WO2016101683A1 - Utilisations d'une protéine insecticide - Google Patents

Utilisations d'une protéine insecticide Download PDF

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WO2016101683A1
WO2016101683A1 PCT/CN2015/092006 CN2015092006W WO2016101683A1 WO 2016101683 A1 WO2016101683 A1 WO 2016101683A1 CN 2015092006 W CN2015092006 W CN 2015092006W WO 2016101683 A1 WO2016101683 A1 WO 2016101683A1
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plant
nucleotide sequence
protein
cry1ab
seq
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PCT/CN2015/092006
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English (en)
Chinese (zh)
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杨旭
丁德荣
张爱红
陶青
李建勇
李梅
张云珠
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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 WO2016101683A1 publication Critical patent/WO2016101683A1/fr

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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
    • 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
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a CrylA protein to control a plant of the millet ash by expressing it in a plant.
  • Agricultural control is a comprehensive and coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, environmental factors, and creating a farmland ecological environment that is conducive to crop growth and is not conducive to pest occurrence.
  • rotation can be used to reduce the density of insect sources, but the economic benefits of different crops, rotation is likely to cause farmers to reduce income, and it is difficult to implement.
  • Chemical control that is, pesticide control
  • pesticide control is the use of chemical pesticides to kill pests. It is an important part of the comprehensive management of pests. It is characterized by rapid, convenient, simple and high economic benefits, especially in the case of large pests. Essential emergency measures. However, chemical control also has its limitations. If improper use, it will lead to phytotoxicity of crops, resistance to pests, killing natural enemies, polluting the environment, destroying farmland ecosystems and threatening the safety of humans and animals. Adverse consequences.
  • Physical control mainly relies on the response of pests to various physical factors in environmental conditions, and uses various physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce pests, radiation infertility and other methods to control pests.
  • the frequency-vibration insecticidal lamp trapping which utilizes the phototaxis of pests, uses light at close range, uses waves at a long distance, attracts insects close to each other, and has certain effects on pest control; however, the frequency-vibration insecticidal lamp It is necessary to clean the dirt on the high-voltage power grid every day in time, otherwise it will affect the insecticidal effect; and it can't turn on the light in thunderstorm days, there is also the danger of electric shock wounding in operation; in addition, the one-time investment of installing the lamp is large.
  • Cry1A insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystal protein produced by Bacillus thuringiensis subsp. kurstaki (B.t.k.).
  • Plants transgenic with the Cry1A gene have been shown to be resistant to Lepidoptera pests such as corn borer, cotton bollworm, and fall armyworm. However, there has been no research on the control of millet by planting transgenic plants expressing the Cry1A protein. Report of the hazard.
  • Chilo infuscatellus also known as sugarcane mites
  • the ash mites are stalk pests, which can cause dead plants after the plants are damaged. Under normal circumstances, the spring valley area and the spring and summer valley mixed area are serious, and the Xiagu area is light. It has been a major pest in North China and South China, mainly affecting sorghum and valley. Son and sugar cane.
  • the object of the present invention is to provide a use of a pesticidal protein, for the first time to provide a method for controlling the damage of a plant by using a transgenic plant expressing a Cry1A protein, and effectively overcoming the prior art agricultural control, chemical control and physical control And other technical defects.
  • the present invention provides a method of controlling a pest of the mites, comprising contacting the mites pest with at least the Cry1A protein.
  • the Cry1A protein is present in a host cell that produces at least the CrylA protein, and the millet worm is in contact with at least the Cry1A protein by ingesting the host cell.
  • the Cry1A protein is present in a bacterium or a transgenic plant which produces at least the Cry1A protein, and the sphagnum pest is contacted with at least the Cry1A protein by a tissue ingesting the bacterium or the transgenic plant, after contact
  • the growth of the worms is inhibited and/or caused to death, in order to achieve control of the plants that are harmful to the ash.
  • the bacterium may be a wild bacterium and/or a recombinant bacterium capable of producing a Cry1A protein.
  • Bacillus thuringiensis subsp. kurstaki Bacillus thuringiensis subsp. kurstaki (B.t.k.).
  • the transgenic plant can be in any growth period.
  • the tissue of the transgenic plant can be various tissues of the plant, such as leaves, stems, fruits, tassels, ears, anthers or filaments.
  • the control of the plants against the ash mites does not change due to changes in the location and/or planting time.
  • the plant may be a variety of gramineous plants that are endangered by the millenium, preferably the plant is corn, sorghum, millet, sugar cane, rice, wheat, barley or oats.
  • the step prior to the contacting step is the planting of a plant containing a polynucleotide encoding the Cry1A protein.
  • the Cry1A protein is a Cry1Ab protein, a Cry1Ac protein or a Cry1Ab/Ac protein.
  • the amino acid sequence of the Cry1A protein has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the nucleotide sequence of the Cry1A protein has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
  • the nucleotide sequence encoding the Cry1A protein is recombined with at least one second nucleotide different from the Cry1A protein coding sequence.
  • the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase or a peroxidase.
  • the second nucleotide encodes a Vip3A protein or a Cry1Ie protein.
  • amino acid sequence of the Vip3A protein has the amino acid sequence of SEQ ID NO: 11
  • amino acid sequence of the Cry1Ie protein has the amino acid sequence of SEQ ID NO: 15.
  • the second nucleotide has the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 16.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • the present invention also provides a use of a Cry1A protein for controlling a mites pest.
  • the present invention also provides a method of producing a plant for controlling a mites pest, comprising introducing a polynucleotide sequence encoding a Cry1A protein into the genome of the plant.
  • the present invention also provides a method of producing a plant propagule for controlling a mites pest, comprising crossing a first plant obtained by the method with a second plant, and/or removing the method by the method
  • the fertile tissue on the obtained plants is cultured to produce a propagule containing a polynucleotide sequence encoding a Cry1A protein.
  • the present invention also provides a method for cultivating a plant for controlling a mites pest, comprising:
  • the plants are grown under conditions in which the artificially inoculated with the mites and/or the mites pests are naturally harmful, and the plants are harvested with reduced plant damage and/or compared to other plants that do not have the polynucleotide sequence encoding the Cry1A protein. Or plants with increased plant yield.
  • Plant propagules as used in the present invention include, but are not limited to, plant sexual propagules and plant asexual propagules.
