WO2016101684A1 - 杀虫蛋白的用途 - Google Patents

杀虫蛋白的用途 Download PDF

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WO2016101684A1
WO2016101684A1 PCT/CN2015/092007 CN2015092007W WO2016101684A1 WO 2016101684 A1 WO2016101684 A1 WO 2016101684A1 CN 2015092007 W CN2015092007 W CN 2015092007W WO 2016101684 A1 WO2016101684 A1 WO 2016101684A1
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protein
cry1a
plant
mites
plants
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PCT/CN2015/092007
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English (en)
French (fr)
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杨旭
丁德荣
张欣馨
康越景
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北京大北农科技集团股份有限公司
北京大北农生物技术有限公司
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Publication of WO2016101684A1 publication Critical patent/WO2016101684A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof

Definitions

  • the present invention relates to the use of a pesticidal protein, and in particular to the use of a CrylA.105 protein to control a plant of a millet ash by expressing it in a plant.
  • Agricultural control is a comprehensive coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, environmental factors, and creating a farmland ecological environment that is conducive to crop growth and is not conducive to pest occurrence.
  • rotation can be used to reduce the density of insect sources, but the economic benefits of different crops, rotation is likely to cause farmers to reduce income, and it is difficult to implement.
  • Chemical control that is, pesticide control
  • pesticide control is the use of chemical pesticides to kill pests. It is an important part of integrated pest management. It is characterized by rapid, convenient, simple and high economic benefits, especially in the case of large pests. Essential emergency measures. However, chemical control also has its limitations. If improper use, it will lead to phytotoxicity of crops, resistance to pests, killing natural enemies, polluting the environment, destroying farmland ecosystems and threatening the safety of humans and animals. Adverse consequences.
  • Physical control mainly relies on the response of pests to various physical factors in environmental conditions, and uses various physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce pests, radiation infertility and other methods to control pests.
  • the most widely used is the frequency-vibration insecticidal lamp trapping, which utilizes the phototaxis of pests, uses light at close range, uses waves at a long distance, attracts insects close to each other, and has certain control effects on pest control;
  • the lamp needs to clean the dirt on the high-voltage power grid every day, otherwise it will affect the insecticidal effect; and it can't turn on the light in thunderstorm days, there is also the danger of electric shock in the operation; in addition, the one-time investment of installing the lamp is large.
  • Cry1A.105 insecticidal protein is one of many insecticidal proteins and is a chimeric protein derived from Cry1Ab protein, Cry1Ac protein and Cry1Fa protein, respectively.
  • Plants transgenic with the Cry1A.105 gene have been shown to be resistant to Lepidoptera pests such as corn borer, cotton bollworm, and fall armyworm. However, there has been no control of millet by producing transgenic plants expressing the Cry1A.105 protein. Report on the hazards of ash to plants.
  • Chilo infuscatellus also known as sugarcane mites
  • the ash mites are stalk pests, which can cause dead plants after the plants are damaged. Under normal circumstances, the spring valley area and the spring and summer valley mixed area occur strictly Heavy, Xiagu District is light.
  • the ash ash was once the main pest in North China and South China, mainly affecting sorghum, millet and sugar cane.
  • the object of the present invention is to provide a use of a pesticidal protein, for the first time, to provide a method for controlling the damage of a plant by using a transgenic plant expressing a Cry1A.105 protein, and effectively overcoming the prior art agricultural control, chemical control and Technical defects such as physical control.
  • the present invention provides a method of controlling a pest of the mites, comprising contacting the mites pest with at least the Cry1A.105 protein.
  • the Cry1A.105 protein is present in a host cell that produces at least the CrylA.105 protein, and the millet worm is in contact with at least the Cry1A.105 protein by ingesting the host cell.
  • the Cry1A.105 protein is present in a bacterium or a transgenic plant which produces at least the Cry1A.105 protein, and the millet worm is at least in contact with the Cry1A by ingesting the bacterium or the tissue of the transgenic plant The .105 protein is contacted, and the growth of the larvae pest is inhibited and/or caused to death after the exposure to achieve control of the plant against the ash mites.
  • the bacterium may be a wild bacterium and/or a recombinant bacterium capable of producing the Cry1A.105 protein.
  • Bacillus thuringiensis subsp. kurstaki Bacillus thuringiensis subsp. kurstaki (B.t.k.).
  • the transgenic plant can be in any growth period.
  • the tissue of the transgenic plant can be various tissues of the plant, such as leaves, stems, fruits, tassels, ears, anthers or filaments.
  • the control of the plants against the ash mites does not change due to changes in the location and/or planting time.
  • the plant may be a variety of gramineous plants that are endangered by the millenium, preferably the plant is corn, sorghum, millet, sugar cane, rice, wheat, barley or oats.
  • the step prior to the contacting step is to plant a plant containing a polynucleotide encoding the Cry1A.105 protein.
  • the amino acid sequence of the Cry1A.105 protein has the amino acid sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence of the Cry1A.105 protein has the nucleotide sequence shown in SEQ ID NO: 2.
  • the plant may further comprise at least one second nucleotide different from the nucleotide encoding the Cry1A.105 protein.
  • the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase or a peroxidase.
  • the second nucleotide encodes a Cry2Ab protein.
  • amino acid sequence of the Cry2Ab protein has the amino acid sequence shown in SEQ ID NO: 3.
  • nucleotide sequence of the Cry2Ab protein has the nucleotide sequence shown in SEQ ID NO: 4.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • the present invention also provides a use of the Cry1A.105 protein for controlling a pest of the worm.
  • the present invention also provides a method of producing a plant for controlling a mites pest, including A polynucleotide sequence encoding a Cry1A.105 protein is introduced into the genome of the plant.
  • the present invention also provides a method of producing a plant propagule for controlling a mites pest, comprising crossing a first plant obtained by the method with a second plant, and/or removing the method by the method
  • the fertile tissue on the obtained plants is cultured to produce a plant propagule containing a polynucleotide sequence encoding the Cry1A.105 protein.
  • the present invention also provides a method for cultivating a plant for controlling a mites pest, comprising:
  • the plants are grown under conditions in which the artificially inoculated with the mites and/or the mites pests are naturally harmful, and the plants are harvested with reduced plant damage compared to other plants that do not have the polynucleotide sequence encoding the Cry1A.105 protein. And/or plants with increased plant yield.
  • Plant propagules as used in the present invention include, but are not limited to, plant sexual propagules and plant asexual propagules.
  • the plant sexual propagule includes, but is not limited to, a plant seed; the plant asexual propagule refers to a vegetative organ of a plant body or a special tissue which can produce a new plant under ex vivo conditions; the vegetative organ or a certain Specific tissues include, but are not limited to, roots, stems and leaves, for example: plants with roots as vegetative propagules including strawberries and sweet potatoes; plants with stems as vegetative propagules including sugar cane and potatoes (tubers), etc.; leaves as asexual Plants of the propagule include aloe vera and begonia.
  • Contact means that insects and/or pests touch, stay and/or ingest plants, plant organs, plant tissues or plant cells, and the plants, plant organs, plant tissues or plant cells can It is a pesticidal protein expressed in the body, and may also be a microorganism having a pesticidal protein on the surface of the plant, plant organ, plant tissue or plant cell and/or having a pesticidal protein.
  • control and/or “control” in the present invention means that the mites are at least in contact with the Cry1A.105 protein, and the growth of the larvae is inhibited and/or causes death after contact. Further, the larvae of the larvae are in contact with at least the Cry1A.105 protein by ingesting plant tissues, and all or part of the growth of the larvae pests is inhibited and/or causes death after the contact. Inhibition refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry1A.105 protein that control the pests of the mites are inoculated under conditions in which the artificially inoculated pests of the mites and/or the mites are naturally harmful.
  • Transgenic wild-type plants have reduced plant damage compared to, but are not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like.
  • the "control” and / or “control” effects of the Cry1A.105 protein on the ash can be independently independent and not attenuated by the presence of other substances that can "control” and/or “control” the worms and/or Or disappear.
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a Cry1A.105 protein
  • Cry1A.105 protein and/or another species that can control the mites pest a substance in which the presence of the other substance neither affects the "control” and/or “control” effect of the Cry1A.105 protein on the ash, nor does it result in the "control” and
  • the "or control” effect is completely and / or partially achieved by the other substance, regardless of the Cry1A.105 protein.
  • any tissue of a plant (containing a polynucleotide sequence encoding a Cry1A.105 protein) is a dead mites pest, and/or a sphagnum pest that inhibits growth growth thereon, and/or a non-transgenic wild type Plants have reduced plant damage compared to the method and/or use of the present invention, i.e., by contacting the Cry1A.105 protein with at least the C. sinensis pest to achieve a method and/or use for controlling the mites.
  • expression of the Cry1A.105 protein in a transgenic plant may be accompanied by expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Co-expression of such more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant first parent
  • the second plant second parent
  • Cry-like insecticidal protein and/or Vip-like insecticidal protein by genetic engineering operation.
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, in the present invention, RNAi technology can be used to specifically knock out or shut down the expression of a specific gene in a target insect pest.
  • the lepidoptera In the classification system, the lepidoptera is generally divided into suborders, superfamily, and family according to the morphological characteristics of the worm's veins, linkages, and types of antennae, while the genus Lepidoptera is the most diverse species of Lepidoptera.
  • One of the departments has found more than 10,000 types in the world, and there are thousands of records in China alone.
  • Most of the moths are pests of crops, most of which are in the form of stolons, such as stem borer and corn borer.
  • the ash mites are similar to the mites and corn borers, they belong to the order Lepidoptera, and there are great differences in other morphological structures except for the similarity in the classification criteria; it is like the strawberry in the plant and the apple ( They belong to the genus Rosaceae, which have the characteristics of flower bisexuality, radiation symmetry, and 5 petals, but their fruits and plant morphology are very different.
  • the ash ash has its unique characteristics in terms of larval morphology and adult morphology.
