WO2016184396A1 - 杀虫蛋白的用途 - Google Patents

杀虫蛋白的用途 Download PDF

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WO2016184396A1
WO2016184396A1 PCT/CN2016/082587 CN2016082587W WO2016184396A1 WO 2016184396 A1 WO2016184396 A1 WO 2016184396A1 CN 2016082587 W CN2016082587 W CN 2016082587W WO 2016184396 A1 WO2016184396 A1 WO 2016184396A1
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sorghum
nucleotide sequence
plant
protein
seq
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PCT/CN2016/082587
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English (en)
French (fr)
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杨旭
张爱红
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北京大北农科技集团股份有限公司
北京大北农生物技术有限公司
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Publication of WO2016184396A1 publication Critical patent/WO2016184396A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G2/00—Vegetative propagation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • 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 protein to control a plant of a sorghum sorghum plant by expression in a plant.
  • Chilo sacchariphagus belongs to the order Lepidoptera, the genus Coleoptera. Also known as sugarcane strips, or sorghum borer. It is mainly distributed in East Asia, South Asia, Southeast Asia and the Indian Ocean. It is widely distributed in China, including in Northeast China, North China, East China and most of southern China. Sorghum can be used to damage corn, sorghum, millet, hemp, sugar cane and other crops. In the northern dryland areas, it mainly harms crops such as corn, sorghum and millet, and often mixes with corn mash, causing dead seedlings; it is also the main pest on sugar cane in the south.
  • the main control methods commonly used are: agricultural control, chemical control and physical control.
  • Agricultural control is to comprehensively coordinate the multi-factors of the whole farmland ecosystem, regulate crops, pests and environmental factors, and create a farmland ecological environment that is conducive to crop growth and is not conducive to the occurrence of sorghum.
  • the sorghum or corn stalks are treated to reduce the overwintering insect source. Straw treatment can be carried out by crushing, burning, manure, mashing, mud sealing and other methods. Since agricultural control must comply with the requirements of crop layout and production increase, its application has certain limitations and cannot be used as an emergency measure. Therefore, it appears to be powerless when the sorghum strip breaks out.
  • Chemical control that is, pesticide control
  • sorghum is the use of chemical pesticides to kill pests. It is an important part of the comprehensive management of sorghum, which is characterized by rapid, convenient, simple and high economic benefits, especially in the occurrence of sorghum. The case is an essential emergency measure.
  • sorghum sorghum is very important for grasping its control period. The best period for the treatment is before the hatching period of the egg to the larvae of the larvae. Otherwise, it will be difficult to achieve the purpose of prevention and control after the young larvae break into the stalk.
  • chemical control methods are mainly liquid spray and application of poisonous soil. However, chemical control also has its limitations. If improper use, it will lead to phytotoxicity of crops, resistance to pests, killing natural enemies, polluting the environment, destroying farmland ecosystems and threatening the safety of humans and animals. Adverse consequences.
  • Physical control is mainly based on the response of pests to various physical factors in environmental conditions, using various physical factors such as light, Electric, color, temperature and humidity, as well as mechanical equipment to induce killing, radiation infertility and other methods to control pests.
  • the most widely used is the frequency-vibration insecticidal lamp trapping, which utilizes the phototaxis of pest adults, uses light at close range, uses waves at a long distance, attracts pests close to each other, and has certain control effects on adults of sorghum; however, frequency vibration
  • the insecticidal lamp needs to clean the dirt on the high-voltage power grid every day, otherwise it will affect the insecticidal effect; and it can't turn on the light in thunderstorm days, there is also the danger of electric shock in the operation; in addition, the one-time investment of installing the lamp is larger .
  • Cry1A insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystalline protein produced by Bacillus thuringiensis subsp. kurstaki.
  • the Cry1A protein is ingested by insects into the midgut, and the protoxin is dissolved in the alkaline pH environment of the insect midgut.
  • the N- and C-termini of the protein are digested with alkaline protease to convert the protoxin into an active fragment; the active fragment specifically binds to the surface receptor on the membrane of the insect midgut epithelial cell, and is inserted into the intestinal membrane, causing perforation of the cell membrane and destruction. Changes in osmotic pressure and pH balance inside and outside the cell membrane disrupt the insect's digestive process and ultimately lead to death.
  • Plants transgenic with Cry1A gene have been shown to be resistant to lepidopteran pests such as corn borer, cotton bollworm, and fall armyworm. However, there have been no reports on the control of plant damage caused by transgenic plants expressing Cry1A protein. .
  • the object of the present invention is to provide a use of a pesticidal protein, for the first time, to provide a method for controlling the damage of sorghum sorghum to plants by producing a transgenic plant expressing the Cry1A protein, and effectively overcome the prior art agricultural control, chemical control and Technical defects such as physical control.
  • the present invention provides a method of controlling a sorghum pest, comprising contacting a sorghum worm with at least a Cry1A protein.
  • the CrylA protein is present in a host cell that produces at least the CrylA protein, and the sorghum pest is at least in contact with the CrylA protein by ingesting the host cell.
  • the Cry1A protein is present in a bacterium or a transgenic plant which produces at least the Cry1A protein, and the sorghum scorpion pest contacts at least the Cry1A protein through a tissue ingesting the bacterium or the transgenic plant, after contact
  • the growth of sorghum scorpion pests is inhibited and/or caused to death, in order to achieve control of sorghum mites.
  • the transgenic plant may be in any growth period; the organization of the transgenic plant is Roots, leaves, stems, fruits, tassels, ears, flower buds, anthers or filaments; the control of sorghum-hit plants is not altered by changes in planting location and/or planting time.
  • the plant is derived from corn, sorghum, sugar cane, millet, hemp or glutinous rice.
  • the step prior to the contacting step is the planting of a plant containing a polynucleotide encoding the Cry1A protein.
  • the Cry1A protein is a Cry1Ab protein, a Cry1Ac protein or a Cry1Ab/Ac protein.
  • the amino acid sequence of the Cry1A protein has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9.
  • the nucleotide sequence of the Cry1A protein has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.
  • the plant may further comprise at least one second nucleotide different from the nucleotide encoding the Cry1A 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 Cry1Fa protein or a Cry1Ie protein.
  • amino acid sequence of the Cry1Ie protein has the amino acid sequence of SEQ ID NO: 11
  • amino acid sequence of the Cry1Fa protein has the amino acid sequence of SEQ ID NO: 13.
  • the second nucleotide has the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • the present invention also provides a use of a Cry1A protein for controlling a sorghum pest.
  • the present invention also provides a method of producing a plant for controlling a sorghum pest, comprising introducing a polynucleotide sequence encoding a Cry1A protein into the genome of the plant.
  • the present invention also provides a method of producing a plant propagule for controlling a sorghum pest, comprising crossing a first plant obtained by the method with a second plant, and/or removing the method by the method
  • the fertile tissue on the obtained plants is cultured to produce a propagule containing a polynucleotide sequence encoding a Cry1A protein.
  • the present invention also provides a method for cultivating a plant for controlling a sorghum pest, comprising:
  • the plants are grown under conditions in which the artificially inoculated sorghum pests and/or sorghum larvae are naturally harmful, and the plants are harvested with reduced plant damage and/or compared with other plants that do not have the polynucleotide sequence encoding the Cry1A protein. Or plants with increased plant yield.
  • Plant propagules as used in the present invention include, but are not limited to, plant sexual propagules and plant asexual propagules.
  • the plant sexual propagule includes, but is not limited to, a plant seed; the plant asexual propagule refers to a vegetative organ of a plant body or a special tissue which can produce a new plant under ex vivo conditions; the vegetative organ or a certain 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.
  • Recombination refers to the form of DNA and/or proteins and/or organisms that are not normally found in nature and are thus produced by human intervention. Such manual intervention can produce recombinant DNA molecules and/or recombinant plants.
