WO2016184387A1 - Utilisation d'une protéine pesticide - Google Patents

Utilisation d'une protéine pesticide Download PDF

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WO2016184387A1
WO2016184387A1 PCT/CN2016/082464 CN2016082464W WO2016184387A1 WO 2016184387 A1 WO2016184387 A1 WO 2016184387A1 CN 2016082464 W CN2016082464 W CN 2016082464W WO 2016184387 A1 WO2016184387 A1 WO 2016184387A1
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sorghum
vip3a
plant
protein
nucleotide sequence
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PCT/CN2016/082464
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English (en)
Chinese (zh)
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张爱红
杨旭
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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 WO2016184387A1 publication Critical patent/WO2016184387A1/fr
Priority to PH12017502083A priority Critical patent/PH12017502083A1/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
    • 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
    • A01H1/123—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide 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
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a Vip3A protein to control a plant of the sorghum sorghum by expression in a plant.
  • Chilo sacchariphagus is a Lepidoptera, a genus. Also known as the sugarcane strip, commonly known as the sorghum heartworm. It is mainly distributed in East Asia, South Asia and the Indian Ocean, including China, India, Indonesia and Malaysia. It is widely distributed in China, and occurs in most parts of Northeast China, North China, East China and South China. Can be harmful to corn, sorghum, millet, hemp, sugar cane and other crops. In the northern dryland areas, mainly crops such as corn, sorghum and millet are often mixed with corn mash, causing dead seedlings; it is also the main pest on sugarcane in the south.
  • the sorghum stalks are damaging the heart leaves in the early stage, while the stalks and leaf sheaths can be eaten in the later stage, which hinders the nutrient transport and makes the stalks susceptible to wind breaking. It has a serious impact on production, usually up to 10%-40%.
  • the main control methods commonly used are: agricultural control, chemical control and physical control.
  • Agricultural control is the comprehensive coordinated management of the multi-factors of the whole farmland ecosystem, regulating crops, pests, environmental factors, and creating 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. Because agricultural control must obey the requirements of crop layout and increase production, the application has certain limitations and cannot be used as an emergency measure. It seems to be powerless when the sorghum strip breaks out.
  • Chemical control that is, pesticide control
  • sorghum sorghum is very important for grasping the period of prevention and treatment. 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 larva breaks into the stalk.
  • the current chemical control methods are mainly medicines. Liquid spray and application of poisonous soil.
  • 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 pest adults, uses light at close range, uses waves at a long distance, attracts insects close to each other, and has certain effects on the prevention and control of adult cockroaches;
  • the insecticidal 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 larger .
  • Vip3A insecticidal protein is one of many insecticidal proteins and is a specific protein produced by Bacillus cereus.
  • the Vip3A protein has a toxic effect on sensitive insects by activating apoptosis-type programmed cell death.
  • the Vip3A protein is hydrolyzed into four major protein products in the insect gut, of which only one protein hydrolysate (33KD) is the toxic core structure of the Vip3A protein.
  • the Vip3A protein binds to the midgut epithelial cells of sensitive insects, initiates programmed cell death, and causes the dissolution of midgut epithelial cells leading to insect death. It does not cause any symptoms to non-sensitive insects and does not cause apoptosis and dissolution of midgut epithelial cells.
  • Plants transfected with the Vip3A gene have been shown to be resistant to Lepidoptera pests such as Lepidoptera, such as the genus Lepidoptera, Spodoptera frugiperda, Euphorbia gracilis, and Spodoptera frugiperda.
  • Lepidoptera such as the genus Lepidoptera, Spodoptera frugiperda, Euphorbia gracilis, and Spodoptera frugiperda.
  • Lepidoptera such as the genus Lepidoptera, Spodoptera frugiperda, Euphorbia gracilis, and Spodoptera frugiperda.
  • sorghum there has been no control of sorghum by producing transgenic plants expressing the Vip3A protein. Report on the hazards of plants.
