WO2016184397A1 - Application d'une protéine insecticide - Google Patents

Application d'une protéine insecticide Download PDF

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WO2016184397A1
WO2016184397A1 PCT/CN2016/082588 CN2016082588W WO2016184397A1 WO 2016184397 A1 WO2016184397 A1 WO 2016184397A1 CN 2016082588 W CN2016082588 W CN 2016082588W WO 2016184397 A1 WO2016184397 A1 WO 2016184397A1
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sorghum
plant
protein
cry2ab
plants
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PCT/CN2016/082588
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English (en)
Chinese (zh)
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杨旭
张爱红
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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 WO2016184397A1 publication Critical patent/WO2016184397A1/fr
Priority to PH12017501966A priority Critical patent/PH12017501966A1/en

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    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/127—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for insect resistance
    • 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
    • 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
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • A01G2/00—Vegetative propagation

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a Cry2Ab protein to control a plant of a sorghum sorghum plant by expression in a plant.
  • Chilo sacchariphagus belongs to the order Lepidoptera, the genus Coleoptera. Also known as sugarcane strips, or sorghum borer. It is mainly distributed in East Asia, South Asia, Southeast Asia and the Indian Ocean. It is widely distributed in China, including in Northeast China, North China, East China and most of southern China. Sorghum can be used to damage corn, sorghum, millet, hemp, sugar cane and other crops. In the northern dryland areas, it mainly harms crops such as corn, sorghum and millet, and often mixes with corn mash, causing dead seedlings; it is also the main pest on sugar cane in the south.
  • the main control methods commonly used are: agricultural control, chemical control and physical control.
  • Agricultural control is to comprehensively coordinate the multi-factors of the whole farmland ecosystem, regulate crops, pests and environmental factors, and create a farmland ecological environment that is conducive to crop growth and is not conducive to the occurrence of sorghum.
  • the sorghum or corn stalks are treated to reduce the overwintering insect source. Straw treatment can be carried out by crushing, burning, manure, mashing, mud sealing and other methods. Since agricultural control must comply with the requirements of crop layout and production increase, its application has certain limitations and cannot be used as an emergency measure. Therefore, it appears to be powerless when the sorghum strip breaks out.
  • Chemical control that is, pesticide control
  • sorghum is the use of chemical pesticides to kill pests. It is an important part of the comprehensive management of sorghum, which is characterized by rapid, convenient, simple and high economic benefits, especially in the occurrence of sorghum. The case is an essential emergency measure.
  • sorghum sorghum is very important for grasping its control period. The best period for the treatment is before the hatching period of the egg to the larvae of the larvae. Otherwise, it will be difficult to achieve the purpose of prevention and control after the young larvae break into the stalk.
  • chemical control methods are mainly liquid spray and application of poisonous soil. 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.
  • Cry2Ab insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystal protein produced by Bacillus thuringiensis.
  • the Cry2Ab protein is ingested by insects into the midgut, and the protoxin is dissolved in the alkaline pH environment of the insect midgut.
  • the N- and C-termini of the protein are digested with alkaline protease to convert the protoxin into an active fragment; the active fragment binds to the receptor on the membrane of the insect midgut epithelial membrane and is inserted into the intestinal membrane, causing perforation of the cell membrane and destroying the osmotic pressure inside and outside the cell membrane. Changes and pH balances, etc., disrupt the insect's digestive process and ultimately lead to its death.
  • Plants transgenic to the Cry2Ab gene have been shown to be resistant to the pests of the genus Spodoptera frugiperda. However, there have been no reports on the control of plant damage by the production of transgenic plants expressing the Cry2Ab protein.
  • 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 sorghum sorghum to plants by producing a transgenic plant expressing the Cry2Ab protein, and effectively overcome the prior art agricultural control, chemical control and Technical defects such as physical control.
  • the present invention provides a method of controlling a sorghum pest, comprising contacting a sorghum pest with at least a Cry2Ab protein.
  • the Cry2Ab protein is present in a host cell that produces at least the Cry2Ab protein, and the sorghum bark pest is in contact with at least the Cry2Ab protein by ingesting the host cell.