  • the "recombinant DNA molecule” is obtained by artificially combining two sequence segments which are otherwise isolated, for example by chemical synthesis or by manipulation of isolated nucleic acid segments by genetic engineering techniques. Techniques for performing nucleic acid manipulation are well known.
  • Contact means that insects and/or pests touch, stay and/or ingest plants, plant organs, plant tissues or plant cells, and the plants, plant organs, plant tissues or plant cells can It is a pesticidal protein expressed in the body, and may also be a microorganism having a pesticidal protein on the surface of the plant, plant organ, plant tissue or plant cell and/or having a pesticidal protein.
  • control and/or "control” in the present invention means that the sorghum scorpion pest is at least in contact with the Cry1A protein, and the growth of the sorghum sorghum pest is inhibited and/or causes death after contact. Further, the sorghum larvae are at least in contact with the Cry1A protein by ingesting plant tissues, and all or part of the sorghum larvae are inhibited from growing and/or causing death after contact. Inhibition refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry1A protein that control sorghum pests are inoculated with artificially inoculated pests of sorghum and/or sorghum pests, and non-transgenic Wild-type plants have reduced plant damage compared to specific manifestations including, but not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like.
  • control and / or “control” effects of the Cry1A protein on sorghum can be independent and not attenuated and/or disappeared by other substances that can "control” and/or “control” the mites. .
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a Cry1A protein
  • the Cry1A protein and/or another substance that can control the sorghum pest At the time, the presence of the other substance does not affect the Cry1A protein to the sorghum
  • the "control” and/or “control” effect also does not cause the "control” and/or “control” effects to be fully and/or partially achieved by the other substance, regardless of the Cry1A protein.
  • any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry1A protein) has a dead sorghum pest, and/or a sorghum pest that inhibits growth growth thereon, and/or a non-transgenic wild type plant
  • the method and/or use of the present invention is achieved by having reduced plant damage, i.e., by contacting the sorghum scorpion pest with at least the Cry1A protein to achieve a method and/or use for controlling mites.
  • expression of the Cry1A protein in a transgenic plant may be accompanied by fusion 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) can express Cry1A protein by genetic engineering operation
  • the second plant second parent
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution.
  • dsRNA double-stranded RNA
  • RNAi technology can be used to specifically knock out or silence the expression of a particular gene in a target insect pest.
  • the lepidoptera In the classification system, the lepidoptera is generally divided into suborders, superfamily, and family according to the morphological characteristics of the worm's veins, linkages, and types of antennae, while the genus Lepidoptera is the most diverse species of Lepidoptera.
  • One of the departments has found more than 10,000 types in the world, and there are thousands of records in China alone.
  • Most of the moths are pests of crops, most of which are in the form of stolons, such as stem borer and corn borer.
  • sorghum sorghum is equivalent to the mites and corn borer, it belongs to the order Lepidoptera, and there is a great difference in other morphological structures except for the similarity in the classification criteria; it is like the strawberry in the plant and the apple ( They belong to the genus Rosaceae, which have the characteristics of flower bisexuality, radiation symmetry, and 5 petals, but their fruits and plant morphology are very different.
  • the sorghum scorpion has its unique characteristics in terms of larval morphology and adult morphology.
  • the back line of the back is three or four five", which means that the sorghum sorghum, corn stalk and ash scorpion belonging to the genus Mothidae have obvious numbers on the top line. difference.
  • the dorsal blood vessel is an important part of the insect circulatory organ. The inside is filled with the hemolymph called the insect "blood”. Therefore, the difference in the number of top lines on the surface of the body appears to reflect the difference in the back vessels, which is the difference in the insect circulation system.
  • Insects belonging to the genus Mothidae not only have large differences in morphological characteristics, but also have differences in feeding habits.
  • the three mites which are also the genus Mothaceae, are mainly harmful to rice, and rarely harm other grass crops.
  • the difference in feeding habits also suggests that the enzymes and receptor proteins produced by the digestive system in the body are different.
  • the enzyme produced in the digestive tract is the key point of the Bt gene function. Only the enzyme or receptor protein that can bind to the specific Bt gene may make a certain Bt gene have an insect resistance effect on the pest.
  • insects of different families and even different families have different sensitivities to the same Bt protein.
  • the Vip3Aa gene showed anti-insect activity against Chilo suppressalis and Ostrinia furnacalis, but for Plodia interpunctella and European corn borer (the same species) Ostrinia nubilalis has no insect resistance.
  • the above four pests belong to the family Lepidoptera, but the same Bt protein shows different resistance effects to the four species of the moth.
  • European corn borer and Asian corn borer are classified in the same species as the genus Corydalis (the same genus), but their response to the same Bt protein is quite different, which is more fully explained.
  • the way in which Bt proteins interact with enzymes and receptors in insects is complex and unpredictable.
  • the genome of a plant, plant tissue or plant cell as referred to in the present invention refers to any genetic material within a plant, plant tissue or plant cell, and includes the nuclear, plastid and mitochondrial genomes.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • DNA is generally present in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • one strand of DNA is generally transcribed into a complementary strand of mRNA, which is used as a template to translate a protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes to the Cry1A gene of the present invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A 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.
  • 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, and then 2 x SSC, 0.1% SDS and 1 x, respectively. The membrane was washed once with SSC and 0.1% SDS.
  • sequences having insect resistance activity and hybridizing under stringent conditions to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 of the present invention is included in the present invention. in.
  • These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. %, 94%, 95%, 96%, 97%, 98%, 99% or more homologous.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or compared to full length proteins). Terminal deletions, variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • variant or “variant” is meant a nucleotide sequence that encodes the same protein or an equivalent protein encoded with insecticidal activity.
  • the "equivalent protein” refers to a protein having the same or substantially the same biological activity as the sorghum scorpion pest of the protein of the claims.
  • a “fragment” or “truncated” of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA sequence or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, suitable for plant expression)
  • the sequence of the foregoing sequence may vary in length, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be readily constructed using standard techniques, for example, techniques for constructing point mutations well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be prepared using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, 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 produced by the most constant part of the protein and the most different part of the other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies of the proteins disclosed herein or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
  • the "substantially identical" sequence refers to a sequence which has an amino acid substitution, deletion, addition or insertion but does not substantially affect the insecticidal activity, and also includes a fragment which retains insecticidal activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R. L.
  • amino acid residues necessary for their activity and thus selected for unsubstituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique 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 protein includes, but is not limited to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, and SEQ ID NO: 1, SEQ ID An amino acid sequence having a certain homology to the amino acid sequence shown by NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 is also included in the present invention.
  • the similarity/identity of these sequences to the sequences of the invention is typically greater than 78%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99%.
  • Preferred polynucleotides and proteins of the invention may also be defined according to a more specific range of identity and/or similarity.
  • sequences of the examples of the present invention are 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity.
  • a transgenic plant producing the Cry1A protein includes, but is not limited to, a Mon810 transgenic maize event and/or a plant material comprising a Mon810 transgenic maize event (as described in US Pat. No.
  • a Bt11 transgenic maize event and/or comprises Bt11 Plant material for the transgenic maize event (as described in USDA APHIS Unregulated Status Application 95-195-01p, which contains the amino acid sequence of the Cry1Ab protein as shown in SEQ ID NO: 3 of the present invention), Bt176 transgenic maize event and / or plant material comprising the Bt176 transgenic maize event (as described in USDA APHIS Unregulated Status Application 94-319-01p, which contains the amino acid sequence of the Cry1Ab protein as described in US 5,625, 136 B2), TT51 transgenic rice events and/or Or plant material comprising TT51 transgenic rice events (as described in CN100582223C and CN101302520B), 223F-S21 transgenic rice lines and/or plant material comprising 223F-S21 transgenic rice lines (as described in CN103773759A), Mon15985 transgene Cotton events and/or plant material containing the Mon15985 GM cotton
  • the methods and/or uses of the invention can also be achieved by expressing the Cry1A protein in the above transgenic events in different plants. More specifically, the Cry1A protein is present in a transgenic plant that produces at least the Cry1A protein, the sorghum bark pest is in contact with at least the Cry1A protein by ingesting the tissue of the transgenic plant, and the sorghum barium is contacted after contact Pest growth is inhibited and/or causes death to achieve control of plants that are damaging to sorghum.