  • An object of the present invention is to provide a use of a pesticidal protein, for the first time, to provide a method for controlling plant damage by using a transgenic plant expressing a Vip3A protein, and effectively overcoming the prior art agricultural control, chemical control and physics Technical defects such as prevention and control.
  • the present invention provides a method of controlling a sorghum pest, comprising contacting a sorghum worm with at least a Vip3A protein.
  • the Vip3A protein is present in a host cell that produces at least the Vip3A protein, and the sorghum pest is at least in contact with the Vip3A protein by ingesting the host cell.
  • the Vip3A protein is present in a bacterium or a transgenic plant which produces at least the Vip3A protein, and the sorghum scorpion pest is contacted with at least the Vip3A protein by ingesting the bacterium or the tissue of the transgenic plant. The growth of the sorghum larvae is then inhibited and/or caused to death, in order to achieve control of the 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 plant is derived from corn, sugar cane, sorghum, millet, hemp or glutinous rice.
  • Plants containing a polynucleotide encoding the Vip3A protein can also be planted prior to the contacting step.
  • the amino acid sequence of the Vip3A protein has the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
  • the nucleotide sequence of the Vip3A protein has the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
  • the plant may further comprise at least one second nucleic acid different from the nucleotide encoding the Vip3A protein.
  • the second nucleic acid encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase or a peroxidase.
  • the second nucleic acid encodes a Cry1Ab protein.
  • amino acid sequence of the Cry1Ab protein has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 11.
  • the second nucleic acid has the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 12.
  • the second nucleic acid is a dsRNA that inhibits important genes in the target insect pest.
  • the present invention also provides a use of a Vip3A protein for controlling a sorghum pest.
  • the Vip3A protein is used in combination with a Cry-like insecticidal protein when used to control a sorghum pest.
  • the present invention also provides a method for producing a plant which controls a pestle of sorghum.
  • the method comprises introducing a polynucleotide sequence encoding a Vip3A protein into the genome of the plant.
  • the method further comprises introducing into the genome of the plant a polynucleotide sequence encoding a Cry-like insecticidal protein.
  • 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 Vip3A 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 larvae 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 Vip3A protein. Or plants with increased plant yield.
  • the polynucleotide of the plant propagule further comprises a polynucleotide sequence encoding a Cry-like insecticidal protein.
  • the invention provides a transgenic plant for controlling a sorghum pest, comprising a nucleic acid encoding a Vip3A protein.
  • the transgenic plant further comprises a nucleic acid encoding a Cry-like insecticidal protein.
  • the invention provides a pesticidal composition for controlling a sorghum pest, comprising a bacterium encoding a Vip3A protein.
  • the bacterium further encodes a Cry-like insecticidal protein
  • the pesticidal composition further comprises a second bacterium encoding a Cry-like insecticidal protein.
  • 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 sorghum scorpion pest is in contact with at least the Vip3A 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 Vip3A protein by ingesting plant tissues, and all or part of the sorghum larvae are inhibited from growing and/or causing death after 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 propagules containing a polynucleotide sequence encoding a Vip3A protein, which are controlled by a sorghum pest and/or a sorghum pest are naturally infested with non-transgenic
  • the 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 Vip3A protein on sorghum can be independent and not attenuated and/or disappeared by other substances that can "control” and/or “control” the mites.
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a Vip3A protein
  • a Vip3A protein and/or another substance that can control the pests of the sorghum The presence of the other substance neither affects the "control” and/or “control” effect of the Vip3A protein on sorghum, nor does it result in complete and/or partial "control” and/or “control” effects.
  • sorghum pests and/or sorghum pests such as genetically modified Any tissue of a plant (containing a polynucleotide sequence encoding a Vip3A protein) is present in a dead sorghum pest, and/or a sorghum pest that inhibits growth growth thereon, and/or with a non-transgenic wild-type plant
  • the method and/or use of the present invention is achieved by having attenuated plant damage, i.e., by contacting the Vip3A protein with at least a pestle of the sorghum, to achieve a method and/or use for controlling the pests of the sorghum.
