WO2016138819A1 - Utilisations d'une protéine insecticide - Google Patents

Utilisations d'une protéine insecticide Download PDF

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WO2016138819A1
WO2016138819A1 PCT/CN2016/074069 CN2016074069W WO2016138819A1 WO 2016138819 A1 WO2016138819 A1 WO 2016138819A1 CN 2016074069 W CN2016074069 W CN 2016074069W WO 2016138819 A1 WO2016138819 A1 WO 2016138819A1
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plant
protein
cry2ab
spodoptera litura
pest
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PCT/CN2016/074069
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English (en)
Chinese (zh)
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李建勇
杨旭
李梅
于彩虹
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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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Priority to AU2016228053A priority Critical patent/AU2016228053B2/en
Publication of WO2016138819A1 publication Critical patent/WO2016138819A1/fr
Priority to PH12017501559A priority patent/PH12017501559A1/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
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • 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 Cry2Ab protein to control a plant of Spodoptera litura by expression in a plant.
  • Spodoptera litura belongs to the family Lepidoptera, and is a omnivorous and gluttonous pest. It is a host of harmful hosts. In addition to corn and soybeans, it can also be used to damage melon, eggplant, beans, onions, leeks, spinach and crucifers. There are nearly 100 families and more than 300 kinds of plants in vegetables, food, cash crops, etc.; Spodoptera litura is a worldwide distribution, which occurs in all parts of the country, mainly in the Yangtze River Basin and the Yellow River Basin. Spodoptera litura mainly kills the whole plant with larvae, and the back of the cluster leaves at the younger age. After 3 years of age, the leaves are scattered, the young stems and the old larvae can feed on the fruit.
  • the annual food loss caused by Spodoptera litura is huge, and even more affects the living conditions of the local population.
  • 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 Spodoptera litura.
  • weeds can be removed, ploughed or irrigated after harvesting to destroy or deteriorate the site of pupation, which can help reduce the source of insects; or combined with the management of the newly hatched larvae that remove the eggs and cluster damage to reduce the source of insects.
  • agricultural control is mostly a preventive measure, its application has certain limitations and cannot be used as an emergency measure. It appears to be powerless when the Spodoptera litura breaks out.
  • Chemical control that is, pesticide control
  • chemical control methods are mainly sprayed with pharmaceuticals. 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 is mainly based on the response of pests to various physical factors in environmental conditions, using various Physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce killing, radiation infertility and other methods to control pests.
  • various Physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce killing, radiation infertility and other methods to control pests.
  • a wide range of methods are mainly used to attract moths, sweet and sour traps and switchgrass 500 times of trichlorfon to trap moths; although the above methods have different degrees of control effects, they have certain difficulties in operation.
  • 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 Spodoptera litura by producing 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 Spodoptera litura to plants by producing a transgenic plant expressing a Cry2Ab protein, and effectively overcoming the prior art agricultural control, chemical control and physical control And other technical defects.
  • the present invention provides the following technical solutions, specifically:
  • the present invention provides a method for controlling a pest of Spodoptera litura, comprising contacting a Spodoptera litura pest at least with a Cry2Ab protein, which is preferably used as a separate insecticidal active ingredient in contact with a Spodoptera litura pest;
  • the Cry2Ab protein is present in a host cell that produces at least the Cry2Ab protein, and the Spodoptera litura 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 that produces at least the Cry2Ab protein
  • the Spodoptera litura pest is at least in contact with the Cry2Ab protein by ingesting the tissue of the bacterium or the transgenic plant, after contact
  • the growth of Spodoptera litura pests is inhibited and/or causes death to achieve control of plants against Spodoptera litura.
  • a method for controlling a pest of Spodoptera litura the transgenic plant may be in any growth period; and/or
  • the tissue of the transgenic plant is a leaf, a stem, a fruit, a tassel, an ear, a flower bud, an anther or a filament; and/or
  • the control of the plants of the Spodoptera litura hazard does not change due to changes in the location and/or planting time.
