concentration of Cry2Ab protein required for ACB-BtS is about 1 ⁇ g/g, and the concentration of Cry2Ab protein required for ACB-AcR is about 5 ⁇ g/g, indicating that Cry2Ab protein is for ACB.
  • -BtS and ACB-AcR have comparable inhibitory activities.
  • Table 1 Toxicity results of Bt protein against ACB-BtS and ACB-AcR newborn larvae
  • ACB-AcR has greater than 5000-fold resistance to Cry1Ac protein (ie, resistance ratio is greater than 5000), and Cry2Ab protein has insecticidal activity against both ACB-BtS and ACB-AcR, so ACB-AcR shows Cry2Ab
  • the combination of Cry2Ab protein and Cry1Ac protein can delay or delay the resistance of Asian corn borer to Cry1Ac protein, and Cry2Ab protein can effectively manage the Cry1Ac protein of Asian corn borer species. The resulting resistance.
  • the Cry2Ab protein and the Cry1Ac protein are expected to be used in combination expression in a transgenic plant such as a corn plant to delay or prevent the resistance of Asian corn borer to the plant.
  • the first method is sequential transformation: wherein a plant into which a first gene (for example, a Cry1Ac gene) has been transformed is retransformed to introduce a second gene (for example, a Cry2Ab gene).
  • This sequential transformation preferably uses two different selectable marker genes, such as a kanamycin resistance gene and a phosphinothricin acetyltransferase gene (e.g., the pat or bar gene) that confers resistance to the glufosinate herbicide.
  • the second method is a co-transformation method: a nucleotide sequence encoding a Cry1Ac protein is expressed in a plant together with a nucleotide sequence encoding a Cry2Ab protein, and the entire selection includes two selection genes by using a selection marker linked to the respective gene. plant.
  • the third method is an independent transformation event, in which each of the two insecticidal protein genes are individually transferred into the genome of a different plant, which can then be combined in a separate plant by hybridization, and can be selected using DNA labeling techniques. These different genetic plants.
  • the present invention effectively utilizes the two insecticidal proteins Cry1Ac protein and Cry2Ab protein to delay or prevent the resistance of Asian corn borer, thereby achieving control or control of Asian corn borer, and maximizing plant protection.
  • Stable yield at the same time, the invention allows Cry1Ac protein and Cry2Ab protein to be expressed in plants, and only needs to be able to grow the transgenic plant without using other measures, thereby saving a lot of manpower, material resources and financial resources, and the effect is stable, thorough.

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Abstract

一种杀虫蛋白组合及其管理昆虫抗性的方法，所述管理昆虫抗性的方法包括将亚洲玉米螟至少与Cry2Ab蛋白和Cry1Ac蛋白接触。通过利用两种杀虫蛋白Cry1Ac蛋白和Cry2Ab蛋白有效延缓或防止亚洲玉米螟产生抗性，从而实现对亚洲玉米螟的控制或防治，使植物更大限度的获得保护并稳定产量。

Description

杀虫蛋白组合及其管理昆虫抗性的方法 技术领域
本发明涉及一种杀虫蛋白组合及其管理昆虫抗性的方法，特别是涉及组合使用Cry1Ac蛋白和Cry2Ab蛋白以管理亚洲玉米螟抗性的方法。
背景技术
昆虫害虫使全球农作物生产遭受着巨大的经济损失，并且农民每年都会面临由于虫害造成产量损失的威胁。亚洲玉米螟(Ostrinia furnacalis)俗名钻心虫，属鳞翅目螟蛾科，是我国玉米生产中的主要害虫，该类昆虫取食玉米叶片，蛀入玉米主茎或果穗内，降低光合作用，影响养分运输，还会导致各种次级病害的产生，致使玉米减产降质。近年来，随着气候条件的变化、耕作制度的改变、玉米种植密度的加大、肥水条件的提高，亚洲玉米螟的危害日益加重。