  • the expression of the Vip3A protein in a transgenic plant may be accompanied by one or more Expression of 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 (the first parent) can express the Vip3A protein by genetic engineering
  • the second plant (the second parent) can express the Cry-like insecticidal protein and/or the Vip-like insecticidal protein by genetic engineering operations.
  • 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 caused by stolons, such as corn borer and stem borer.
  • sorghum sorghum is equivalent to corn borer and stem 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 that are also the genus Mothaceae are only 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 can make a certain Bt gene It has an insect resistance effect.
  • insects of different families and even different families have different sensitivities to the same Bt protein.
  • the Vip3A gene showed anti-insect activity against the Chilo suppressalis and the Asian corn borer Ostrinia furnacalis, but there was no insect resistance to the Plodia interpunctella and the European corn borer Ostrinia nubilalis. .
  • the above four pests belong to the family Lepidoptera, but the same kind of Bt protein has different resistance effects to the four species of the moth.
  • European corn borer and Asian corn borer are classified in the same species as the genus Corydalis (the same genus), but their response to the same Bt protein is quite different, which further explains Bt.
  • the way 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 nucleus and plastid and mitochondrial genomes.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • To express a protein in vivo one strand of DNA is typically transcribed into a complementary strand of mRNA that is used as a template to translate the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes to the Vip3A gene of the present invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Vip3A 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 a nucleic acid is divided When each nucleotide of a subunit 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.
  • sequence having insect resistance activity and hybridizing under stringent conditions to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 of the present invention is included in the present invention.
  • These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or end ratios compared to full length proteins). Deletions), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • the "variant” or “variation” refers to encoding the same protein or encoding a pesticidal 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 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.
  • an antibody to a pesticidal protein disclosed and claimed herein can be used to identify and isolate a Vip3A protein from a protein mixture.
  • 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 Vip3A protein includes, but is not limited to, SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: Amino acid sequences having a certain homology to the amino acid sequence are 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.
  • transgenic plants producing the Vip3A protein include, but are not limited to, a COT102 transgenic cotton event and/or a plant material comprising a COT102 transgenic cotton event (as described in CN1004395507 C), a COT202 transgenic cotton event and/or inclusion Plant material of the COT202 transgenic cotton event (as described in CN1886513 A), or MIR162 transgenic maize event and/or plant material comprising the MIR162 transgenic cotton event (as described in CN101548011 A), all of which can implement the invention
  • Method and/or use i.e., by contacting at least a Vip3A protein with a sorghum scorpion pest to achieve a method and/or use for controlling a sorghum pest, more specifically, the Vip3A protein is present in a transgenic plant that produces at least the Vip3A protein
  • the sorghum scorpion pest is at least in contact with the Vip3A protein by ingesting the tissue of the
  • 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 Vip3A 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 (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 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 so that the resulting translation product is translated with the desired protein
  • the relationship of the open reading frame is in accordance with the reading frame.
  • 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 Vip3A protein is toxic to the sorghum cockroach pest.
  • 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 Vip3A 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 Vip3A 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 Vip3A protein may have the amino acid sequence shown by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 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 Vip3A 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), benzoin resistance genes (such as Pmph gene), glyphosate resistance gene (such as EPSPS gene), bromoxynil resistance gene, sulfonylurea resistance gene, resistance gene to herbicide tortoise, resistance gene to cyanamide Or a resistance gene of a glutamine synthetase inhibitor (such as PPT), thereby obtaining a transgenic plant having both high insecticidal activity and herbicide resistance.
  • oxalic acid Phospho-resistant genes such as bar gene, pat gene
  • benzoin resistance genes such as Pmph gene
  • glyphosate resistance gene such as EPSPS gene
  • bromoxynil resistance gene sulfonylurea resistance gene
  • a foreign DNA is introduced into a plant, such as a gene encoding the Vip3A 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, such as agricultural control, chemical control and physical control, through external effects, ie, external factors; and the present invention controls sorghum strips by producing Vip3A protein in plants which can kill sorghum sorghum.