  • a method for controlling a pest of Spodoptera litura the plant being derived from corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, sugar beet, cabbage or eggplant.
  • the step prior to the contacting step is planting a plant containing a polynucleotide encoding the Cry2Ab protein.
  • the method for controlling a pest of Spodoptera litura the amino acid sequence of the Cry2Ab protein having the amino acid sequence shown in SEQ ID NO:
  • 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 and the second nucleoside Acid does not encode the Cry1A.105 protein;
  • the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase, a peroxidase or an important gene for inhibiting a target insect pest.
  • dsRNA dsRNA
  • the second nucleotide encodes a Cry1Fa protein or a Vip3Aa protein
  • amino acid sequence of the Cry1Fa protein has the amino acid sequence shown in SEQ ID NO:3;
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO:4.
  • the invention provides the use of a Cry2Ab protein for controlling Spodoptera litura pests, preferably, in said use, said Cry2Ab protein as a separate insecticidal active ingredient.
  • the present invention provides a plant cell, a plant tissue, a plant, or a bacterium for transducing a Cry2Ab gene, wherein the Cry2Ab gene is used as a separate insecticidal active ingredient, and preferably, in the use, the Cry2Ab gene is used as a separate insecticidal active ingredient. Encoding the gene.
  • the present invention provides a method of producing a plant for controlling a pest of Spodoptera litura, comprising introducing a polynucleotide sequence encoding a Cry2Ab protein into the genome of the plant.
  • the present invention provides a method of producing a plant seed for controlling a Spodoptera litura pest, comprising hybridizing a first plant obtained by the method as described above to a second plant, thereby producing a polynucleotide sequence encoding the Cry2Ab protein. Seed.
  • the invention provides a plant seed for controlling a Spodoptera litura pest produced by the method described above.
  • the present invention provides a method of cultivating a plant for controlling a pest of Spodoptera litura, comprising:
  • the plants are grown under conditions in which the artificially inoculated pests of Spodoptera litura and/or Spodoptera litura pests are naturally harmful, and harvested with reduced plant damage and/or plants other than those having no polynucleotide sequence encoding the Cry2Ab protein. Or plants with increased plant yield.
  • the invention provides a plant for controlling Spodoptera litura pests produced by the method as described above.
  • the present invention provides a plant cell, plant tissue, plant or bacterium which controls a Spodoptera litura pest, the plant cell, plant tissue, plant or bacterium comprising a polynucleotide encoding a Cry2Ab protein, preferably, The polynucleotide sequence encoding the Cry2Ab protein is encoded as a separate insecticidal active ingredient;
  • the amino acid sequence of the Cry2Ab protein has the amino acid sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence of the Cry2Ab protein has the nucleotide sequence shown in SEQ ID NO: 2.
  • a plant cell, plant tissue, plant or bacterium which controls a Spodoptera litura pest the plant cell, plant tissue, plant or bacterium further comprising at least one nucleus different from the Cry2Ab protein encoding a second nucleotide of the nucleoside and the second nucleotide does not encode a Cry1A.105 protein;
  • the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase, a peroxidase or an important gene for inhibiting a target insect pest.
  • dsRNA dsRNA
  • the second nucleotide encodes a Cry1Fa protein or a Vip3Aa protein
  • amino acid sequence of the Cry1Fa protein has the amino acid sequence shown in SEQ ID NO:3;
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO:4.
  • the plant cell, plant tissue, plant or bacteria controlling the Spodoptera litura pest as described above the plant being corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, beet, cabbage or eggplant
  • the plant tissue is a leaf, a stem, a fruit, a tassel, an ear, a flower bud, an anther or a filament.