  • Regulatory sequences of the invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Cry1A protein.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • the promoter expressible in the plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, in other tissues of the plant ( As determined by conventional RNA assays, such as the PEP carboxylase promoter.
  • the promoter expressible in the plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, cauliflower mosaic virus (CaMV) enhancer, Scrophulari mosaic virus (FMV) Enhancer, carnation weathering ring virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, purple jasmine mosaic virus (MMV) enhancer, night fragrant yellow leaf curl virus (CmYLCV) enhancer, Multan Cotton leaf curl virus (CLCuMV), comfrey yellow mottle virus (CoYMV) and peanut chlorotic mosaic virus (PCLSV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV Scrophulari mosaic virus
  • FMV carnation weathering ring virus
  • CERVMV carnation weathering ring virus
  • CsVMVMV cassava vein mosaic virus
  • MMV purple jasmine mosaic virus
  • CmYLCV night fragrant yellow leaf curl virus
  • CLCuMV Multan Cotton leaf curl virus
  • CoYMV comfrey yellow mottle virus
  • PCLSV peanut chlorotic 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.
  • “operably linked” means that the promoter is ligated to the sequence in a manner such that the resulting transcript is efficiently translated.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • Insecticide or "insect-resistant” as used in the present invention means toxic to crop pests, thereby achieving "control” And / or “control” crop pests.
  • said "insecticide” or “insect-resistant” means killing crop pests.
  • the target insect is a sorghum pest.
  • the Cry1A protein is toxic to sorghum.
  • the plants of the present invention particularly sorghum, corn and sugar cane, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1A protein, and the sorghum pest is in contact with the protein by feeding plant tissue. After exposure, the growth of pests is inhibited and/or caused to death. Inhibition refers to death or sub-lethal death.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the plant substantially eliminates the need for chemical or biological insecticides that are insecticides against the sorghum pests targeted by the Cry1A protein.
  • the expression level of insecticidal crystal protein (ICP) in plant material can be detected by various methods described in the art, for example, by using specific primers to quantify the mRNA encoding the insecticidal protein produced in the tissue, or directly specific The amount of insecticidal protein produced is detected.
  • the target insect is mainly sorghum.
  • the Cry1A protein may have the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 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 protein of the invention may also be expressed in a plant together with at least one protein encoding a herbicide resistance gene including, but not limited to, oxalic acid Phospho-resistant genes (such as bar gene, pat gene), benthamiana resistance genes (such as pmph gene), glyphosate resistance genes (such as EPSPS gene), bromoxynil resistance gene, sulfonylurea Resistance gene, resistance gene to herbicide tortoise, resistance gene to cyanamide or glutamine synthetase inhibitor (such as PPT), thereby obtaining high insecticidal activity and weeding Agent-resistant transgenic plants.
  • oxalic acid Phospho-resistant genes such as bar gene, pat gene
  • benthamiana resistance genes such as pmph gene
  • glyphosate resistance genes such as EPSPS gene
  • bromoxynil resistance gene sulfonylurea Resistance gene
  • the foreign DNA is introduced into a plant, such as a gene encoding the Cry1A protein or an expression cassette or a recombinant vector
  • the conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-shot bombardment, Direct DNA uptake into protoplast, electroporation or whisker silicon-mediated DNA introduction.
  • the prior art mainly controls the hazards of sorghum pests, such as agricultural control, chemical control and physical control, through external effects, ie, external factors; and the present invention controls sorghum strips by producing Cry1A protein in plants which can kill sorghum sorghum.
  • the pests are controlled by internal factors.
  • the effect is stable.
  • Both the agricultural control method and the physical control method used in the prior art require the use of environmental conditions to control pests, and there are many variable factors; the present invention is to express the Cry1A protein in plants, thereby effectively avoiding environmental conditions.
  • Unstable defects, and the control effect of the transgenic plants (Cry1A protein) of the present invention are stable and consistent at different locations, at different times, and in different genetic backgrounds.
  • the frequency-active insecticidal lamp used in the prior art has a large one-time investment, and the operation is improper and there is a danger of electric shock wounding; the invention only needs to plant a transgenic plant capable of expressing the Cry1A protein, and does not need other measures. , which saves a lot of manpower, material resources and financial resources.
  • the method for controlling mites and pests used in the prior art has an effect that is incomplete and only serves to alleviate the effect; whereas the transgenic plant of the present invention (Cry1A protein) can cause a large number of deaths of the larvae of the mites, a small number of surviving larvae. It also basically stopped development. After 3 days, the larvae were still in the initial hatching state, all of which were obviously dysplastic, and had stopped developing, and could not survive in the natural environment of the field, while the transgenic plants were generally only slightly damaged.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Cry1Ab-01 nucleotide sequence for use of the insecticidal protein of the present invention
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100124 containing a Cry1Ab-01 nucleotide sequence for use of the insecticidal protein of the present invention
  • Figure 3 is a diagram showing the damage of leaves of transgenic maize plants inoculated with sorghum sorghum using the insecticidal protein of the present invention.
  • the amino acid sequence of Cry1Ab-01 insecticidal protein (818 amino acids), as shown in SEQ ID NO: 1 in the Sequence Listing; encoding the Cry1Ab-01 nucleotide sequence corresponding to the amino acid sequence of the Cry1Ab-01 insecticidal protein ( 2457 nucleotides) as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the amino acid sequence of Cry1Ab-02 insecticidal protein (615 amino acids), as shown in SEQ ID NO: 3 in the Sequence Listing; encoding the Cry1Ab-02 nucleotide sequence corresponding to the amino acid sequence of the Cry1Ab-02 insecticidal protein ( 1848 nucleotides) as shown in SEQ ID NO: 4 in the Sequence Listing.
  • Cry1Ac-01 insecticidal protein (1156 amino acids), as shown in SEQ ID NO: 5 in the Sequence Listing; encoding the Cry1Ac-01 nucleotide sequence corresponding to the amino acid sequence of the Cry1Ac-01 insecticidal protein ( 3471 nucleotides) as shown in SEQ ID NO: 6 in the Sequence Listing.
  • Cry1Ac-02 insecticidal protein (616 amino acids), as shown in SEQ ID NO: 7 in the Sequence Listing; encoding the Cry1Ac-02 nucleotide sequence corresponding to the amino acid sequence of the Cry1Ac-02 insecticidal protein ( 1851 nucleotides) as shown in SEQ ID NO: 8 in the Sequence Listing.
  • Cry1Ab/Ac insecticidal protein (609 amino acids), as shown in SEQ ID NO: 9 in the Sequence Listing; encoding a Cry1Ab/Ac nucleotide sequence corresponding to the amino acid sequence of the Cry1Ab/Ac insecticidal protein ( 1830 nucleotides) as shown in SEQ ID NO: 10 in the Sequence Listing.
  • Cry1Ie insecticidal protein (719 amino acids), as shown in SEQ ID NO: 11 in the Sequence Listing; Cry1Ie nucleotide sequence (2160 nucleotides) encoding the amino acid sequence corresponding to the Cry1Ie insecticidal protein , as shown in SEQ ID NO: 12 in the Sequence Listing.
  • Cry1Fa insecticidal protein (605 amino acids), as shown in SEQ ID NO: 13 in the Sequence Listing; Cry1Fa nucleotide sequence (1818 nucleotides) encoding the amino acid sequence corresponding to the Cry1Fa insecticidal protein , as shown in SEQ ID NO: 14 in the Sequence Listing.