  • the Cry2Ab protein is present in a bacterium or a transgenic plant which produces at least the Cry2Ab protein, and the sorghum scorpion pest is contacted with at least the Cry2Ab protein by the tissue ingesting the bacterium or the transgenic plant, after contact
  • the growth of sorghum scorpion pests is inhibited and/or caused to death, in order to achieve control of sorghum mites.
  • the transgenic plant may be in any growth period; the tissue of the transgenic plant is a root, a leaf, a stem, a fruit, a tassel, an ear, a flower bud, an anther or a filament; Control of hazardous plants does not change due to changes in planting location and/or planting time.
  • the plants are from grass crops such as corn, sorghum, sugar cane, millet, hemp or glutinous rice.
  • the step prior to the contacting step is to plant a plant containing a polynucleotide encoding the Cry2Ab protein.
  • the amino acid sequence of the Cry2Ab protein has the amino acid sequence set forth in SEQ ID NO: 1.
  • the nucleotide sequence of the Cry2Ab 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 Cry2Ab 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 Cry1A.105 protein.
  • amino acid sequence of the Cry1A.105 protein has the amino acid sequence shown in SEQ ID NO: 3.
  • the second nucleotide 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 a Cry2Ab protein for controlling a sorghum pest.
  • the present invention also provides a method of producing a plant for controlling a sorghum pest, comprising introducing a polynucleotide sequence encoding a Cry2Ab protein into the genome of the plant.
  • the present invention also provides a method of producing a plant propagule for controlling a sorghum 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 a Cry2Ab protein.
  • the present invention also provides a method for cultivating a plant for controlling a sorghum pest, comprising:
  • the plants are grown under conditions in which the artificially inoculated sorghum pests and/or sorghum scorpion pests are naturally harmful, and the plants are harvested with reduced plant damage and/or compared with other plants that do not have the polynucleotide sequence encoding the Cry2Ab 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 Special tissues include, but are not limited to, roots, stems and leaves, for example: plants with roots as vegetative propagules including strawberries and sweet potatoes; stems as vegetative propagules Plants include sugar cane and potato (tubers); plants with leaves as vegetative propagules 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.
  • 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.
  • control and/or "control” in the present invention means that the sorghum scorpion pest is in contact with at least the Cry2Ab protein, and the growth of the sorghum sorghum pest is inhibited and/or causes death after contact. Further, the sorghum larvae are at least in contact with the Cry2Ab protein by ingesting plant tissues, and all or part of the sorghum larvae are inhibited from growing and/or causing 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 Cry2Ab protein that control mites and pests are artificially inoculated with sorghum pests and/or sorghum pests, and non-transgenic 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.
  • control and / or “control” effects of the Cry2Ab protein on sorghum can be independent and not attenuated and/or disappeared by other substances that can "control” and/or “control” the mites. .
  • tissue of a transgenic plant containing a polynucleotide sequence encoding a Cry2Ab protein
  • the Cry2Ab protein and/or another substance that can control the sorghum pest At the time, the presence of the other substance does not affect the "control” and/or “control” effect of the Cry2Ab protein on sorghum, nor does it cause the "control” and/or “control” effects to be complete and / Or partially achieved by the other substance, regardless of the Cry2Ab protein.
  • sorghum and/or sorghum pests such as natural hazards, such as Any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry2Ab protein) is present in a dead sorghum pest, and/or a sorghum pest, on 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 sorghum scorpion pest with at least the Cry2Ab protein to achieve a method and/or use for controlling mites.
  • expression of a Cry2Ab protein in a transgenic plant can be accompanied by fusion expression of one or more Cry-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 (the first parent) can express the Cry2Ab protein by genetic engineering operation
  • the second plant (the second parent) can express the Cry-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.
  • dsRNA double-stranded RNA
  • RNAi technology can be used to specifically knock out or silence the expression of a particular 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.
  • sorghum sorghum is equivalent to the mites and corn borer, it belongs to the order Lepidoptera, and there is a great difference 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 sorghum scorpion 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 three mites which are also the genus Mothaceae, are mainly harmful to rice, and rarely harm 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) is not resistant to insects effect.
  • the above four pests belong to the family Lepidoptera, but the same Bt protein shows 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 genus Corydalis (the same genus), but their response to the same Bt protein is quite different, which is more fully explained.
  • the way in which Bt proteins interact with enzymes and receptors 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 nuclear, 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 is generally present 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.
