WO2016101684A1 - Utilisations d'une protéine insecticide - Google Patents

Utilisations d'une protéine insecticide Download PDF

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WO2016101684A1
WO2016101684A1 PCT/CN2015/092007 CN2015092007W WO2016101684A1 WO 2016101684 A1 WO2016101684 A1 WO 2016101684A1 CN 2015092007 W CN2015092007 W CN 2015092007W WO 2016101684 A1 WO2016101684 A1 WO 2016101684A1
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protein
cry1a
plant
mites
plants
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PCT/CN2015/092007
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English (en)
Chinese (zh)
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杨旭
丁德荣
张欣馨
康越景
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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 WO2016101684A1 publication Critical patent/WO2016101684A1/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
    • 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

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a CrylA.105 protein to control a plant of a millet ash by expressing it in a plant.
  • Agricultural control is a comprehensive 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 integrated pest management. 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 most widely used is 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 control effects on pest control;
  • the lamp needs to clean the dirt on the high-voltage power grid every day, 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 in the operation; in addition, the one-time investment of installing the lamp is large.
  • Cry1A.105 insecticidal protein is one of many insecticidal proteins and is a chimeric protein derived from Cry1Ab protein, Cry1Ac protein and Cry1Fa protein, respectively.
  • Plants transgenic with the Cry1A.105 gene have been shown to be resistant to Lepidoptera pests such as corn borer, cotton bollworm, and fall armyworm. However, there has been no control of millet by producing transgenic plants expressing the Cry1A.105 protein. Report on the hazards of ash to plants.
  • 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 occur strictly Heavy, Xiagu District is light.
  • the ash ash was once the main pest in North China and South China, mainly affecting sorghum, millet 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.105 protein, and effectively overcoming the prior art agricultural control, chemical control and Technical defects such as physical control.
  • the present invention provides a method of controlling a pest of the mites, comprising contacting the mites pest with at least the Cry1A.105 protein.
  • the Cry1A.105 protein is present in a host cell that produces at least the CrylA.105 protein, and the millet worm is in contact with at least the Cry1A.105 protein by ingesting the host cell.
  • the Cry1A.105 protein is present in a bacterium or a transgenic plant which produces at least the Cry1A.105 protein, and the millet worm is at least in contact with the Cry1A by ingesting the bacterium or the tissue of the transgenic plant The .105 protein is contacted, and the growth of the larvae pest is inhibited and/or caused to death after the exposure to achieve control of the plant against the ash mites.
  • the bacterium may be a wild bacterium and/or a recombinant bacterium capable of producing the Cry1A.105 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 to plant a plant containing a polynucleotide encoding the Cry1A.105 protein.
  • the amino acid sequence of the Cry1A.105 protein has the amino acid sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence of the Cry1A.105 protein has the nucleotide sequence shown in SEQ ID NO: 2.
  • the plant may further comprise at least one second nucleotide different from the nucleotide encoding the Cry1A.105 protein.
  • 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 Cry2Ab protein.
  • amino acid sequence of the Cry2Ab protein has the amino acid sequence shown in SEQ ID NO: 3.
  • nucleotide sequence of the Cry2Ab protein has the nucleotide sequence shown in SEQ ID NO: 4.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • the present invention also provides a use of the Cry1A.105 protein for controlling a pest of the worm.
  • the present invention also provides a method of producing a plant for controlling a mites pest, including A polynucleotide sequence encoding a Cry1A.105 protein is introduced 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 plant propagule containing a polynucleotide sequence encoding the Cry1A.105 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 compared to other plants that do not have the polynucleotide sequence encoding the Cry1A.105 protein. And/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.
  • 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 mites are at least in contact with the Cry1A.105 protein, and the growth of the larvae is inhibited and/or causes death after contact. Further, the larvae of the larvae are in contact with at least the Cry1A.105 protein by ingesting plant tissues, and all or part of the growth of the larvae pests 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 containing a polynucleotide sequence encoding a Cry1A.105 protein that control the pests of the mites are inoculated under conditions in which the artificially inoculated pests of the mites and/or the mites are naturally harmful.
  • 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.
  • the "control” and / or “control” effects of the Cry1A.105 protein on the ash can be independently independent and not attenuated by the presence of other substances that can "control” and/or “control” the worms and/or Or disappear.
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a Cry1A.105 protein
  • Cry1A.105 protein and/or another species that can control the mites pest a substance in which the presence of the other substance neither affects the "control” and/or “control” effect of the Cry1A.105 protein on the ash, nor does it result in the "control” and
  • the "or control” effect is completely and / or partially achieved by the other substance, regardless of the Cry1A.105 protein.