  • the Cry1Ab-01 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing), the Cry1Ab-02 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing), the Cry1Ac- 01 nucleotide sequence (as shown in SEQ ID NO: 6 in the Sequence Listing), the Cry1Ac-02 nucleotide sequence (as shown in SEQ ID NO: 8 in the Sequence Listing), the Cry1Ab/Ac nucleotide a sequence (as shown in SEQ ID NO: 10 in the Sequence Listing), the Cry1Ie nucleotide sequence (as shown in SEQ ID NO: 12 in the Sequence Listing), and the Cry1Fa nucleotide sequence (such as SEQ ID in the Sequence Listing) NO: 14) synthesized by Nanjing Kingsray Biotechnology Co., Ltd.; the 5' end of the synthesized Cry1Ab-01 nucleotide sequence (SEQ ID NO
  • the synthesized Cry1Ac-01 nucleotide sequence (SEQ ID NO: 6) has a Sac I restriction site linked to the 5' end and a Kas I restriction site at the 3' end;
  • the 5' end of the Cry1Ac-02 nucleotide sequence (SEQ ID NO: 8) is ligated with Sac I
  • the cleavage site has a Kas I restriction site linked to the 3' end;
  • the 5' end of the synthesized Cry1Ab/Ac nucleotide sequence (SEQ ID NO: 10) is ligated with a Spe I restriction site, 3'
  • the terminal is ligated with a Kas I restriction site;
  • the synthetic Cry1Ab-01 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • 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 initiated by T7 RNA polymerase).
  • Cry1Ab-01 is the Cry1Ab-01 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) in LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) 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 Cry1Ab-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was SEQ ID NO: 2 in the sequence listing.
  • the synthesized Cry1Ab-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1Ab-02 was Cry1Ab-02. Nucleotide sequence (SEQ ID NO: 4).
  • the Cry1Ab-02 nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ac-01 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein Cry1Ac-01 was Cry1Ac-01.
  • Nucleotide sequence SEQ ID NO: 6
  • the Cry1Ac-01 nucleotide sequence in the recombinant cloning vector DBN03-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ac-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein Cry1Ac-02 was Cry1Ac-02. Nucleotide sequence (SEQ ID NO: 8).
  • the Cry1Ac-02 nucleotide sequence in the recombinant cloning vector DBN04-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab/Ac nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN05-T, wherein Cry1Ab/Ac was Cry1Ab/Ac.
  • Nucleotide sequence SEQ ID NO: 10
  • the Cry1Ab/Ac nucleotide sequence in the recombinant cloning vector DBN05-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ie nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN06-T, wherein Cry1Ie was a Cry1Ie nucleotide sequence (SEQ ID NO: 12).
  • the Cry1Ie nucleotide sequence in the recombinant cloning vector DBN06-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Fa nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN07-T, wherein Cry1Fa is a Cry1Fa nucleotide sequence (SEQ ID NO: 14).
  • Cry1Fa nucleotide sequence in the recombinant cloning vector DBN07-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 were digested with restriction endonucleases Spe I and Kas I, respectively, and the cut Cry1Ab-01 nucleotide sequence was cleaved. The fragment was inserted between the Spe I and Kas I sites of the expression vector DBNBC-01, and the vector was constructed by a conventional restriction enzyme digestion method.
  • the recombinant expression vector DBN100124 was constructed.
  • the recombinant expression vector DBN100124 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 DBN100124), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake at 100 rpm); then at 50 mg/L Kana Kanamycin LB solid plate (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, adjusted to pH 7.5 with NaOH) and incubated at 37 ° C for 12 hours , pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, kanamycin 50mg / L, pH adjusted to 7.5 with NaOH) at 37 ° C Incubate overnight under conditions.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases Spe I and Kas I, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100124 between Spe I and Kas I sites was sequenced.
  • the Cry1Ab-02 nucleotide sequence excised from the Spe I and Kas I recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100743.
  • the nucleotide sequence in the recombinant expression vector DBN100743 contains the nucleotide sequence shown as SEQ ID NO: 4 in the sequence listing, that is, the Cry1Ab-02 nucleotide sequence, and the Cry1Ab-02 nucleotide sequence is digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Ac-01 nucleotide sequence excised by Sac I and Kas I digestion of the recombinant cloning vector DBN03-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100741.
  • the nucleotide sequence in the recombinant expression vector DBN100741 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 6 in the sequence listing, that is, the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-01 nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the nucleotide sequence in the recombinant expression vector DBN100734 contains the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 12 in the sequence listing, namely the Cry1Ac-02 nucleotide sequence and the Cry1Ie nucleoside.
  • the acid sequence, the Cry1Ac-02 nucleotide sequence and the Cry1Ie nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the Cry1Ab/Ac nucleotide sequence excised by the Spe I and Kas I digestion recombinant cloning vector DBN05-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100737.
  • the nucleotide sequence in the recombinant expression vector DBN100737 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, that is, the Cry1Ab/Ac nucleotide sequence, and the Cry1Ab/Ac nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Fa nucleotide sequence excised by the Asc I and BamH I digestion recombinant cloning vector DBN07-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100735.
  • the nucleotide sequence in the recombinant expression vector DBN100735 contains the nucleotide sequence shown as SEQ ID NO: 14 in the sequence listing, that is, the Cry1Fa nucleotide sequence, and the Cry1Fa nucleotide sequence can be ligated. Ubi promoter and Nos terminator.
  • the nucleotide sequence in the recombinant expression vector DBN100013 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 2 and SEQ ID NO: 14 in the sequence listing, that is, the Cry1Ab-01 nucleotide sequence and the Cry1Fa nucleoside.
  • the acid sequence, the Cry1Ab-01 nucleotide sequence and the Cry1Fa nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737, DBN100735 and DBN100013, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were : 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube at a temperature of 28 ° C, 200 rpm Incubate for 2 hours, apply to LB plates containing 50 mg/L of rifampicin and 100 mg/L of kanamycin until a positive monoclonal grows, pick a monoclonal culture and extract it.
  • Agrobacterium LBA4404 Invitrgen, Chicago, USA, CAT:
  • the recombinant plasmids DBN100124, DBN100743, DBN100741, DBN100734, DBN100737, DBN100735 and DBN100013 were digested with restriction endonucleases Ahd I and Xho I, and the results showed that the recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734 The DBN100737, DBN100735, and DBN100013 are completely correct.
  • T-DNA in recombinant expression vectors DBN100124, DBN100743, DBN100741, DBN100734, DBN100737, DBN100735 and DBN100013 including the promoter sequence of maize Ubiquitin gene, Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ac-01
  • the nucleotide sequence, the Cry1Ac-02 nucleotide sequence, the Cry1Ie nucleotide sequence, the Cry1Ab/Ac nucleotide sequence, the Cry1Fa nucleotide sequence, the Hpt gene, and the Nos terminator sequence are transferred into the maize genome to obtain A maize plant transformed with the Cry1Ab-01 nucleotide
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can express Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide
  • Agrobacterium can express Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide
  • the sequence, the Cry1Ac-01 nucleotide sequence, the Cry1Ac-02-Cry1Ie nucleotide sequence, the Cry1Ab/Ac nucleotide sequence, the Cry1Fa nucleotide sequence, and the Cry1Ab-01-Cry1Fa nucleotide sequence are delivered to one of the young embryos.
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, plant gel 3g / L, pH 5.8) at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250mg / L), no addition of plant transformant selection agent (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the grown transformed callus is selected (step 4: selection step).
  • a selection agent hygromycin
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, hygromycin 50 mg/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8, resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, M. 50 mg/L, vegetal gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel 3 g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.
  • the medium formulation is referred to Molecular Biology and Genetic Engineering ISSN 2053-5767, wherein the screening agent is replaced with hygromycin (30-50 mg/L) according to the transgenic vector of the present invention.