通过转化Bt(苏云金芽孢杆菌，Bacillus thuringiensis)杀虫蛋白基因产生昆虫抗性植物的能力给现代农业带来了革命，并提高了杀虫蛋白及其基因的重要性和价值。已有数种Bt蛋白用于产生昆虫抗性的转基因植物中，包括Cry1Ab蛋白、Cry1Ac蛋白、Cry1F蛋白、Cry2Ab蛋白、Cry3Bb蛋白和Vip3A蛋白等。然而随着转基因作物的推广应用，昆虫在持续的选择压力下将进化出针对转基因植物中表达的Bt蛋白的抗性，这样的抗性一旦产生并且不能被有效控制的话，无疑将限制含有Bt蛋白的转基因作物品种的商业价值。只有实施合理的抗性管理策略，才能使这一现代化技术成果得以持久的利用。
生产上为了减少昆虫抗性的产生主要采用以下几种抗性管理策略：
1)田间设置庇护所。设置庇护所的目的在于保持一定比例非抗性的等位基因，以延缓耐受高剂量抗虫蛋白的昆虫的产生，但此种策略会使得农民在实际操作上较为繁琐，并且在一定程度上降低总产量。
2)提高单一抗虫蛋白的使用剂量，此种策略可持续性较差，昆虫在持续的选择压力下将对单一蛋白产生更高的抗性。
3)不同抗虫蛋白交替使用或共使用。鉴于Bt蛋白特异地结合敏感昆虫的受体，因此叠加使用抗虫蛋白需评估昆虫对不同抗虫蛋白是否存在交互抗性，即是否共享或竞争结合位点，具有较高的不确定性，故至今尚无关于Cry2Ab蛋白针对亚洲玉米螟与Cry1Ac蛋白是否具有交互抗性的相关报道。
发明概述
本发明的目的是提供一种杀虫蛋白组合及其管理昆虫抗性的方法，不仅首次提出了通过Cry2Ab蛋白和Cry1Ac蛋白组合控制抗性亚洲玉米螟的方法，而且有效延缓了亚洲玉米螟对单一蛋白产生抗性。
为实现上述目的，本发明提供了一种管理昆虫抗性的方法，所述方法包括将所述昆虫至少与Cry2Ab蛋白和Cry1Ac蛋白接触，其中所述昆虫包括亚洲玉米螟。
进一步地，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理亚洲玉米螟的抗性。
更进一步地，所述亚洲玉米螟为对Cry1Ac蛋白产生抗性的亚洲玉米螟，将对Cry1Ac蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry1Ac蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry1Ac蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理所述对Cry1Ac蛋白产生抗性的亚洲玉米螟的抗性。
再进一步地，所述亚洲玉米螟为对Cry2Ab蛋白产生抗性的亚洲玉米螟，将对Cry2Ab蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry2Ab蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry2Ab蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理所述对Cry2Ab蛋白产生抗性的亚洲玉米螟的抗性。
在上述技术方案中，所述植物为转基因植物，处于任意生育期。
进一步地，所述植物部分为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
更进一步地，所述对亚洲玉米螟抗性的管理不因植物种植地点和/或种植时间的改变而改变。
可选择地，所述植物为玉米、小麦、高粱、谷子、水稻、向日葵或大豆。
在上述技术方案中，所述Cry2Ab蛋白：(1)具有SEQ ID NO:1所示的氨基酸序列；(2)与SEQ ID NO:1所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或(3)与SEQ ID NO:1所示的氨基酸序列相比具有至少80％以上的同源性。
进一步地，编码所述Cry2Ab蛋白的多核苷酸：(1)具有SEQ ID NO:3所示的核苷酸序列；或(2)与SEQ ID NO:3所示的核苷酸序列相比具有至少80％以上的同源性。
优选地，所述Cry1Ac蛋白：(1)具有SEQ ID NO:2所示的氨基酸序列；(2)与SEQ ID NO:2所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或(3)与SEQ ID NO:2所示的氨基酸序列相比具有至少80％以上的同源性。
进一步地，编码所述Cry1Ac蛋白的多核苷酸：(1)具有SEQ ID NO:4所示的核苷酸序列；或(2)与SEQ ID NO:4所示的核苷酸序列相比具有至少80％以上的同源性。
为实现上述目的，本发明还提供了一种控制亚洲玉米螟的方法，所述方法包括将亚洲玉米螟至少与Cry2Ab蛋白和Cry1Ac蛋白接触，从而实现对亚洲玉米螟的控制。
进一步地，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述亚洲玉米螟生长受到抑制和/或导致死亡，以实现对亚洲玉米螟危害植物的控制。
更进一步地，所述亚洲玉米螟为对Cry1Ac蛋白产生抗性的亚洲玉米螟，将对Cry1Ac蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry1Ac蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry1Ac蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现对所述对Cry1Ac蛋白产生抗性的亚洲玉米螟危害植物的控制。
再进一步地，所述亚洲玉米螟为对Cry2Ab蛋白产生抗性的亚洲玉米螟，将对Cry2Ab蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry2Ab蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry2Ab蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现对所述对Cry2Ab蛋白产生抗性的亚洲玉米螟危害植物的控制。
在上述技术方案中，所述植物为转基因植物，处于任意生育期。
进一步地，所述植物部分为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
优选地，所述对亚洲玉米螟危害植物的控制不因植物种植地点和/或种植时间的改变而改变。
可选择地，所述植物为玉米、小麦、高粱、谷子、水稻、向日葵或大豆。
在上述技术方案中，所述Cry2Ab蛋白：(1)具有SEQ ID NO:1所示的氨基酸序列；(2)与SEQ ID NO:1所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或(3)与SEQ ID NO:1所示的氨基酸序列相比具有至少80％以上的同源性。
进一步地，编码所述Cry2Ab蛋白的多核苷酸：(1)具有SEQ ID NO:3所示的核苷酸序列；或(2)与SEQ ID NO:3所示的核苷酸序列相比具有至少80％以上的同源性。
优选地，所述Cry1Ac蛋白：(1)具有SEQ ID NO:2所示的氨基酸序列；(2)与SEQ ID NO:2所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或(3)与SEQ ID NO:2所示的氨基酸序列相比具有至少80％以上的同源性。
进一步地，编码所述Cry1Ac蛋白的多核苷酸：(1)具有SEQ ID NO:4所示的核苷酸序列；或(2)与SEQ ID NO:4所示的核苷酸序列相比具有至少80％以上的同源性。
为实现上述目的，本发明提供了一种Cry2Ab蛋白和Cry1Ac蛋白组合使用以防止或延缓亚洲玉米螟群体对Cry1Ac蛋白或Cry2Ab蛋白产生抗性的用途。