  • the back line of the back is three or four five", which means that the sorghum sorghum, corn stalk and ash scorpion belonging to the genus Mothidae have obvious numbers on the top line. difference.
  • the dorsal blood vessel is an important part of the insect circulatory organ. The inside is filled with the hemolymph called the insect "blood”. Therefore, the difference in the number of top lines on the surface of the body appears to reflect the difference in the back vessels, which is the difference in the insect circulation system.
  • Insects belonging to the genus Mothidae not only have large differences in morphological characteristics, but also have differences in feeding habits.
  • the stem borer of the same family is mainly harmful to rice, and rarely harms other grass crops.
  • the difference in feeding habits also suggests that the enzymes and receptor proteins produced by the digestive system in the body are different.
  • the enzyme produced in the digestive tract is the key point of the Bt gene function. Only the enzyme or receptor protein that can bind to the specific Bt gene may make a certain Bt gene have an insect resistance effect on the pest.
  • European corn borer and Asian corn borer are classified in the same species as the Ostrinia genus (the same genus), but their response to the same Bt protein is quite different, which further demonstrates the Bt protein and The way enzymes and receptors interact in insects is complex and unpredictable.
  • the genome of a plant, plant tissue or plant cell as referred to in the present invention refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • To express a protein in vivo one strand of DNA is typically transcribed into a complementary strand of mRNA that is used as a template to translate the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention is hybridized under stringent conditions to the Cry1A.105 gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A.105 gene of the present invention.
  • a nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes.
  • the stringent conditions of the present invention may be specific hybridization with SEQ ID NO: 2 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1% SDS and 1 x SSC. 0.1% SDS was washed once each time.
  • sequences having insect resistance and hybridizing to SEQ ID NO: 2 of the present invention under stringent conditions are included in the present invention. These sequences have at least about 40%-50% homology to the sequences of the invention, about 60%, 65% or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater homology.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or end ratios compared to full length proteins). Deletions), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • variant or “variant” is meant a nucleotide sequence that encodes the same protein or an equivalent protein encoded with insecticidal activity.
  • the "equivalent protein” refers to a protein having the same or substantially the same biological activity as the anti-mite pest of the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • the length of the aforementioned sequence may vary, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A gene encoding an active fragment can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
  • the present invention may derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries.
  • antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from protein mixtures.
  • antibodies may be caused by protein portions that are most constant in protein and most different from other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies of the proteins disclosed herein or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
  • the "substantially identical" sequence refers to a sequence which has an amino acid substitution, deletion, addition or insertion but does not substantially affect the insecticidal activity, and also includes a fragment which retains insecticidal activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R. L.
  • substitutions can occur outside of the regions that are important for molecular function and still produce active polypeptides.
  • amino acids from the polypeptides of the invention that are essential for their activity and are therefore selected for unsubstitution they can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique introduces a mutation at each positively charged residue in the molecule, and detects the insecticidal activity of the resulting mutant molecule, thereby determining an amino acid residue important for the activity of the molecule.
  • the substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, eg, de Vos et al., 1992, Science 255). : 306-312; Smith et al, 1992, J. Mol. Biol 224: 899-904; Wlodaver et al, 1992, FEBS Letters 309: 59-64).
  • the Cry1A.105 protein includes, but is not limited to, SEQ ID NO: 1, and an amino acid sequence having a certain homology with the amino acid sequence shown by SEQ ID NO: 1 is also included in the present invention.
  • the homology (similarity/identity) of these sequences to the sequences of the invention is typically greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%.
  • Preferred polynucleotides and proteins of the invention may also be defined in terms of a more specific range of homology.
  • the sequence of the example of the present invention is 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% homology.
  • the insecticidal activity or the insecticidal activity remaining means the insecticidal activity of the protein having a certain amino acid sequence with the amino acid sequence of SEQ ID NO: 1 (resistance total score, according to the fifth embodiment)
  • the method described herein is obtained by 80% or more, or 90% or more, or 92% or more, or 95% or more, or 98, of the insecticidal activity (total resistance score) of the Cry1A.105 protein represented by SEQ ID NO: 1. More than %, or 100%.
  • the nucleotide sequence of the Cry1A.105 protein is capable of encoding a Cry1A.105 protein having an insecticidal activity that satisfies the above requirements.
  • the transgenic plants producing the Cry1A.105 protein include, but are not limited to, the MON89034 transgenic maize event and/or plant material comprising the MON89034 transgenic maize event (as described in CN101495635A) or the MON87751 transgenic soybean event and/or Plant material comprising the MON87751 genetically modified soybean event (as described in USDA APHIS Unregulated Status Application 13-337-01p), each of which can achieve the method and/or use of the present invention, ie, through the pestle of at least Cry1A. 105 protein contact to achieve a method and/or use for controlling millet mites.
  • the method and/or use of the present invention can also be achieved by expressing the Cry1A.105 protein in the above transgenic events in different plants. More specifically, the Cry1A.105 protein is present in a transgenic plant that produces at least the Cry1A.105 protein, the contact with at least the Cry1A.105 protein by contacting the tissue of the transgenic plant. Subsequent growth of the larvae pest is inhibited and/or causes death to achieve control of the plants against the ash mites.
  • Regulatory sequences in the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Cry1A.105 protein.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pinI and pinII) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • Insecticide or "insect-resistant” as used in the present invention means toxic to crop pests, thereby achieving "control” and/or “control” of crop pests.
  • said "insecticide” or “insect-resistant” means killing crop pests.
  • the target insect is a mites pest.
  • the Cry1A.105 protein is toxic to the mites.
  • the plants of the present invention particularly millet, sugar cane, sorghum and corn, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1A.105 protein, and the planting of plant tissues by the fungus In contact with the protein, the growth of the larvae is inhibited and/or causes death after contact. Inhibition refers to death or sub-lethal death.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the plant can be substantially eliminated from chemistry or The need for a biocide (the chemical or biocide is an insecticide against the mites of the Cry1A.105 protein).
  • the expression level of insecticidal crystal protein (ICP) in plant material can be detected by various methods described in the art, for example, by using specific primers to quantify the mRNA encoding the insecticidal protein produced in the tissue, or directly specific The amount of insecticidal protein produced is detected. Different tests can be applied to determine the insecticidal effect of ICP in plants.
  • the target insects in the present invention are mainly milled ash.
  • the Cry1A.105 protein may have the amino acid sequence shown by SEQ ID NO: 1 in the Sequence Listing.
  • other elements may be included, such as a protein encoding a selectable marker.
  • an expression cassette comprising a nucleotide sequence encoding a Cry1A.105 protein of the present invention may also be expressed in a plant together with at least one protein encoding a herbicide resistance gene including, but not limited to, Glufosinate resistance gene (such as bar gene, pat gene), benzoin resistance gene (such as pmph gene), glyphosate resistance gene (such as EPSPS gene), bromoxynil resistance gene, sulfonate a resistance gene of a ureide resistance gene, a resistance gene against a herbicide, a tobacco, a resistance gene to a cyanamide or a glutamine synthetase inhibitor (such as PPT), thereby obtaining high insecticidal activity and Herbicide-resistant transgenic plants.
  • Glufosinate resistance gene such as bar gene, pat gene
  • benzoin resistance gene such as pmph gene
  • glyphosate resistance gene such as EPSPS gene
  • bromoxynil resistance gene sulfonate
  • exogenous DNA is introduced into a plant, such as a gene encoding the Cry1A.105 protein or an expression cassette or a recombinant vector
  • conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-emission Bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the harm of the mites and pests through external action, ie external factors, such as agricultural control, chemical control and physical control; and the invention is controlled by the production of Cry1A.105 protein in the plant capable of killing the ash mites.
  • the worms are pests, which are controlled by internal factors.
  • the frequency-vibration insecticidal lamp used in the prior art not only needs to clean the dirt of the high-voltage power grid every day, but also cannot be used in thunderstorm days; the invention is to express the Cry1A.105 protein in plants, effectively overcoming the frequency vibration.
  • the effect of the insecticidal lamp is affected by external factors, and the control effect of the transgenic plant (Cry1A.105 protein) of the present invention is stable at different locations, at different times, and in different genetic backgrounds.
  • the method for controlling the mites of the larvae used in the prior art has an effect which is incomplete and only serves to alleviate the effect; and the transgenic plant of the present invention (Cry1A.105 protein) can cause a large number of deaths of the newly hatched larvae, and The development progress of a small number of surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and stopped developing, and could not survive in the natural environment of the field, while the transgenic plants were generally only affected. Minor damage.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing the Cry1A.105 nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 2 is a flow chart showing the construction of a recombinant expression vector DBN100745 containing the Cry1A.105 nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the damage of leaves of the transgenic corn plants inoculated with the ash mites according to the use of the insecticidal protein of the present invention
  • Figure 4 is a diagram showing the damage of the leaves of the transgenic sugarcane plants inoculated with the worms of the present invention
  • Figure 5 is a diagram showing the damage of the leaves of the transgenic sorghum plant inoculated with the ash sorghum of the present invention
  • Figure 6 is a diagram showing the damage of leaves of transgenic millet plants inoculated with millet mites for the use of the insecticidal protein of the present invention.
  • Cry1A.105 insecticidal protein (1177 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; Cry1A.105 encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein Nucleotide sequence (3534 nucleotides) as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing) and the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing) are manufactured by Nanjing Kingsray Biotech Synthesized by the company; the 5' end of the synthesized Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also ligated with an NcoI cleavage site, the Cry1A.105 nucleotide sequence (SEQ ID NO: 2) a HindIII cleavage site is also ligated to the 3' end; the 5' end of the synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 4) is further ligated with an NcoI cleavage site, the Cry2Ab nucleotide sequence The 3' end of (SEQ ID NO: 4) is also ligated with a SpeI cleavage site.
  • the synthetic Cry1A.105 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • the construction process is shown in Figure 1 (wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; and T7 is T7 RNA polymerization).
  • Cry1A.105 is the Cry1A.105 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) in LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) was grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ l of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose.