  • the plant sexual propagule includes, but is not limited to, a plant seed; the plant asexual propagule refers to a vegetative organ of a plant body or a special tissue which can produce a new plant under ex vivo conditions; the vegetative organ or a certain Specific tissues include, but are not limited to, roots, stems and leaves, for example: plants with roots as vegetative propagules including strawberries and sweet potatoes; plants with stems as vegetative propagules including sugar cane and potatoes (tubers), etc.; leaves as asexual Plants of the propagule include aloe vera and begonia.
  • Recombination refers to the form of DNA and/or proteins and/or organisms that are not normally found in nature and are thus 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.
  • 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.
  • control and/or “control” in the present invention means that the mulberry pest is at least in contact with the Cry1A protein, and the growth of the larvae is inhibited and/or causes death after contact. Further, the larvae pests are at least contacted with the Cry1A protein by ingesting plant tissues, and all or part of the larvae 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. At the same time, the plants should be normal in morphology and can be used under conventional methods. Culture for consumption and/or production of the product.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry1A protein that control the pests of the mites are inoculated with non-transgenic under conditions in which the artificially inoculated pests of the mites and/or the mites are naturally harmful.
  • Wild-type plants have reduced plant damage compared to specific manifestations including, but not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like.
  • the "control" and / or “control” effects of the Cry1A protein on the ash can be independently independent and not attenuated and/or disappeared by other substances that can "control" and/or "control” the worms. .
  • any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry1A protein) is present and/or asynchronously, present and/or produced, a Cry1A protein and/or another substance that can control the pest of the mites,
  • the presence of the other substance neither affects the "control” and/or "control” effect of the Cry1A protein on the ash, nor does it result in a complete and/or partial effect of the "control" and/or “control” It is achieved by the other substance, but not by the Cry1A protein.
  • the process of feeding on plant tissues by the larvae is short-lived and difficult to observe with the naked eye.
  • any tissue of a plant (containing a polynucleotide sequence encoding a Cry1A protein) is present in a dead mites pest, and/or a sphagnum pest in which growth growth is inhibited, and/or with a non-transgenic wild-type plant
  • the method and/or use of the present invention is achieved by having reduced plant damage, i.e., by contacting the Cry1A protein with at least the C. sinensis pest to achieve a method and/or use for controlling the mites.
  • expression of the Cry1A protein in a transgenic plant may be accompanied by fusion expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins.
  • 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 (first parent) can express Cry1A protein by genetic engineering operation
  • the second plant second parent
  • 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.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, in the present invention, RNAi technology can be used to specifically knock out or shut down the expression of a specific gene in a target insect pest.
  • the lepidoptera In the classification system, the lepidoptera is generally divided into suborders, superfamily, and family according to the morphological characteristics of the worm's veins, linkages, and types of antennae, while the genus Lepidoptera is the most diverse species of Lepidoptera.
  • One of the departments has found more than 10,000 types in the world, and there are thousands of records in China alone.
  • Most of the moths are pests of crops, most of which are in the form of stolons, such as stem borer and corn borer.
  • the ash mites are similar to the mites and corn borers, they belong to the order Lepidoptera, and there are great differences in other morphological structures except for the similarity in the classification criteria; it is like the strawberry in the plant and the apple ( They belong to the genus Rosaceae, which have the characteristics of flower bisexuality, radiation symmetry, and 5 petals, but their fruits and plant morphology are very different. Millet ash has its unique characteristics in terms of larval morphology and adult morphology.
  • the back line of the back is three or four five", which means that the sorghum sorghum, corn stalk and ash scorpion belonging to the genus Mothidae have obvious numbers on the top line. difference.
  • the dorsal blood vessel is an important part of the insect circulatory organ. The inside is filled with the hemolymph called the insect "blood”. Therefore, the difference in the number of top lines on the surface of the body appears to reflect the difference in the back vessels, which is the difference in the insect circulation system.
  • Insects belonging to the genus Mothidae not only have large differences in morphological characteristics, but also have differences in feeding habits.
  • the stem borer of the same family is mainly harmful to rice, and rarely harms other grass crops.
  • the difference in feeding habits also suggests that the enzymes and receptor proteins produced by the digestive system in the body are different.
  • the enzyme produced in the digestive tract is the key point of the Bt gene function. Only the enzyme or receptor protein that can bind to the specific Bt gene may make a certain Bt gene have an insect resistance effect on the pest.
  • insects of different families and even different families have different sensitivities to the same Bt protein.
  • the Vip3Aa gene showed anti-insect activity against Chilo suppressalis and Ostrinia furnacalis, but for Plodia interpunctella and European corn borer (the same species) Ostrinia nubilalis has no insect resistance.
  • the above four pests belong to the family Lepidoptera, but the same kind of Bt protein has different resistance effects to the four species of the moth.
  • European corn borer and Asian corn borer are classified in the same species as the Ostrinia genus (the same genus), but their response to the same Bt protein is quite different, which further demonstrates the Bt protein and The way enzymes and receptors interact in insects is complex and unpredictable.
  • 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.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • 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 “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes to the Cry1A gene of the present invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A 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. In the present invention, if 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 have “mutually” if they are capable of hybridizing to each other with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other. "Complementary".
  • 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 act as a primer or probe, it is only necessary to ensure that it has a sequence Sufficient complementarity to form a stable double-stranded structure at the particular solvent and salt concentrations 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: 2 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1% SDS and 1 x SSC. 0.1% SDS was washed once each time.
  • sequences having insect resistance activity and hybridizing under stringent conditions to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 of the present invention is included in the present invention. in.
  • These sequences have at least about 40%-50% homology to the sequences of the invention, about 60%, 65% or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater homology.
  • 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.
  • variant or “variant” is meant a nucleotide sequence that encodes the same protein or an equivalent protein encoded with insecticidal activity.
  • the "equivalent protein” refers to a protein having the same or substantially the same biological activity as the anti-mite pest of the protein of the claims.
  • 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 or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • the length of the aforementioned sequence may vary, 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 B.t. isolates and/or DNA libraries.
  • 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 B.t. proteins.
  • Immunoprecipitation, enzyme-linked immunosorbent assay The assay (ELISA) or Western blot method uses these antibodies to uniquely identify a characteristically active equivalent protein.
  • Antibodies of the proteins disclosed herein 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.
  • the "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.
  • substitutions can occur outside of the regions that are important for molecular function and still produce active polypeptides.
  • amino acids from the polypeptides of the invention that are essential for their activity and are therefore selected for unsubstitution they 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 Cry1A protein includes, but is not limited to, a Cry1Ab, a Cry1Ac or a Cry1Ab/Ac protein, or an insecticidal fragment having at least 70% homology with the amino acid sequence of the above protein and still having insecticidal activity against the millet or Functional Area.