  • 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 Spodoptera litura pest is in contact with at least the Cry2Ab protein, and growth of Spodoptera litura pests is inhibited and/or causes death after contact. Further, the Spodoptera litura pest is at least in contact with the Cry2Ab protein by ingesting plant tissue, and all or part of the Spodoptera litura pest growth is inhibited and/or causes death after the contact. Inhibition refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry2Ab protein for controlling Spodoptera litura pests under conditions in which artificially inoculated pests of Spodoptera litura and/or Spodoptera litura pests are naturally harmful, 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 Spodoptera litura can be independently and not attenuated and/or disappeared by other substances that can "control” and/or “control” the pests of Spodoptera litura. .
  • any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry2Ab protein) is present and/or asynchronously, present and/or produced, a Cry2Ab protein and/or another substance that can control a Spodoptera litura pest,
  • the presence of the other substance neither affects the "control” and/or "control” effect of the Cry2Ab protein on Spodoptera litura, nor does it result in complete and/or partial "control” and/or “control” effects. It is achieved by the other substance, but not by the Cry2Ab protein.
  • the process of feeding on plant tissues by Spodoptera litura pests is short and difficult to observe with the naked eye.
  • any tissue of a plant (containing a polynucleotide sequence encoding a Cry2Ab protein) is present in a dead Spodoptera litura pest, and/or a Spodoptera litura pest having a growth inhibition thereon, and/or a non-transgenic base
  • the method and/or use of the present invention is achieved by the fact that the wild type plant has reduced plant damage, that is, the method and/or use for controlling the Spodoptera litura pest by contacting the Spodoptera litura pest with at least the Cry2Ab protein.
  • expression of a Cry2Ab protein in a transgenic plant may be accompanied by expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Co-expression of such more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant (the first parent) can express the Cry2Ab protein by genetic engineering operation
  • the second plant second parent
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, in the present invention, RNAi technology can be used to specifically knock out or shut down the expression of a specific gene in a target insect pest.
  • the Lepidoptera In the classification system, the Lepidoptera is generally classified into suborders, superfamily, and family according to the morphological characteristics of the worms, linkages, and types of antennae, while the Noctuidae is the most diverse species in the Lepidoptera.
  • Spodoptera litura and the cotton bollworm, the small tiger, the giant salamander, and the genus Spodoptera belong to the Noctuidae family, in addition to the similarity in the classification criteria, there are great differences in other morphological structures; Strawberries are the same as apples (both of which belong to the genus Rosaceae). They are characterized by bisexuality, radiation symmetry, and 5 petals, but their fruits and plant morphology vary widely. Spodoptera litura has its unique characteristics both in terms of larval morphology and adult morphology.
  • the head of Spodoptera litura larvae is dark brown and has a variety of chests, ranging from khaki to black-green; the adult body of Spodoptera litura is dark brown, with white tufts on the back of the chest, grayish brown front wings and many patterns.
  • the larvae of Spodoptera frugiperda belonging to the genus Noctuidae were all green when they first hatched; the adult larvae of the genus Spodoptera was gray-brown, the front wings of the females were gray to gray-brown, and the males had darker fore wings with dark spots and light-colored dark lines.
  • Insects belonging to the genus Noctuidae not only have large differences in morphological characteristics, but also have differences in feeding habits.
  • the cotton bollworm of the same family, the cotton boll or the corn ear of the cotton-sucking type, the base of the stem of the small tiger, the corn, etc. the method of feeding the leaves, cutting the roots or drilling into the ear of corn. It is harmful to corn, while Spodoptera litura is more inclined to bite the leaves, leaving only the main vein; the old larvae can eat the fruit.
  • 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 a key point in the action of the Bt gene.
  • the Cry2Ab protein is highly virulence to the Helicoverpa armigera Hubner of the Noctuidae family, but not to the Spodoptera exigua Hiibner of the genus Noctuidae.
  • the Cry2Ac protein showed high virulence against Helicoverpa armigera Hubner and the cabbage looper, but only showed inhibition on Spodoptera exigua Hiibner.
  • 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 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".