  • the TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was dissolved in the precipitate; the RNA was digested in a water bath at 37 ° C for 30 min; and stored at -20 ° C until use.
  • the synthesized Cry2Ab nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein the Cry2Ab was a Cry2Ab nucleotide sequence (SEQ ID NO: 4).
  • the Cry2Ab nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases NcoI and HindIII, respectively, and the cut Cry1A.105 nucleotide sequence fragment was inserted. Between the NcoI and HindIII sites of the expression vector DBNBC-01, using conventional enzyme digestion Methods Construction vectors are well known to those skilled in the art and constructed into a recombinant expression vector DBN100745.
  • the recombinant expression vector DBN100745 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ l of E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant expression vector DBN100745), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50 mg/L was adjusted to pH 7.5 with NaOH and incubated overnight at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases NcoI and HindIII, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100745 between NcoI and HindIII was SEQ ID NO in the sequence listing.
  • the nucleotide sequence shown in 2 the Cry1A.105 nucleotide sequence.
  • the Cry2Ab nucleotide sequence excised from the recombinant cloning vector DBN02-T by NcoI and SpeI was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100744.
  • the nucleotide sequence in the recombinant expression vector DBN100744 contains the nucleotide sequence shown as SEQ ID NO: 4 in the sequence listing, that is, the Cry2Ab nucleotide sequence, and the Cry2Ab nucleotide sequence can be ligated. Ubi promoter and Nos terminator.
  • NcCI and HindIII, NcoI and SpeI were respectively inserted into the recombinant cloning vectors DBN01-T and DBN02-T, and the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence were inserted.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100029.
  • the nucleotide sequence in the recombinant expression vector DBN100029 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing, that is, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleoside.
  • the acid sequence, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100745, DBN100744 and DBN100029 which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube and incubated at a temperature of 28 ° C, 200 rpm for 2 hours, applied to On a LB plate containing 50 mg/L of rifampicin and 100 mg/L of kanamycin until a positive monoclonal grows, pick a monoclonal culture and extract the plasmid with restriction endonuclease
  • the recombinant expression vectors DBN100745, DBN100744 and DBN100029 were digested
  • the immature embryo of the aseptically cultured maize variety Heisei 31 was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the second embodiment.
  • T-DNA including the promoter sequence of maize Ubiquitin gene, Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence, Hpt gene and Nos terminator sequence
  • DBN100745, DBN100744 and DBN100029 was transferred to maize chromosome
  • a maize plant transformed with the Cry1A.105 nucleotide sequence, a maize plant transformed with the Cry2Ab nucleotide sequence, and a maize plant transformed with the Cry1A.105-Cry2Ab nucleotide sequence were obtained; and the wild type maize plant was simultaneously obtained. as comparison.
  • Agrobacterium can express the Cry1A.105 nucleotide sequence, the Cry2
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the grown transformed callus is selected (step 4: selection step).
  • a selection agent hygromycin
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8, resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, M. 50 mg/L, vegetal gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel 3 g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. Cultivate at 28 ° C per day in the greenhouse After an hour, it was further cultured at 20 ° C for 8 hours.
  • the medium formulation is referred to Molecular Biology and Genetic Engineering ISSN 2053-5767, wherein the screening agent is replaced with hygromycin according to the transgenic vector of the present invention.
  • a sorghum plant transformed into the Cry1A.105 nucleotide sequence, a sorghum plant transformed into the Cry2Ab nucleotide sequence, and a sorghum plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence were obtained; Control.
  • the conversion method mainly refers to the 22nd to 24th pages of the 2012 Master's degree of Guangxi University. Take the fresh stem section of the cane top, remove the cane tip and leaf sheath, leaving the stem tip growth cone and the heart leaf stem segment. On the ultra-clean workbench, wipe the surface with a 75% (v/v) alcohol cotton ball, carefully peel off the outer layer of the heart leaf with the sterilized tweezers, and take the heart leaf segment 5-7 cm long in the middle. A sheet cut into a thickness of about 3 mm was inoculated on an induction medium, and cultured in the dark at a temperature of 26 ° C for 20 days.
  • the callus pieces were cut into small pieces of 0.6*0.6 cm, and then transferred to MR solid medium containing 100 ⁇ mol/L acetosyringone (AS), and cultured in a dark state at 23 ° C for 3 days; the callus after infection
  • the tissue was clipped, placed on a filter paper and dried on a clean bench until the surface of the material was dry.
  • the culture medium was once removed, and the contaminated callus was removed.
  • the seedlings were about 3 cm long, they were transferred to a rooting medium containing hygromycin screening agent to induce rooting.
  • a sugarcane plant transformed into the Cry1A.105 nucleotide sequence a sugarcane plant transformed into the Cry2Ab nucleotide sequence, and a sugarcane plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence were obtained; and the wild type sugarcane plant was used as the Control.
  • the conversion method refers to the Hebei University of Agriculture 2012 Master Wang Hanyu's dissertation on pages 9 to 10.
  • the mature seeds were immersed in 0.1% (v/v) Tween-20 solution, washed with 70% (v/v) ethanol, then transferred to 0.1% (w/v) HgCl 2 solution, and finally sterilized water. Wash 2-3 times.
  • the sterilized seeds were transferred to MS medium and incubated at a temperature of 25 ° C for 2-3 days. When the stem tip of the seed grows to 4-6 mm, the shoot tip is transferred to the callus induction medium under aseptic conditions.
  • Induction of shoot tip callus The induced shoot tip callus was immersed in the Agrobacterium suspension for 30 minutes, and the shoot tip callus was taken out on the sterilized filter paper, and the excess bacterial solution was aspirated and transferred to co-culture.
  • the medium (MS + 100 mol / L AS + 2, 4-D) was co-cultured in the dark at a temperature of 28 ° C for 2-4 days. Then, the callus after co-cultivation was transferred to MS callus induction medium (2,4-D 4.5 ⁇ mol/L, 2.25 ⁇ mol/L Kn, cephalosporin 500 mg/L), and cultured at 25 ° C in dark. Two weeks of healing the callus.
  • the dense yellow-white callus was transferred to the differentiation medium for differentiation, and the screening medium hygromycin was added to the differentiation medium. After about five weeks, the callus formed a nodular structure, and the callus with the knot structure was transferred to the MS differentiation rooting medium (thiazolyl phenylurea (TDZ) 4.5 ⁇ mol/L, sucrose 120 ⁇ mol/L).
  • MS differentiation rooting medium thiazolyl phenylurea (TDZ) 4.5 ⁇ mol/L, sucrose 120 ⁇ mol/L).
  • a millet plant into which the Cry1A.105 nucleotide sequence was transferred a millet plant into which the Cry2Ab nucleotide sequence was transferred, and a millet plant into which the Cry1A.105-Cry2Ab nucleotide sequence was transferred were obtained; and the wild-type millet plant was used as the plant. Control.
  • Maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry2Ab nucleotide sequence, and maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence were used as samples, respectively, using Qiagen
  • the DNeasy Plant Maxi Kit was used to extract the genomic DNA, and the copy number of the Cry1A.105 gene and the Cry2Ab gene was detected by Taqman probe fluorescent quantitative PCR.
  • the wild type corn plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.
  • the specific method for detecting the copy number of the Cry1A.105 gene and the Cry2Ab gene is as follows:
  • Step 11 The maize plants transformed with the Cry1A.105 nucleotide sequence, the maize plants transformed with the Cry2Ab nucleotide sequence, the maize plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, and the leaves of the wild-type maize plants were respectively taken. Each 100 mg was separately homogenized with liquid nitrogen in a mortar, and each sample was taken in 3 replicates;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ l;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Primer 2 GTTCTGGACGGCGAAGAGTG is shown in SEQ ID NO: 9 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ l of each primer at a concentration of 1 mM, 50 ⁇ l of a probe at a concentration of 100 ⁇ M, and 860 ⁇ l of 1 ⁇ TE buffer, and stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Transgenic sorghum plants, transgenic sugarcane plants and transgenic millet plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan. The results showed that the Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence and Cry1A.105-Cry2Ab nucleotide sequence were integrated into the genome of the tested sorghum, sugarcane and millet plants, respectively.
  • a maize plant transformed into a Cry1A.105 nucleotide sequence a maize plant transformed into a Cry2Ab nucleotide sequence, a maize plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, and a Cry1A.105 nucleotide sequence
  • Sorghum plants, sorghum plants transferred to the Cry2Ab nucleotide sequence, and sorghum plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence a sugarcane plant transformed into a Cry1A.105 nucleotide sequence, a sugarcane plant transformed into a Cry2Ab nucleotide sequence, a sugarcane plant transformed into a Cry1A.105-Cry2Ab nucleotide sequence, and a Cry1A.105 nucleotide sequence
  • Maize plants transfected with Cry1A.105 nucleotide sequence, maize plants transfected with Cry2Ab nucleotide sequence, maize plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, wild-type maize plants and Taqman were identified as Fresh leaves of non-transgenic corn plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the corn leaves into strips of about 1 cm ⁇ 2 cm, take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / total number of insects) + 10 ⁇ ( Negative control number of insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S1, S2 and S3) transferred into the Cry1A.105 nucleotide sequence, and transferred to the Cry2Ab nucleotide sequence for a total of 3 transformation event lines (S4, S5 and S6), transferred to A total of three transformation event lines (S7, S8 and S9) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM1) strain by Taqman, and one wild type (CK1). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 1 and Figure 3.
  • Table 1 The results in Table 1 indicate that maize plants transferred to the Cry1A.105 nucleotide sequence were transferred to Cry1A.105-Cry2Ab
  • the nucleotide sequence of maize plants had good insecticidal effect on the millet, and the average mortality rate of the millet was more than 80%, and the total score of resistance was also about 280; and it was identified by Taqman as The total resistance score of non-transgenic corn plants and wild-type corn plants is generally around 20 minutes.
  • Maize plants transferred to the Cry2Ab nucleotide sequence showed no control effect on millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic corn identified by Taqman. Plants did not show differences compared to wild-type maize plants.