  • one strand of DNA is generally transcribed into a complementary strand of mRNA, which is used as a template to translate a 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 under stringent conditions to the Cry2Ab gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry2Ab 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". If two nucleic acid molecules are capable of hybridizing to each other with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other, then the two nucleic acid molecules are said to be "minimum degree of mutual Similarly, if two nucleic acid molecules are capable of hybridizing to each other with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other, the two nucleic acid molecules are said to be “complementary.” Deviation in complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence. This results in a stable double
  • 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, and then 2 x SSC, 0.1% SDS and 1 x, respectively. The membrane was washed once with SSC and 0.1% SDS.
  • 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 are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. %, 94%, 95%, 96%, 97%, 98%, 99% or more homologous.
  • 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 compared to full length proteins). Terminal 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 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 sequence or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, suitable for plant expression) Sequence), the length of the foregoing sequences 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 constructing 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 prepared using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically excise from the ends of these genes. Nucleotide. 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 produced by the most constant part of the protein and the most different part of the 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.
  • amino acid residues necessary for their activity and thus selected for unsubstituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique is in the molecule A mutation is introduced at a positively charged residue, and the insecticidal activity of the resulting mutant molecule is detected to determine 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 Cry2Ab 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. These sequences are typically greater than 78%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99%, similar to the sequences of the present invention. Preferred polynucleotides and proteins of the invention may also be defined according to a more specific range of identity and/or similarity.
  • sequences of the examples of the present invention are 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity.
  • the transgenic plants producing the Cry2Ab protein include, but are not limited to, the Mon89034 transgenic maize event and/or plant material comprising the Mon89034 transgenic maize event (as described in CN101495635A), the MON87751 transgenic soybean event, and/or comprise MON87751 Plant material for the transgenic soybean event (as described in USDA APHIS Unregulated Status Application 13-337-01p), or Mon15985 transgenic cotton event and/or plant material containing the Mon15985 transgenic cotton event (as described in CN101413028B), It is possible to carry out the method and/or use of the invention by contacting the pestle with at least the Cry2Ab protein to achieve a method and/or use for controlling the pests of the sorghum.
  • the methods and/or uses of the present invention can also be achieved by expressing the Cry2Ab protein in the above transgenic events in different plants. More specifically, the Cry2Ab protein is present in a transgenic plant that produces at least the Cry2Ab protein, the sorghum bark pest contacting at least the Cry2Ab protein by tissue ingesting the transgenic plant, the sorghum barium after contact Pest growth is inhibited and/or causes death to achieve control of plants that are damaging to sorghum.
  • 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 Cry2Ab 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.
  • the promoter expressible in the plant may be a tissue-specific promoter, ie the promoter is in some tissues of the plant, such as in green tissue than in other plants.
  • the level of expression of the coding sequence in the tissue is high (can be determined by routine RNA assays), such as the PEP carboxylase promoter.
  • the promoter expressible in the plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • 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 linked to the sequence of interest such that Transcription of the sequence of interest is controlled and regulated by the promoter.
  • “operably linked” means that the promoter is ligated to the sequence in a manner such that the resulting transcript is efficiently translated.
  • 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 sorghum pest.
  • the Cry2Ab protein is toxic to sorghum pests.
  • the plants of the present invention particularly maize, sugar cane and sorghum, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry2Ab protein, and the sorghum pest is in contact with the protein by feeding plant tissue. After exposure, the growth of pests is inhibited and/or caused to death. 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 sorghum pests targeted by the Cry2Ab 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 insect is mainly sorghum.
  • the Cry2Ab 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 Cry2Ab 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
  • a foreign DNA is introduced into a plant, such as a gene encoding the Cry2Ab protein or an expression cassette or a recombinant vector
  • conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-launch bombardment, Direct DNA uptake into protoplast, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the hazards of sorghum pests and diseases, such as agricultural control, chemical control and physical control, through external effects, ie, external factors; and the present invention controls sorghum strips by producing Cry2Ab protein capable of killing sorghum scorpion in plants.
  • the pests are controlled by internal factors.
  • the effect is stable.
  • Both agricultural control methods and physical control methods used in the prior art require the use of environmental conditions to control pests, and there are many variable factors; the present invention is to express the Cry2Ab protein in plants, thereby effectively avoiding environmental conditions.