  • an amino acid sequence having a certain homology to the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 is also included in the present invention.
  • the homology (similarity/identity) of these sequences to the sequences of the invention is typically greater than 60%, preferably greater than 75%, more preferably greater than 80%, Even more preferred is greater than 90% and may be greater than 95%.
  • Preferred polynucleotides and proteins of the invention may also be defined in terms of a more specific range of homology.
  • the sequence of the example of the present invention is 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% homology.
  • still having insecticidal activity or retaining insecticidal activity means amino acid sequence and amino acid represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the insecticidal activity of the protein having a certain homology of the sequence is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID 80% or more, or 90% or more, or 92% or more, or 95% or more, or 98% or more of the insecticidal activity (total score of resistance) of the Cry1A protein represented by NO: 7 or SEQ ID NO: 9, or 100%.
  • the nucleotide sequence of the Cry1A protein is capable of encoding a Cry1A protein having an insecticidal activity that satisfies the above requirements.
  • a transgenic plant producing the Cry1A protein includes, but is not limited to, a Mon810 transgenic maize event and/or a plant material comprising a Mon810 transgenic maize event (as described in US Pat. No.
  • a Bt11 transgenic maize event and/or comprises Bt11 Plant material for the transgenic maize event (as described in USDA APHIS Unregulated Status Application 95-195-01p, which contains the amino acid sequence of the Cry1Ab protein as shown in SEQ ID NO: 3 of the present invention), Bt176 transgenic maize event and / or plant material comprising the Bt176 transgenic maize event (as described in USDA APHIS Unregulated Status Application 94-319-01p, which contains the amino acid sequence of the Cry1Ab protein as described in US 5,625, 136 B2), TT51 transgenic rice events and/or Or plant material comprising TT51 transgenic rice events (as described in CN100582223C and CN101302520B), 223F-S21 transgenic rice lines and/or plant material comprising 223F-S21 transgenic rice lines (as described in CN103773759A), Mon15985 transgene Cotton events and/or plant material containing the Mon15985 GM cotton
  • the methods and/or uses of the invention can also be achieved by expressing the Cry1A protein in the above transgenic events in different plants. More specifically, the Cry1A protein is present in a transgenic plant that produces at least the Cry1A protein, and the millet worm is in contact with at least the Cry1A protein by ingesting tissue of the transgenic plant, and the millet is contacted after contact The growth of pests is inhibited and/or caused to death, in order to achieve control of the plants that are harmful to the ash.
  • Regulatory sequences of the invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Cry1A 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, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, the promoter of the 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 (pinI and pinII) 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
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked 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 a mites pest.
  • the Cry1A protein is toxic to the mites.
  • the plants of the present invention particularly millet, sorghum, corn and sugar cane, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1A protein, which is fed by a plant tissue The protein is contacted and the growth of the insects is inhibited and/or causes death after exposure. 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 that are insecticides against the mites pests targeted by the Cry1A protein.
  • the expression level of insecticidal crystal protein (ICP) in plant material can be detected by various methods described in the art, for example, by using specific primers to quantify the mRNA encoding the insecticidal protein produced in the tissue, or directly specific The amount of insecticidal protein produced is detected.
  • the target insects in the present invention are mainly milled ash.
  • the Cry1A protein may have the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 in the Sequence Listing.
  • other elements may be included, such as a protein encoding a selectable marker.
  • an expression cassette comprising a nucleotide sequence encoding a Cry1A 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, oxalic acid Phospho-resistant genes (such as bar gene, pat gene), benthamiana 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 weeding Agent-resistant transgenic plants.
  • oxalic acid Phospho-resistant genes such as bar gene, pat gene
  • benthamiana resistance genes such as pmph gene
  • glyphosate resistance genes such as EPSPS gene
  • bromoxynil resistance gene sulfonylurea Resistance gene
  • the foreign DNA is introduced into a plant, such as a gene encoding the Cry1A protein or an expression cassette or a recombinant vector
  • the conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-shot bombardment, Direct DNA uptake into protoplast, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the harm of the mites and pests by external action, ie, external factors, such as agricultural control, chemical control and physical control; and the present invention controls the ash by using the Cry1A protein which can kill the ash mites in the plant.
  • the pests are controlled by internal factors.
  • the frequency-vibration insecticidal lamp used in the prior art not only needs to clean the dirt of the high-voltage power grid every day, but also cannot be used in thunderstorm days; the invention makes the Cry1A protein express in the plant body, effectively overcomes the frequency vibration type killing
  • the effect of the insect lamp is affected by external factors, and the control effect of the transgenic plant (Cry1A protein) of the invention is stable and consistent at different locations, at different times, and in different genetic backgrounds.
  • the frequency-active insecticidal lamp used in the prior art has a large one-time investment, and the operation is improper and there is a danger of electric shock wounding; the invention only needs to plant a transgenic plant capable of expressing the Cry1A protein, and does not need other measures. , which saves a lot of manpower, material resources and financial resources.
  • the method for controlling the mites and pests used in the prior art has the effect of being incomplete and only reducing the effect; and the transgenic plant of the invention (Cry1A protein) has almost 100% control effect on the newly hatched larvae of the larvae. Individual surviving larvae also basically stopped development. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and had stopped developing, and could not survive in the natural environment of the field, while the transgenic plants were generally only slightly damaged.
  • Figure 1 is a recombinant cloning vector containing the Cry1Ab-01 nucleotide sequence for use of the insecticidal protein of the present invention DBN01-T build flow chart;
  • Figure 2 is a flow chart showing the construction of a recombinant expression vector DBN100124 containing the Cry1Ab-01 nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the damage of leaves of the transgenic corn plants inoculated with the ash mites according to the use of the insecticidal protein of the present invention
  • Figure 4 is a diagram showing the damage of the leaves of the transgenic sugarcane plants inoculated with the worms of the present invention
  • Figure 5 is a diagram showing the damage of the leaves of the transgenic sorghum plant inoculated with the ash sorghum of the present invention
  • Figure 6 is a diagram showing the damage of leaves of transgenic millet plants inoculated with millet mites for the use of the insecticidal protein of the present invention.
  • the amino acid sequence of Cry1Ab-02 insecticidal protein (615 amino acids), as shown in SEQ ID NO: 3 in the Sequence Listing; Cry1Ab-02 encoding the amino acid sequence (615 amino acids) corresponding to the Cry1Ab-02 insecticidal protein
  • the nucleotide sequence (1848 nucleotides) is shown as SEQ ID NO: 4 in the Sequence Listing.