  • a sorghum plant transformed into the Cry1Ab-01 nucleotide sequence, a sorghum plant transformed into the Cry1Ab-02 nucleotide sequence, a sorghum plant transformed into the Cry1Ac-01 nucleotide sequence, and transferred to Cry1Ac-02-Cry1Ie were obtained.
  • a sorghum plant having a nucleotide sequence, a sorghum plant transformed into a Cry1Ab/Ac nucleotide sequence, a sorghum plant transformed into a Cry1Fa nucleotide sequence, and a sorghum plant transformed into a Cry1Ab-01-Cry1Fa nucleotide sequence; Sorghum plants were used as controls.
  • the conversion method mainly refers to the 22nd to 24th pages of the 2012 Master's degree of Guangxi University. Take the fresh stem section of the cane top, remove the cane tip and leaf sheath, leaving the stem tip growth cone and the heart leaf stem segment. On the ultra-clean workbench, wipe the surface with a 75% (v/v) alcohol cotton ball, carefully peel off the outer layer of the heart leaf with the sterilized tweezers, and take the heart segment of the 5-7 cm length in the middle. A sheet cut into a thickness of about 3 mm was inoculated on an induction medium, and cultured in the dark at a temperature of 26 ° C for 20 days.
  • the callus pieces were cut into small pieces of 0.6*0.6 cm, and then transferred to MR solid medium containing 100 ⁇ mol/L acetosyringone (AS), and cultured in a dark state at 23 ° C for 3 days;
  • the wound tissue was clipped, placed on a filter paper and dried on a clean bench until the surface of the material was dry, and the material was transferred to a differentiation medium containing 500 mg/L cephalosporin and 30-50 mg/L hygromycin.
  • the medium was changed every 2 weeks, during which the contaminated calli was removed, and when the seedlings were about 3 cm long, they were transferred to a rooting medium containing hygromycin screening agent to induce rooting.
  • a sugarcane plant transformed with the Cry1Ab-01 nucleotide sequence a sugarcane plant transformed with the Cry1Ab-02 nucleotide sequence, a sugarcane plant transformed with the Cry1Ac-01 nucleotide sequence, and a Cry1Ac-02-Cry1Ie were obtained.
  • the acid sequence of the maize plant, the maize plant transformed with the Cry1Ab/Ac nucleotide sequence, the maize plant transformed with the Cry1Fa nucleotide sequence, and the maize plant transfected with the Cry1Ab-01-Cry1Fa nucleotide sequence were approximately 100 mg as a sample.
  • Genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of Cry1A gene and Cry1Fa gene was detected by Taqman probe fluorescent quantitative PCR.
  • the wild type corn plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.
  • Step 11 The maize plants transfected into the Cry1Ab-01 nucleotide sequence, the maize plants transformed into the Cry1Ab-02 nucleotide sequence, the maize plants transformed into the Cry1Ac-01 nucleotide sequence, and transferred to Cry1Ac-02- Maize plants with Cry1Ie nucleotide sequence, maize plants transfected with Cry1Ab/Ac nucleotide sequence, maize plants transfected with Cry1Fa nucleotide sequence, maize plants transfected with Cry1Ab-01-Cry1Fa nucleotide sequence and wild type
  • the leaves of the corn plants were each 100 mg, and they were homogenized by liquid nitrogen in a mortar, and each sample was taken in 3 replicates;
  • Step 12 The genomic DNA of the above sample is extracted using Qiagen's DNeasy Plant Mini Kit, and the specific method is referred to the product specification;
  • Step 13 measuring 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 copy number of the sample was identified by Taqman probe real-time PCR method, and the sample with the known copy number was used as a standard, and the sample of the wild type corn plant was used as a control, and each sample was repeated 3 times, and the average was taken. Values; fluorescent PCR primers and probe sequences are as follows:
  • Primer 2 GTAGATTTCGCGGGTCAGTTG, as shown in SEQ ID NO: 19 in the Sequence Listing;
  • Probe 1 CTACCCGATCCGCACCGTGTCC, as shown in SEQ ID NO: 20 in the Sequence Listing;
  • Probe 2 CAGCGCCTTGACCACAGCTATCCC, as shown in SEQ ID NO: 23 in the Sequence Listing;
  • Probe 3 TCCGCTCTTCGCCGTTCAGAATTACC, as shown in SEQ ID NO: 26 in the Sequence Listing;
  • Primer 7 GGTTACACTCCCATCGACATCTC, as shown in SEQ ID NO: 27 in the Sequence Listing;
  • Primer 8 CACAAGACCAAGCACGAAACC, as shown in SEQ ID NO: 28 in the Sequence Listing;
  • Probe 4 CCTTACCCAGTTCCTTCTTTCCGAGTTCG, as shown in SEQ ID NO: 29 in the Sequence Listing;
  • Primer 9 CAGTCAGGAACTACAGTTGTAAGAGGG, as shown in SEQ ID NO: 30 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 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 and transgenic sugarcane plants were tested and analyzed according to the above method for verifying transgenic maize plants with TaqMan.
  • the results showed that the Cry1Ab-01 nucleotide sequence, Cry1Ab-02 nucleotide sequence, Cry1Ac-01 nucleotide sequence, Cry1Ac-02-Cry1Ie nucleotide sequence, Cry1Ab/Ac nucleotide sequence, Cry1Fa nucleotide
  • the sequence, Cry1Ab-01-Cry1Fa nucleotide sequence has been integrated into the genome of the tested sorghum and sugarcane plants, respectively, and transferred to the sorghum plant of the Cry1Ab-01 nucleotide sequence, and transferred to the Cry1Ab-02 nucleoside.
  • a piece of cut long strips of leaves are placed on a moisturizing filter paper at the bottom of a round plastic petri dish, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22 After 3-4°C, relative humidity 70%-80%, photoperiod (light/dark) 0:24, after 3 days, the resistance was obtained according to the three indicators of larval larvae development progress, mortality and leaf damage rate.
  • a total of 3 transformation event lines (S1, S2 and S3) transfected into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence for a total of 3 transformation event lines (S4, S5 and S6).
  • the results in Table 1 indicate that a maize plant transformed with the Cry1Ab-01 nucleotide sequence, a maize plant transformed with the Cry1Ab-02 nucleotide sequence, a maize plant transformed with the Cry1Ab-01-Cry1Fa nucleotide sequence, and transferred to Cry1Ac
  • the maize plant with -01 nucleotide sequence, the maize plant transformed with the Cry1Ac-02-Cry1Ie nucleotide sequence, and the maize plant transformed with the Cry1Ab/Ac nucleotide sequence have good insecticidal effects on sorghum.
  • the average mortality rate of sorghum strips is basically over 80%, and some even reach 100%.
  • the total score of resistance is also above 270 points, and some even reach 299 points (out of 300 points); and the total resistance of non-transgenic corn plants and wild-type corn plants identified by Taqman The score is generally around 20 points.
  • the maize plant transformed into the Cry1Ab-01 nucleotide sequence, the maize plant transformed into the Cry1Ab-02 nucleotide sequence, the maize plant transformed into the Cry1Ab-01-Cry1Fa nucleotide sequence, and the Cry1Ac-01 nucleus were transferred.
  • the maize plant with a nucleotide sequence, the maize plant transformed with the Cry1Ac-02-Cry1Ie nucleotide sequence, and the maize plant transformed with the Cry1Ab/Ac nucleotide sequence all showed high activity against sorghum, which is sufficient for The growth of sorghum strips produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the sorghum strip it is also possible to reduce the occurrence of diseases on the corn and greatly improve the yield and quality of the corn.
  • the sugarcane plants transformed into the Cry1Ab-01 nucleotide sequence, the sugarcane plants transformed into the Cry1Ab-02 nucleotide sequence, the sugarcane plants transformed into the Cry1Ac-01 nucleotide sequence, and the Cry1Ac-02-Cry1Ie nucleoside were transferred.