  • Sugarcane plants transfected into Cry1A.105 nucleotide sequence, sugarcane plants transfected into Cry2Ab nucleotide sequence, sugarcane plants transfected with Cry1A.105-Cry2Ab nucleotide sequence, wild-type sugarcane plants and Taqman were identified as Fresh leaves of non-transgenic sugarcane plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the sugarcane leaves into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / total number of insects) + 10 ⁇ ( Negative control number of insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • the results in Figure 4 indicate that sugarcane plants transfected with the Cry1A.105 nucleotide sequence and sugarcane plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence can cause larvae of the larvae of the larvae compared to wild-type sugarcane plants. A large number of deaths, and greatly inhibited the development of a very small number of surviving larvae. After 3 days, the larvae were still in the initial hatching state, and showed extremely weak vitality, and transferred to the Cry1A.105 nucleotide sequence of sugarcane plants and transferred. The sugarcane plants entering the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the naked eye could hardly distinguish the feeding traces of the millet, and the leaf damage rate was below 3%.
  • sugarcane plants which were transferred into the Cry1A.105 nucleotide sequence and sugarcane plants which were transferred into the Cry1A.105-Cry2Ab nucleotide sequence showed high activity against the mulberry, which was sufficient for the growth of the ash mites.
  • the adverse effects thus allow it to be controlled in the field.
  • by controlling the drill collar of the ash ash it is also possible to reduce the occurrence of diseases on the sugar cane, and greatly improve the yield and quality of sugar cane.
  • Sorghum plants transfected into the Cry1A.105 nucleotide sequence, sorghum plants transfected with the Cry2Ab nucleotide sequence, sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, wild-type sorghum plants and Taqman were identified as Fresh leaves of non-transgenic sorghum plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the sorghum leaves into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting Long strips of leaves
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / total number of insects) + 10 ⁇ (negative control number of insects / insects) Total)] + 100 ⁇ (1 - blade damage rate).
  • the results in Table 3 indicate that the sorghum plants transfected with the Cry1A.105 nucleotide sequence and the sorghum plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence have good insecticidal effects on the ash mites, and the ash mites
  • the average mortality rate is about 90%, and the total score of resistance is also above 280 points.
  • the total resistance score of sorghum plants and wild-type sorghum plants identified by Taqman as non-transgenic is generally about 20 points.
  • the sorghum plants transferred to the Cry2Ab nucleotide sequence showed no control effect on the millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic identification by Taqman. There was no difference in sorghum plants compared to wild-type sorghum plants.
  • the sorghum plant transformed into the Cry1A.105 nucleotide sequence and the sorghum plant transformed into the Cry1A.105-Cry2Ab nucleotide sequence showed high activity against the mulberry, which was sufficient for the growth of the ash mites.
  • the adverse effects thus allow it to be controlled in the field.
  • by controlling the drill collar of the ash mites it is also possible to reduce the occurrence of diseases on sorghum and greatly improve the yield and quality of sorghum.
  • the millet plants transformed with the Cry1A.105 nucleotide sequence, the millet plants transferred to the Cry2Ab nucleotide sequence, the millet plants transfected with the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type millet plants, and Taqman were identified as Fresh leaves of non-transgenic millet plants (expanded young leaves), rinsed with sterile water and blotted the water on the leaves with gauze, then cut the leaves of the millet into strips of about 1 cm ⁇ 2 cm, and take 1 piece after cutting The long strips of leaves are placed on the moisturizing filter paper at the bottom of the round plastic Petri dish.
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (number of initial hatching - negative control insects / total number of insects) + 10 ⁇ ( Negative control number of insects / total number of insects)] + 100 ⁇ (1 - blade damage rate).
  • a total of 3 transformation event lines (S28, S29 and S30) transfected into the Cry1A.105 nucleotide sequence, and transferred to the Cry2Ab nucleotide sequence for a total of 3 transformation event lines (S31, S32 and S33), transferred to A total of 3 transformation event lines (S34, S35 and S36) of the Cry1A.105-Cry2Ab nucleotide sequence were identified as one non-transgenic (NGM4) strain by Taqman and 1 wild type (CK4). Strains; 3 strains were selected from each strain and tested 6 times per plant. The results are shown in Table 4 and Figure 6.
  • the results in Table 4 indicate that the millet plant transferred to the Cry1A.105 nucleotide sequence was transferred to Cry1A.105-Cry2Ab
  • the nucleotide sequence of the millet plant has good insecticidal effect on the millet, and the average mortality rate of the millet is about 90%, and the total resistance score is also above 280;
  • the total score of resistance of non-transgenic millet plants and wild-type millet plants is generally around 20 points.
  • the results in Figure 6 indicate that the millet plants transferred to the Cry1A.105 nucleotide sequence and the millet plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence can cause the larvae of the larvae of the larvae to be larvae compared to the wild-type millet plants. A large number of deaths, and greatly inhibited the development of a very small number of surviving larvae. After 3 days, the larvae were still in the initial hatching state, and showed extremely weak vitality, and the millet plants and the Cry1A.105 nucleotide sequence were transferred. The millet plants that entered the Cry1A.105-Cry2Ab nucleotide sequence were only slightly damaged, and the naked eye could hardly discern the feeding traces of the millet, and the leaf damage rate was below 5%.