  • Cry1Ac-01 insecticidal protein (1156 amino acids), as shown in SEQ ID NO: 5 in the Sequence Listing; Cry1Ac-01 encoding the amino acid sequence (1156 amino acids) corresponding to the Cry1Ac-01 insecticidal protein Nucleotide sequence (3471 nucleotides) as shown in SEQ ID NO: 6 in the Sequence Listing.
  • the amino acid sequence of Cry1Ac-02 insecticidal protein (616 amino acids), as shown in SEQ ID NO: 7 in the Sequence Listing; Cry1Ac-02 encoding the amino acid sequence (616 amino acids) corresponding to the Cry1Ac-02 insecticidal protein
  • the nucleotide sequence (1851 nucleotides) is shown as SEQ ID NO: 8 in the Sequence Listing.
  • the nucleotide sequence (1830 nucleotides) is shown as SEQ ID NO: 10 in the Sequence Listing.
  • the amino acid sequence of the Cry1Ab+Vip3A insecticidal protein (1436 amino acids), as set forth in SEQ ID NO: 13 in the Sequence Listing; the Cry1Ab+Vip3A nucleotide sequence encoding the amino acid sequence corresponding to the Cry1Ab+Vip3A insecticidal protein ( 4311 nucleotides), as shown in SEQ ID NO: 14 of the Sequence Listing; wherein the amino acid sequence of Vip3A insecticidal protein (788 amino acids) is as set forth in SEQ ID NO: 11 in the Sequence Listing;
  • the Vip3A nucleotide sequence (2364 nucleotides) of the amino acid sequence of the Vip3A insecticidal protein as shown in SEQ ID NO: 12 in the sequence listing; the amino acid sequence of the Cry1Ab effective fragment (648 amino acids), as in the sequence listing ID NO: 35; a nucleotide sequence (1944 nucleotides) encoding a Cry1Ab effective fragment
  • Cry1Ie insecticidal protein (719 amino acids), as shown in SEQ ID NO: 15 of the Sequence Listing
  • the Cry1Ab-01 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing), the Cry1Ab-02 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing), the Cry1Ac- 01 nucleotide sequence (as shown in SEQ ID NO: 6 in the Sequence Listing), the Cry1Ac-02 nucleotide sequence (as shown in SEQ ID NO: 8 in the Sequence Listing), the Cry1Ab/Ac nucleotide a sequence (as set forth in SEQ ID NO: 10 in the Sequence Listing), a Cry1Ab+Vip3A nucleotide sequence (as set forth in SEQ ID NO: 14 in the Sequence Listing), and the Cry1Ie nucleotide sequence (eg, in the Sequence Listing) SEQ ID NO: 16) was synthesized by Nanjing Kingsray Biotechnology Co., Ltd.; the 5' end of the synthesized Cry1Ab-01 nu
  • the 3' end of the Cry1Ab-01 nucleotide sequence (SEQ ID NO: 2) is also ligated with a KasI cleavage site; the 5' of the synthesized Cry1Ab-02 nucleotide sequence (SEQ ID NO: 4) The SpeI cleavage site is also ligated to the end, and the 3' end of the Cry1Ab-02 nucleotide sequence (SEQ ID NO: 4) is further ligated with a KasI cleavage site; the synthesized Cry1Ac-01 nucleotide sequence The 5' end of (SEQ ID NO: 6) is also ligated with a SacI cleavage site, said Cry The 3' end of the 1Ac-01 nucleotide sequence (SEQ ID NO: 6) is also ligated with a KasI cleavage site; the 5' end of the synthesized Cry1Ac-02 nucleotide sequence (SEQ ID NO: 8) is also The SacI clea
  • the synthetic Cry1Ab-01 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • the construction process is shown in Figure 1 (wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; and T7 is T7 RNA polymerization).
  • Enzyme promoter; Cry1Ab-01 is the Cry1Ab-01 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking 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) in LB plate (tryptone 10 g/L, leaven The mother extract was grown overnight at 5 g/L, NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH.
  • 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 in the precipitate; the RNA was digested in a water bath at 37 ° C for 30 min; and stored at -20 ° C until use.
  • the Cry1Ab-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 2 in the sequence listing.
  • the synthesized Cry1Ab-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1Ab-02 was Cry1Ab-02. Nucleotide sequence (SEQ ID NO: 4).
  • the Cry1Ab-02 nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ac-01 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein Cry1Ac-01 was Cry1Ac-01.
  • Nucleotide sequence SEQ ID NO: 6
  • the Cry1Ac-01 nucleotide sequence in the recombinant cloning vector DBN03-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ac-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein Cry1Ac-02 was Cry1Ac-02. Nucleotide sequence (SEQ ID NO: 8).
  • the Cry1Ac-02 nucleotide sequence in the recombinant cloning vector DBN04-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab/Ac nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN05-T, wherein Cry1Ab/Ac was Cry1Ab/Ac.
  • Nucleotide sequence SEQ ID NO: 10
  • the Cry1Ab/Ac nucleotide sequence in the recombinant cloning vector DBN05-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab+Vip3A nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN06-T, wherein Cry1Ab+Vip3A was Cry1Ab+Vip3A.
  • Nucleotide sequence SEQ ID NO: 14
  • the Cry1Ab+Vip3A nucleotide sequence was correctly inserted into the recombinant cloning vector DBN06-T by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ie nucleotide sequence was ligated according to the above method for constructing the recombinant cloning vector DBN01-T.
  • the cloning vector pGEM-T was introduced to obtain a recombinant cloning vector DBN07-T, wherein Cry1Ie was a Cry1Ie nucleotide sequence (SEQ ID NO: 16).
  • Cry1Ie nucleotide sequence in the recombinant cloning vector DBN07-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the restriction endonuclease SpeI and KasI were used to digest the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)), and the cut Cry1Ab-01 nucleotide sequence fragment was inserted.
  • DBNBC-01 vector backbone: pCAMBIA2301 (available from CAMBIA)
  • the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100124 is constructed.
  • FIG. 2 Kanamycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 17); Cry1Ab-01: Cry1Ab-01 nucleotide sequence (SEQ ID NO: 2); Nos : terminator of the nopaline synthase gene (SEQ ID NO: 18); Hpt: hygromycin phosphotransferase (SEQ ID NO: 19); LB: left border).
  • the recombinant expression vector DBN100124 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ l of E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant expression vector DBN100124), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50 mg/L was adjusted to pH 7.5 with NaOH and incubated overnight at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100124 was SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 2, that is, the Cry1Ab-01 nucleotide sequence.