  • 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 and hybridizing to SEQ ID NO: 2 of the present invention under stringent conditions is included in the present invention.
  • These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93.
  • 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 in comparison to full length proteins and/or 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 Spodoptera litura pest of the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • the length of the aforementioned sequence may vary, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A gene encoding an active fragment can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
  • the present invention may derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries.
  • antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from protein mixtures.
  • antibodies may be caused by protein portions that are most constant in protein and most different from other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies of the proteins disclosed herein or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
  • the "substantially identical" sequence refers to a sequence which has an amino acid substitution, deletion, addition or insertion but does not substantially affect the insecticidal activity, and also includes a fragment which retains insecticidal activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been, 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 Cry2Ab protein includes, but is not limited to, Sequence 1, and an amino acid sequence having a certain homology with the amino acid sequence shown in 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), Both can achieve the methods and/or uses of the present invention by contacting at least the Cry2Ab protein with Spodoptera litura pests to achieve a method and/or use for controlling Spodoptera litura pests.
  • 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 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 Spodoptera litura pest is in contact with at least the Cry2Ab protein by ingesting tissue of the transgenic plant, the Spodoptera litura after contact Harm Insect growth is inhibited and/or causes death to achieve control of the plants of the Spodoptera litura.
  • 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.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pinI and pinII) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, CAT-1 introns, pKANNIBAL intron, PIV2 intron and "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • Insecticide or "insect-resistant” as used in the present invention means toxic to crop pests, thereby achieving "control” and/or "control” of crop pests.
  • said "insecticide” or “insect-resistant” means killing crop pests.
  • the target insect is a Spodoptera litura pest.
  • the Cry2Ab protein is toxic to Spodoptera litura pests.
  • the plants of the present invention particularly corn and soybean, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry2Ab protein, and the Spodoptera litura pest is contacted with the protein by feeding plant tissue, contacting The growth of Spodoptera litura pests is inhibited and/or causes 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 can substantially eliminate the need for chemical or biological pesticides.
  • the chemical or biological insecticide is an insecticide against Spodoptera litura 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 in the present invention is mainly Spodoptera litura.
  • 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 damage of Spodoptera litura pests through external action, ie, external factors, such as agricultural control, chemical control and physical control; and the present invention controls the twill night by producing a Cry2Ab protein capable of killing Spodoptera litura in plants. Moth 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 method for controlling Spodoptera litura pests used in the prior art has an effect that is incomplete and only plays a mitigating effect; whereas the transgenic plant of the present invention (Cry2Ab protein) can cause a large number of deaths of the larvae of Spodoptera litura, and is small
  • the developmental progress of some surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state or between the initial hatching-negative control state. This severely inhibited development of the pests could not survive and harm under natural conditions.
  • 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 DBN100033 containing a Cry2Ab nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the damage of the leaves of the transgenic maize plants inoculated with the insecticidal protein of the present invention
  • Figure 4 is a diagram showing the damage of leaves of a transgenic soybean plant inoculated with Spodoptera litura in the use of 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.
  • Cry1Fa insecticidal protein (605 amino acids), as shown in SEQ ID NO: 3 in the Sequence Listing; Cry1Fa nucleotide sequence (1818 nucleotides) encoding the amino acid sequence corresponding to the Cry1Fa insecticidal protein , 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 Cry1Fa nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing) are manufactured by Nanjing Jinsrui Biotechnology Co., Ltd.
  • 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 Cry2Ab nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleus represented by SEQ ID NO: 2 in the sequence listing.
  • the nucleotide sequence, the Cry2Ab nucleotide sequence, was correctly inserted.
  • the synthesized Cry1Fa nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1Fa is a Cry1Fa nucleotide sequence (SEQ ID NO: 4).
  • Cry1Fa nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases NcoI and SpeI, respectively, and the excised Cry2Ab nucleotide sequence fragment was inserted into the expression.