  • the millet plants transferred to the Cry2Ab nucleotide sequence showed no control effect on the millet ash, regardless of mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic millet identified by Taqman. Plants did not show differences compared to wild-type millet plants.
  • the control and control of the millet and the millet plant transferred into the Cry1A.105-Cry2Ab nucleotide sequence is apparently because the plant itself can produce the Cry1A.105 protein, so, as is well known to those skilled in the art, according to the Cry1A.105 protein
  • the same toxic effect on the ash mites can produce similar transgenic plants expressing the Cry1A.105 protein that can be used to control/control the damage of the ash.
  • the Cry1A.105 protein of the present invention includes, but is not limited to, the Cry1A.105 protein of the amino acid sequence given in the specific embodiment, and the transgenic plant can also produce at least one second insecticidal protein different from the Cry1A.105 protein, such as Vip-like protein, Cry-like protein.
  • the use of the insecticidal protein of the present invention controls the C. sinensis pest by producing a Cry1A.105 protein capable of killing the ash mites in the plant; the agricultural control method, the chemical control method and the physical control used in the prior art Compared with the method, the invention protects the plant from the whole growth period and the whole plant to prevent the damage of the worm and the pest, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economical.

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Abstract

本发明涉及一种杀虫蛋白的用途，所述控制粟灰螟害虫的方法包括：将粟灰螟害虫至少与Cry1A.105蛋白接触。本发明通过植物体内产生能够杀死粟灰螟的Cry1A.105蛋白来控制粟灰螟害虫；与现有技术使用的农业防治方法、化学防治方法和物理防治方法相比，本发明对植物进行全生育期、全植株的保护以防治粟灰螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。

Description

杀虫蛋白的用途 技术领域
本发明涉及一种杀虫蛋白的用途，特别是涉及一种Cry1A.105蛋白质通过在植物中表达来控制粟灰螟为害植物的用途。
背景技术
随着全球温室效应的加强，温度不断上升，粮食作物虫害发生种类及数量都有所提高。目前针对虫害，主要依靠农业防治、化学防治和物理防治。
农业防治是对整个农田生态***中的多因素的综合协调管理，调控作物、害虫、环境因素、创造一个有利于作物生长而不利于虫害发生的农田生态环境。同时可采用轮作的方式以降低虫源密度，但不同作物的经济效益的不同，轮作很可能造成农民收入的降低，而很难得到实行。
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是虫害综合治理的重要组成部分，它具有快速、方便、简单和高经济效益的特点，特别是虫害大发生的情况下，是必不可少的应急措施。但化学防治也有其局限性，如使用不当往往会导致农作物发生药害、害虫产生抗药性，以及杀伤天敌、污染环境，使农田生态***遭到破坏和农药残留对人、畜的安全构成威胁等不良后果。
物理防治主要根据害虫对环境条件中各种物理因素的反应，利用各种物理因素如光、电、色、温湿度等以及机械设备进行诱杀、辐射不育等方法来防治害虫。目前应用最广泛的是频振式杀虫灯诱杀，它利用害虫成虫的趋光性，近距离用光，远距离用波，引诱害虫靠近，对虫害防治具有一定的防治效果；但是频振式杀虫灯需要每天及时清理高压电网上的污垢，否则会影响杀虫效果；并且在雷雨天不能开灯，在操作上还有电击伤人的危险；此外安装灯的一次性投入较大。
为了解决农业防治、化学防治和物理防治在实际应用中的局限性，科学家们经过研究发现将来自于苏云金芽孢杆菌(Bacillus thuringiensis subsp.kurstaki，B.t.k.)的编码杀虫蛋白的抗虫基因转入植物中，可获得一些抗虫转基因植物以防治植物虫害。Cry1A.105杀虫蛋白是众多杀虫蛋白中的一种，是一种嵌合蛋白，分别来自于Cry1Ab蛋白、Cry1Ac蛋白和Cry1Fa蛋白。
已证明转Cry1A.105基因的植株可以抵抗玉米螟、棉铃虫、秋粘虫等鳞翅目(Lepidoptera)害虫的侵害，然而，至今尚无关于通过产生表达Cry1A.105蛋白的转基因植株来控制粟灰螟对植物危害的报道。
栗灰螟(Chilo infuscatellus)又名甘蔗二点螟，属于鳞翅目螟蛾科，广泛分布于东南亚、南亚及中国，在中国主要为害禾本科作物，如玉米、高粱、谷子和甘蔗等。粟灰螟为蛀茎害虫，植株受害后可造成枯心株。一般情况下，春谷区和春夏谷混播区发生严 重，夏谷区为害轻。粟灰螟曾经是华北地区和华南地区的主要害虫，主要为害高粱、谷子和甘蔗。但近年来，出现了大量关于粟灰螟为害玉米的报道，且该为害呈上升趋势。
发明内容
本发明的目的是提供一种杀虫蛋白的用途，首次提供了通过产生表达Cry1A.105蛋白的转基因植株来控制粟灰螟对植物危害的方法，且有效克服现有技术农业防治、化学防治和物理防治等技术缺陷。
为实现上述目的，本发明提供了一种控制粟灰螟害虫的方法，包括将粟灰螟害虫至少与Cry1A.105蛋白接触。
进一步地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的宿主细胞中，所述粟灰螟害虫通过摄食所述宿主细胞至少与所述Cry1A.105蛋白接触。
更进一步地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的细菌或转基因植物中，所述粟灰螟害虫通过摄食所述细菌或所述转基因植物的组织至少与所述Cry1A.105蛋白接触，接触后所述粟灰螟害虫生长受到抑制和/或导致死亡，以实现对粟灰螟危害植物的控制。
所述细菌可以为能够产生Cry1A.105蛋白的野生菌和/或重组菌。例如苏云金芽孢杆菌库斯塔基亚种(Bacillus thuringiensis subsp.kurstaki，B.t.k.)。
所述转基因植物可以处于任意生育期。
所述转基因植物的组织可以为植物的各种组织，如叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
所述对粟灰螟危害植物的控制不因种植地点和/或种植时间的改变而改变。
所述植物可以为各种受粟灰螟危害的禾本科植物，优选地，所述植物为玉米、高粱、谷子、甘蔗、水稻、小麦、大麦或燕麦。
所述接触步骤之前的步骤为种植含有编码所述Cry1A.105蛋白的多核苷酸的植物。
优选地，所述Cry1A.105蛋白的氨基酸序列具有SEQ ID NO:1所示的氨基酸序列。所述Cry1A.105蛋白的核苷酸序列具有SEQ ID NO:2所示的核苷酸序列。
在上述技术方案的基础上，所述植物还可以包括至少一种不同于编码所述Cry1A.105蛋白的核苷酸的第二种核苷酸。
进一步地，所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶。
优选地，所述第二种核苷酸编码Cry2Ab蛋白。
进一步地，所述Cry2Ab蛋白的氨基酸序列具有SEQ ID NO:3所示的氨基酸序列。
更进一步地，所述Cry2Ab蛋白的核苷酸序列具有SEQ ID NO:4所示的核苷酸序列。
可选择地，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
为实现上述目的，本发明还提供了一种Cry1A.105蛋白质控制粟灰螟害虫的用途。
为实现上述目的，本发明还提供了一种产生控制粟灰螟害虫的植物的方法，包括 向所述植物的基因组中引入编码Cry1A.105蛋白的多核苷酸序列。
为实现上述目的，本发明还提供了一种产生控制粟灰螟害虫的植物繁殖体的方法，包括将由所述方法获得的第一植株与第二植株杂交，和/或取下由所述方法获得的植株上具有繁殖能力的组织进行培养，从而产生含有编码Cry1A.105蛋白的多核苷酸序列的植物繁殖体。
为实现上述目的，本发明还提供了一种培养控制粟灰螟害虫的植物的方法，包括：
种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括编码Cry1A.105蛋白的多核苷酸序列；
使所述植物繁殖体长成植株；
使所述植株在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下生长，收获与其他不具有编码Cry1A.105蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
本发明中所述的“植物繁殖体”包括但不限于植物有性繁殖体和植物无性繁殖体。所述植物有性繁殖体包括但不限于植物种子；所述植物无性繁殖体是指植物体的营养器官或某种特殊组织，其可以在离体条件下产生新植株；所述营养器官或某种特殊组织包括但不限于根、茎和叶，例如：以根为无性繁殖体的植物包括草莓和甘薯等；以茎为无性繁殖体的植物包括甘蔗和马铃薯(块茎)等；以叶为无性繁殖体的植物包括芦荟和秋海棠等。
本发明中所述的“接触”，是指昆虫和/或害虫触碰、停留和/或摄食植物、植物器官、植物组织或植物细胞，所述植物、植物器官、植物组织或植物细胞既可以是其体内表达杀虫蛋白，还可以是所述植物、植物器官、植物组织或植物细胞的表面具有杀虫蛋白和/或具有产生杀虫蛋白的微生物。
本发明术语“控制”和/或“防治”是指粟灰螟害虫至少与Cry1A.105蛋白接触，接触后粟灰螟害虫生长受到抑制和/或导致死亡。进一步地，粟灰螟害虫通过摄食植物组织至少与Cry1A.105蛋白接触，接触后全部或部分粟灰螟害虫生长受到抑制和/或导致死亡。抑制是指亚致死，即尚未致死但能引起生长发育、行为、生理、生化和组织等方面的某种效应，如生长发育缓慢和/或停止。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，含有编码Cry1A.105蛋白的多核苷酸序列的控制粟灰螟害虫的植物和/或植物种子，在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下，与非转基因的野生型植株相比具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量、和/或增产等。Cry1A.105蛋白对粟灰螟的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”粟灰螟害虫的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码Cry1A.105蛋白的多核苷酸序列)的任何组织同时和/或不同步地，存在和/或产生，Cry1A.105蛋白和/或可控制粟灰螟害虫的另一种物质，则所述另一种物质的存在既不影响Cry1A.105蛋白对粟灰螟的“控制”和/或“防治”作用，也不能导致所述“控制”和 /或“防治”作用完全和/或部分由所述另一种物质实现，而与Cry1A.105蛋白无关。通常情况下，在大田，粟灰螟害虫摄食植物组织的过程短暂且很难用肉眼观察到，因此，在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下，如转基因植物(含有编码Cry1A.105蛋白的多核苷酸序列)的任何组织存在死亡的粟灰螟害虫、和/或在其上停留生长受到抑制的粟灰螟害虫、和/或与非转基因的野生型植株相比具有减弱的植物损伤，即为实现了本发明的方法和/或用途，即通过粟灰螟害虫至少与Cry1A.105蛋白接触以实现控制粟灰螟害虫的方法和/或用途。
在本发明中，Cry1A.105蛋白在一种转基因植物中的表达可以伴随着一个或多个Cry类杀虫蛋白质和/或Vip类杀虫蛋白质的表达。这种超过一种的杀虫毒素在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达Cry1A.105蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达Cry类杀虫蛋白质和/或Vip类杀虫蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。因此在本发明中可以使用RNAi技术特异性剔除或关闭目标昆虫害虫中特定基因的表达。
在分类***上，一般主要根据成虫翅的脉序、连锁方式和触角的类型等形态特征，将鳞翅目分为亚目、总科、科等，而螟蛾科是鳞翅目中种类最多的科之一，全世界已发现1万种以上，仅中国记录就有几千条。大部分螟蛾科昆虫是农作物的害虫，多数以蛀茎形式为害，如二化螟和玉米螟。尽管粟灰螟与二化螟、玉米螟等同属于鳞翅目螟蛾科，除了在分类标准上存在相似性，在其它形态结构上则存在极大差异；就好比植物中的草莓与苹果一样(同属于蔷薇目蔷薇科)，它们都有花两性，辐射对称，花瓣5片等特征，但是其果实以及植株形态却是千差万别。而粟灰螟不管是从幼虫形态还是成虫形态上来看，都具有其独特的特征。如背部纵线，在农民中就有流传着“高粱玉米谷，背线三四五”，表示同属于螟蛾科的高粱条螟、玉米螟和粟灰螟在背线数量上就存在明显的差异。而背部纵线下就是背血管，背血管是昆虫循环器官的重要组成部分，内里充满了有昆虫“血液”之称的血淋巴。因此体表形态上看似细微的背线数量的差异，体现的却是背血管的差异，是昆虫循环***上的差异。