  • the Cry1Ab-02 nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100743.
  • the nucleotide sequence in the recombinant expression vector DBN100743 contains the nucleotide sequence shown as SEQ ID NO: 4 in the sequence listing, that is, the Cry1Ab-02 nucleotide sequence, and the Cry1Ab-02 nucleotide sequence is digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Ac-01 nucleotide sequence excised from the SacI and KasI recombinant cloning vector DBN03-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100741.
  • the nucleotide sequence in the recombinant expression vector DBN100741 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, that is, the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-01 nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • nucleotide sequence in the recombinant expression vector DBN100734 contains SEQ ID NO: 8 in the sequence listing and The nucleotide sequence shown in SEQ ID NO: 16, that is, the Cry1Ac-02 nucleotide sequence and the Cry1Ie nucleotide sequence, the Cry1Ac-02 nucleotide sequence and the Cry1Ie nucleotide sequence can be linked to the Ubi promoter Sub and Nos terminators.
  • the Cry1Ab/Ac nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN05-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100737.
  • the nucleotide sequence in the recombinant expression vector DBN100737 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, that is, the Cry1Ab/Ac nucleotide sequence, and the Cry1Ab/Ac nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Ab+Vip3A nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN06-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100736.
  • the nucleotide sequence in the recombinant expression vector DBN100736 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 14 in the sequence listing, that is, the Cry1Ab+Vip3A nucleotide sequence, and the Cry1Ab+Vip3A nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737 and DBN100736, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L.
  • Agrobacterium LBA4404 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube at a temperature of 28 ° C, 200 rpm After culturing for 2 hours, it was applied to LB plates containing 50 mg/L of rifampicin and 100 mg/L of kanamycin until a positive monoclonal was grown, and the monoclonal culture was picked and the plasmid was extracted.
  • Recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737 and DBN100736 were digested with restriction endonucleases AhdI and XhoI, and the results showed that the recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737 and DBN100736 were completely structurally intact. correct.
  • T-DNA of recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737 and DBN100736 (including the promoter sequence of maize Ubiquitin gene, Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ac-01 nucleotide)
  • the sequence, Cry1Ac-02 nucleotide sequence, Cry1Ie nucleotide sequence, Cry1Ab/Ac nucleotide sequence, Cry1Ab+Vip3A nucleotide sequence, Hpt gene and Nos terminator sequence were transferred into the maize genome and obtained Maize plants transformed with Cry1Ab-01 nucleotide sequence, maize plants transfected
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can express Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the grown transformed callus is selected (step 4: selection step).
  • a selection agent hygromycin
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8, resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, M. 50 mg/L, vegetal gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.
  • the medium formulation is referred to Molecular Biology and Genetic Engineering ISSN 2053-5767, wherein the screening agent is replaced with hygromycin according to the transgenic vector of the present invention.
  • the sorghum plant transformed into the Cry1Ab-01 nucleotide sequence, the sorghum plant transformed into the Cry1Ab-02 nucleotide sequence, the sorghum plant transformed into the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-02-Cry1Ie nucleoside were transferred.
  • the sorghum plant of the acid sequence The sorghum plant of the acid sequence, the sorghum plant transformed into the Cry1Ab/Ac nucleotide sequence, the sorghum plant transformed into the Cry1Ab+Vip3A nucleotide sequence, and the wild type sorghum plant as a control.
  • the conversion method mainly refers to the 22nd to 24th pages of the 2012 Master's degree of Guangxi University. Take the fresh stem section of the cane top, remove the cane tip and leaf sheath, leaving the stem tip growth cone and the heart leaf stem segment. On the ultra-clean workbench, wipe the surface with a 75% (v/v) alcohol cotton ball, carefully peel off the outer layer of the heart leaf with the sterilized tweezers, and take the heart leaf segment 5-7 cm long in the middle. A sheet cut into a thickness of about 3 mm was inoculated on an induction medium, and cultured in the dark at a temperature of 26 ° C for 20 days.
  • the callus pieces were cut into small pieces of 0.6*0.6 cm, and then transferred to MR solid medium containing 100 ⁇ mol/L acetosyringone (AS), and cultured in a dark state at 23 ° C for 3 days; the callus after infection
  • the tissue was clipped, placed on a filter paper and dried on a clean bench until the surface of the material was dry.
  • the culture medium was once removed, and the contaminated callus was removed.
  • the seedlings were about 3 cm long, they were transferred to a rooting medium containing hygromycin screening agent to induce rooting.
  • a sugarcane plant transformed into the Cry1Ab-01 nucleotide sequence, a sugarcane plant transformed into the Cry1Ab-02 nucleotide sequence, a sugarcane plant transformed into the Cry1Ac-01 nucleotide sequence, and a Cry1Ac-02-Cry1Ie nucleoside were obtained.
  • the conversion method refers to the Hebei University of Agriculture 2012 Master Wang Hanyu's dissertation on pages 9 to 10. After soaking the mature seeds in 0.1% (v/v) Tween-20 solution, wash them with 70% (v/v) ethanol, then transfer to 0.1% (w/v) HgCl 2 solution, and finally use sterile water. Wash 2-3 times. The sterilized seeds were transferred to MS medium and incubated at a temperature of 25 ° C for 2-3 days. When the stem tip of the seed grows to 4-6 mm, the shoot tip is transferred to the callus induction medium under aseptic conditions.
  • Induction of shoot tip callus The induced shoot tip callus was immersed in the Agrobacterium suspension for 30 minutes, and the shoot tip callus was taken out on the sterilized filter paper, and the excess bacterial solution was aspirated and transferred to co-culture.
  • the medium (MS + 100 mol / L AS + 2, 4-D) was co-cultured in the dark at a temperature of 28 ° C for 2-4 days.
  • the callus after co-cultivation was then transferred to MS callus induction medium (2,4-D 4.5 ⁇ mol/L, 2.25 ⁇ mol/L Kn, cephalosporin 500 mg/L) at a temperature of 25 ° C. Cultivate, and the callus is subcultured every two weeks.
  • the dense yellow-white callus was transferred to the differentiation medium for differentiation, and the screening medium hygromycin was added to the differentiation medium. After about five weeks, the callus formed a nodular structure, and the callus with the knot structure was transferred to the MS differentiation rooting medium (thiazolyl phenylurea (TDZ) 4.5 ⁇ mol/L, sucrose 120 ⁇ mol/L).
  • MS differentiation rooting medium thiazolyl phenylurea (TDZ) 4.5 ⁇ mol/L, sucrose 120 ⁇ mol/L).