  • any tissue of a plant (containing a polynucleotide sequence encoding a Cry1A.105 protein) is a dead mites pest, and/or a sphagnum pest that inhibits growth growth thereon, and/or a non-transgenic wild type Plants have reduced plant damage compared to the method and/or use of the present invention, i.e., by contacting the Cry1A.105 protein with at least the C. sinensis pest to achieve a method and/or use for controlling the mites.
  • expression of the Cry1A.105 protein in a transgenic plant may be accompanied by 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
  • the second plant second parent
  • Cry-like insecticidal protein and/or Vip-like insecticidal protein by genetic engineering operation.
  • 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.
  • the ash 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.
  • 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 is hybridized under stringent conditions to the Cry1A.105 gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A.105 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 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: 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 and hybridizing to SEQ ID NO: 2 of the present invention under stringent conditions are included in the present invention. 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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.105 protein includes, but is not limited to, SEQ ID NO: 1, and an amino acid sequence having a certain homology with the amino acid sequence shown by SEQ ID NO: 1 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 preferably 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.
  • the insecticidal activity or the insecticidal activity remaining means the insecticidal activity of the protein having a certain amino acid sequence with the amino acid sequence of SEQ ID NO: 1 (resistance total score, according to the fifth embodiment)
  • the method described herein is obtained by 80% or more, or 90% or more, or 92% or more, or 95% or more, or 98, of the insecticidal activity (total resistance score) of the Cry1A.105 protein represented by SEQ ID NO: 1. More than %, or 100%.
  • the nucleotide sequence of the Cry1A.105 protein is capable of encoding a Cry1A.105 protein having an insecticidal activity that satisfies the above requirements.
  • the transgenic plants producing the Cry1A.105 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) or the MON87751 transgenic soybean event and/or Plant material comprising the MON87751 genetically modified soybean event (as described in USDA APHIS Unregulated Status Application 13-337-01p), each of which can achieve the method and/or use of the present invention, ie, through the pestle of at least Cry1A. 105 protein contact to achieve a method and/or use for controlling millet mites.
  • the method and/or use of the present invention can also be achieved by expressing the Cry1A.105 protein in the above transgenic events in different plants. More specifically, the Cry1A.105 protein is present in a transgenic plant that produces at least the Cry1A.105 protein, the contact with at least the Cry1A.105 protein by contacting the tissue of the transgenic plant. Subsequent growth of the larvae pest is inhibited and/or causes death to achieve control of the plants against the ash mites.
  • Regulatory sequences in the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Cry1A.105 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
  • 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.105 protein is toxic to the mites.
  • the plants of the present invention particularly millet, sugar cane, sorghum and corn, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1A.105 protein, and the planting of plant tissues by the fungus In contact with the protein, the growth of the larvae is inhibited and/or causes death after contact. 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 can be substantially eliminated from chemistry or The need for a biocide (the chemical or biocide is an insecticide against the mites of the Cry1A.105 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. Different tests can be applied to determine the insecticidal effect of ICP in plants.
  • the target insects in the present invention are mainly milled ash.
  • the Cry1A.105 protein may have the amino acid sequence shown by SEQ ID NO: 1 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.105 protein of the present invention may also be expressed in a plant together with at least one protein encoding a herbicide resistance gene including, but not limited to, Glufosinate resistance gene (such as bar gene, pat gene), benzoin resistance gene (such as pmph gene), glyphosate resistance gene (such as EPSPS gene), bromoxynil resistance gene, sulfonate a resistance gene of a ureide resistance gene, a resistance gene against a herbicide, a tobacco, a resistance gene to a cyanamide or a glutamine synthetase inhibitor (such as PPT), thereby obtaining high insecticidal activity and Herbicide-resistant transgenic plants.
  • Glufosinate resistance gene such as bar gene, pat gene
  • benzoin resistance gene such as pmph gene
  • glyphosate resistance gene such as EPSPS gene
  • bromoxynil resistance gene sulfonate
  • exogenous DNA is introduced into a plant, such as a gene encoding the Cry1A.105 protein or an expression cassette or a recombinant vector
  • conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-emission Bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the harm of the mites and pests through external action, ie external factors, such as agricultural control, chemical control and physical control; and the invention is controlled by the production of Cry1A.105 protein in the plant capable of killing the ash mites.
  • the worms are pests, which 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 is to express the Cry1A.105 protein in plants, effectively overcoming the frequency vibration.
  • the effect of the insecticidal lamp is affected by external factors, and the control effect of the transgenic plant (Cry1A.105 protein) of the present invention is stable at different locations, at different times, and in different genetic backgrounds.