  • Unstable defects, and the control effect of the transgenic plants (Cry2Ab protein) of the present invention are stable and consistent at different locations, at different times, and in different genetic backgrounds.
  • the effect is thorough.
  • the method used in the prior art for controlling cockroaches and pests is not thorough, only To reduce the effect; the transgenic plant (Cry2Ab protein) of the present invention can cause a large number of deaths of the newly hatched larvae, and greatly inhibit the development of a small number of surviving larvae. After 3 days, the larvae are still in the initial hatching state, Significantly stunted, and has stopped developing, unable to survive in the natural environment of the field, while transgenic plants are generally only slightly damaged.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Cry2Ab 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 a Cry2Ab nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the damage of leaves of a transgenic maize plant inoculated with sorghum sorghum using the insecticidal protein of the present invention.
  • Cry2Ab insecticidal protein (634 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; Cry2Ab nucleotide sequence (1905 nucleotides) encoding the amino acid sequence corresponding to the Cry2Ab insecticidal protein , as shown in SEQ ID NO: 2 in the Sequence Listing.
  • Cry1A.105 insecticidal protein (1177 amino acids), as shown in SEQ ID NO: 3 in the Sequence Listing; encoding the Cry1A.105 nucleotide sequence corresponding to the amino acid sequence of the Cry1A.105 insecticidal protein ( 3534 nucleotides) as shown in SEQ ID NO: 4 in the Sequence Listing.
  • the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing) and the Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing) are manufactured by Nanjing Kingsray Biotech Synthetic; synthetically synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 2) also has a NcoI cleavage site at the 5' end and a SpeI cleavage site at the 3' end; the synthesized Cry1A
  • the 5' end of the .105 nucleotide sequence (SEQ ID NO: 4) is also ligated with a NcoI cleavage site, and the 3' end is also ligated with a Hind III cleavage site.
  • the synthetic Cry2Ab 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.
  • FIG. 1 wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; and T7 is the T7 RNA polymerase promoter; Cry2Ab is the Cry2Ab 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 Cry2Ab nucleotide sequence inserted into the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 2 in the sequence listing.
  • the nucleotide sequence, ie the Cry2Ab nucleotide sequence, is correctly inserted.
  • the synthesized Cry1A.105 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1A.105 was Cry1A.105.
  • Nucleotide sequence SEQ ID NO: 4
  • Enzyme digestion and sequencing confirmed Cry1A.105 in recombinant cloning vector DBN02-T The nucleotide sequence is inserted correctly.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases Nco I and Spe I, respectively, and the cut Cry2Ab nucleotide sequence fragment was inserted. Between the Nco I and Spe I sites of the expression vector DBNBC-01, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100744 is constructed, and the construction process thereof is shown in FIG.
  • Kan kanamycin gene
  • RB right border
  • Ubi maize Ubiquitin (ubiquitin) gene promoter
  • Cry2Ab Cry2Ab nucleotide sequence (SEQ ID NO: 2); Nos: rouge Terminator of the base synthase gene (SEQ ID NO: 6); Hpt: hygromycin phosphotransferase gene (SEQ ID NO: 7); LB: left border).
  • the recombinant expression vector DBN100744 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 DBN100744), 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 Nco I and Hind III, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100744 between Nco I and Spe I sites was sequenced.
  • the Cry1A.105 nucleotide sequence excised by the Nco I and Hind III digestion cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100745.
  • the nucleotide sequence in the recombinant expression vector DBN100745 contains the nucleotide sequence shown in 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.
  • Nco I and Hind III, Nco I and Spe I were respectively digested into the Cry2Ab nucleotide sequence and the Cry1A.105 core cut by the recombinant cloning vectors DBN01-T and DBN02-T, respectively.
  • the nucleotide sequence was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100029.
  • the nucleotide sequence in the recombinant expression vector DBN100029 contains the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing, namely the Cry2Ab nucleotide sequence and the Cry1A.105 nucleoside. Acid sequence, said The Cry2Ab nucleotide sequence and the Cry1A.105 nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100744, DBN100745 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 DBN100744, DBN100745 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 promoter sequence of maize Ubiquitin gene, Cry2Ab nucleotide sequence, Cry1A.105 nucleotide sequence, Hpt gene and Nos terminator sequence
  • DBN100744, DBN100745 and DBN100029 was transferred to maize chromosome
  • a maize plant transformed with the Cry2Ab nucleotide sequence, a maize plant transformed with the Cry1A.105 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.