同属螟蛾科的昆虫不仅在形态特征上存在较大差异，同时在取食习性上，也存在差异。例如同为螟蛾科的二化螟主要为害水稻，极少为害其它禾本科作物。而粟灰螟未见有报道对水稻造成为害，更多的是对南方的甘蔗，北方的高粱、谷子与玉米造成为害。取食习性的不同，也暗示着体内消化***所产生的酶和受体蛋白不同。而消化道中产生的酶是Bt基因起作用的关键点，只有能够与特异性Bt基因相结合的酶或受体蛋白，才有可能使得某个Bt基因对该害虫具有抗虫效果。越来越多的研究表明，同目不同科、甚至同科不同种的昆虫对同种Bt蛋白的敏感性表现不同。例如Vip3Aa基因对螟蛾科的二化螟(Chilo suppressalis)、亚洲玉米螟(Ostrinia furnacalis)都表现出了抗虫活性， 但是对于同属螟蛾科的印度谷螟(Plodia interpunctella)以及欧洲玉米螟(Ostrinia nubilalis)却没有抗虫效果。上述四种害虫均属于鳞翅目螟蛾科，但同种Bt蛋白对四种螟蛾科害虫表现出不同的抗性效果。尤其是欧洲玉米螟和亚洲玉米螟在分类上甚至同属于螟蛾科Ostrinia属(同目同科同属)，但是其对同种Bt蛋白的反应却是截然不同的，更加充分说明了Bt蛋白与昆虫体内酶和受体的相互作用方式是复杂且难以预料的。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA。
本发明中核酸分子或其片段在严格条件下与本发明Cry1A.105基因杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定本发明Cry1A.105基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。 此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:2发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
因此，具有抗虫活性并在严格条件下与本发明SEQ ID NO:2杂交的序列包括在本发明中。这些序列与本发明序列至少具有大约40％-50％的同源性，大约60％、65％或70％的同源性，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的同源性。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的杀虫活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“变异”是指编码同一蛋白或编码有杀虫活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与权利要求的蛋白具有相同或基本相同的抗粟灰螟害虫的生物活性的蛋白。
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸或氨基酸)的一部分或其人工改造形式(例如适合植物表达的序列)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为昆虫毒素。
使用标准技术可以修饰基因和容易的构建基因变异体。例如，本领域熟知制造点突变的技术。又例如美国专利号5605793描述了在随机断裂后使用DNA重装配产生其它分子多样性的方法。可以使用商业化核酸内切酶制造全长基因的片段，并且可以按照标准程序使用核酸外切酶。例如，可以使用酶诸如Bal31或定点诱变从这些基因的末端***地切除核苷酸。还可以使用多种限制性内切酶获取编码活性片段的基因。可以使用蛋白酶直接获得这些毒素的活性片段。
本发明可以从B.t.分离物和/或DNA文库衍生出等价蛋白和/或编码这些等价蛋白的基因。有多种方法获取本发明的杀虫蛋白。例如，可以使用本发明公开和要求保护的杀虫蛋白的抗体从蛋白质混合物鉴定和分离其它蛋白。特别地，抗体可能是由蛋白最恒定和与其它B.t.蛋白最不同的蛋白部分引起的。然后可以通过免疫沉淀、酶联免疫吸附测定(ELISA)或Western印迹方法使用这些抗体专一地鉴定有特征活性的等价蛋白。可使用本领域标准程序容易的制备本发明中公开的蛋白或等价蛋白或这类蛋白的片段的抗体。然后可以从微生物中获得编码这些蛋白的基因。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或***但实质上不影响杀虫活性的序列，亦包括保留杀虫活性的片段。
本发明中氨基酸序列的取代、缺失或添加是本领域的常规技术，优选这种氨基酸变化为：小的特性改变，即不显著影响蛋白的折叠和/或活性的保守氨基酸取代；小的缺失，通常约1-30个氨基酸的缺失；小的氨基或羧基端延伸，例如氨基端延伸一个甲硫氨酸残基；小的连接肽，例如约20-25个残基长。
保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。通常不改变特定活性的那些氨基酸取代在本领域内是众所周知的，并且已由，例如，N.Neurath和R.L.Hill在1979年纽约学术出版社(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn，Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的抗虫活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol 224:899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。
在本发明中，Cry1A.105蛋白包括但不限于SEQ ID NO:1，与SEQ ID NO:1所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列的同源性(类似性/相同性)典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的同源性范围定义本发明的优选的多核苷酸和蛋白质。例如与本发明示例的序列有49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同源性。
其中，仍具有杀虫活性或者保留杀虫活性意指氨基酸序列与SEQ ID NO:1所示的氨基酸序列具有一定同源性的蛋白的杀虫活性(抗性总分，按照第五实施例中记载的方法得到)为SEQ ID NO:1所示的Cry1A.105蛋白的杀虫活性(抗性总分)的80％以上，或90％以上，或92％以上，或95％以上，或98％以上，或100％。相应地，所述Cry1A.105蛋白的核苷酸序列能够编码杀虫活性满足上述要求的Cry1A.105蛋白。
可以通过Smith-Waterman算法对序列之间的“同源性”进行测量(Smith TF，Waterman MS(1981)，“Identification of Common Molecular Subsequences”，Journal of Molecular Biology 147：195-197)，通过引用其全部内容将其与本文结合。
在本发明中，产生所述Cry1A.105蛋白的转基因植物包括但不限于MON89034转基因玉米事件和/或包含MON89034转基因玉米事件的植物材料(如在CN101495635A所描述的)或者MON87751转基因大豆事件和/或包含MON87751转基因大豆事件的植物材料(如在USDA APHIS非管制状态申请13-337-01p所描述的)，其均可以实现本发明的方法和/或用途，即通过粟灰螟害虫至少与Cry1A.105蛋白接触以实现控制粟灰螟害虫的方法和/或用途。本领域技术人员所理解的，使上述转基因事件中的Cry1A.105蛋白在不同植物中表达亦能实现本发明的方法和/或用途。更具体地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的转基因植物中，所述粟灰螟害虫通过摄食所述转基因植物的组织至少与所述Cry1A.105蛋白接触，接触后所述粟灰螟害虫生长受到抑制和/或导致死亡，以实现对粟灰螟危害植物的控制。
本发明中所述调控序列包括但不限于启动子、转运肽、终止子、增强子、前导序列，内含子以及其它可操作地连接到所述Cry1A.105蛋白的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pinⅠ和pinⅡ)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫 茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明中所述的“杀虫”或“抗虫”是指对农作物害虫是有毒的，从而实现“控制”和/或“防治”农作物害虫。优选地，所述“杀虫”或“抗虫”是指杀死农作物害虫。更具体地，目标昆虫是粟灰螟害虫。
本发明中Cry1A.105蛋白对粟灰螟害虫具有毒性。本发明中的植物，特别是谷子、甘蔗、高粱和玉米，在其基因组中含有外源DNA，所述外源DNA包含编码Cry1A.105蛋白的核苷酸序列，粟灰螟害虫通过摄食植物组织与该蛋白接触，接触后粟灰螟害虫生长受到抑制和/或导致死亡。抑制是指致死或亚致死。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，该植物可基本消除对化学或 生物杀虫剂的需要(所述化学或生物杀虫剂为针对Cry1A.105蛋白所靶向的粟灰螟害虫的杀虫剂)。植物材料中杀虫晶体蛋白(ICP)的表达水平可通过本领域内所描述的多种方法进行检测，例如通过应用特异引物对组织内产生的编码杀虫蛋白质的mRNA进行定量，或直接特异性检测产生的杀虫蛋白质的量。可以应用不同的试验测定植物中ICP的杀虫效果。本发明中目标昆虫主要为粟灰螟。
本发明中，所述Cry1A.105蛋白可以具有序列表中SEQ ID NO:1所示的氨基酸序列。除了包含Cry1A.105蛋白的编码区外，也可包含其他元件，例如编码选择性标记的蛋白质。
此外，包含编码本发明Cry1A.105蛋白的核苷酸序列的表达盒在植物中还可以与至少一种编码除草剂抗性基因的蛋白质一起表达，所述除草剂抗性基因包括但不限于，草胺膦抗性基因(如bar基因、pat基因)、苯敌草抗性基因(如pmph基因)、草甘膦抗性基因(如EPSPS基因)、溴苯腈(bromoxynil)抗性基因、磺酰脲抗性基因、对除草剂茅草枯的抗性基因、对氨腈的抗性基因或谷氨酰胺合成酶抑制剂(如PPT)的抗性基因，从而获得既具有高杀虫活性、又具有除草剂抗性的转基因植物。
本发明中，将外源DNA导入植物，如将编码所述Cry1A.105蛋白的基因或表达盒或重组载体导入植物细胞，常规的转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。
本发明提供了一种杀虫蛋白的用途，具有以下优点：
1、内因防治。现有技术主要是通过外部作用即外因来控制粟灰螟害虫的危害，如农业防治、化学防治和物理防治；而本发明是通过植物体内产生能够杀死粟灰螟的Cry1A.105蛋白来控制粟灰螟害虫的，即通过内因来防治。
2、无污染、无残留。现有技术使用的化学防治方法虽然对控制粟灰螟害虫的危害起到了一定作用，但同时也对人、畜和农田生态***带来了污染、破坏和残留；使用本发明控制粟灰螟害虫的方法，可以消除上述不良后果。
3、全生育期防治。现有技术使用的控制粟灰螟害虫的方法都是阶段性的，而本发明是对植物进行全生育期的保护，转基因植物(Cry1A.105蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受粟灰螟的侵害。
4、全植株防治。现有技术使用的控制粟灰螟害虫的方法大多是局部性的，如叶面喷施；而本发明是对整个植株进行保护，如转基因植物(Cry1A.105蛋白)的叶片、茎秆、果实、雄穗、雌穗、花药或花丝等都是可以抵抗粟灰螟侵害的。
5、效果稳定。现有技术使用的频振式杀虫灯不仅需要每天及时清理高压电网的污垢，而且在雷雨天不能使用；本发明是使所述Cry1A.105蛋白在植物体内进行表达，有效地克服了频振式杀虫灯的效果受外界因素影响的缺陷，且本发明转基因植物(Cry1A.105蛋白)的防治效果在不同地点、不同时间、不同遗传背景也都是稳定一致的。
6、简单、方便、经济。现有技术使用的频振式杀虫灯的一次性投入较大，且操作 不当还有电击伤人的危险；本发明只需种植能够表达Cry1A.105蛋白的转基因植物即可，而不需要采用其它措施，从而节省了大量人力、物力和财力。
7、效果彻底。现有技术使用的控制粟灰螟害虫的方法，其效果是不彻底的，只起到减轻作用；而本发明转基因植物(Cry1A.105蛋白)可以造成初孵粟灰螟幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，都是明显的发育不良，且已停止发育，在田间自然环境中无法存活，而转基因植物大体上只受到轻微损伤。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明杀虫蛋白的用途的含有Cry1A.105核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本发明杀虫蛋白的用途的含有Cry1A.105核苷酸序列的重组表达载体DBN100745构建流程图；
图3为本发明杀虫蛋白的用途的转基因玉米植株接种粟灰螟的叶片损伤图；
图4为本发明杀虫蛋白的用途的转基因甘蔗植株接种粟灰螟的叶片损伤图；
图5为本发明杀虫蛋白的用途的转基因高粱植株接种粟灰螟的叶片损伤图；
图6为本发明杀虫蛋白的用途的转基因谷子植株接种粟灰螟的叶片损伤图。
具体实施方式
下面通过具体实施例进一步说明本发明杀虫蛋白的用途的技术方案。
第一实施例、基因的获得和合成
1、获得核苷酸序列
Cry1A.105杀虫蛋白质的氨基酸序列(1177个氨基酸)，如序列表中SEQ ID NO:1所示；编码相应于所述Cry1A.105杀虫蛋白质的氨基酸序列(1177个氨基酸)的Cry1A.105核苷酸序列(3534个核苷酸)，如序列表中SEQ ID NO:2所示。
编码Cry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)，如序列表中SEQ ID No:3所示；编码相应于所述Cry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)的Cry2Ab核苷酸序列(1905个核苷酸)，如序列表中SEQ ID NO:4所示。
2、合成上述核苷酸序列
所述Cry1A.105核苷酸序列(如序列表中SEQ ID NO:2所示)和所述Cry2Ab核苷酸序列(如序列表中SEQ ID NO:4所示)由南京金斯瑞生物科技有限公司合成；合成的所述Cry1A.105核苷酸序列(SEQ ID NO:2)的5’端还连接有NcoI酶切位点，所述Cry1A.105核苷酸序列(SEQ ID NO:2)的3’端还连接有HindIII酶切位点；合成的所述Cry2Ab核苷酸序列(SEQ ID NO:4)的5’端还连接有NcoI酶切位点，所述Cry2Ab核苷酸序列(SEQ ID NO:4)的3’端还连接有SpeI酶切位点。