为实现上述目的，本发明还提供了一种Cry2Ab蛋白和Cry1Ac蛋白组合使用以控制对Cry1Ac蛋白或Cry2Ab蛋白产生抗性的亚洲玉米螟群体的用途。
为实现上述目的，本发明还提供了一种Cry2Ab蛋白与Cry1Ac蛋白组合在生产抗虫转基因植物中的用途。
发明详述
定义
本发明中所述的“接触”，是指昆虫和/或害虫触碰、停留和/或摄食植物、植物器官、植物组织或植物细胞，所述植物、植物器官、植物组织或植物细胞既可以是其体内表达杀虫蛋白，还可以是所述植物、植物器官、植物组织或植物细胞的表面具有杀虫蛋白和/或具有产生杀虫蛋白的微生物。
本发明所述的“管理昆虫抗性”或“昆虫抗性管理”是指为了减轻害虫对杀虫剂(例如Bt蛋白)产生抗性，而采用至少两种毒素、优选双毒素管理植物，所述植物优选为转基因 植物，更优选转基因作物，最优选地，所述植物为抗虫转基因作物或杀虫转基因作物。
本发明所述的“控制”和/或“防治”是指亚洲玉米螟害虫至少与Cry2Ab蛋白和Cry1Ac蛋白接触，接触后亚洲玉米螟害虫生长受到抑制和/或导致死亡。进一步地，亚洲玉米螟害虫通过摄食植物组织至少与Cry2Ab蛋白和Cry1Ac蛋白接触，接触后全部或部分亚洲玉米螟害虫生长受到抑制和/或导致死亡。“抑制”是指亚致死，即尚未致死但能引起生长发育、行为、生理、生化和组织等方面的某种效应，如生长发育缓慢和/或停止。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，含有编码Cry2Ab蛋白和Cry1Ac蛋白的多核苷酸的控制亚洲玉米螟害虫的植物和/或植物种子，在人工接种亚洲玉米螟害虫和/或亚洲玉米螟害虫自然发生危害的条件下，与非转基因的野生型植株相比具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量、和/或增产等。Cry2Ab蛋白和Cry1Ac蛋白对亚洲玉米螟的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”亚洲玉米螟害虫的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码Cry2Ab蛋白和Cry1Ac蛋白的多核苷酸)的任何组织同时和/或不同步地，存在和/或产生，Cry2Ab蛋白和Cry1Ac蛋白、以及任选地可控制亚洲玉米螟害虫的另一种物质，则所述另一种物质的存在既不影响Cry2Ab蛋白和Cry1Ac蛋白对亚洲玉米螟的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全和/或部分由所述另一种物质实现，而与Cry2Ab蛋白和Cry1Ac蛋白无关。通常情况下，在大田，亚洲玉米螟害虫摄食植物组织的过程短暂且很难用肉眼观察到，因此，在人工接种亚洲玉米螟害虫和/或亚洲玉米螟害虫自然发生危害的条件下，如转基因植物(含有编码Cry2Ab蛋白和Cry1Ac蛋白的多核苷酸序列)的任何组织存在死亡的亚洲玉米螟害虫、和/或在其上停留生长受到抑制的亚洲玉米螟害虫、和/或与非转基因的野生型植株相比具有减弱的植物损伤，即为实现了本发明的方法和/或用途，即通过亚洲玉米螟害虫至少与Cry2Ab蛋白和Cry1Ac蛋白接触以实现控制亚洲玉米螟害虫的方法和/或用途。
术语“植物”包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物丛(plant clumps)和植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎秆、根、根尖、花药等。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
本发明中所述的“重组”是指通常不能在自然界中发现并且因此通过人工干预产 生的DNA和/或蛋白和/或生物体的形式。这种人工干预可产生重组DNA分子和/或重组植物。所述“重组DNA分子”是通过人工组合两种在其它情况下是分离的序列区段而获得的，例如通过化学合成或通过遗传工程技术操作分离的核酸区段。进行核酸操作的技术是众所周知的。
本发明术语“原毒素”指在中肠中发生任何断裂之前编码杀虫蛋白的全长基因的最初翻译产物。本发明术语“毒素”或“杀虫毒素”或“最小毒性片段”应理解为杀虫蛋白例如Cry2Ab或Cry1Ac蛋白的部分，其可以通过胰蛋白酶消化或通过在(目标昆虫，例如亚洲玉米螟)中肠液中水解而获得、且仍具有杀昆虫活性。通常在SDS-PAGE凝胶上，Cry1毒素具有约60-65kD的分子量，Cry2A毒素具有约50-58kD的分子量。
转基因植物
在本发明中，Cry2Ab蛋白和Cry1Ac蛋白在一种转基因植物中表达。这种超过一种的杀虫毒素在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达Cry2Ab蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达Cry1Ac蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
在本发明中，产生所述Cry1Ac蛋白的转基因植物包括但不限于TT51转基因水稻事件和/或包含TT51转基因水稻事件的植物材料(如在CN100582223C和CN101302520B所描述的)、223F-S21转基因水稻品系和/或包含223F-S21转基因水稻品系的植物材料(如在CN103773759A所描述的)、Mon15985转基因棉花事件和/或包含Mon15985转基因棉花事件的植物材料(如在CN101413028B所描述的)、Mon531转基因棉花事件和/或包含Mon531转基因棉花事件的植物材料(如在USDA APHIS非管制状态申请00-342-01p所描述的)、COT67B转基因棉花事件和/或包含COT67B转基因棉花事件的植物材料(如在USDA APHIS非管制状态申请07-108-01p所描述的)或者3006-210-23转基因棉花事件和/或包含3006-210-23转基因棉花事件的植物材料(如在USDA APHIS非管制状态申请03-036-02p所描述的)。
在本发明中，产生所述Cry2Ab蛋白的转基因植物包括但不限于Mon89034转基因玉米事件和/或包含Mon89034转基因玉米事件的植物材料(如在CN101495635A所描述的)、Mon87751转基因大豆事件和/或包含Mon87751转基因大豆事件的植物材料(如在USDA APHIS非管制状态申请13-337-01p所描述的)、或者Mon15985转基因棉花事件和/或包含Mon15985转基因棉花事件的植物材料(如在CN101413028B所描述的)。
DNA和蛋白质
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链由一系列密码子组成(密码子是三个核苷酸，一次读取三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA。
任何常规的核酸杂交或扩增方法都可以用于鉴定本发明杀虫基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:3和SEQ ID NO:4发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜 1次。