  • the pests are controlled by internal factors.
  • the frequency-vibration insecticidal lamp used in the prior art not only needs to clean the dirt of the high-voltage power grid every day, but also cannot be used in thunderstorm days; the invention makes the Vip3A protein express in the plant body, effectively overcomes the frequency vibration type killing
  • the effect of the insect lamp is affected by external factors, and the control effect of the transgenic plant (Vip3A protein) of the invention is stable and consistent at different locations, at different times, and in different genetic backgrounds.
  • the method for controlling mites and pests used in the prior art has an effect that is incomplete and only serves to alleviate the effect; and the transgenic plant of the present invention (Vip3A protein) can cause a large number of deaths of the larvae of the mites, and a small amount of The developmental progress of the surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and had stopped developing, and could not survive in the natural environment of the field, while the transgenic plants were generally only slightly damaged.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Vip3A 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 DBN100002 containing a Vip3A 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.
  • Cry1Ab insecticidal protein (615 amino acids), as shown in SEQ ID NO: 7 in the Sequence Listing; Cry1Ab nucleotide sequence (1848 nucleotides) encoding the amino acid sequence corresponding to the Cry1Ab insecticidal protein , as shown in SEQ ID NO: 8 in the Sequence Listing.
  • the amino acid sequence of the Cry1Ab+Vip3A insecticidal protein (1436 amino acids), as shown in SEQ ID NO: 9 in the Sequence Listing; the Cry1Ab+Vip3A nucleotide sequence encoding the amino acid sequence corresponding to the Cry1Ab+Vip3A insecticidal protein ( 4311 nucleotides), as shown in SEQ ID NO: 10 in the Sequence Listing; wherein the amino acid sequence of Vip3A insecticidal protein (788 amino acids) is as shown in SEQ ID NO: 5 in the Sequence Listing; The Vip3A nucleotide sequence (2364 nucleotides) of the amino acid sequence of the Vip3A insecticidal protein, as shown in SEQ ID NO: 6 in the sequence listing; the amino acid sequence of the Cry1Ab effective fragment (648 amino acids), as in the sequence listing ID NO: 11; a nucleotide sequence (1944 nucleotides) encoding a Cry1Ab effective fragment corresponding to
  • the Vip3A-01 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing), the Vip3A-02 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing), the Cry1Ab core
  • the nucleotide sequence (as shown in SEQ ID NO: 8 in the Sequence Listing) and the Cry1Ab + Vip3A nucleotide sequence (as shown in SEQ ID NO: 10 in the Sequence Listing) were synthesized by Nanjing Kingsray Biotechnology Co., Ltd.;
  • the 5' end of the synthesized Vip3A-01 nucleotide sequence (SEQ ID NO: 2) is also ligated with a ScaI cleavage site, and the Vip3A-01 nucleotide sequence (SEQ ID NO: 2) is 3' The SpeI cleavage site is also ligated to the end; the 5' end of the synthesized Vip3A-02 nucleotide sequence (SEQ ID
  • the 5' end of the synthesized Cry1Ab nucleotide sequence (SEQ ID NO: 8) is further ligated with an NcoI cleavage site, and the 3' end of the Cry1Ab nucleotide sequence (SEQ ID NO: 8) is also ligated BamHI cleavage site.
  • the synthetic Vip3A-01 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • FIG. 1 wherein Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation density
  • the coding SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; Vip3A-01 is the Vip3A-01 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) in LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g / L, agar 15 g / L, adjusted to pH 7.5 with NaOH) overnight growth.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured overnight at 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. .
  • 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 positive clone was verified by sequencing, and the result showed that the Vip3A-01 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 2 in the sequence listing.
  • the synthesized Vip3A-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Vip3A-02 was Vip3A-02. Nucleotide sequence (SEQ ID NO: 4).
  • the correct insertion of the Vip3A-02 nucleotide sequence in the recombinant cloning vector DBN02-T was confirmed by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab nucleus was synthesized according to the above method for constructing the recombinant cloning vector DBN01-T.