第二实施例、重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有Cry1A.105基因的重组克隆载体
将合成的Cry1A.105核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图1所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6 RNA聚合酶启动子；T7为T7 RNA聚合酶启动子；Cry1A.105为Cry1A.105核苷酸序列(SEQ ID NO:2)；MCS为多克隆位点)。
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100毫克/升)的LB平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl，10mM EDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入200μl新配制的溶液II(0.2M NaOH，1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μl冰冷的溶液III(3M醋酸钾，5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μl含RNase(20μg/ml)的TE(10mM Tris-HCl，1mM EDTA，PH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。
提取的质粒经AhdI和XhoI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中***的所述Cry1A.105核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即Cry1A.105核苷酸序列正确***。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry2Ab核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN02-T，其中，Cry2Ab为Cry2Ab核苷酸序列(SEQ ID NO:4)。酶切和测序验证重组克隆载体DBN02-T中所述Cry2Ab核苷酸序列正确***。
2、构建含有Cry1A.105基因的重组表达载体
用限制性内切酶NcoI和HindIII分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的Cry1A.105核苷酸序列片段插到表达载体DBNBC-01的NcoI和HindIII位点之间，利用常规的酶切 方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100745，其构建流程如图2所示(Kan：卡那霉素基因；RB：右边界；Ubi：玉米Ubiquitin(泛素)基因启动子(SEQ ID NO:5)；Cry1A.105：Cry1A.105核苷酸序列(SEQ ID NO:2)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:6)；Hpt：潮霉素磷酸转移酶基因(SEQ ID NO:7)；LB：左边界)。
将重组表达载体DBN100745用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组表达载体DBN100745)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，卡那霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶NcoI和HindIII酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100745在NcoI和HindIII位点间的核苷酸序列为序列表中SEQ IDNO:2所示核苷酸序列，即Cry1A.105核苷酸序列。
按照上述构建重组表达载体DBN100745的方法，将NcoI和SpeI酶切重组克隆载体DBN02-T切下的所述Cry2Ab核苷酸序列***表达载体DBNBC-01，得到重组表达载体DBN100744。酶切和测序验证重组表达载体DBN100744中的核苷酸序列含有为序列表中SEQ ID NO:4所示核苷酸序列，即Cry2Ab核苷酸序列，所述Cry2Ab核苷酸序列可以连接所述Ubi启动子和Nos终止子。
按照上述构建重组表达载体DBN100745的方法，将NcoI和HindIII、NcoI和SpeI分别酶切重组克隆载体DBN01-T和DBN02-T切下的所述Cry1A.105核苷酸序列和Cry2Ab核苷酸序列***表达载体DBNBC-01，得到重组表达载体DBN100029。酶切和测序验证重组表达载体DBN100029中的核苷酸序列含有为序列表中SEQ ID NO:2和SEQ ID NO:4所示核苷酸序列，即Cry1A.105核苷酸序列和Cry2Ab核苷酸序列，所述Cry1A.105核苷酸序列和所述Cry2Ab核苷酸序列可以连接所述Ubi启动子和Nos终止子。
3、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100745、DBN100744和DBN100029用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和100mg/L的卡那霉素(Kanamycin)的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶AhdI和XhoI对重组表达载体DBN100745、DBN100744和DBN100029酶切后进行酶切验证，结果表明重组表达载体DBN100745、DBN100744和 DBN100029结构完全正确。
第三实施例、转基因植株的获得
1、获得转基因玉米植株
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与第二实施例中3所述的农杆菌共培养，以将第二实施例中2构建的重组表达载体DBN100745、DBN100744和DBN100029中的T-DNA(包括玉米Ubiquitin基因的启动子序列、Cry1A.105核苷酸序列、Cry2Ab核苷酸序列、Hpt基因和Nos终止子序列)转入到玉米染色体组中，获得了转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株；同时以野生型玉米植株作为对照。
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)，在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(潮霉素)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、潮霉素50mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。
筛选得到的抗性愈伤组织转移到所述MS分化培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、6-苄基腺嘌呤2mg/L、潮霉素50mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养分化。分化出来的小苗转移到所述MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16 小时，再于20℃下培养8小时。
2、获得转基因高粱植株
参考Molecular Biology and Genetic Engineering ISSN 2053-5767的高粱转化方法。收集高粱品种APKI的种子，并用清水冲洗数次；浸泡于tween-20浸润液中5分钟；之后用双蒸水悬浮清洗，并在通风橱中干燥；种子表面用70％(v/v)乙醇消毒30秒，紧接着用0.1％(w/v)HgCl2消毒6分钟；再用双蒸水清洗5-6次；将种子铺于含有MS基础固体培养基(pH5.8)的培养皿中，将培养皿摆放于温度为24±2℃、相对湿度为70％、光周期(光/暗)为12:12的培养间中；3-5天后，种子发芽，取茎尖外植体浸泡于农杆菌中30分钟；取出浸泡后的外植体摆放于已灭菌的滤纸上；黑暗条件下共培养72小时；愈伤组织用含有500mg/L头孢霉素的无菌水清洗3-5次；将清洗后的愈伤组织转移至诱导培养基上培养7天；再转移至筛选培养基上2-3周，重复筛选3次；抗性愈伤被转移至再生培养基上；再生出叶片等，将小苗移至生根培养基上，待生根后移栽至温室中。培养基配方参考Molecular Biology and Genetic Engineering ISSN 2053-5767，其中筛选剂根据本发明中转基因载体所用，更换为潮霉素。由此获得了转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株；同时以野生型高粱植株作为对照。
3、获得转基因甘蔗植株
转化方法主要参考广西大学2012级硕士李粲学位论文第22页至24页。取甘蔗顶端新生茎节，去掉蔗梢和叶鞘，留下茎尖生长锥及心叶茎段。在超净工作台上，用75％(v/v)酒精棉球对表面进行擦拭消毒，用已灭菌的镊子小心剥去心叶外层，取中间5—7cm长的心叶段，横切成厚度约3mm的薄片接种于诱导培养基上，温度26℃条件下，黑暗培养20天。挑选生长情况良好的愈伤组织转移到新的MS培养基中预培养4天，再用于转化试验；转化时，在超净工作台中将待侵染的愈伤组织用已灭菌的镊子夹出，放在干净的滤纸上面静置2小时，至表面完全干燥，稍有收缩；将干燥的甘蔗愈伤组织放入侵染液中浸泡30分钟，同时放在摇床上缓慢摇动；将愈伤组织捞出并转移到干净的滤纸上，在超净工作台中完全吹干，直至愈伤组织表面干燥、无水膜。把愈伤组织块切成0.6*0.6cm的小块，之后转移到含有100μmol/L乙酰丁香酮(AS)的MR固体培养基中，温度23℃暗培养3天；把侵染后的愈伤组织夹出，置于滤纸上在超净工作台上吹干，直到材料表面干爽后，将材料转移到含有500mg/L头孢霉素和潮霉素筛选的分化培养基中；每隔2周更换一次培养基，期间把被污染的愈伤组织剔除，当幼苗长约3cm高的时候，转移到含有潮霉素筛选剂的生根培养基中诱导生根。由此获得了转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株；同时以野生型甘蔗植株作为对照。
4、获得转基因谷子植株
转化方法参考河北农业大学2012级硕士王寒玉学位论文第9页至第10页。将成熟种子在0.1％(v/v)Tween-20溶液中浸泡后，用70％(v/v)乙醇清洗，然后转移到 0.1％(w/v)HgCl2溶液中，最后用灭菌水清洗2-3次。将经过灭菌处理的种子转移到MS培养基上，温度25℃暗培养2-3天。待种子茎尖长到4-6mm时，在无菌条件下将茎尖转移到愈伤诱导培养基上。
茎尖愈伤组织的诱导：将诱导后的茎尖愈伤组织浸取农杆菌菌悬液30分钟，取出茎尖愈伤组织放于已灭菌滤纸上，吸出多余菌液，转移到共培养培养基上(MS+100mol/L AS+2,4-D)，温度28℃黑暗中共培养2-4天。之后将共培养后的愈伤组织转到MS愈伤诱导培养基(2,4-D 4.5μmol/L、2.25μmol/L Kn、头孢霉素500mg/L)上，温度25℃暗培养，每两周对愈伤进行一次继代。幼穗在愈伤诱导培养基上培养5周后，选取结构致密的黄白色愈伤组织转移到分化培养基上进行分化，分化培养基中加入筛选剂潮霉素。约五周后，愈伤形成节状结构，将有节状结构的愈伤转移到MS分化生根培养基(噻二唑苯基脲(TDZ)4.5μmol/L、蔗糖120μmol/L)上。由此获得了转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株；同时以野生型谷子植株作为对照。
第四实施例、用TaqMan验证转基因植株
分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测Cry1A.105基因和Cry2Ab基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测Cry1A.105基因和Cry2Ab基因拷贝数的具体方法如下：
步骤11、分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μl；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型玉米植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测Cry1A.105核苷酸序列：
引物1：GCGCATCCAGTTCAACGAC如序列表中SEQ ID NO:8所示；
引物2：GTTCTGGACGGCGAAGAGTG如序列表中SEQ ID NO:9所示；
探针1：TGAACAGCGCCCTGACCACCG如序列表中SEQ ID NO:10所示；
以下引物和探针用来检测Cry2Ab核苷酸序列：
引物3：CTGATACCCTTGCTCGCGTC如序列表中SEQ ID NO:11所示；
引物4：CACTTGGCGGTTGAACTCCTC如序列表中SEQ ID NO:12所示；
探针2：CGCTGAGCTGACGGGTCTGCAAG如序列表中SEQ ID NO:13所示；
PCR反应体系为：
Figure PCTCN2015092007-appb-000001
所述50×引物/探针混合物包含1mM浓度的每种引物各45μl，100μM浓度的探针50μl和860μl 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2015092007-appb-000002
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株均获得了单拷贝的转基因玉米植株。
按照上述用TaqMan验证转基因玉米植株的方法，对转基因高粱植株、转基因甘蔗植株和转基因谷子植株进行检测分析。实验结果表明，Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列均己分别整合到所检测的高粱植株、甘蔗植株和谷子植株的染色体组中，而且转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株均获得了单拷贝的转基因植株。
第五实施例、转基因植株的抗虫效果检测