因此，具有抗虫活性并在严格条件下与本发明SEQ ID NO:3或SEQ ID NO:4杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。优选地，编码Cry2Ab蛋白的多核苷酸的核苷酸序列如SEQ ID NO:3所示，编码Cry1Ac蛋白的多核苷酸的核苷酸序列如SEQ ID NO:4所示。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的杀虫活性特征的部分和/片段(包括与全长蛋白质相比在内部和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“突变体”是指编码同一蛋白或编码有杀虫活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与Cry2Ab蛋白和Cry1Ac蛋白具有相同或基本相同的抗鳞翅目昆虫害虫(如亚洲玉米螟)的生物活性的蛋白。
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸序列或氨基酸序列)的一部分或其人工改造形式(例如适合植物表达的序列)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为昆虫毒素。
使用标准技术可以修饰基因和容易地构建基因变异体。例如，本领域熟知制造点突变的技术。又例如美国专利号5605793描述了在随机断裂后使用DNA重装配产生其它分子多样性的方法。可以使用商业化核酸内切酶制造全长基因的片段，并且可以按照标准程序使用核酸外切酶。例如，可以使用酶诸如Bal31或定点诱变从这些基因的末端系统地切除核苷酸。还可以使用多种限制性内切酶获取编码活性片段的基因。可以使用蛋白酶直接获得这些毒素的活性片段。
本发明可以从苏云金芽孢杆菌(B.t.)分离物和/或DNA文库衍生出等价蛋白和/或编码这些等价蛋白的基因。有多种方法获取本发明的杀虫蛋白。例如，可以使用本发明公开和要求保护的杀虫蛋白的抗体从蛋白质混合物鉴定和分离其它蛋白。特别地，抗体可能是由蛋白最恒定和与其它Bt蛋白最不同的蛋白部分引起的。然后可以通过免疫沉淀、酶联免疫吸附测定(ELISA)或western印迹方法使用这些抗体专一地鉴定有特征活性的等价蛋白。可使用本领域标准程序容易地制备特异性结合本发明中公开的蛋白或等价蛋白或这类蛋白的片段的抗体。然后可以从微生物中获得编码这些蛋白的基因。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生 这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或插入但实质上不影响杀虫活性的序列，亦包括保留杀虫活性的片段。
本发明中氨基酸序列的取代、缺失或添加是本领域的常规技术，优选这种氨基酸变化为：小的特性改变，即不显著影响蛋白的折叠和/或活性的保守氨基酸取代；小的缺失，通常约1-30个氨基酸的缺失；小的氨基或羧基端延伸，例如氨基端延伸一个甲硫氨酸残基；小的连接肽，例如约20-25个残基长。
保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。通常不改变特定活性的那些氨基酸取代在本领域内是众所周知的，并且已由，例如，N.Neurath和R.L.Hill在1979年纽约学术出版社(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn，Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的抗虫活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol 224:899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。
优选地，本发明的Cry2Ab蛋白的氨基酸序列如SEQ ID NO:1所示，本发明的Cry1Ac蛋白的氨基酸序列如SEQ ID NO:2所示。此外，与SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于78％，优选的大于85％，更优选的大于90％，甚至更优选的大于95％，并且可以大于99％。也可以根据更特定的相同性和/或类似性范围定义本发明的 优选的多核苷酸和蛋白质。例如与本发明示例的序列有78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
昆虫抗性
本发明术语“交互抗性”是指昆虫的一个品系由于相同抗性机理或相似作用机理或类似化学结构，对于选择药剂以外的其它从未使用过的一种药剂或一类药剂也产生抗药性的现象。特别的，本发明中“交互抗性”指对选择的杀虫蛋白以外的其它从未使用过的一种杀虫蛋白或一类杀虫蛋白也产生抗性的现象。被选择用于昆虫抗性管理的蛋白质需要独立地发挥其杀虫效果，从而对一种蛋白质产生的抗性不会赋予对第二种蛋白质的抗性(即对于第二种蛋白质无交叉抗性或有较低的交叉抗性)。例如对“蛋白A”有抗性的昆虫群体对“蛋白B”敏感，则人们可以得出结论，蛋白A和蛋白B无交叉抗性并且它们的组合可有效延迟对单一蛋白A的抗性。
本发明术语“抗性比例”是指同一种杀虫蛋白在昆虫的Bt抗性群体相对Bt敏感群体的LC50值(半数致死浓度)提高的倍数，即抗性比例＝Bt抗性株LC50值/Bt敏感株LC50值。
本发明中所使用的，昆虫物种群体对杀虫蛋白和/或表达杀虫蛋白植物(该植物以前控制或杀死所述昆虫群体)“产生抗性”、“已经产生抗性”或“已成为抗性的”是指，与首次引入该杀虫蛋白和/或该植物时对相同昆虫物种造成的控制效果和/或由相同昆虫物种造成的该植物的产量损失水平相比，在该植物中检测到由该昆虫群体控制效果减弱和/或造成的重复的且显著的产量损失。
亚洲玉米螟
亚洲玉米螟(Ostriniafurnacalis)与棉铃虫(HelicoverpaarmigeraHubner)虽然同属鳞翅目，但是亚洲玉米螟与棉铃虫在生物学上是清晰的、截然不同的两个物种，至少存在以下主要区别：1)亚洲玉米螟属螟蛾科，棉铃虫属夜蛾科。2)食性不同。亚洲玉米螟最常危害玉米，可危害玉米植株地上的各个部位，包括叶片、雄穗、茎秆、穗柄、穗轴，对各地的春、夏、秋播玉米都有不同程度危害，尤以夏播玉米最重；棉铃虫是危害棉花蕾铃期的重要钻蛀性害虫，主要蛀食蕾、花、铃，也取食嫩叶。3)分布区域不同。亚洲玉米螟分布在中国东部及西南主要玉米、高粱产区；而棉铃虫广泛分布在中国及世界各地，中国棉区和蔬菜种植区均有发生，黄河流域棉区、长江流域棉区受害较重；近年来，新疆棉区也时有发生。4)形态特征不同。亚洲玉米螟与棉铃虫的卵、幼虫、蛹、成虫的形态特征均有较大差异。5)生长习性和发生规律不同。玉米螟的发生代数随纬度而有显著的差异： 在中国，北纬45°以北1代，45°-40°2代，40°-30°3代，30°-25°4代，25°-20°5-6代。海拔越高，发生代数越少；在四川省一年发生2-4代，温度高、海拔低，发生代数较多，通常以老熟幼虫在玉米茎秆、穗轴内或高粱、向日葵的秸秆中越冬，次年4-5月化蛹，蛹经过10天左右羽化。成虫夜间活动，飞翔力强，有趋光性，寿命5～10天，喜欢在离地50厘米以上、生长较茂盛的玉米叶背面中脉两侧产卵，一个雌蛾可产卵350-700粒，卵期3-5天；玉米螟适合在高温、高湿条件下发育，冬季气温较高，天敌寄生量少，有利于玉米螟的繁殖，危害较重；卵期干旱，玉米叶片卷曲，卵块易从叶背面脱落而死亡，危害较轻。而棉铃虫发生的代数因年份因地区而异，在山东省莱州市每年发生4代，九月下旬成长幼虫陆续下树入土，在苗木附近或杂草下5-10cm深的土中化蛹越冬；立春气温回升15℃以上时开始羽化，4月下旬至5月上旬为羽化盛期，成虫出现第一代在6月中下旬，第二代在7月中下旬，第三代在8月中下旬至9月上旬至10月上旬尚有棉铃虫出现，成虫有趋光性，羽化后即在夜间闪配产卵，卵散产，较分散，一头雌蛾一生可产卵500-1000粒，最高可达2700粒，卵多产在叶背面，也有产在正面、顶芯、叶柄、嫩茎上或农作的、杂草等其它植物上；幼虫孵化后有取食卵壳习性，初孵幼虫有群集限食习性，二三头、三五头在叶片正面或背面，头向叶缘排列、自叶缘向内取食，结果叶片被吃光，只剩主脉和叶柄，或成网状枯萎，造成干叶；1-2龄幼虫沿柄下行至银杏苗顶芽处自一侧蛀食或沿顶芽处下蛀入嫩枝，造成顶梢或顶部簇生叶死亡，危害十分严重；3龄前的幼虫食量较少，较集中，随着幼虫生长而逐渐分散，进入4龄食量大增，可食光叶片，只剩叶柄；幼虫7-8月份为害最盛；棉铃虫有转移危害的习性，一只幼虫可危害多株苗木；各龄幼虫均有食掉蜕下旧皮留头壳的习性，给鉴别虫龄造成一定困难，虫龄不整齐；棉铃虫发生的最适宜温度为25-28℃，相对湿度70-90％；第二代、第三代为害最为严重，严重地片虫口密度达98头/百叶，虫株率60-70％，个别地片达100％，受害叶片达1/3以上，影响叶产量20％，质量下降至少1个等级，苗木生长量影响很大。