  • Acid sequence of sugarcane plants sugarcane plants transformed into Cry1Ab/Ac nucleotide sequence, sugarcane plants transformed into Cry1Fa nucleotide sequence, sugarcane plants transformed into Cry1Ab-01-Cry1Fa nucleotide sequence, wild type sugarcane plants and Fresh leaves of non-transgenic sugarcane plants (expanded young leaves) identified by Taqman were rinsed with sterile water and the water on the leaves was blotted with gauze, and then the sugarcane leaves were cut into strips of about 1 cm ⁇ 2 cm.
  • a piece of cut long strips of leaves are placed on a moisturizing filter paper at the bottom of a round plastic petri dish, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22 After 3-4°C, relative humidity 70%-80%, photoperiod (light/dark) 0:24, after 3 days, the resistance was obtained according to the three indicators of larval larvae development progress, mortality and leaf damage rate.
  • a total of 3 transformation event lines (S22, S23 and S24) transfected into the Cry1Ab-01 nucleotide sequence were transferred into the Cry1Ab-02 nucleotide sequence for a total of 3 transformation event lines (S25, S26 and S27).
  • the results in Table 2 indicate that sugarcane plants transformed into the Cry1Ab-01 nucleotide sequence, sugarcane plants transformed into the Cry1Ab-02 nucleotide sequence, sugarcane plants transformed into the Cry1Ab-01-Cry1Fa nucleotide sequence, and transferred to Cry1Ac -01 nucleotide sequence of sugarcane plants, sugarcane plants transferred into Cry1Ac-02-Cry1Ie nucleotide sequence and transferred into Cry1Ab/Ac nucleus
  • the sugarcane plants with glycosidic acid sequence have good insecticidal effects on sorghum sorghum.
  • the average mortality of sorghum sorghum is basically above 80%, and some even reach 100%.
  • the total score of resistance is basically More than 270 points, some even reached 299 points (out of 300 points); and the total resistance scores of non-transgenic sugarcane plants and wild-type sugarcane plants identified by Taqman were below 60 points.
  • sugarcane plants transformed into Cry1Ab-01 nucleotide sequence Compared with wild-type sugarcane plants, sugarcane plants transformed into Cry1Ab-01 nucleotide sequence, sugarcane plants transformed into Cry1Ab-02 nucleotide sequence, sugarcane plants transformed into Cry1Ab-01-Cry1Fa nucleotide sequence, and transgenic Sugarcane plants that enter the Cry1Ac-01 nucleotide sequence, sugarcane plants that are transferred into the Cry1Ac-02-Cry1Ie nucleotide sequence, and sugarcane plants that are transferred into the Cry1Ab/Ac nucleotide sequence can cause a large number of deaths of the newly hatched larvae, and a small amount The surviving larva also basically stopped development.
  • sugarcane plants were generally only slightly damaged, and the naked eye could hardly distinguish the feeding traces of the sorghum scorpion, and the leaf damage rate was below 10%.
  • sugarcane plants transferred to the Cry1Fa nucleotide sequence there was no obvious control effect on sorghum sorghum, whether it was mortality, leaf damage rate, larval development progress, or total resistance score, and non-transgenic as identified by Taqman.
  • the sugarcane plants showed no significant difference compared with the wild-type sugarcane plants.
  • the sugarcane plant transformed into the Cry1Ab-01 nucleotide sequence, the sugarcane plant transformed into the Cry1Ab-02 nucleotide sequence, the sugarcane plant transformed into the Cry1Ab-01-Cry1Fa nucleotide sequence, and the Cry1Ac-01 nucleus were transferred.
  • the sugarcane plant of the nucleotide sequence, the sugarcane plant transformed with the Cry1Ac-02-Cry1Ie nucleotide sequence, and the sugarcane plant transformed with the Cry1Ab/Ac nucleotide sequence all showed high activity against sorghum, which is sufficient for The growth of sorghum strips produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the sorghum strip it is also possible to reduce the occurrence of diseases on the sugarcane and greatly improve the yield and quality of the sugarcane.
  • Sorghum plants transfected into the Cry1Ab-01 nucleotide sequence Sorghum plants transfected with the Cry1Ab-02 nucleotide sequence, sorghum plants transfected with the Cry1Ac-01 nucleotide sequence, and transfected into Cry1Ac-02-Cry1Ie nucleoside Sorghum plants with acid sequence, sorghum plants transfected with Cry1Ab/Ac nucleotide sequence, sorghum plants transfected with Cry1Fa nucleotide sequence, sorghum plants transfected with Cry1Ab-01-Cry1Fa nucleotide sequence, wild-type sorghum plants and Fresh leaves identified by Taqman as non-transgenic sorghum plants (expanded young leaves), rinsed with sterile water and blotted with water on the leaves, then the sorghum leaves were cut into strips of about 1 cm ⁇ 2 cm.
  • a piece of cut long strips of leaves are placed on a moisturizing filter paper at the bottom of a round plastic petri dish, and 10 sorghum strips (initial hatching larvae) are placed in each dish, and the insect test dishes are capped at a temperature of 22 After 3-4°C, relative humidity 70%-80%, photoperiod (light/dark) 0:24, after 3 days, the resistance was obtained according to the three indicators of larval larvae development progress, mortality and leaf damage rate.
  • Turn A total of 3 transformation event lines (S43, S44 and S45) into the Cry1Ab-01 nucleotide sequence were transferred to the Cry1Ab-02 nucleotide sequence for a total of 3 transformation event lines (S46, S47 and S48).
  • a total of 3 transformation event lines (S61, S62 and S63) transferred into the Cry1Fa nucleotide sequence identified as one non-transgenic (NGM3) strain by Taqman, and one wild type (CK3)
  • NMM3 non-transgenic
  • CK3 wild type
  • the average mortality rate of sorghum sorghum is basically above 80%, and some even reach 100%. At 270 points or more, some even reached 299 points (out of 300 points); the total score of resistance of sorghum plants and wild-type sorghum plants identified by Taqman as non-transgenic was generally around 20 points.
  • Sorghum plants transferred to the Cry1Ab-01 nucleotide sequence, sorghum plants transfected with the Cry1Ab-02 nucleotide sequence, and sorghum plants transfected with the Cry1Ab-01-Cry1Fa nucleotide sequence were transfected with wild-type sorghum plants.
  • Sorghum plants into the Cry1Ac-01 nucleotide sequence, sorghum plants transfected with the Cry1Ac-02-Cry1Ie nucleotide sequence, and sorghum plants transfected with the Cry1Ab/Ac nucleotide sequence can cause a large number of deaths of the newly hatched larvae And a small number of surviving larvae also basically stopped development.
  • the sorghum plant transformed into the Cry1Ab-01 nucleotide sequence the sorghum plant transformed into the Cry1Ab-02 nucleotide sequence, the sorghum plant transformed into the Cry1Ab-01-Cry1Fa nucleotide sequence, and the Cry1Ac-01 nucleus were transferred.
  • Sorghum plants with a nucleotide sequence, sorghum plants transfected with the Cry1Ac-02-Cry1Ie nucleotide sequence, and sorghum plants transfected with the Cry1Ab/Ac nucleotide sequence all showed high activity against sorghum, which is sufficient for
  • the growth of sorghum strips produces undesirable effects that allow it to be controlled in the field.
  • by controlling the drill collar of the sorghum strip it is also possible to reduce the occurrence of diseases on the sorghum and greatly improve the yield and quality of the sorghum.
  • the Cry1A protein of the present invention includes, but is not limited to, the Cry1A 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 protein, such as a Cry-like protein or a Vip3A protein. Wait.