  • the method for controlling the mites of the larvae used in the prior art has an effect which is incomplete and only serves to alleviate the effect; and the transgenic plant of the present invention (Cry1A.105 protein) can cause a large number of deaths of the newly hatched larvae, and The development progress of a small number of surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and stopped developing, and could not survive in the natural environment of the field, while the transgenic plants were generally only affected. Minor damage.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing the Cry1A.105 nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 2 is a flow chart showing the construction of a recombinant expression vector DBN100745 containing the Cry1A.105 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.
  • Cry1A.105 insecticidal protein (1177 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; Cry1A.105 encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein Nucleotide sequence (3534 nucleotides) as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing) and the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing) are manufactured by Nanjing Kingsray Biotech Synthesized by the company; the 5' end of the synthesized Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also ligated with an NcoI cleavage site, the Cry1A.105 nucleotide sequence (SEQ ID NO: 2) a HindIII cleavage site is also ligated to the 3' end; the 5' end of the synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 4) is further ligated with an NcoI cleavage site, the Cry2Ab nucleotide sequence The 3' end of (SEQ ID NO: 4) is also ligated with a SpeI cleavage site.
  • the synthetic Cry1A.105 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).
  • Cry1A.105 is the Cry1A.105 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, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) was 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 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 synthesized Cry2Ab nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein the Cry2Ab was a Cry2Ab nucleotide sequence (SEQ ID NO: 4).
  • the Cry2Ab nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases NcoI and HindIII, respectively, and the cut Cry1A.105 nucleotide sequence fragment was inserted. Between the NcoI and HindIII sites of the expression vector DBNBC-01, using conventional enzyme digestion Methods Construction vectors are well known to those skilled in the art and constructed into a recombinant expression vector DBN100745.
  • the recombinant expression vector DBN100745 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 DBN100745), 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 plasmid was digested with restriction endonucleases NcoI and HindIII, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100745 between NcoI and HindIII was SEQ ID NO in the sequence listing.
  • the nucleotide sequence shown in 2 the Cry1A.105 nucleotide sequence.
  • the Cry2Ab nucleotide sequence excised from the recombinant cloning vector DBN02-T by NcoI and SpeI was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100744.
  • the nucleotide sequence in the recombinant expression vector DBN100744 contains the nucleotide sequence shown as SEQ ID NO: 4 in the sequence listing, that is, the Cry2Ab nucleotide sequence, and the Cry2Ab nucleotide sequence can be ligated. Ubi promoter and Nos terminator.
  • NcCI and HindIII, NcoI and SpeI were respectively inserted into the recombinant cloning vectors DBN01-T and DBN02-T, and the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence were inserted.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100029.
  • the nucleotide sequence in the recombinant expression vector DBN100029 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing, that is, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleoside.
  • the acid sequence, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100745, DBN100744 and DBN100029 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 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 and incubated at a temperature of 28 ° C, 200 rpm for 2 hours, applied to On a LB plate containing 50 mg/L of rifampicin and 100 mg/L of kanamycin until a positive monoclonal grows, pick a monoclonal culture and extract the plasmid with restriction endonuclease
  • the recombinant expression vectors DBN100745, DBN100744 and DBN100029 were digested
  • the immature embryo of the aseptically cultured maize variety Heisei 31 was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the second embodiment.
  • T-DNA including the promoter sequence of maize Ubiquitin gene, Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence, Hpt gene and Nos terminator sequence
  • DBN100745, DBN100744 and DBN100029 was transferred to maize chromosome
  • a maize plant transformed with the Cry1A.105 nucleotide sequence, a maize plant transformed with the Cry2Ab nucleotide sequence, and a maize plant transformed with the Cry1A.105-Cry2Ab nucleotide sequence were obtained; and the wild type maize plant was simultaneously obtained. as comparison.
  • Agrobacterium can express the Cry1A.105 nucleotide sequence, the Cry2
  • 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 3 g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. Cultivate at 28 ° C per day in the greenhouse After an hour, it was further cultured 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.
  • a sorghum plant transformed into the Cry1A.105 nucleotide sequence, a sorghum plant transformed into the Cry2Ab nucleotide sequence, and a sorghum plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence were obtained; 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 Cry1A.105 nucleotide sequence a sugarcane plant transformed into the Cry2Ab nucleotide sequence, and a sugarcane plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence were obtained; and the wild type sugarcane plant was used as the Control.
  • the conversion method refers to the Hebei University of Agriculture 2012 Master Wang Hanyu's dissertation on pages 9 to 10.