综合上述，亚洲玉米螟与棉铃虫虽然同属鳞翅目，但是仅在形态特征和为害习性上就存在诸多方面的不同，且二者亲缘关系较远，无法交配产生后代。因此，二者中肠上皮细胞膜上表面上与Bt毒素结合的受体也是不同的。
昆虫对Bt蛋白的抗性机制
昆虫对Bt蛋白的抗性机制不是单一的，Heckel(1994)等分析了昆虫对Bt蛋白产生抗性的潜在机理，认为抗性的产生主要和以下因素有关：1)Bt杀虫蛋白的溶解性：原毒素不能溶解或溶解性降低；2)Bt原毒素的蛋白水解性：水解不充分或过度水解；3)Bt 蛋白与细胞膜上受体的结合：Bt蛋白与受体的结合由于竞争性抑制而受阻、受体的一级结构或二级结构的修饰作用发生改变，导致Bt蛋白与受体的结合位点减少；4)细胞膜上空洞的形成：空洞形成受阻或阻塞；5)中肠上皮的修复作用；6)行为机理等。其中毒素与细胞膜上受体结合能力的改变是主要抗性机理。
当两种或以上不同的Bt蛋白在昆虫中共享结合位点时，它们不能够提供用于昆虫抗性管理目的的良好组合。实际情况下，针对不同的昆虫采取何种抗性管理策略具有较高的不确定性。氨基酸序列差异较大的两个Bt蛋白也可能在某一昆虫物种中以高亲和力结合共同的结合位点，例如Cry1Ab和Cry1F蛋白在菜蛾(Plutella xylostella)中。而且，已发现在一个昆虫物种中不具有共享的结合位点的两种蛋白可以在另一个昆虫物种中共享结合位点，例如Fiuza等人(1996)发现Cry1Ac和Cry1Ba蛋白在二化螟(Chilo suppressalis)中共享结合位点，而Ballester等人(1999)发现以上两种蛋白在菜蛾中结合不同的结合位点。
构建体
本发明中，所述Cry2Ab蛋白可以具有序列表中SEQ ID NO:1所示的氨基酸序列；所述Cry1Ac蛋白可以具有序列表中SEQ ID NO:2所示的氨基酸序列。除了包含Cry2Ab蛋白和Cry1Ac蛋白的编码区外，也可包含其他元件，例如编码转运肽的编码区或编码选择性标记的蛋白质，本发明提供的Cry2Ab蛋白和Cry1Ac蛋白的核苷酸序列可以通过常规手段形成构建体。
此外，包含编码本发明Cry2Ab蛋白和Cry1Ac蛋白的构建体在植物中还可以与至少一种编码除草剂抗性基因的蛋白质一起表达，所述除草剂抗性基因包括但不限于，草胺膦抗性基因(如bar基因、pat基因)、苯敌草抗性基因(如pmph基因)、草甘膦抗性基因(如EPSPS基因)、溴苯腈(bromoxynil)抗性基因、磺酰脲抗性基因、对除草剂茅草枯的抗性基因、对氨腈的抗性基因或谷氨酰胺合成酶抑制剂(如PPT)的抗性基因，从而获得既具有高杀虫活性、又具有除草剂抗性的转基因植物。
本发明中所述调控序列包括但不限于启动子、转运肽、终止子、增强子、前导序列、内含子以及其它可操作地连接到编码所述Cry2Ab蛋白或所述Cry1Ac蛋白的核苷酸序列的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S 启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pin Ⅰ和pin Ⅱ)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pin Ⅱ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”或“可操作地连接”表示核酸序列的联结，所述联结使得一 条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明中所述的“杀虫”或“抗虫”是指对农作物害虫是有毒的，从而实现“控制”和/或“防治”农作物害虫。优选地，所述“杀虫”或“抗虫”是指杀死农作物害虫。更具体地，目标昆虫是亚洲玉米螟害虫。
本发明中的植物，特别是玉米，在其基因组中含有外源DNA，所述外源DNA包含编码Cry1Ac蛋白和Cry2Ab蛋白的核苷酸序列，亚洲玉米螟害虫通过摄食植物组织与Cry1Ac蛋白和Cry2Ab蛋白接触，接触后亚洲玉米螟害虫生长受到抑制和/或导致死亡，同时亚洲玉米螟会延缓产生对于Cry1Ac蛋白的抗性。抑制是指致死或亚致死。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，该植物可基本消除对化学或生物杀虫剂的需要。
本发明中，通过转化方法，将外源DNA导入植物，如将编码所述Cry2Ab蛋白和/或所述Cry1Ac蛋白的基因或表达盒或重组载体导入植物细胞。常规的转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。
本发明提供了一种杀虫蛋白组合及其管理昆虫抗性的方法，具有以下优点：
1、延缓抗性。昆虫在持续的选择压力下，会对单一Bt蛋白产生抗性，本发明通过利 用两种杀虫蛋白Cry1Ac蛋白和Cry2Ab蛋白有效延缓或防止亚洲玉米螟产生抗性，从而实现对亚洲玉米螟的控制或防治。
2、有效控制抗性害虫。针对已经对Cry1Ac蛋白产生抗性的亚洲玉米螟施用Cry2Ab蛋白可有效控制抗性亚洲玉米螟为害植物，从而使植物更大限度地获得保护并稳定产量。
3、本发明是使Cry1Ac蛋白和Cry2Ab蛋白在植物体内进行表达，且只需能够种植该转基因植物即可，而不需要采用其它措施，从而节省了大量人力、物力和财力，同时效果稳定、彻底。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图简述
图1为本发明杀虫蛋白组合及其管理昆虫抗性的方法的含有Cry2Ab核苷酸序列的重组表达载体Cry2Ab-pET30的结构示意图；
图2为本发明杀虫蛋白组合及其管理昆虫抗性的方法的饲喂Cry1Ac蛋白处理的人工饲料的ACB-BtS和ACB-AcR的幼虫校正死亡率示意图；
图3为本发明杀虫蛋白组合及其管理昆虫抗性的方法的饲喂Cry2Ab蛋白处理的人工饲料的ACB-BtS和ACB-AcR的幼虫校正死亡率示意图；
图4为本发明杀虫蛋白组合及其管理昆虫抗性的方法的饲喂Cry1Ac蛋白处理的人工饲料的ACB-BtS和ACB-AcR的幼虫生长抑制率示意图；
图5为本发明杀虫蛋白组合及其管理昆虫抗性的方法的饲喂Cry2Ab蛋白处理的人工饲料的ACB-BtS和ACB-AcR的幼虫生长抑制率示意图。
具体实施方式
下面通过具体实施例进一步说明本发明杀虫蛋白组合及其管理昆虫抗性的方法的技术方案。
第一实施例、Cry2Ab蛋白的表达、纯化及活化
1、含有Cry2Ab核苷酸序列的重组表达载体的构建