  • a millet plant transformed into the Cry1Ab-01 nucleotide sequence, a millet plant transformed into the Cry1Ab-02 nucleotide sequence, a millet plant transformed with the Cry1Ac-01 nucleotide sequence, and a Cry1Ac-02-Cry1Ie nucleoside were obtained.
  • Step 11 The maize plants transformed with the Cry1Ab-01 nucleotide sequence, the maize plants transformed with the Cry1Ab-02 nucleotide sequence, the maize plants transformed with the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-02- Corn plants of Cry1Ie nucleotide sequence, maize plants transfected with Cry1Ab/Ac nucleotide sequence, maize plants transfected with Cry1Ab+Vip3A nucleotide sequence and leaves of wild-type maize plants were each 100 mg, respectively, used in mortars The liquid nitrogen was ground into a homogenate, and each sample was taken in 3 replicates;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ L;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Primer 2 GTAGATTTCGCGGGTCAGTTG is shown in SEQ ID NO: 21 in the Sequence Listing;
  • Probe 2 CAGCGCCTTGACCACAGCTATCCC is shown in SEQ ID NO: 25 in the Sequence Listing;
  • Probe 3 TCCGCTCTTCGCCGTTCAGAATTACC is shown in SEQ ID NO: 28 in the Sequence Listing;
  • Primer 7 GGTTACACTCCCATCGACATCTC is shown in SEQ ID NO: 29 in the Sequence Listing;
  • Primer 8 CACAAGACCAAGCACGAAACC is shown in SEQ ID NO: 30 in the Sequence Listing;
  • Probe 4 CCTTACCCAGTTCCTTCTTTCCGAGTTCG as shown in SEQ ID NO: 31 in the Sequence Listing;
  • Primer 9 CCGAGCTTCATCGACTACTTCAAC is shown in SEQ ID NO: 32 in the Sequence Listing;
  • Probe 5 CCACCGGCATCAAGGACATCATGAAC is shown in SEQ ID NO: 34 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ l of each primer at a concentration of 1 mM, 50 ⁇ l of a probe at a concentration of 100 ⁇ M, and 860 ⁇ l of 1 ⁇ TE buffer, and stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ac-01 nucleotide sequence, Cry1Ac-02-Cry1Ie nucleotide sequence, Cry1Ab/Ac nucleotide sequence, Cry1Ab+Vip3A nucleus The nucleotide sequence has been integrated into the genome of the tested maize plant, and the maize plant transformed into the Cry1Ab-01 nucleotide sequence, the maize plant transformed into the Cry1Ab-02 nucleotide sequence, and the Cry1Ac-01 core were transferred.
  • the maize plant with the nucleotide sequence, the maize plant transformed with the Cry1Ac-02-Cry1Ie nucleotide sequence, the maize plant transformed with the Cry1Ab/Ac nucleotide sequence, and the maize plant transformed with the Cry1Ab+Vip3A nucleotide sequence were obtained. Single copy of transgenic maize plants.
  • Transgenic sorghum plants, transgenic sugarcane, according to the above method for verifying transgenic maize plants with TaqMan Plants and transgenic millet plants were tested and analyzed.
  • the results showed that Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ac-01 nucleotide sequence, Cry1Ac-02-Cry1Ie nucleotide sequence, Cry1Ab/Ac nucleotide sequence, Cry1Ab+Vip3A nucleus
  • the nucleotide sequences have been integrated into the genomes of the tested sorghum, sugarcane and millet plants, respectively, and the sorghum plants transferred to the Cry1Ab-01 nucleotide sequence and the sorghum plants transferred to the Cry1Ab-02 nucleotide sequence.
  • Total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / total number of insects) + 10 ⁇ (negative Control number of insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S1, S2 and S3) transfected into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence for a total of 3 transformation event lines (S4, S5 and S6).
  • a total of 3 transformation event lines (S7, S8 and S9) transfected into the Cry1Ab+Vip3A nucleotide sequence were transferred into the Cry1Ac-01 nucleotide sequence for a total of 3 transformation event lines (S10, S11 and S12).
  • the results in Table 1 indicate that a maize plant transformed with the Cry1Ab-01 nucleotide sequence, a maize plant transformed with the Cry1Ab-02 nucleotide sequence, a maize plant transformed with the Cry1Ab+Vip3A nucleotide sequence, and transferred to Cry1Ac-01
  • the maize plant with nucleotide sequence, the maize plant transformed with the Cry1Ac-02-Cry1Ie nucleotide sequence, and the maize plant transformed with the Cry1Ab/Ac nucleotide sequence have good insecticidal effects on the millet, and the gray ash
  • the average mortality rate of cockroaches is basically over 90%, and some even reach 100%.
  • the total score of resistance is basically above 280 points, and some even reach a maximum of 300 points.
  • the corn plants identified as non-transgenic by Taqman The total score of resistance to wild-type maize plants is generally around 20 points.
  • the results in Figure 3 indicate that maize plants transfected with the Cry1Ab-01 nucleotide sequence, maize plants transfected with the Cry1Ab-02 nucleotide sequence, and transferred to the Cry1Ab+Vip3A nucleotide sequence were compared to wild-type maize plants.
  • the control effect is almost 100%, and very few surviving larvae basically stop developing.
  • the larvae After 3 days, the larvae are still in the initial hatching state, and at the same time show very weak vitality, and the corn plants transferred to the Cry1Ab-01 nucleotide sequence are transferred.
  • Maize plants into the Cry1Ab-02 nucleotide sequence maize plants transfected with the Cry1Ab+Vip3A nucleotide sequence, maize plants transfected with the Cry1Ac-01 nucleotide sequence, and transferred into the Cry1Ac-02-Cry1Ie nucleotide sequence
  • Maize plants and maize plants transferred to the Cry1Ab/Ac nucleotide sequence were only slightly damaged, and the naked eye could hardly distinguish the feeding traces of the ash, and the leaf damage rate was below 3%.
  • the maize plant transformed into the Cry1Ab-01 nucleotide sequence, the maize plant transformed into the Cry1Ab-02 nucleotide sequence, the maize plant transformed into the Cry1Ab+Vip3A nucleotide sequence, and the Cry1Ac-01 nucleotide were transferred.
  • the sequence of the maize plant, the maize plant transformed into the Cry1Ac-02-Cry1Ie nucleotide sequence, and the maize plant transformed into the Cry1Ab/Ac nucleotide sequence all showed high anti-mulberry activity, which was sufficient for the ash ash Growth produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the ash it is also possible to reduce the occurrence of diseases on the corn and greatly improve the yield and quality of the corn.