  • the use of the insecticidal protein of the present invention controls the sorghum scorpion pest by producing a Cry1A protein capable of killing sorghum scorpion in the plant; and the agricultural control method, the chemical control method and the physical control method used in the prior art Compared with the invention, the plant protects the whole growth period and the whole plant to prevent the infestation of the cockroach pest, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economic.

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Abstract

一种杀虫蛋白的用途，通过植物体内产生能够杀死高粱条螟的Cry1A蛋白来控制高粱条螟害虫；所述控制高粱条螟害虫的方法包括：将高粱条螟害虫至少与Cry1A蛋白接触。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，该杀虫蛋白对植物进行全生育期、全植株的保护以防治高粱条螟害虫的侵害，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蛋白质通过在植物中表达来控制高粱条螟为害植物的用途。
背景技术
高粱条螟(Chilo sacchariphagus)属鳞翅目(Lepidoptera)，螟蛾科。又名甘蔗条螟，或高粱钻心虫。其主要分布在东亚、南亚、东南亚及印度洋地区，在国内分布极广，包括在东北、华北、华东和华南大部分地区都有发生。高粱条螟可为害玉米、高粱、粟、麻、甘蔗等作物。其在北方旱粮地区，主要为害玉米、高粱和粟等作物，并常和玉米螟混合发生，造成枯心苗；同时也是南方甘蔗上的主要害虫。
玉米是中国重要的粮食作物，随着全球温室效应的加强，近两年温度不断上升，虫害发生种类及数量都有所提高。高粱条螟的幼虫在初期为害心叶，后期则可蛀食茎秆、叶鞘，阻碍养分输送，使茎秆易受风折断。并且其对产量有严重影响，通常损失可达10％-40％。为了防治高粱条螟，人们通常采用的主要防治方法有：农业防治、化学防治和物理防治。
农业防治是把整个农田生态系统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高粱条螟发生的农田生态环境。在越冬幼虫化蛹与羽化之前，将高粱或玉米秸秆处理完毕，以减少越冬虫源。秸秆处理可采用粉碎、烧毁、沤肥、铡碎、泥封等不同方法。由于农业防治必须服从作物布局和增产的要求，其应用有一定的局限性，不能作为应急措施，因此在高粱条螟爆发时就显得无能为力。
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是高粱条螟综合治理的重要组成部分，它具有快速、方便、简便和高经济效益的特点，特别是在高粱条螟大发生的情况下，是必不可少的应急措施。高粱条螟作为蛀茎害虫，对其防治时期的把握非常重要，用药最佳时期是卵孵盛期至幼虫蛀茎之前，否则高龄幼虫蛀入茎秆之后，将很难达到防治的目的。目前化学防治方法主要是药液喷雾和施撒毒土。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，使农田生态系统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，对高粱条螟成虫的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杀虫蛋白是众多杀虫蛋白中的一种，是由苏云金芽孢杆菌库斯塔基亚种(Bacillus thuringiensis subsp.kurstaki)产生的不溶性伴孢结晶蛋白。
Cry1A蛋白被昆虫摄入进入中肠，毒蛋白原毒素被溶解在昆虫中肠的碱性pH环境下。蛋白N-和C-末端被碱性蛋白酶消化，将原毒素转变成活性片段；活性片段和昆虫中肠上皮细胞膜上的表面受体特异性地结合，插入肠膜，导致细胞膜出现穿孔病灶，破坏细胞膜内外的渗透压变化及pH平衡等，扰乱昆虫的消化过程，最终导致其死亡。
已证明转Cry1A基因的植株可以抵抗玉米螟、棉铃虫、秋粘虫等鳞翅目害虫的侵害，然而，至今尚无关于通过产生表达Cry1A蛋白的转基因植株来控制高粱条螟对植物危害的报道。
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蛋白的转基因植株来控制高粱条螟对植物危害的方法，且有效克服了现有技术中农业防治、化学防治和物理防治等的技术缺陷。
为实现上述目的，本发明提供了一种控制高粱条螟害虫的方法，包括将高粱条螟害虫至少与Cry1A蛋白接触。
进一步地，所述Cry1A蛋白存在于至少产生所述Cry1A蛋白的宿主细胞中，所述高粱条螟害虫通过摄食所述宿主细胞至少与所述Cry1A蛋白接触。
更进一步地，所述Cry1A蛋白存在于至少产生所述Cry1A蛋白的细菌或转基因植物中，所述高粱条螟害虫通过摄食所述细菌或转基因植物的组织至少与所述Cry1A蛋白接触，接触后所述高粱条螟害虫生长受到抑制和/或导致死亡，以实现对高粱条螟危害植物的控制。
在上述技术方案中，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为 根、叶片、茎秆、果实、雄穗、雌穗、花蕾、花药或花丝；所述对高粱条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。
所述植物来自玉米、高粱、甘蔗、粟、麻或薏米。
所述接触步骤之前的步骤为种植含有编码所述Cry1A蛋白的多核苷酸的植物。
在上述技术方案的基础上，所述Cry1A蛋白为Cry1Ab蛋白、Cry1Ac蛋白或Cry1Ab/Ac蛋白。
优选地，所述Cry1A蛋白的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:9所示的氨基酸序列。所述Cry1A蛋白的核苷酸序列具有SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8或SEQ ID NO:10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Cry1Fa蛋白或Cry1Ie蛋白。
进一步地，所述Cry1Ie蛋白的氨基酸序列具有SEQ ID NO:11所示的氨基酸序列，所述Cry1Fa蛋白的氨基酸序列具有SEQ ID NO:13所示的氨基酸序列。
更进一步地，所述第二种核苷酸具有SEQ ID NO:12或SEQ ID NO:14所示的核苷酸序列。
可选地，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蛋白质控制高粱条螟害虫的用途。
为实现上述目的，本发明还提供了一种产生控制高粱条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高粱条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蛋白的多核苷酸序列的繁殖体。
为实现上述目的，本发明还提供了一种培养控制高粱条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高粱条螟害虫和/或高粱条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“重组”是指通常不能在自然界中发现并且因此通过人工干预产生的DNA和/或蛋白和/或生物体的形式。这种人工干预可产生重组DNA分子和/或重组植物。所述“重组DNA分子”是通过人工组合两种在其它情况下是分离的序列区段而获得的，例如通过化学合成或通过遗传工程技术操作分离的核酸区段。进行核酸操作的技术是众所周知的。
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”是指高粱条螟害虫至少与Cry1A蛋白接触，接触后高粱条螟害虫生长受到抑制和/或导致死亡。进一步地，高粱条螟害虫通过摄食植物组织至少与Cry1A蛋白接触，接触后全部或部分高粱条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蛋白的多核苷酸序列的控制高粱条螟害虫的植物和/或植物种子，在人工接种高粱条螟害虫和/或高粱条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蛋白对高粱条螟的“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”高粱条螟害虫的物质的存在而减弱和/或消失。具体地，当转基因植物(含有编码Cry1A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蛋白和/或可控制高粱条螟害虫的另一种物质时，所述另一种物质的存在既不影响Cry1A蛋白对高粱条螟的 “控制”和/或“防治”作用，也不会导致所述“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蛋白无关。通常情况下，在大田中，高粱条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，在人工接种高粱条螟害虫和/或高粱条螟害虫自然发生危害的条件下，如转基因植物(含有编码Cry1A蛋白的多核苷酸序列)的任何组织存在死亡的高粱条螟害虫、和/或在其上停留生长受到抑制的高粱条螟害虫、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，即通过将高粱条螟害虫至少与Cry1A蛋白接触以实现控制高粱条螟害虫的方法和/或用途。
在本发明中，Cry1A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类杀虫蛋白质的融合表达。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技术特异性敲除或沉默目标昆虫害虫中特定基因的表达。
在分类系统上，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。尽管高粱条螟与二化螟、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。高粱条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，是昆虫循环系统上的差异。