Cry2Ab杀虫蛋白的氨基酸序列(634个氨基酸)，如序列表中SEQ ID NO:1所示；根据大肠杆菌密码子的偏好性对Cry2Ab杀虫蛋白的核苷酸序列进行优化，获得编码相应于所述Cry2Ab杀虫蛋白的氨基酸序列的Cry2Ab核苷酸序列(1905个核苷酸)，如序列表中SEQ ID NO:3所示。
所述Cry2Ab核苷酸序列(如序列表中SEQ ID NO:3所示)由南京金斯瑞生物科技 有限公司合成，且由其构建的重组表达载体Cry2Ab-pET30的结构示意图如图1所示(其中，Kan表示卡那霉素抗性基因；f1表示噬菌体f1的复制起点；Cry2Ab为Cry2Ab核苷酸序列(SEQ ID NO:3)；Lac I表示操纵子；ori表示复制起点)。
然后将重组表达载体Cry2Ab-pET30用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组表达载体Cry2Ab-pET30)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100mg/L)的LB平板(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl、10mM EDTA(乙二胺四乙酸)、50mM葡萄糖，pH8.0)悬浮；向管中加入150μl新配制的溶液II(0.2M NaOH、1％SDS(十二烷基硫酸钠))，颠倒4次，混合，置冰上3-5min；加入150μl冰冷的溶液III(4M醋酸钾、2M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μl含RNase(20μg/ml)的TE(10mM Tris-HCl，1mM EDTA，pH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。
提取的质粒经KpnI和HindIII酶切鉴定后，对阳性克隆进行测序验证，结果表明重组表达载体Cry2Ab-pET30中插入的所述Cry2Ab核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即Cry2Ab核苷酸序列正确插入。
2、Cry2Ab蛋白的表达
将测序正确的重组表达载体Cry2Ab-pET30转化到大肠杆菌表达宿主BL21(DE3)(购自北京天根生化科技有限公司)中，具体转化方法为：取50μL冰上融化的BL21(DE3)感受态细胞，加入质粒DNA(重组表达载体Cry2Ab-pET30)并轻轻混匀，于冰上静置25min；于温度42℃下水浴90s，然后迅速放回冰上并静置2min；向离心管中加入800μL不含抗生素的LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L，用NaOH调pH至7.5)，混匀后在温度37℃、转速150rpm条件下复苏60min；接着在转速4000 rpm条件下离心1min，收集菌体，留取100μL左右上清轻轻吹打后重悬并涂布在含有50μg/mL卡那霉素的LB平板(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、琼脂15g/L，用NaOH调pH至7.5)上生长过夜。获得表达菌株后，按照下述步骤进行诱导表达：
步骤121、挑取一个含有重组表达载体Cry2Ab-pET30的阳性单克隆，在5mL的LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、卡那霉素50μg/mL，用NaOH调pH至7.5)中于温度37℃条件下震荡培养，使其OD600值达到0.5-0.6；
步骤122、取0.5mL上述步骤121的菌液于转速12000rpm条件下离心10min，分别取上清和沉淀作为未诱导表达的阴性对照；
步骤123、在剩余的4.5mL上述步骤121的菌液中加入异丙基硫代半乳糖苷(IPTG)至终浓度为1mM，于转速150rpm、温度16℃条件下继续诱导培养20h；
步骤124、将继续诱导后的菌液于转速12000rpm条件下离心10min后收集菌体，并加入10mM Tris(pH 8.0)悬浮；
步骤125、超声波破碎悬浮后的菌体，于转速12000rpm条件下离心10min，分别收集上清和沉淀并进行SDS-PAGE蛋白电泳检测(参考《蛋白质电泳实验技术(第二版)》，郭尧君编著，科学出版社出版，2005)Cry2Ab蛋白的表达，检测结果表明：上清和沉淀中均有Cry2Ab蛋白表达(72KDa)。
3、Cry2Ab蛋白的纯化
根据上述步骤121-125大量诱导表达Cry2Ab蛋白，将收集到的粗蛋白过镍柱纯化(His-Trap，FFGE Healthcare)，具体步骤如下：
步骤131、将上述步骤124中收集的菌体溶于结合缓冲液(50mM NaH2PO4、500mM NaCl、20mM咪唑，pH 8.0)中，再置于冰上超声破碎，于转速12000rpm条件下离心30min后收集上清，将上清过0.45uM滤膜除杂；
步骤132、用所述结合缓冲液平衡镍柱，洗脱5个柱体积，流速为2mL/min；
步骤133、将过滤后的上清上样，流速为0.5mL/min；
步骤134、用所述结合缓冲液再洗脱5个柱体积，流速为2mL/min，使Cry2Ab蛋白充分挂在柱上；
步骤135、用所述结合缓冲液继续冲洗，洗去杂蛋白，流速为2mL/min；
步骤136、用洗脱缓冲液(50mM NaH2PO4、500mM NaCl、500mM咪唑，pH 8.0)进行梯度洗脱(依次为：浓度百分比为10％的所述洗脱缓冲液洗脱3个柱体积，浓度百 分比为20％的所述洗脱缓冲液洗脱3个柱体积，浓度百分比为40％的所述洗脱缓冲液洗脱3个柱体积，浓度百分比为100％的所述洗脱缓冲液洗脱5个柱体积)，流速为2mL/min，分别收集洗脱峰和洗脱液，经过SDS-PAGE蛋白电泳检测后获得纯化后的Cry2Ab蛋白。
4、Cry2Ab蛋白的活化
将上述纯化后的Cry2Ab蛋白溶于50mM碳酸钠缓冲液(pH 10.0)中；将胰蛋白酶配制成浓度为1mg/mL的水溶液，并按100:1(Cry2Ab蛋白:胰蛋白酶)的质量比向Cry2Ab蛋白中加入胰蛋白酶水溶液；混匀后在温度37℃下消化1-3小时。对活化后的Cry2Ab蛋白进行SDS-PAGE蛋白电泳检测，结果表明获得了酶切活化后的Cry2Ab蛋白。
用于下述实验的Cry1Ac蛋白购买自北京乐士宁科技有限公司。
第二实施例、Bt敏感型亚洲玉米螟株系及Cry1Ac蛋白亚洲玉米螟抗性株系的生物测定
1、供试昆虫来源
供试昆虫为亚洲玉米螟(Ostrinia furnacalis)的Bt敏感株系(下文以ACB-BtS代表亚洲玉米螟Bt敏感株系)和亚洲玉米螟(Ostrinia furnacalis)的Bt抗性株系(下文以ACB-AcR代表亚洲玉米螟Bt抗性株系)，上述两种株系的昆虫均来自于中国农业科学院植物保护研究所。
2、亚洲玉米螟生物测定方法