  • the sugarcane plants transformed into the Cry1Ab-01 nucleotide sequence, the sugarcane plants transformed into the Cry1Ab-02 nucleotide sequence, the sugarcane plants transformed into the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-02-Cry1Ie nucleoside were transferred.
  • Acid sequence of sugarcane plants sugarcane plants transformed into Cry1Ab/Ac nucleotide sequence, sugarcane plants transformed into Cry1Ab+Vip3A nucleotide sequence, wild-type sugarcane plants and non-transgenic sugarcane plants identified by Taqman (expanded young leaves)
  • Fresh leaves rinsed with sterile water and blotted the water on the leaves with gauze, then cut the cane leaves into strips of about 1 cm ⁇ 2 cm, and take a piece of the cut long leaves into a circle
  • On the moisturizing filter paper at the bottom of the plastic petri dish put 10 ash larvae (initial hatching larvae) in each petri dish, and cover the petri dish at the temperature of 22-26 ° C, relative humidity 70%-80%, light
  • the strains (S34, S35 and S36) were identified as one non-transgenic (NGM2) strain by Taqman and one strain of wild-type (CK2); three strains were selected from each strain for testing. The strain was repeated 6 times. The results are
  • the results in Table 2 indicate that sugarcane plants transformed into the Cry1Ab-01 nucleotide sequence, sugarcane plants transformed into the Cry1Ab-02 nucleotide sequence, sugarcane plants transformed into the Cry1Ab+Vip3A nucleotide sequence, and transferred into Cry1Ac-01
  • the sugarcane plant with nucleotide sequence, the sugarcane plant transformed into the Cry1Ac-02-Cry1Ie nucleotide sequence, and the sugarcane plant transformed into the Cry1Ab/Ac nucleotide sequence have good insecticidal effect on the millet, and the gray ash
  • the average mortality rate of cockroaches is basically above 90%, and some even reach 100%.
  • the total score of resistance is basically around 290 points, and some even reach Up to 300 points; the total resistance score of sugarcane plants and wild-type sugarcane plants identified by Taqman as non-transgenic is generally around 20 points.
  • the results in Figure 4 indicate that the sugarcane plant transformed into the Cry1Ab-01 nucleotide sequence, the sugarcane plant transformed into the Cry1Ab-02 nucleotide sequence, and the Cry1Ab+Vip3A nucleotide sequence were transduced compared to the wild-type sugarcane plant.
  • Sugarcane plant, sugarcane plant transformed into Cry1Ac-01 nucleotide sequence, sugarcane plant transformed into Cry1Ac-02-Cry1Ie nucleotide sequence, and sugarcane plant transformed into Cry1Ab/Ac nucleotide sequence The control effect is almost 100%, and very few surviving larvae basically stop developing.
  • sugarcane plants transferred to the Cry1Ab-01 nucleotide sequence are transferred.
  • Sugarcane plants into the Cry1Ab-02 nucleotide sequence sugarcane plants transformed into the Cry1Ab+Vip3A nucleotide sequence, sugarcane plants transformed into the Cry1Ac-01 nucleotide sequence, and transferred into the Cry1Ac-02-Cry1Ie nucleotide sequence
  • the sugarcane plants and the sugarcane plants transferred into the Cry1Ab/Ac nucleotide sequence were only slightly damaged, and the naked eye could hardly distinguish the feeding traces of the millet, and the leaf damage rate was below 2%.
  • the sugarcane plant transformed into the Cry1Ab-01 nucleotide sequence, the sugarcane plant transformed into the Cry1Ab-02 nucleotide sequence, the sugarcane plant transformed into the Cry1Ab+Vip3A nucleotide sequence, and the Cry1Ac-01 nucleotide were transferred.
  • the sequence of sugarcane plants, sugarcane plants transformed into the Cry1Ac-02-Cry1Ie nucleotide sequence, and sugarcane plants transformed into the Cry1Ab/Ac nucleotide sequence all showed high activity against the mulberry, which was sufficient for the ash mites. Growth produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the ash ash it is also possible to reduce the occurrence of diseases on the sugar cane, and greatly improve the yield and quality of sugar cane.
  • Sorghum plants transfected into the Cry1Ab-01 nucleotide sequence Sorghum plants transfected with the Cry1Ab-02 nucleotide sequence, sorghum plants transfected with the Cry1Ac-01 nucleotide sequence, and transfected into Cry1Ac-02-Cry1Ie nucleoside Sorghum plants with acid sequence, sorghum plants transfected with Cry1Ab/Ac nucleotide sequence, sorghum plants transfected with Cry1Ab+Vip3A nucleotide sequence, wild-type sorghum plants and sorghum plants identified by Taqman as non-transgenic (expanded young leaves)
  • the fresh leaves are rinsed with sterile water and the water on the leaves is blotted dry with gauze, then the sorghum leaves are cut into strips of about 1 cm x 2 cm, and one piece of the cut long strips is placed in a circle.
  • total score 100 ⁇ Mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / Total number of insects) +10 ⁇ (negative control number of insects / total number of insects)] +100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S37, S38 and S39) transfected into the Cry1Ab-01 nucleotide sequence were transformed into a total of 3 transformation event lines (S40, S41 and S42) of the Cry1Ab-02 nucleotide sequence.
  • a total of 3 transformation event lines (S43, S44 and S45) transfected into the Cry1Ab+Vip3A nucleotide sequence were transferred to the Cry1Ac-01 nucleotide sequence for a total of 3 transformation event lines (S46, S47 and S48).
  • Table 3 indicate that sorghum plants transfected with the Cry1Ab-01 nucleotide sequence, sorghum plants transfected with the Cry1Ab-02 nucleotide sequence, sorghum plants transfected with the Cry1Ab+Vip3A nucleotide sequence, and transferred to Cry1Ac-01 Sorghum plants with nucleotide sequence, sorghum plants transfected with Cry1Ac-02-Cry1Ie nucleotide sequence and sorghum plants transfected with Cry1Ab/Ac nucleotide sequence have good insecticidal effect on millet ash, The average mortality rate of cockroaches is basically above 90%, and some even reach 100%. The total score of resistance is about 290 points, and some even reach a maximum of 300 points. The sorghum plants and wild type identified by Taqman as non-transgenic. The total resistance score of sorghum plants is generally around 20 points.