  • the mature seeds were immersed in 0.1% (v/v) Tween-20 solution, washed with 70% (v/v) ethanol, then transferred to 0.1% (w/v) HgCl 2 solution, and finally sterilized 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. Then, the callus after co-cultivation was transferred to MS callus induction medium (2,4-D 4.5 ⁇ mol/L, 2.25 ⁇ mol/L Kn, cephalosporin 500 mg/L), and cultured at 25 ° C in dark. Two weeks of healing the callus.
  • 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 into which the Cry1A.105 nucleotide sequence was transferred a millet plant into which the Cry2Ab nucleotide sequence was transferred, and a millet plant into which the Cry1A.105-Cry2Ab nucleotide sequence was transferred were obtained; and the wild-type millet plant was used as the plant. Control.
  • Maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry2Ab nucleotide sequence, and maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence were used as samples, respectively, using Qiagen
  • the DNeasy Plant Maxi Kit was used to extract the genomic DNA, and the copy number of the Cry1A.105 gene and the Cry2Ab gene was detected by Taqman probe fluorescent quantitative PCR.
  • the wild type corn plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.
  • the specific method for detecting the copy number of the Cry1A.105 gene and the Cry2Ab gene is as follows:
  • Step 11 The maize plants transformed with the Cry1A.105 nucleotide sequence, the maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, and the leaves of the wild-type maize plants were respectively taken. Each 100 mg was separately homogenized with liquid nitrogen in a mortar, 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 GTTCTGGACGGCGAAGAGTG is shown in SEQ ID NO: 9 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:
  • Transgenic sorghum plants, transgenic sugarcane plants and transgenic millet plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan. The results showed that the Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence and Cry1A.105-Cry2Ab nucleotide sequence were integrated into the genome of the tested sorghum, sugarcane and millet plants, respectively.
  • a maize plant transformed into a Cry1A.105 nucleotide sequence a maize plant transformed into a Cry2Ab nucleotide sequence, a maize plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, and a Cry1A.105 nucleotide sequence
  • Sorghum plants, sorghum plants transferred to the Cry2Ab nucleotide sequence, and sorghum plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence a sugarcane plant transformed into a Cry1A.105 nucleotide sequence, a sugarcane plant transformed into a Cry2Ab nucleotide sequence, a sugarcane plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, and a Cry1A.105 nucleotide sequence
  • Maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry2Ab nucleotide sequence, maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, wild-type maize plants and Taqman were identified as Fresh leaves of non-transgenic corn plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the corn leaves into strips of about 1 cm ⁇ 2 cm, take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • 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) transferred into the Cry1A.105 nucleotide sequence, and transferred to the Cry2Ab nucleotide sequence for a total of 3 transformation event lines (S4, S5 and S6), transferred to A total of three transformation event lines (S7, S8 and S9) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM1) strain by Taqman, and one wild type (CK1). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 1 and Figure 3.
  • Table 1 The results in Table 1 indicate that maize plants transferred to the Cry1A.105 nucleotide sequence were transferred to Cry1A.105-Cry2Ab
  • the nucleotide sequence of maize plants had good insecticidal effect on the millet, and the average mortality rate of the millet was more than 80%, and the total score of resistance was also about 280; and it was identified by Taqman as The total resistance score of non-transgenic corn plants and wild-type corn plants is generally around 20 minutes.
  • Maize plants transferred to the Cry2Ab nucleotide sequence showed no control effect on millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic corn identified by Taqman. Plants did not show differences compared to wild-type maize plants.
  • Sugarcane plants transfected into Cry1A.105 nucleotide sequence, sugarcane plants transfected into Cry2Ab nucleotide sequence, sugarcane plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, wild-type sugarcane plants and Taqman were identified as Fresh leaves of non-transgenic sugarcane plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the sugarcane leaves into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • 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).
  • the results in Figure 4 indicate that sugarcane plants transfected with the Cry1A.105 nucleotide sequence and sugarcane plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence can cause larvae of the larvae of the larvae compared to wild-type sugarcane plants. A large number of deaths, and greatly inhibited the development of a very small number of surviving larvae. After 3 days, the larvae were still in the initial hatching state, and showed extremely weak vitality, and transferred to the Cry1A.105 nucleotide sequence of sugarcane plants and transferred. The sugarcane plants entering the Cry1A.105-Cry2Ab 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 3%.
  • sugarcane plants which were transferred into the Cry1A.105 nucleotide sequence and sugarcane plants which were transferred into the Cry1A.105-Cry2Ab nucleotide sequence showed high activity against the mulberry, which was sufficient for the growth of the ash mites.