针对每种Bt蛋白使用不同浓度进行生物测定，测定Cry1Ac蛋白的浓度范围为0μg/g至50μg/g，测定Cry2Ab蛋白的浓度范围为0μg/g至100μg/g。Bt蛋白溶液的制备方式为：将Cry1Ac蛋白与蒸馏水(或20mM Tris-HCl缓冲液，pH8.0)按照一定比例混合制成浓度分别为0μg/g、0.01μg/g、0.05μg/g、0.1μg/g、0.5μg/g、1μg/g、5μg/g、10μg/g和50μg/g的Cry1Ac蛋白溶液。将Cry2Ab蛋白与蒸馏水(或20mM Tris-HCl缓冲液，pH8.0)按照一定比例混合制成浓度分别为0μg/g、0.01μg/g、0.05μg/g、0.1μg/g、0.5μg/g、1μg/g、5μg/g、10μg/g、50μg/g和100μg/g的Cry2Ab蛋白溶液。将不同浓度的Bt蛋白溶液(Cry1Ac蛋白溶液或Cry2Ab蛋白溶液)与人工饲料(人工饲料来自于中国农业科学院植物保护研究所)按质量比为1.5:1分别混合成混合饲料，将混合饲料分散放置于48孔的生物测定板中，每个孔中放入约0.5g混合饲料，将与Bt蛋白溶液(Cry1Ac蛋白溶液或Cry2Ab蛋白溶液)等量的蒸馏水(或20mM Tris-HCl缓冲液，pH8.0)处理的人工饲料作为空白对照。在生物测定板每个孔的混合饲料表面上接种1只亚洲玉米螟的新生幼 虫(存活<24h)，用封口膜覆盖孔。将生物测定板在温度28℃、相对湿度80％、光周期(光/暗)16h:8h的条件下放置6天，接种后第7天开始记录幼虫死亡率和幼虫生长抑制率。昆虫株系与Bt蛋白溶液(Cry1Ac蛋白溶液或Cry2Ab蛋白溶液)浓度的组合每组重复3次，每个重复包括48只幼虫。
幼虫死亡率以“实际死亡率”(下文简称为死亡率)体现，计算死亡率需考虑实际死亡的幼虫和不显示体重显著增加(<0.1mg/幼虫)的存活幼虫。亚洲玉米螟的死亡率用如下的公式计算：死亡率(％)＝100×[死亡幼虫数目+不显示体重显著增加(<0.1mg/幼虫)的存活幼虫数目]/测试昆虫的总数。每个幼虫的死亡率根据饲喂空白对照的幼虫死亡率进行校正。然后对经过校正的剂量和死亡率数据进行机率分析，应用POLO统计软件确定导致50％死亡率的Bt蛋白溶液(Cry1Ac蛋白溶液或Cry2Ab蛋白溶液)浓度(LC50)和相应的95％置信区间。通过将ACB-AcR的LC50值除以ACB-BtS的LC50值计算抗性比例。
饲喂混合饲料的亚洲玉米螟的幼虫生长抑制率用以下公式计算：幼虫生长抑制(％)＝100×(用空白对照饲喂的幼虫体重-用混合饲料饲喂的幼虫体重)/(用空白对照饲喂的幼虫体重)。如果没有体重显著增加(<0.1mg/幼虫)的幼虫，则指定该重复为100％的幼虫生长抑制。生长抑制数据用双向ANOVA进行分析，以昆虫株系和Bt蛋白溶液浓度作为两个主要因子。使用LSMEANS检验确定α＝0.05水平的处理差异。
3、饲喂混合饲料的昆虫株系的幼虫死亡率及生长抑制率测定结果
饲喂Cry1Ac蛋白处理的混合饲料的ACB-BtS和ACB-AcR的幼虫死亡率均随着Cry1Ac蛋白浓度的增加而提高，且在同一浓度下ACB-BtS的幼虫死亡率显著高于ACB-AcR。如表1和图2所示，基于ACB-BtS的幼虫死亡率计算的LC50值为0.21μg/g；基于ACB-AcR的幼虫死亡率计算的LC50值大于1000μg/g，因此ACB-BtS和ACB-AcR的LC50值的差异是极显著的，两者对于Cry1Ac蛋白的抗性差异也是极显著的，即Cry1Ac蛋白对于ACB-AcR具有极弱的杀虫活性。
饲喂Cry2Ab蛋白处理的混合饲料的ACB-BtS和ACB-AcR的幼虫死亡率均随着Cry2Ab蛋白浓度的增加而提高，且在同一浓度下ACB-BtS的幼虫死亡率高于ACB-AcR。如表1和图3所示，基于ACB-BtS的幼虫死亡率计算的LC50值为1.23μg/g；基于ACB-AcR的幼虫死亡率计算的LC50值6.71μg/g，因此ACB-BtS和ACB-AcR的LC50值的差异是显著的，但Cry2Ab蛋白对于ACB-BtS和ACB-AcR均具有杀虫活性。
如图4所示，横坐标为所用Bt蛋白浓度(μg/g)的对数值，纵坐标为对应亚洲玉米螟的生长抑制率，饲喂Cry1Ac蛋白处理的混合饲料的ACB-BtS和ACB-AcR的幼虫生长 抑制率均随着Cry1Ac蛋白浓度的增加而提高，且在同一浓度下ACB-BtS的幼虫生长抑制率显著高于ACB-AcR；达到70％生长抑制率时，ACB-BtS所需Cry1Ac蛋白的浓度为约0.1μg/g，ACB-AcR所需Cry1Ac蛋白的浓度为约500μg/g，表明Cry1Ac蛋白对于ACB-AcR具有较弱的抑制活性。
如图5所示，饲喂Cry2Ab蛋白处理的混合饲料的ACB-BtS和ACB-AcR的幼虫生长抑制率均随着Cry2Ab蛋白浓度的增加而提高，且在同一浓度下ACB-BtS的幼虫生长抑制率高于ACB-AcR；达到100％生长抑制率时，ACB-BtS所需Cry2Ab蛋白的浓度为约1μg/g，ACB-AcR所需Cry2Ab蛋白的浓度为约5μg/g，表明Cry2Ab蛋白对于ACB-BtS和ACB-AcR具有相当的抑制活性。
表1：Bt蛋白对ACB-BtS和ACB-AcR新生幼虫的毒性结果
Figure PCTCN2017102442-appb-000001
上述结果表明，ACB-AcR对Cry1Ac蛋白具有大于5000倍抗性(即抗性比例大于5000)，且Cry2Ab蛋白对于ACB-BtS和ACB-AcR均具有杀虫活性，故ACB-AcR显示出对Cry2Ab蛋白较低的交互抗性，抗性比例显著降低为5.4。由此可见，Cry2Ab蛋白与Cry1Ac蛋白的交互抗性较低，Cry2Ab蛋白与Cry1Ac蛋白组合应用可以延缓或推迟亚洲玉米螟对Cry1Ac蛋白产生抗性，同时Cry2Ab蛋白可以有效管理亚洲玉米螟物种对Cry1Ac蛋白产生的抗性。
第三实施例、Cry2Ab蛋白与Cry1Ac蛋白在生产抗虫转基因植物中的用途
通过第二实施例的实验结果可知，Cry2Ab蛋白与Cry1Ac蛋白预期可以用于在转基因植物例如玉米植物中组合表达以延缓或防止亚洲玉米螟对该植物产生抗性。
第一种方法为相继转化：其中对已转化了第一基因(例如Cry1Ac基因)的植物进行再转化，以引入第二基因(例如Cry2Ab基因)。该相继转化优选地使用两个不同的选择标记基因，例如卡那霉素抗性基因和赋予对草铵膦除草剂的抗性的膦丝菌素乙酰转移酶基因(例如pat或bar基因)。
第二种方法为共转化方法：编码Cry1Ac蛋白的核苷酸序列在植物中与编码Cry2Ab蛋白的核苷酸序列一起表达，可以通过利用与各自基因连接的选择标记，整体筛选包含两个选择基因的植物。
第三种方法为独立的转化事件，将两个杀虫蛋白基因各自单独地转移至不同植物的基因组中，随后可以通过杂交将其组合在单独的植物中，并且可以使用DNA标记技术来选择包含这些不同基因的植物。
综上所述，本发明通过利用两种杀虫蛋白Cry1Ac蛋白和Cry2Ab蛋白有效延缓或防止亚洲玉米螟产生抗性，从而实现对亚洲玉米螟的控制或防治，使植物更大限度地获得保护并稳定产量；同时本发明使Cry1Ac蛋白和Cry2Ab蛋白在植物体内进行表达，且只需能够种植该转基因植物即可，而不需要采用其它措施，从而节省了大量人力、物力和财力，同时效果稳定、彻底。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (27)

  1. 一种管理昆虫抗性的方法，其特征在于，包括将所述昆虫至少与Cry2Ab蛋白和Cry1Ac蛋白接触，其中所述昆虫包括亚洲玉米螟。
  2. 根据权利要求1所述管理昆虫抗性的方法，其特征在于，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理亚洲玉米螟的抗性。
  3. 根据权利要求1所述管理昆虫抗性的方法，其特征在于，所述亚洲玉米螟为对Cry1Ac蛋白产生抗性的亚洲玉米螟，将对Cry1Ac蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry1Ac蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物的组织至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry1Ac蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理所述对Cry1Ac蛋白产生抗性的亚洲玉米螟的抗性。