  • the sorghum plant of the nucleotide sequence of 01, the sorghum plant transformed into the nucleotide sequence of Cry1Ac-02-Cry1Ie, and the sorghum plant transformed into the nucleotide sequence of Cry1Ab/Ac are generally only slightly damaged, and the corn is almost indistinguishable from the naked eye.
  • the damage rate of the ash is less than 1%.
  • the sorghum plant transformed into the Cry1Ab-01 nucleotide sequence was transferred.
  • Sequence sorghum plants, sorghum plants transfected with the Cry1Ac-02-Cry1Ie nucleotide sequence, and sorghum plants transfected with the Cry1Ab/Ac nucleotide sequence all showed high anti-mulg activity, which was sufficient for the ash mites Growth produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the ash mites it is also possible to reduce the occurrence of diseases on sorghum and greatly improve the yield and quality of sorghum.
  • the millet plant transformed into the Cry1Ab-01 nucleotide sequence, the millet plant transferred into the Cry1Ab-02 nucleotide sequence, the millet plant transferred into the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-02-Cry1Ie nucleoside were transferred.
  • the fresh leaves are rinsed with sterile water and the water on the leaves is blotted dry with gauze, then the millet leaves are cut into strips of about 1 cm x 2 cm, and one piece of cut long strips is placed in a circle.
  • total score 100 ⁇ Mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / Total number of insects) +10 ⁇ (negative control number of insects / total number of insects)] +100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S55, S56 and S57) transfected into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence for a total of 3 transformation event lines (S58, S59 and S60).
  • a total of 3 transformation event lines (S61, S62 and S63) transfected into the Cry1Ab+Vip3A nucleotide sequence were transferred into the Cry1Ac-01 nucleotide sequence for a total of 3 transformation event lines (S64, S65 and S66).
  • the results in Table 4 indicate that the millet plant transferred into the Cry1Ab-01 nucleotide sequence, the millet plant transferred into the Cry1Ab-02 nucleotide sequence, the millet plant transformed into the Cry1Ab+Vip3A nucleotide sequence, and transferred to Cry1Ac-01
  • the nucleotide sequence of the millet plant, the millet plant transformed into the Cry1Ac-02-Cry1Ie nucleotide sequence, and the millet plant transformed into the Cry1Ab/Ac nucleotide sequence have good insecticidal effects on the millet ash, and the gray ash
  • the average mortality rate of cockroaches is mostly over 90%, and some even reach 100%.
  • the total score of resistance is about 290 points, and some even reach a maximum of 300 points.
  • the non-transgenic millet plants and wild type identified by Taqman The total score of resistance of millet plants is generally around 20 points.
  • Fig. 6 indicate that the millet plant transformed with the Cry1Ab-01 nucleotide sequence, the millet plant transferred into the Cry1Ab-02 nucleotide sequence, and the Cry1Ab+Vip3A nucleotide sequence were transferred to the wild-type millet plant.
  • the control effect is almost 100%, and very few surviving larvae basically stop developing.
  • the larvae After 3 days, the larvae are still basically in the initial hatching state. Very low vitality, and the millet plant transferred to the Cry1Ab-01 nucleotide sequence, the millet plant transferred to the Cry1Ab-02 nucleotide sequence, the millet plant transferred to the Cry1Ab+Vip3A nucleotide sequence, and transferred to Cry1Ac
  • the millet plant of the -01 nucleotide sequence, the millet plant transferred to the Cry1Ac-02-Cry1Ie nucleotide sequence, and the millet plant transferred to the Cry1Ab/Ac nucleotide sequence are generally only slightly damaged, and are almost indistinguishable to the naked eye.
  • the leaf damage rate of the millet ash is less than 2%.
  • the millet plant transformed into the Cry1Ab-01 nucleotide sequence, the millet plant transformed into the Cry1Ab-02 nucleotide sequence, the millet plant transformed into the Cry1Ab+Vip3A nucleotide sequence, and the Cry1Ac-01 nucleotide were transferred.
  • the sequence of the millet plant, the millet plant transferred into the Cry1Ac-02-Cry1Ie nucleotide sequence, and the millet plant transformed into the Cry1Ab/Ac nucleotide sequence all showed high activity against the millworm, which was sufficient for the millet ash Growth produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the ash it is also possible to reduce the occurrence of diseases on the millet and greatly improve the yield and quality of the millet.
  • the above experimental results also showed that the maize plant transformed into the Cry1Ab-01 nucleotide sequence, the maize plant transformed into the Cry1Ab-02 nucleotide sequence, the maize plant transformed into the Cry1Ac-01 nucleotide sequence, and transferred to Cry1Ac-02- a maize plant having a Cry1Ie nucleotide sequence, a maize plant transformed with a Cry1Ab/Ac nucleotide sequence, a maize plant transformed with a Cry1Ab+Vip3A nucleotide sequence; a sorghum plant transformed into a Cry1Ab-01 nucleotide sequence, transferred into A sorghum plant of the Cry1Ab-02 nucleotide sequence, a sorghum plant transformed into a Cry1Ac-01 nucleotide sequence, a sorghum plant transformed into a Cry1Ac-02-Cry1Ie nucleotide sequence, and a sorghum
  • the Cry1A protein of the present invention includes, but is not limited to, the Cry1Ab protein and its fusion protein, Cry1Ac protein and Cry1Ab/Ac protein of the amino acid sequence given in the specific embodiment, and the transgenic plant can also produce at least one second different from the Cry1A protein.
  • Insecticidal proteins such as Vip-like proteins, Cry-like proteins.
  • the use of the insecticidal protein of the present invention controls the C. sinensis pest by producing a Cry1A protein capable of killing the ash mites in the plant; and the agricultural control method, the chemical control method and the physical control method used in the prior art Compared with the invention, the plant protects the whole growth period and the whole plant to prevent the damage of the worm and the pest, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economical.

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Abstract

La présente invention concerne des utilisations d'une protéine insecticide. Ce procédé de lutte contre Chilo infuscatellus, un insecte nuisible, consiste à mettre en contact Chilo infuscatellus avec la protéine Cry1A. La présente invention permet de lutter contre Chilo infuscatellus à l'aide de la protéine Cry1A qui est produite in vivo par une plante et qui est capable de tuer Chilo infuscatellus. Par comparaison avec l'art antérieur qui utilise des procédés de prévention agricole, chimique et physique, la présente invention protège l'intégralité de la plante tout au long de sa période de croissance pour empêcher tout dommage dû à Chilo infuscatellus, ne pollue pas, ne laisse pas de résidus, produit ses effets de façon stable et aboutie, et est simple, pratique et économique.
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