  • 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 / L, plant gel 3g / L, pH 5.8) at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250mg / L), no addition of plant transformant selection agent (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 5 g/L, hygromycin 50 mg/L, sucrose 30 g/L, 2, Incubation of 4-dichlorophenoxyacetic 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 MS rooting medium) Recycled plants.
  • 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, plant coagulation)
  • the gel was cultured at 25 ° C for 3 g/L, pH 5.8).
  • 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. 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 (30-50 mg/L) according to the transgenic vector of the present invention.
  • a sorghum plant transformed into a Cry2Ab nucleotide sequence a sorghum plant transformed into a Cry1A.105 nucleotide sequence, and a sorghum plant transformed into a 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 segment of the 5-7 cm length 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 wound tissue was clipped, placed on a filter paper and dried on a clean bench until the surface of the material was dry, and the material was transferred to a differentiation medium containing 500 mg/L cephalosporin and 30-50 mg/L hygromycin.
  • the medium was changed every 2 weeks, during which the contaminated calli was removed, and when 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 Cry2Ab nucleotide sequence a sugarcane plant transformed into the Cry1A.105 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.
  • Maize plants transfected with Cry2Ab nucleotide sequence, maize plants transfected with Cry1A.105 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 Cry2Ab gene and the Cry1A.105 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.
  • Step 11 The maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transfected with the Cry1A.105 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. Refer to its product manual for the law;
  • 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. Values; fluorescent PCR primers and probe sequences are as follows:
  • Probe 1 CGCTGAGCTGACGGGTCTGCAAG, as shown in SEQ ID NO: 10 in the Sequence Listing;
  • Primer 4 GTTCTGGACGGCGAAGAGTG, as shown in SEQ ID NO: 12 in the Sequence Listing;
  • Probe 2 TGAACAGCGCCCTGACCACCG, as shown in SEQ ID NO: 13 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 was stored in an amber tube at 4°C.
  • the PCR reaction conditions are:
  • Jade A single copy of the transgenic maize plant was obtained from the rice plants, the maize plants transformed into the Cry1A.105 nucleotide sequence, and the maize plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence.
  • Transgenic sorghum plants and transgenic sugarcane plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan. The results showed that the Cry2Ab nucleotide sequence, Cry1A.105 nucleotide sequence and Cry1A.105-Cry2Ab nucleotide sequence were integrated into the genome of the tested sorghum and sugarcane plants, respectively, and transferred to the Cry2Ab nucleus.
  • Sorghum plants of the nucleotide sequence Sorghum plants of the nucleotide sequence, sorghum plants transfected with the Cry1A.105 nucleotide sequence, sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, sugarcane plants transfected with the Cry2Ab nucleotide sequence, and transferred into Cry1A.
  • a single copy of the transgenic plant was obtained from the sugarcane plant of the 105 nucleotide sequence and the sugarcane plant transformed into the nucleotide sequence of the Cry1A.105-Cry2Ab.
  • a maize plant transformed into a Cry2Ab nucleotide sequence a maize plant transformed into a Cry1A.105 nucleotide sequence, a maize plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, and a sorghum plant transformed into a Cry2Ab nucleotide sequence a sorghum plant transformed into a Cry1A.105 nucleotide sequence, a sorghum plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, a sugarcane plant transformed into a Cry2Ab nucleotide sequence, and a Cry1A.105 nucleotide sequence
  • Sugarcane plants, sugarcane plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence corresponding wild-type maize plants, sorghum plants and sugarcane plants, and non-transgenic maize plants, sorghum plants and sugarcane plants identified by Taqman
  • Maize plants transfected with Cry2Ab nucleotide sequence, maize plants transfected with Cry1A.105 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, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22-26 ° C.