  4. 根据权利要求1所述管理昆虫抗性的方法，其特征在于，所述亚洲玉米螟为对Cry2Ab蛋白产生抗性的亚洲玉米螟，将对Cry2Ab蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry2Ab蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物的组织至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry2Ab蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现管理所述对Cry2Ab蛋白产生抗性的亚洲玉米螟的抗性。
  5. 根据权利要求2-4任一项所述管理昆虫抗性的方法，其特征在于，所述植物为转基因植物，处于任意生育期。
  6. 根据权利要求2-4任一项所述管理昆虫抗性的方法，其特征在于，所述植物部分为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
  7. 根据权利要求2-4任一项所述管理昆虫抗性的方法，其特征在于，所述对亚洲玉米螟抗性的管理不因植物种植地点和/或种植时间的改变而改变。
  8. 根据权利要求2-7任一项所述管理昆虫抗性的方法，其特征在于，所述植物为玉米、小麦、高粱、谷子、水稻、向日葵或大豆。
  9. 根据权利要求1-8任一项所述管理昆虫抗性的方法，其特征在于，所述Cry2Ab蛋白：
    (1)具有SEQ ID NO:1所示的氨基酸序列；
    (2)与SEQ ID NO:1所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或
    (3)与SEQ ID NO:1所示的氨基酸序列相比具有至少80％以上的同源性。
  10. 根据权利要求9所述管理昆虫抗性的方法，其特征在于，编码所述Cry2Ab蛋白的多核苷酸：
    (1)具有SEQ ID NO:3所示的核苷酸序列；或
    (2)与SEQ ID NO:3所示的核苷酸序列相比具有至少80％以上的同源性。
  11. 根据权利要求1-10任一项所述管理昆虫抗性的方法，其特征在于，所述Cry1Ac蛋白：
    (1)具有SEQ ID NO:2所示的氨基酸序列；
    (2)与SEQ ID NO:2所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或
    (3)与SEQ ID NO:2所示的氨基酸序列相比具有至少80％以上的同源性。
  12. 根据权利要求11所述管理昆虫抗性的方法，其特征在于，编码所述Cry1Ac蛋白的多核苷酸：
    (1)具有SEQ ID NO:4所示的核苷酸序列；或
    (2)与SEQ ID NO:4所示的核苷酸序列相比具有至少80％以上的同源性。
  13. 一种控制亚洲玉米螟的方法，其特征在于，包括将亚洲玉米螟至少与Cry2Ab蛋白和Cry1Ac蛋白接触，从而实现对亚洲玉米螟的控制。
  14. 根据权利要求13所述控制亚洲玉米螟的方法，其特征在于，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述亚洲玉米螟生长受到抑制和/或导致死亡，以实现对亚洲玉米螟危害植物的控制。
  15. 根据权利要求13所述控制亚洲玉米螟的方法，其特征在于，所述亚洲玉米螟为对Cry1Ac蛋白产生抗性的亚洲玉米螟，将对Cry1Ac蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产 生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry1Ac蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry1Ac蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现对所述对Cry1Ac蛋白产生抗性的亚洲玉米螟危害植物的控制。
  16. 根据权利要求13所述控制亚洲玉米螟的方法，其特征在于，所述亚洲玉米螟为对Cry2Ab蛋白产生抗性的亚洲玉米螟，将对Cry2Ab蛋白产生抗性的亚洲玉米螟至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，所述Cry2Ab蛋白和Cry1Ac蛋白存在于至少产生所述Cry2Ab蛋白和Cry1Ac蛋白的细菌或植物中，所述对Cry2Ab蛋白产生抗性的亚洲玉米螟通过摄食所述细菌或所述植物或其部分至少与所述Cry2Ab蛋白和Cry1Ac蛋白接触，接触后所述对Cry2Ab蛋白产生抗性的亚洲玉米螟生长受到抑制和/或导致死亡，以实现对所述对Cry2Ab蛋白产生抗性的亚洲玉米螟危害植物的控制。
  17. 根据权利要求14-16任一项所述控制亚洲玉米螟的方法，其特征在于，所述植物为转基因植物，处于任意生育期。
  18. 根据权利要求14-16任一项所述控制亚洲玉米螟的方法，其特征在于，所述植物部分为根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
  19. 根据权利要求14-16任一项所述控制亚洲玉米螟的方法，其特征在于，所述对亚洲玉米螟危害植物的控制不因植物种植地点和/或种植时间的改变而改变。
  20. 根据权利要求14-19任一项所述控制亚洲玉米螟的方法，其特征在于，所述植物为玉米、小麦、高粱、谷子、水稻、向日葵或大豆。
  21. 根据权利要求13-20任一项所述控制亚洲玉米螟的方法，其特征在于，所述Cry2Ab蛋白：
    (1)具有SEQ ID NO:1所示的氨基酸序列；
    (2)与SEQ ID NO:1所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或
    (3)与SEQ ID NO:1所示的氨基酸序列相比具有至少80％以上的同源性。
  22. 根据权利要求21所述控制亚洲玉米螟的方法，其特征在于，编码所述Cry2Ab蛋白的多核苷酸：
    (1)具有SEQ ID NO:3所示的核苷酸序列；或
    (2)与SEQ ID NO:3所示的核苷酸序列相比具有至少80％以上的同源性。
  23. 根据权利要求13-22任一项所述控制亚洲玉米螟的方法，其特征在于，所述Cry1Ac 蛋白：
    (1)具有SEQ ID NO:2所示的氨基酸序列；
    (2)与SEQ ID NO:2所示的氨基酸序列相比具有一个或几个氨基酸取代、缺失、添加或插入；或
    (3)与SEQ ID NO:2所示的氨基酸序列相比具有至少80％以上的同源性。
  24. 根据权利要求23所述控制亚洲玉米螟的方法，其特征在于，编码所述Cry1Ac蛋白的多核苷酸：
    (1)具有SEQ ID NO:4所示的核苷酸序列；或
    (2)与SEQ ID NO:4所示的核苷酸序列相比具有至少80％以上的同源性。
  25. 一种Cry2Ab蛋白和Cry1Ac蛋白组合使用以防止或延缓亚洲玉米螟群体对Cry1Ac蛋白或Cry2Ab蛋白产生抗性的用途。
  26. 一种Cry2Ab蛋白和Cry1Ac蛋白组合使用以控制对Cry1Ac蛋白或Cry2Ab蛋白产生抗性的亚洲玉米螟群体的用途。
  27. 一种Cry2Ab蛋白与Cry1Ac蛋白组合在生产抗虫转基因植物中的用途。
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