  • NcoI and SpeI sites of the 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 DBN100033 is constructed, and the construction process thereof is shown in Fig.
  • the recombinant expression vector DBN100033 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 DBN100033), 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 NcoI and SpeI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the NcoI and SpeI sites of the recombinant expression vector DBN100033 was SEQ ID in the sequence listing. NO: The nucleotide sequence shown by 2, that is, the Cry2Ab nucleotide sequence.
  • the Cry2Ab nucleotide sequence and the Cry1Fa nucleotide sequence excised from the recombinant cloning vectors DBN01-T and DBN02-T were separately digested with BamHI to insert the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100076.
  • the nucleotide sequence in the recombinant expression vector DBN100076 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 Cry1Fa nucleotide sequence.
  • the Cry2Ab nucleotide sequence and the Cry1Fa nucleotide sequence can be ligated to the CaMV35S promoter and the Nos terminator.
  • the recombinant expression vectors DBN100033 and DBN100076 which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA ( Recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube and incubated at a temperature of 28 ° C, 200 rpm for 2 hours, coated with 50 mg /L rifampicin and 100 mg/L Kanamycin on LB plates until a positive monoclonal grows, picking up monoclonal cultures and extracting the plasmid with restriction endonuclease AhdI and XhoI was digested with the recombinant expression vectors DBN100033 and DBN100076, and the results showed that the re
  • 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.
  • the T-DNAs in the recombinant expression vectors DBN100033 and DBN100076 (including the promoter sequence of the CaMV35S gene, the Cry2Ab nucleotide sequence, the Cry1Fa nucleotide sequence, the Hpt gene and the Nos terminator sequence) were transferred into the maize genome, and obtained.
  • Maize plants transformed with the Cry2Ab nucleotide sequence and maize plants transfected with the Cry2Ab-Cry1Fa nucleotide sequence; wild type maize plants were used as controls.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of expressing the Cry2Ab nucleotide sequence and/or the Cry2Ab-Cry1Fa nucleotide
  • infecting medium MS salt 4.3 g) /L
  • MS vitamin case
  • 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 a solid medium (MS salt 4.3 g/L, after the infusing step, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, agar Incubate on 8g/L, pH 5.8).
  • MS salt MS salt 4.3 g/L
  • MS vitamin, casein 300mg/L sucrose 20g/L, glucose 10g/L
  • acetosyringone (AS) 100mg/L
  • 2,4-D 2,4-dichlorophenoxyacetic acid
  • 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 cotyledonary node tissue of the aseptically cultivated soybean variety Zhonghuang 13 is co-cultured with the Agrobacterium described in the third embodiment, to reconstitute the second embodiment.
  • the T-DNAs including the CaMV35S promoter sequence, the Cry2Ab nucleotide sequence, the Cry1Fa nucleotide sequence, the Hpt gene and the Nos terminator sequence
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5.6.
  • the seeds were inoculated on a germination medium and cultured under the following conditions: temperature 25 ⁇ 1 ° C; photoperiod (light/dark) was 16/8 h.
  • photoperiod light/dark
  • infecting medium MS salt 2.15 g/L
  • Cotyledonary node tissue is co-cultured with Agrobacterium for a period of time (3 days) (Step 2: Co-cultivation step).
  • cotyledonary node tissue is in solid medium (MS salt 4.3 g/L, B5 vitamin, sucrose after infection step) 20 g/L, glucose 10 g/L, 2-morpholine ethanesulfonic acid (MES) 4 g/L, zeatin 2 mg/L, agar 8 g/L, pH 5.6).
  • MS salt 4.3 g/L, B5 vitamin, sucrose after infection step
  • the medium is restored (B5 salt 3.1 g/L, B5 vitamins, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, corn) Prime (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH5.6) at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin) Selective agent for plant transformants (step 3: recovery step).