  • total resistance 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 Cry2Ab nucleotide sequence, and transferred to the Cry1A.105 nucleotide sequence for a total of 3 transformation event lines (S4, S5 and S6), transferred to Cry1A.105-Cry2Ab nucleotide sequence total 3
  • One transformation event strain (S7, S8 and S9), identified by Taqman as a non-transgenic (NGM1) strain, and one wild type (CK1); 3 strains from each strain Test, repeat 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 transfected with the Cry2Ab nucleotide sequence, maize plants transfected with the Cry1A.105 nucleotide sequence, and maize plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence have sorghum
  • the better insecticidal effect, the average mortality rate of sorghum scorpion is basically over 70%, and the total score of resistance is basically about 250 points; and the non-transgenic corn plants and wild plants identified by Taqman The total resistance score of type corn plants is generally around 20 minutes.
  • Maize plants can cause a large number of deaths of the newly hatched larvae, and greatly inhibit the development of a small number of surviving larvae, stunting, and exhibit extremely weak vitality; and corn plants that are transferred into the Cry2Ab nucleotide sequence, Maize plants transformed with the Cry1A.105 nucleotide sequence and maize plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the feeding area was small, and the leaf damage rate was below 10%. .
  • the maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transformed with the Cry1A.105 nucleotide sequence, and the maize plants transformed with the Cry1A.105-Cry2Ab nucleotide sequence all showed high resistance to sorghum Activity, this activity is sufficient to have an adverse effect on the growth of sorghum sorghum so that it can be controlled in the field. At the same time by controlling the sorghum It is also possible to reduce the occurrence of diseases on corn and greatly increase the yield and quality of corn.
  • the sugarcane plants transformed into the Cry2Ab nucleotide sequence, the sugarcane plants transformed into the Cry1A.105 nucleotide sequence, the sugarcane plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type sugarcane plants and the 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, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22-26 ° C.
  • total resistance 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 (S10, S11 and S12) transfected into the Cry2Ab nucleotide sequence, and transferred to the Cry1A.105 nucleotide sequence for a total of 3 transformation event lines (S13, S14 and S15), transferred into A total of three transformation event lines (S16, S17 and S18) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM2) strain by Taqman and one wild type (CK2). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 2.
  • the results in Table 2 indicate that the sugarcane plant transformed into the Cry2Ab nucleotide sequence was transferred to the Cry1A.105 nucleotide sequence.
  • the sugarcane plants and the sugarcane plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence have good insecticidal effects on the sorghum sorghum.
  • the average mortality of sorghum sorghum is basically above 80%, and the total resistance is
  • the scores are also basically around 250 points; the total resistance score of sugarcane plants and wild-type sugarcane plants identified by Taqman as non-transgenic is generally less than 50 points.
  • sugarcane plants transformed into the Cry2Ab nucleotide sequence can cause sorghum A large number of larvae larvae died, and the development of a small number of surviving larvae was significantly inhibited, growth retardation, while showing weak vitality, and the sugarcane plants transferred to the Cry2Ab nucleotide sequence were transferred to the Cry1A.105 nucleus.
  • the sugarcane plants with the nucleotide sequence and the sugarcane plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the feeding area was small, and the leaf damage rate was below 10%.
  • sugarcane plants transformed into the Cry2Ab nucleotide sequence sugarcane plants transformed into the Cry1A.105 nucleotide sequence, and sugarcane plants transformed into the Cry1A.105-Cry2Ab nucleotide sequence all showed high resistance to sorghum Activity, this activity is sufficient to have an adverse effect on the growth of sorghum sorghum so that it can be controlled in the field.
  • by controlling the drill collar of the sorghum strip it is also possible to reduce the occurrence of diseases on the sugarcane and greatly improve the yield and quality of the sugarcane.
  • Sorghum plants transfected into the Cry2Ab nucleotide sequence 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 The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic petri dish, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22-26 ° C.
  • total resistance 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 (S19, S20 and S21) transfected into the Cry2Ab nucleotide sequence, and transferred to the Cry1A.105 nucleotide sequence for a total of 3 transformation event lines (S22, S23 and S24), transferred into A total of three transformation event lines (S25, S26 and S27) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM3) strain by Taqman, and one wild type (CK3). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 3.
  • Table 3 The results in Table 3 indicate that sorghum plants transformed into the Cry2Ab nucleotide sequence, sorghum plants transfected with the Cry1A.105 nucleotide sequence, and sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence have sorghum sorghum
  • the total resistance score of plants is generally around 20 minutes.