将转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株；转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植 株；转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株；转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株，转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株；相应的野生型玉米植株、高粱植株、甘蔗植株和谷子植株，以及经Taqman鉴定为非转基因的玉米植株、高粱植株、甘蔗植株和谷子植株对粟灰螟进行抗虫效果检测。
1、转基因玉米植株的抗虫效果检测
分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株、野生型玉米植株和经Taqman鉴定为非转基因的玉米植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将玉米叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S1、S2和S3)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S4、S5和S6)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S7、S8和S9)，经Taqman鉴定为非转基因的(NGM1)共1个株系，野生型的(CK1)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表1和图3所示。
表1、转基因玉米植株接种粟灰螟的抗虫实验结果
Figure PCTCN2015092007-appb-000003
表1的结果表明：转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab 核苷酸序列的玉米植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在80％以上，其抗性总分也均在280分左右；而经Taqman鉴定为非转基因的玉米植株和野生型玉米植株的抗性总分一般在20分左右。图3的结果表明：与野生型玉米植株相比，转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。
对于转入Cry2Ab核苷酸序列的玉米植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的玉米植株和野生型玉米植株相比，均未表现出差异。
由此证明转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低玉米上病害的发生，极大的提高玉米的产量及品质。
2、转基因甘蔗植株的抗虫效果检测
分别取转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、野生型甘蔗植株和经Taqman鉴定为非转基因的甘蔗植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将甘蔗叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S10、S11和S12)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S13、S14和S15)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S16、S17和S18)，经Taqman鉴定为非转基因的(NGM2)共1个株系，野生型的(CK2)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表2和图4所示。
表2、转基因甘蔗植株接种粟灰螟的抗虫实验结果
Figure PCTCN2015092007-appb-000004
表2的结果表明：转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在80％以上，其抗性总分也均在280分左右；而经Taqman鉴定为非转基因的甘蔗植株和野生型甘蔗植株的抗性总分一般在20分以下。图4的结果表明：与野生型甘蔗植株相比，转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。
对于转入Cry2Ab核苷酸序列的甘蔗植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的甘蔗植株和野生型甘蔗植株相比，均未表现出差异。
由此证明转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低甘蔗上病害的发生，极大的提高甘蔗的产量及品质。
3、转基因高粱植株的抗虫效果检测
分别取转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、野生型高粱植株和经Taqman鉴定为非转基因的高粱植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将高粱叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入 圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S19、S20和S21)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S22、S23和S24)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S25、S26和S27)，经Taqman鉴定为非转基因的(NGM3)共1个株系，野生型的(CK3)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表3和图5所示。
表3、转基因高粱植株接种粟灰螟的抗虫实验结果
Figure PCTCN2015092007-appb-000005
表3的结果表明：转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在90％左右，其抗性总分也均在280分以上；而经Taqman鉴定为非转基因的高粱植株和野生型高粱植株的抗性总分一般在20分左右。图5的结果表明：与野生型高粱植株相比，转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。
对于转入Cry2Ab核苷酸序列的高粱植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的 高粱植株和野生型高粱植株相比，均未表现出差异。
由此证明转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低高粱上病害的发生，极大的提高高粱的产量及品质。
4、转基因谷子植株的抗虫效果检测
分别取转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株、转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株、野生型谷子植株和经Taqman鉴定为非转基因的谷子植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将谷子叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S28、S29和S30)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S31、S32和S33)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S34、S35和S36)，经Taqman鉴定为非转基因的(NGM4)共1个株系，野生型的(CK4)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表4和图6所示。
表4、转基因谷子植株接种粟灰螟的抗虫实验结果
Figure PCTCN2015092007-appb-000006
表4的结果表明：转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab 核苷酸序列的谷子植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在90％左右，其抗性总分也均在280分以上；而经Taqman鉴定为非转基因的谷子植株和野生型谷子植株的抗性总分一般在20分左右。图6的结果表明：与野生型谷子植株相比，转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在5％以下。
对于转入Cry2Ab核苷酸序列的谷子植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的谷子植株和野生型谷子植株相比，均未表现出差异。
由此证明转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低谷子上病害的发生，极大的提高谷子的产量及品质。
上述实验结果还表明转入Cry1A.105核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株对粟灰螟的控制/防治显然是因为植物本身可产生Cry1A.105蛋白，所以，本领域技术人员熟知的，根据Cry1A.105蛋白对粟灰螟的相同毒杀作用，可产生类似的可表达Cry1A.105蛋白的转基因植株能够用于控制/防治粟灰螟的危害。本发明中Cry1A.105蛋白包括但不限于具体实施方式中所给出氨基酸序列的Cry1A.105蛋白，同时转基因植株还可以产生至少一种不同于Cry1A.105蛋白的第二种杀虫蛋白质，如Vip类蛋白、Cry类蛋白。
综上所述，本发明杀虫蛋白的用途通过植物体内产生能够杀死粟灰螟的Cry1A.105蛋白来控制粟灰螟害虫；与现有技术使用的农业防治方法、化学防治方法和物理防治方法相比，本发明对植物进行全生育期、全植株的保护以防治粟灰螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (20)

  1. 一种控制粟灰螟害虫的方法，其特征在于，包括将粟灰螟害虫至少与Cry1A.105蛋白接触。
  2. 根据权利要求1所述的控制粟灰螟害虫的方法，其特征在于，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的宿主细胞中，所述粟灰螟害虫通过摄食所述宿主细胞至少与所述Cry1A.105蛋白接触。
  3. 根据权利要求2所述的控制粟灰螟害虫的方法，其特征在于，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的细菌或转基因植物中，所述粟灰螟害虫通过摄食所述细菌或所述转基因植物的组织至少与所述Cry1A.105蛋白接触，接触后所述粟灰螟害虫生长受到抑制和/或导致死亡，以实现对粟灰螟危害植物的控制。
  4. 根据权利要求3所述的控制粟灰螟害虫的方法，其特征在于，所述转基因植物可以处于任意生育期。
  5. 根据权利要求3所述的控制粟灰螟害虫的方法，其特征在于，所述转基因植物的组织为叶片、茎秆、果实、雄穗、雌穗、花药或花丝。
  6. 根据权利要求3所述的控制粟灰螟害虫的方法，其特征在于，所述对粟灰螟危害植物的控制不因种植地点和/或种植时间的改变而改变。
  7. 根据权利要求3至6任一项所述的控制粟灰螟害虫的方法，其特征在于，所述植物为玉米、高粱、谷子、甘蔗、水稻、小麦、大麦或燕麦。
  8. 根据权利要求2至7任一项所述的控制粟灰螟害虫的方法，其特征在于，所述接触步骤之前的步骤为种植含有编码所述Cry1A.105蛋白的多核苷酸的植物。
  9. 根据权利要求1至8任一项所述的控制粟灰螟害虫的方法，其特征在于，所述Cry1A.105蛋白的氨基酸序列具有与SEQ ID NO:1所示的氨基酸序列有大于60％的同源性的序列。
  10. 根据权利要求9所述的控制粟灰螟害虫的方法，其特征在于，所述Cry1A.105蛋白的核苷酸序列具有与SEQ ID NO:2所示的核苷酸序列有大于60％的同源性的序列。
  11. 根据权利要求2至10任一项所述的控制粟灰螟害虫的方法，其特征在于，所述植物还可以包括至少一种不同于编码所述Cry1A.105蛋白的核苷酸的第二种核苷酸。
  12. 根据权利要求11所述的控制粟灰螟害虫的方法，其特征在于，所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶。
  13. 根据权利要求12所述的控制粟灰螟害虫的方法，其特征在于，所述第二种核苷酸编码Cry2Ab蛋白。
  14. 根据权利要求13所述的控制粟灰螟害虫的方法，其特征在于，所述Cry2Ab蛋白的氨基酸序列具有SEQ ID NO:3所示的氨基酸序列。
  15. 根据权利要求14所述的控制粟灰螟害虫的方法，其特征在于，所述Cry2Ab蛋白的核苷酸序列具有SEQ ID NO:4所示的核苷酸序列。
  16. 根据权利要求11所述的控制粟灰螟害虫的方法，其特征在于，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
  17. 一种Cry1A.105蛋白质控制粟灰螟害虫的用途。
  18. 一种产生控制粟灰螟害虫的植物的方法，其特征在于，包括向所述植物的基因组中引入编码Cry1A.105蛋白的多核苷酸序列。
  19. 一种产生控制粟灰螟害虫的植物繁殖体的方法，其特征在于，包括将由权利要求18所述方法获得的第一植株与第二植株杂交，和/或取下由权利要求18所述方法获得的植株上具有繁殖能力的组织进行培养，从而产生含有编码Cry1A.105蛋白的多核苷酸序列的植物繁殖体。
  20. 一种培养控制粟灰螟害虫的植物的方法，其特征在于，包括：
    种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括编码Cry1A.105蛋白的多核苷酸序列；
    使所述植物繁殖体长成植株；
    使所述植株在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下生长，收获与其他不具有编码Cry1A.105蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。
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