  • B5 salt 3.1 g/L B5 vitamins, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, corn) Prime (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH5.6
  • the tissue blocks of the cotyledonary node regeneration are cultured on a solid medium with antibiotics but no selective agent to eliminate Agrobacterium and invade The stained cells provide a recovery period.
  • the cotyledonary node-regenerated tissue pieces are cultured on a medium containing a selection agent (hygromycin) and the grown transformed callus is selected (step 4: selection step).
  • the cotyledonary node The regenerated tissue block was selected from solid media with selective agent (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, 6-benzyl adenine ( 6-BAP) 1 mg / L, agar 8 g / L, cephalosporin 150 mg / L, glutamic acid 100 mg / L, aspartic acid 100 mg / L, hygromycin 50 mg / L, pH 5.6) culture, resulting in The transformed cells are selectively grown.
  • selective agent B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, 6-benzyl adenine ( 6-BAP) 1 mg / L, agar 8 g / L, cephalosporin 150 mg / L, glutamic acid 100 mg / L,
  • the transformed cells regenerate the plants (step 5: regeneration step), preferably, cotyledonary node regeneration grown on the medium containing the selection agent
  • the tissue pieces were cultured on a solid medium (B5 differentiation medium and B5 rooting medium) to regenerate the plants.
  • the selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, hygromycin 50mg/L , pH 5.6), culture differentiation at 25 ° C.
  • B5 differentiation medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)
  • MES 2-morpholine ethanesulfonic acid
  • ZT zeatin
  • the differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin) 150 mg/L, indole-3-butyric acid (IBA) 1 mg/L), cultured in rooting culture at 25 ° C to a height of about 10 cm, and transferred to a greenhouse for cultivation to firmness. In the greenhouse, the cells were cultured at 26 ° C for 16 hours and then at 20 ° C for 8 hours.
  • B5 rooting medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin
  • IBA indole-3-butyric acid
  • the specific method for detecting the copy number of the Cry2Ab gene and the Cry1Fa gene is as follows:
  • Step 11 Take 100 mg of each of the maize plants transformed with the Cry1Fa nucleotide sequence, the maize plants transformed with the Cry2Ab-Cry1Fa nucleotide sequence, and the wild-type maize plants, respectively, and homogenize them with liquid nitrogen in a mortar. , each sample takes 3 repetitions;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ l;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Primer 1 As shown in SEQ ID NO: 8 in the sequence listing;
  • Primer 2 As shown in SEQ ID NO: 9 in the sequence listing;
  • Probe 1 As shown in SEQ ID NO: 10 in the sequence listing;
  • Primer 3 As shown in SEQ ID NO: 11 in the sequence listing;
  • Primer 4 As shown in SEQ ID NO: 12 in the sequence listing;
  • Probe 2 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 stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Transgenic soybean plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan.
  • Soybean plants of the nucleotide sequence obtained a single copy of the transgenic plants.
  • a maize plant transformed into a Cry2Ab nucleotide sequence a maize plant transformed into a Cry2Ab-Cry1Fa nucleotide sequence, a soybean plant transformed into a Cry2Ab nucleotide sequence, and a soybean plant transformed into a Cry2Ab-Cry1Fa nucleotide sequence; Wild-type maize plants and soybean plants, as well as non-transgenic maize plants and soybean plants identified by Taqman were tested for insect resistance to Spodoptera litura.
  • the maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transformed with the Cry2Ab-Cry1Fa nucleotide sequence, the wild-type maize plants, and the fresh leaves of the non-transgenic maize plants (V3-V4 phase) identified by Taqman were taken. Rinse with sterile water and blot the water on the leaves with gauze. Then cut the corn leaves into strips about 1cm ⁇ 4cm. Take 3 pieces of cut long leaves into the bottom of the round plastic dish.
  • Table 1 indicate that the maize plants transformed with the Cry2Ab nucleotide sequence and the maize plants transfected with the Cry2Ab-Cry1Fa nucleotide sequence have good insecticidal effects on Spodoptera litura, and the average mortality rate of Spodoptera litura is Above 60%, the total score of resistance is also about 250 points; and the total score of resistance of non-transgenic corn plants and wild-type corn plants identified by Taqman is generally about 70 points.