  • the nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein the Cry1Ab was a Cry1Ab nucleotide sequence (SEQ ID NO: 8).
  • the Cry1Ab nucleotide sequence in the recombinant cloning vector DBN03-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab+Vip3A nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein Cry1Ab+Vip3A was Cry1Ab+Vip3A.
  • Nucleotide sequence SEQ ID NO: 10
  • the Cry1Ab+Vip3A nucleotide sequence was correctly inserted into the recombinant cloning vector DBN04-T 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 ScaI and SpeI, respectively, and the cut Vip3A-01 nucleotide sequence fragment was inserted. Between the ScaI and SpeI 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 DBN100002 is constructed.
  • FIG. 2 Kanamycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 13); Vip3A-01: Vip3A-01 nucleotide sequence (SEQ ID NO: 2); Nos : terminator of the nopaline synthase gene (SEQ ID NO: 14); Hpt: hygromycin phosphotransferase gene (SEQ ID NO: 15); LB: left border).
  • the recombinant expression vector DBN100002 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant expression vector DBN100002), water bath at 42 ° C 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 ScaI and SpeI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the ScaI and SpeI sites of the recombinant expression vector DBN100002 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 2, that is, the Vip3A-01 nucleotide sequence.
  • the Vip3A-01 nucleotide sequence and the Cry1Ab nucleotide sequence excised from the recombinant cloning vectors DBN01-T and DBN03-T were digested with BamHI and inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100003.
  • the nucleotide sequence in the recombinant expression vector DBN100003 contains the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 8 in the sequence listing, namely the Vip3A-01 nucleotide sequence and the Cry1Ab nucleoside.
  • the acid sequence, the Vip3A-01 nucleotide sequence and the Cry1Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the Vip3A-02 nucleotide sequence excised from the ScaI and SpeI recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100740.
  • the nucleotide sequence in the recombinant expression vector DBN100740 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 4 in the sequence listing, that is, the Vip3A-02 nucleotide sequence, and the Vip3A-02 nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Ab+Vip3A nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN04-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100736.
  • the nucleotide sequence in the recombinant expression vector DBN100736 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, that is, the Cry1Ab+Vip3A nucleotide sequence, and the Cry1Ab+Vip3A nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the recombinant expression vectors DBN100002 and DBN100003, DBN100740 and DBN100736, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours.
  • Agrobacterium LBA4404 Invitrgen, Chicago, USA, CAT: 18313-015
  • the callus 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 in the recombinant expression vectors DBN100002 and DBN100003, DBN100740 and DBN100736 including the promoter sequence of the maize Ubiquitin gene, the Vip3A-01 nucleotide sequence, the Vip3A-02 nucleotide sequence, the Cry1Ab+Vip3A nucleotide sequence, The Cry1Ab nucleotide sequence, the Hpt gene and the Nos terminator sequence were transferred into the maize genome, and the maize plant into which the Vip3A-01 nucleotide sequence was transferred, the Vip3A-02 nucleotide sequence, and the maize plant were transferred.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can administer Vip3A-01 nucleotide sequence, Vip3A-02 nucleotide
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the grown transformed callus is selected (step 4: selection step).
  • a selection agent hygromycin
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8, resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, M. 50 mg/L, vegetal gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel 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 according to the transgenic vector of the present invention.
  • a sorghum plant transformed into the Vip3A-01 nucleotide sequence a sorghum plant transformed into the Vip3A-02 nucleotide sequence, a sorghum plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and transferred into Sorghum plants of the Cry1Ab+Vip3A nucleotide sequence; and wild-type sorghum plants were used as controls.
  • 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 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 Vip3A-01 nucleotide sequence a sugarcane plant transformed into the Vip3A-02 nucleotide sequence, a sugarcane plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and transformed into Cry1Ab+Vip3A were obtained.