  • sorghum plants transformed into the Cry2Ab nucleotide sequence can cause sorghum A large number of larvae larvae die, and the development of a small number of surviving larvae is significantly inhibited, growth retardation, while showing very weak vitality, and transferred to the Cry2Ab nucleotide sequence of sorghum plants, transferred to the Cry1A.105 nucleus
  • the sorghum plants of the nucleotide sequence and the sorghum plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the feeding area was small, and the leaf damage rate was below 10%.
  • the sorghum plant transformed into the Cry2Ab nucleotide sequence, the sorghum plant transformed into the Cry1A.105 nucleotide sequence, and the sorghum plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence all showed high resistance to sorghum Activity, this activity is sufficient to have an adverse effect on the growth of sorghum sorghum so that it can be controlled in the field.
  • by controlling the drill collar of the sorghum strip it is also possible to reduce the occurrence of diseases on the sorghum and greatly improve the yield and quality of the sorghum.
  • the Cry2Ab protein of the present invention includes, but is not limited to, the Cry2Ab 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 Cry2Ab protein, such as Cry1Fa protein, Cry1A.105. Protein or Vip3A protein, etc.
  • the use of the insecticidal protein of the present invention controls the sorghum scorpion pest by producing a Cry2Ab protein capable of killing sorghum scorpion 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 infestation of the cockroach pest, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economic.

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Abstract

Cette invention concerne l'application d'une protéine insecticide. Le procédé de lutte contre l'insecte nuisible Chilo sacchariphagus selon l'invention comprend : la mise en contact de l'insecte nuisible Chilo sacchariphagus avec au moins une protéine Cry2Ab. L'invention lutte contre l'insecte nuisible Chilo sacchariphagus par génération de protéines Cry2Ab capables de détruire le Chilo sacchariphagus dans le corps d'une plante. Comparativement à un procédé de prévention et de lutte de type agricole, à un procédé de prévention et de lutte de type chimique et à un procédé de prévention et de lutte de type physique utilisés dans l'état de la technique, la présente invention protège la plante entière pendant toute sa période de croissance de façon à prévenir et à lutter contre les dommages occasionnés par l'insecte Chilo sacchariphagus, ne génère pas de pollution et ne laisse aucun résidu, a un effet stable et complet et est simple, pratique et économique.
PCT/CN2016/082588 2015-05-20 2016-05-19 Application d'une protéine insecticide WO2016184397A1 (fr)

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CN106591352B (zh) * 2016-11-21 2020-05-05 北京大北农科技集团股份有限公司 杀虫蛋白组合及其管理昆虫抗性的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101268094A (zh) * 2005-08-31 2008-09-17 孟山都技术有限公司 编码杀虫蛋白的核苷酸序列
CN101495635A (zh) * 2006-05-26 2009-07-29 孟山都技术有限公司 对应于转基因事件mon89034的玉米植物和种子及其检测和使用方法
CN104286014A (zh) * 2014-08-27 2015-01-21 北京大北农科技集团股份有限公司 杀虫蛋白的用途
CN104920425A (zh) * 2015-05-20 2015-09-23 北京大北农科技集团股份有限公司 杀虫蛋白的用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101268094A (zh) * 2005-08-31 2008-09-17 孟山都技术有限公司 编码杀虫蛋白的核苷酸序列
CN101495635A (zh) * 2006-05-26 2009-07-29 孟山都技术有限公司 对应于转基因事件mon89034的玉米植物和种子及其检测和使用方法
CN104286014A (zh) * 2014-08-27 2015-01-21 北京大北农科技集团股份有限公司 杀虫蛋白的用途
CN104920425A (zh) * 2015-05-20 2015-09-23 北京大北农科技集团股份有限公司 杀虫蛋白的用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, TANLIN: "Prevention and Control Technique for Common Maize Diseases and Insect Pests", AGRICULTURAL TECHNOLOGY & EQUIPMENT, 25 January 2011 (2011-01-25) *
WU, LILI: "Construction of Recombinant Plasmids Carrying Cry2ab and Its Expression in Bacillus Subtilis", AGRICULTURE, CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 January 2013 (2013-01-15) *

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