  • the maize plants transformed with the Cry2Ab nucleotide sequence and the maize plants transformed with the Cry2Ab-Cry1Fa nucleotide sequence showed high activity against Spodoptera litura, which was sufficient to have an adverse effect on the growth of Spodoptera litura. So that it can be controlled in the field.
  • Soybean plants transformed with the Cry2Ab nucleotide sequence, soybean plants transformed with the Cry2Ab-Cry1Fa nucleotide sequence, wild-type soybean plants, and fresh leaves identified by Taqman as non-transgenic soybean plants (V3-V4 phase) were taken. Rinse with sterile water and blot the water on the leaves with gauze. Then cut the soybean leaves into strips of about 1cm ⁇ 4cm. Take one piece of the cut long leaves into the bottom of the round plastic dish.
  • Soy plants that were harmed and transferred to the Cry2Ab nucleotide sequence and soybean plants that were transferred into the Cry2Ab-Cry1Fa nucleotide sequence were only slightly damaged, and only a small amount of damage was observed on the leaves, and the leaf damage rate was 20 About about %, especially the damage formed on soybean plants that were transferred into the Cry2Ab-Cry1Fa nucleotide sequence, was almost invisible to the naked eye.
  • soybean plants transformed with the Cry2Ab nucleotide sequence and soybean plants transformed with the Cry2Ab-Cry1Fa nucleotide sequence showed high activity against Spodoptera litura, which was sufficient to have an adverse effect on the growth of Spodoptera litura. So that it can be controlled in the field.
  • the above experimental results also showed that the maize plant transformed with the Cry2Ab nucleotide sequence, the maize plant transformed with the Cry2Ab-Cry1Fa nucleotide sequence, the soybean plant transformed with the Cry2Ab nucleotide sequence, and the Cry2Ab-Cry1Fa nucleotide sequence were transferred.
  • the control/control of soybean plants against Spodoptera litura is apparently because the plants themselves can produce Cry2Ab protein, so, as is well known to those skilled in the art, similar Cry2Ab proteins can be produced according to the same toxicity of Cry2Ab protein to Spodoptera litura.
  • the transgenic plants can be used to control/control the hazards of Spodoptera litura.
  • 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 Spodoptera litura pest by producing a Cry2Ab protein capable of killing Spodoptera litura in the plant; and the agricultural control method, chemical control method and physical control method used in the prior art Compared with the invention, the plant protects the whole growth period and the whole plant to prevent the damage of the pests of Spodoptera litura, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economical.

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Abstract

La présente invention concerne un procédé de contrôle de Prodenia litura. Le procédé comprend : le fait de permettre à Prodenia litura de se trouver en contact avec une protéine Cry2Ab. Le procédé comprend spécifiquement : la culture de plants génétiquement modifiés en utilisant un polynucléotide codé par une protéine Cry2Ab ; et le fait de laisser Prodenia litura se trouver en contact avec la protéine Cry2Ab en laissant Prodenia litura manger des plants génétiquement modifiés, où après le contact, Prodenia litura voit sa croissance restreinte et/ou finit par mourir, afin d'empêcher Prodenia litura de détériorer les plants.
PCT/CN2016/074069 2015-03-04 2016-02-18 Utilisations d'une protéine insecticide WO2016138819A1 (fr)

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CN106749566B (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
CN108148841B (zh) * 2017-12-14 2020-12-29 云南大学 氨基酸序列在用于使昆虫Dip3蛋白失活中的应用
CN108676813B (zh) * 2018-03-30 2019-10-18 北京大北农生物技术有限公司 杀虫蛋白的用途
CN108611362B (zh) * 2018-03-30 2020-08-28 北京大北农生物技术有限公司 杀虫蛋白的用途
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