  • Maize plants transfected with Vip3A-01 nucleotide sequence, maize plants transfected with Vip3A-02 nucleotide sequence, maize plants transfected with Vip3A-01-Cry1Ab nucleotide sequence, and transfected into Cry1Ab+Vip3A nucleoside About 100 mg of the leaves of the acid sequence of the maize plants were used as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of the Vip3A gene and the Cry1Ab gene was detected by Taqman probe fluorescent quantitative PCR. At the same time, 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 Take the maize plant transformed into the Vip3A-01 nucleotide sequence, the maize plant into which the Vip3A-02 nucleotide sequence is transferred, the maize plant into which the Vip3A-01-Cry1Ab nucleotide sequence is transferred, and transfer to Cry1Ab+.
  • the maize plants of the Vip3A nucleotide sequence and the leaves of the wild-type maize plants were each 100 mg, and respectively ground in a mortar with liquid nitrogen, 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:
  • Probe 1 CTCCTGAGCCCCGAGCTGATTAACACC as shown in SEQ ID NO: 18 in the Sequence Listing;
  • Probe 2 CAGCGCCTTGACCACAGCTATCCC is shown in SEQ ID NO: 21 in the Sequence Listing;
  • Primer 5 CCGAGCTTCATCGACTACTTCAAC is shown in SEQ ID NO: 22 in the Sequence Listing;
  • Probe 3 CCACCGGCATCAAGGACATCATGAAC is shown in SEQ ID NO: 24 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:
  • the results showed that the Vip3A-01 nucleotide sequence, the Vip3A-02 nucleotide sequence, the Vip3A-01-Cry1Ab nucleotide sequence and the Cry1Ab+Vip3A nucleotide sequence were integrated into the genome of the tested maize plants.
  • the maize plant transformed into the Vip3A-01 nucleotide sequence, the maize plant transformed into the Vip3A-02 nucleotide sequence, the maize plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and the Cry1Ab+Vip3A nucleoside A single copy of the transgenic maize plant was obtained from the acid sequence of the maize plants.
  • 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 Vip3A-01 nucleotide sequence, Vip3A-02 nucleotide sequence, Vip3A-01-Cry1Ab nucleotide sequence and Cry1Ab+Vip3A nucleotide sequence were integrated into the tested sorghum plants and sugarcane plants, respectively.
  • the sorghum plant transformed into the Vip3A-01 nucleotide sequence the sorghum plant transformed into the Vip3A-02 nucleotide sequence, the sorghum plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and transferred to the Cry1Ab +Vip3A nucleotide sequence
  • Sorghum plants sugarcane plants transfected with Vip3A-01 nucleotide sequence, sugarcane plants transfected with Vip3A-02 nucleotide sequence, sugarcane plants transfected with Vip3A-01-Cry1Ab nucleotide sequence and transferred into Cry1Ab+Vip3A nucleus
  • Each of the sugarcane plants of the nucleotide sequence obtained a single copy of the transgenic plant.
  • Maize plants transformed with the Vip3A-01 nucleotide sequence maize plants transfected with the Vip3A-02 nucleotide sequence, maize plants transfected with the Vip3A-01-Cry1Ab nucleotide sequence, and transfected into Cry1Ab+Vip3A nucleotides Sequence of maize plants; sorghum plants transfected with the Vip3A-01 nucleotide sequence, sorghum plants transfected with the Vip3A-02 nucleotide sequence, sorghum plants transfected with the Vip3A-01-Cry1Ab nucleotide sequence, and transferred to Cry1Ab+ Sorghum plants of the Vip3A-01 nucleotide sequence; sugarcane plants transformed into the Vip3A-01 nucleotide sequence, sugarcane plants transformed into the Vip3A-02 nucleotide sequence, and sugarcane transformed into the Vip3A-01-Cry1Ab nucleotide sequence Plants and sugar
  • 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 ⁇ (number of negative control insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • Table 1 The results in Table 1 indicate that maize plants transfected with the Vip3A-01 nucleotide sequence, maize plants transfected with the Vip3A-02 nucleotide sequence, maize plants transfected with the Vip3A-01-Cry1Ab nucleotide sequence, and transformed into Cry1Ab
  • the corn plants with +Vip3A nucleotide sequence have good insecticidal effects on sorghum, and the average mortality of sorghum scorpion is basically above 70%, and some even reach 100%.