  • the adverse effects thus 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 Cry1A.105 nucleotide sequence, sorghum plants transfected with the Cry2Ab nucleotide sequence, sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, wild-type sorghum plants and Taqman were identified as Fresh leaves of non-transgenic sorghum plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the sorghum leaves into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting Long strips of leaves
  • 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 / insects) Total)] + 100 ⁇ (1 - blade damage rate).
  • the results in Table 3 indicate that the sorghum plants transfected with the Cry1A.105 nucleotide sequence and the sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence have good insecticidal effects on the ash mites, and the ash mites
  • the average mortality rate is about 90%, and the total score of resistance is also above 280 points.
  • the total resistance score of sorghum plants and wild-type sorghum plants identified by Taqman as non-transgenic is generally about 20 points.
  • the sorghum plants transferred to the Cry2Ab nucleotide sequence showed no control effect on the millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic identification by Taqman. There was no difference in sorghum plants compared to wild-type sorghum plants.
  • the sorghum plant transformed into the Cry1A.105 nucleotide sequence and the sorghum plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence showed high activity against the mulberry, which was sufficient for the growth of the ash mites.
  • the adverse effects thus 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 plants transformed with the Cry1A.105 nucleotide sequence, the millet plants transferred to the Cry2Ab nucleotide sequence, the millet plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type millet plants, and Taqman were identified as Fresh leaves of non-transgenic millet plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the leaves of the millet into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • 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 (S28, S29 and S30) transfected into the Cry1A.105 nucleotide sequence, and transferred to the Cry2Ab nucleotide sequence for a total of 3 transformation event lines (S31, S32 and S33), transferred to A total of 3 transformation event lines (S34, S35 and S36) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM4) strain by Taqman and 1 wild type (CK4). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 4 and Figure 6.
  • the results in Table 4 indicate that the millet plant transferred to the Cry1A.105 nucleotide sequence was transferred to Cry1A.105-Cry2Ab
  • the nucleotide sequence of the millet plant has good insecticidal effect on the millet, and the average mortality rate of the millet is about 90%, and the total resistance score is also above 280;
  • the total score of resistance of non-transgenic millet plants and wild-type millet plants is generally around 20 points.
  • the results in Figure 6 indicate that the millet plants transferred to the Cry1A.105 nucleotide sequence and the millet plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence can cause the larvae of the larvae of the larvae to be larvae compared to the wild-type millet plants. A large number of deaths, and greatly inhibited the development of a very small number of surviving larvae. After 3 days, the larvae were still in the initial hatching state, and showed extremely weak vitality, and the millet plants and the Cry1A.105 nucleotide sequence were transferred. The millet plants that entered the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the naked eye could hardly discern the feeding traces of the millet, and the leaf damage rate was below 5%.
  • the millet plants transferred to the Cry2Ab nucleotide sequence showed no control effect on the millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic millet identified by Taqman. Plants did not show differences compared to wild-type millet plants.
  • the control and control of the millet and the millet plant transferred into the Cry1A.105-Cry2Ab nucleotide sequence is apparently because the plant itself can produce the Cry1A.105 protein, so, as is well known to those skilled in the art, according to the Cry1A.105 protein
  • the same toxic effect on the ash mites can produce similar transgenic plants expressing the Cry1A.105 protein that can be used to control/control the damage of the ash.
  • the Cry1A.105 protein of the present invention includes, but is not limited to, the Cry1A.105 protein of the amino acid sequence given in the specific embodiment, and the transgenic plant can also produce at least one second insecticidal protein different from the Cry1A.105 protein, such as Vip-like protein, Cry-like protein.
  • the use of the insecticidal protein of the present invention controls the C. sinensis pest by producing a Cry1A.105 protein capable of killing the ash mites in the plant; the agricultural control method, the chemical control method and the physical control used in the prior art Compared with the method, the invention protects the plant from 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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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Biotechnology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

La présente invention concerne des utilisations d'une protéine insecticide. Le procédé de lutte contre Chilo infuscatellus, un insecte nuisible, consiste à mettre Chilo infuscatellus en contact avec la protéine Cry1A.105. La présente invention permet de lutter contre Chilo infuscatellus à l'aide de la protéine Cry1A.105 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 de façon à 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.
PCT/CN2015/092007 2014-12-22 2015-10-15 Utilisations d'une protéine insecticide WO2016101684A1 (fr)

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CN104522033B (zh) * 2014-12-22 2016-09-14 北京大北农科技集团股份有限公司 杀虫蛋白的用途
CN104824010B (zh) * 2015-05-20 2018-06-19 北京大北农科技集团股份有限公司 杀虫蛋白的用途
CN104886111B (zh) * 2015-05-20 2017-01-18 北京大北农科技集团股份有限公司 杀虫蛋白的用途

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