  • the average score is about 270 points, and some even reach 299 points; and the total score of resistance of non-transgenic corn plants and wild-type corn plants identified by Taqman is generally about 20 points.
  • the maize plant with a nucleotide sequence and the maize plant transformed into the Cry1Ab+Vip3A nucleotide sequence 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 simultaneously Very weak vitality; and maize plants that have been transferred to the Vip3A-01 nucleotide sequence, maize plants that have been transferred to the Vip3A-02 nucleotide sequence, maize plants that have been transferred to the Vip3A-01-Cry1Ab nucleotide sequence, and transferred to Cry1Ab
  • the maize plants with the +Vip3A nucleotide sequence were only slightly damaged, and the feeding area was small, and the leaf damage rate was below 10%.
  • the maize plant transformed into the Vip3A-01 nucleotide sequence the maize plant transformed into the Vip3A-02 nucleotide sequence, the maize plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and the Cry1Ab+Vip3A nucleus were transformed.
  • Maize plants with a glycoside sequence have been shown to be highly resistant to sorghum, which is sufficient to have an adverse effect on the growth of sorghum sorghum and thus to 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 corn and greatly improve the yield and quality of the corn.
  • 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 ⁇ (number of negative control insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • NMM2 non-transgenic
  • CK2 wild type
  • the results in Table 2 indicate that sugarcane plants transformed into the Vip3A-01 nucleotide sequence, sugarcane plants transformed into the Vip3A-02 nucleotide sequence, sugarcane plants transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and transferred into Cry1Ab
  • the +Vip3A nucleotide sequence of sugarcane plants has good insecticidal effect on sorghum sorghum.
  • the average mortality of sorghum sorghum is basically above 70%, and some even reach 100%.
  • the average score is above 250 points, and some even reach 299 points; and the total score of resistance of non-transgenic sugarcane plants and wild-type sugarcane plants identified by Taqman is generally about 50 points.
  • sugarcane plants transformed into Vip3A-01 nucleotide sequence, sugarcane plants transformed into Vip3A-02 nucleotide sequence, sugarcane plants transformed into Vip3A-01-Cry1Ab nucleotide sequence and transgenic Sugarcane plants that enter the Cry1Ab+Vip3A nucleotide sequence can cause sorghum A large number of insects die, and significantly inhibit the development of a small number of surviving larvae, growth retardation, while showing weak vitality, and transferred to the Vip3A-01 nucleotide sequence of sugarcane plants, transferred to Vip3A-02 nucleoside
  • the acid sequence of sugarcane plants, the sugarcane plants transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and the sugarcane plants transformed into the Cry1Ab+Vip3A nucleotide sequence were only slightly damaged, and the feeding area was small, and the leaf damage was The rate is below 20%.
  • the sugarcane plant transformed into the Vip3A-01 nucleotide sequence, the sugarcane plant transformed into the Vip3A-02 nucleotide sequence, the sugarcane plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and the Cry1Ab+Vip3A nucleus were transferred.
  • the sugarcane plants of the glycoside sequence showed high activity against sorghum, which was sufficient to have an adverse effect on the growth of sorghum and thus allowed to 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 with Vip3A-01 nucleotide sequence Sorghum plants transfected with Vip3A-02 nucleotide sequence, sorghum plants transfected with Vip3A-01-Cry1Ab nucleotide sequence, and transferred into Cry1Ab+Vip3A nucleoside Acidic sorghum plants, wild-type sorghum plants, and fresh leaves identified by Taqman as non-transgenic sorghum plants (expanded young leaves), rinsed with sterile water and blotted with water on the leaves, then sorghum leaves Into a long strip of about 1cm ⁇ 2cm, take a piece of cut long strips of leaves into the moisturizing filter paper on the bottom of a round plastic dish, put 10 sorghum strips (initial hatching larvae) in each dish.
  • 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 ⁇ (number of negative control insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S25, S26 and S27) transfected into the Vip3A-01 nucleotide sequence were transferred to the Vip3A-02 nucleotide sequence for a total of 3 transformation event lines (S28, S29 and S30).
  • a total of 3 transformation event lines (S31, S32 and S33) transfected into the Vip3A-01-Cry1Ab nucleotide sequence, and 3 transformation event lines (S34, S35 and S36) transfected into the Cry1Ab+Vip3A nucleotide sequence A total of 1 strain of non-transgenic (NGM3) was identified by Taqman, and 1 strain of wild type (CK3); 3 strains were selected from each strain, and each plant was repeated 6 times. The results are shown in Table 3.
  • the results in Table 3 indicate that sorghum plants transformed into the Vip3A-01 nucleotide sequence, sorghum plants transfected with the Vip3A-02 nucleotide sequence, sorghum plants transfected with the Vip3A-01-Cry1Ab nucleotide sequence, and transferred into Cry1Ab
  • the sorghum plant with +Vip3A nucleotide sequence has good insecticidal effect on sorghum sorghum.
  • the average mortality rate of sorghum sorghum is basically above 60%, and some even reach 100%.
  • the average score is above 240 points, and some even reach 299 points; and the total score of resistance of sorghum plants and wild-type sorghum plants identified by Taqman as non-transgenic is generally about 20 points.
  • Sorghum plants transferred to the Vip3A-01 nucleotide sequence Sorghum plants transferred to the Vip3A-01 nucleotide sequence, sorghum plants transfected with the Vip3A-02 nucleotide sequence, sorghum plants transfected with the Vip3A-01-Cry1Ab nucleotide sequence and transgenic plants were compared with wild-type sorghum plants.
  • Sorghum plants that enter the Cry1Ab+Vip3A nucleotide sequence can cause sorghum A large number of insects die, and significantly inhibit the development of a small number of surviving larvae, growth retardation, while showing very weak vitality, and transferred to the Vip3A-01 nucleotide sequence of sorghum plants, transferred to Vip3A-02 nucleoside
  • the acid sequence of the sorghum plant, the transferred feeding area is small, the sorghum plant of the nucleotide sequence and the sorghum plant transformed into the Cry1Ab+Vip3A nucleotide sequence are generally only slightly damaged, and the feeding area is small, The blade damage rate is below 20%.
  • the sorghum plant transformed into the Vip3A-01 nucleotide sequence, the sorghum plant transformed into the Vip3A-02 nucleotide sequence, the sorghum plant transformed into the Vip3A-01-Cry1Ab nucleotide sequence, and the Cry1Ab+Vip3A nucleus were transferred.
  • the sorghum plants of the glucosinolate sequence showed high activity against sorghum, which was sufficient to have an adverse effect on the growth of sorghum sorghum so that it could 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 Vip3A protein of the present invention includes, but is not limited to, the Vip3A 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 Vip3A protein, such as a Vip-like protein or a Cry-like protein. protein.
  • the use of the insecticidal protein of the present invention controls the cockroach pests by producing a Vip3A protein capable of killing sorghum scorpion in the plant; and the agricultural control methods, chemical control methods and physical control methods 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

La présente invention concerne l'utilisation d'une protéine pesticide pour lutter contre le Chilo sacchariphagus, par : mise en contact du Chilo sacchariphagus au moins avec la protéine Vip3A et lutte contre le Chilo sacchariphagus par production de la protéine Vip3A dans des plantes pouvant tuer le Chilo sacchariphagus. La présente invention peut protéger la plante entière pendant toute la période de croissance.
PCT/CN2016/082464 2015-05-20 2016-05-18 Utilisation d'une protéine pesticide WO2016184387A1 (fr)

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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
CN105660674B (zh) * 2016-01-04 2018-06-26 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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