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。 例如同为螟蛾科的三化螟主要为害水稻，极少为害其它禾本科作物。而高粱条螟未见有报道对水稻造成为害，更多的是对南方的甘蔗，北方的玉米、高粱和谷子造成为害。取食习性的不同，也暗示着体内消化系统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蛋白对这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杆野螟属(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，由此更加充分说明了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、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一般都以双链形式存在。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，一般将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，然后分别用2×SSC、0.1％SDS和1×SSC、0.1％SDS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、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8或SEQ ID NO:10杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多同源。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保留了所述特定示例的蛋白质的杀虫活性特征的部分和/或片段(包括与全长蛋白质相比在内部和/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”是指与权利要求所述的蛋白具有相同或基本相同的抗高粱条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“截短体”是指涉及的原始DNA序列或蛋白序列(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。
使用标准技术可修饰基因和容易地构建基因变异体，例如，本领域熟知的构建点突变的技术。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备全长基因的片段，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系统地切除核苷酸。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的部分和与其它B.t.蛋白最不同的部分刺激产生的。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特异性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地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、添加或插入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蛋白包括但不限于SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:9，与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:9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相似性/同一性典型地大于78％，优选地大于85％，更优选地大于90％，甚至更优选地大于95％，并且可以大于99％。也可以根据更特定的同一性和/或相似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％的同一性和/或相似性。
在本发明中，产生所述Cry1A蛋白的转基因植物包括但不限于Mon810转基因玉米事件和/或包含Mon810转基因玉米事件的植物材料(如在US6713259B2所描述的)、Bt11转基因玉米事件和/或包含Bt11转基因玉米事件的植物材料(如在USDA APHIS非管制状态申请95-195-01p所描述的，其所包含的Cry1Ab蛋白的氨基酸序列如本发明SEQ ID NO:3所示)、Bt176转基因玉米事件和/或包含Bt176转基因玉米事件的植物材料(如在USDA APHIS非管制状态申请94-319-01p所描述的，其所包含的Cry1Ab蛋白的氨基酸序列如US5625136B2所描述的)、TT51转基因水稻事件和/或包含TT51转基因水稻事件的植物材料(如在CN100582223C和CN101302520B所描述的)、223F-S21转基因水稻品系和/或包含223F-S21转基因水稻品系的植物材料(如在CN103773759A所描述的)、Mon15985转基因棉花事件和/或包含Mon15985转基因棉花事件的植物材料(如在CN101413028B所描述的)、MON531转基因棉花事件和/或包含MON531转基因棉花事件的植物材料(如在USDA APHIS非管制状态申请00-342-01p所描述的)、COT67B转基因棉花事件和/或包含COT67B转基因棉花事件的植物材料(如在USDA APHIS非管制 状态申请07-108-01p所描述的)或者3006-210-23转基因棉花事件和/或包含3006-210-23转基因棉花事件的植物材料(如在USDA APHIS非管制状态申请03-036-02p所描述的)，其均可以实现本发明的方法和/或用途，即通过使高粱条螟害虫至少与Cry1A蛋白接触以实现控制高粱条螟害虫的方法和/或用途。如本领域技术人员所理解的，使上述转基因事件中的Cry1A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蛋白存在于至少产生所述Cry1A蛋白的转基因植物中，所述高粱条螟害虫通过摄食所述转基因植物的组织至少与所述Cry1A蛋白接触，接触后所述高粱条螟害虫生长受到抑制和/或导致死亡，以实现对高粱条螟危害植物的控制。
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。可选地，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比在植物的其他组织中指导编码序列的表达水平高(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。可选地，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(pin I和pin II)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”可以为将启动子与目的序列相连，使得该目的序列的转录受到该启动子控制和调控。当目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。可选地，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，目标昆虫是高粱条螟害虫。
本发明中Cry1A蛋白对高粱条螟害虫具有毒性。本发明中的植物，特别是高粱、玉米和甘蔗，在其基因组中含有外源DNA，所述外源DNA包含编码Cry1A蛋白的核苷酸序列，高粱条螟害虫通过摄食植物组织与该蛋白接触，接触后高粱条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蛋白所靶向的高粱条螟害虫的杀虫剂)。
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高粱条螟。
本发明中，所述Cry1A蛋白可以具有序列表中SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:9所示的氨基酸序列。除了包含Cry1A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高粱条螟害虫的危害，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高粱条螟的Cry1A蛋白来控制高粱条螟害虫的，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高粱条螟害虫的危害 起到了一定作用，但同时也对人、畜和农田生态系统带来了污染、破坏和残留；使用本发明控制高粱条螟害虫的方法，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高粱条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蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受高粱条螟的侵害。
4、全植株防治。现有技术使用的控制高粱条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蛋白)的根、叶片、茎秆、果实、雄穗、雌穗、花蕾、花药或花丝等都是可以抵抗高粱条螟侵害的。
5、效果稳定。现有技术使用的无论是农业防治方法还是物理防治方法都需要利用环境条件对害虫进行防治，可变因素较多；本发明是使所述Cry1A蛋白在植物体内进行表达，有效地避免了环境条件不稳定的缺陷，且本发明转基因植物(Cry1A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高粱条螟害虫的方法，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蛋白)可以造成高粱条螟初孵幼虫的大量死亡，少量存活幼虫也基本上停止发育，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为本发明的杀虫蛋白的用途的含有Cry1Ab-01核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本发明的杀虫蛋白的用途的含有Cry1Ab-01核苷酸序列的重组表达载体DBN100124构建流程图；
图3为本发明的杀虫蛋白的用途的转基因玉米植株接种高粱条螟的叶片损伤图。
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、目的基因的获得和合成
1、获得核苷酸序列
Cry1Ab-01杀虫蛋白质的氨基酸序列(818个氨基酸)，如序列表中SEQ ID NO:1所示；编码相应于所述Cry1Ab-01杀虫蛋白质的氨基酸序列的Cry1Ab-01核苷酸序列(2457个核苷酸)，如序列表中SEQ ID NO:2所示。Cry1Ab-02杀虫蛋白质的氨基酸序列(615个氨基酸)，如序列表中SEQ ID NO:3所示；编码相应于所述Cry1Ab-02杀虫蛋白质的氨基酸序列的Cry1Ab-02核苷酸序列(1848个核苷酸)，如序列表中SEQ ID NO:4所示。
Cry1Ac-01杀虫蛋白质的氨基酸序列(1156个氨基酸)，如序列表中SEQ ID NO:5所示；编码相应于所述Cry1Ac-01杀虫蛋白质的氨基酸序列的Cry1Ac-01核苷酸序列(3471个核苷酸)，如序列表中SEQ ID NO:6所示。Cry1Ac-02杀虫蛋白质的氨基酸序列(616个氨基酸)，如序列表中SEQ ID NO:7所示；编码相应于所述Cry1Ac-02杀虫蛋白质的氨基酸序列的Cry1Ac-02核苷酸序列(1851个核苷酸)，如序列表中SEQ ID NO:8所示。
Cry1Ab/Ac杀虫蛋白质的氨基酸序列(609个氨基酸)，如序列表中SEQ ID NO:9所示；编码相应于所述Cry1Ab/Ac杀虫蛋白质的氨基酸序列的Cry1Ab/Ac核苷酸序列(1830个核苷酸)，如序列表中SEQ ID NO:10所示。
Cry1Ie杀虫蛋白质的氨基酸序列(719个氨基酸)，如序列表中SEQ ID NO:11所示；编码相应于所述Cry1Ie杀虫蛋白质的氨基酸序列的Cry1Ie核苷酸序列(2160个核苷酸)，如序列表中SEQ ID NO:12所示。
Cry1Fa杀虫蛋白质的氨基酸序列(605个氨基酸)，如序列表中SEQ ID NO:13所示；编码相应于所述Cry1Fa杀虫蛋白质的氨基酸序列的Cry1Fa核苷酸序列(1818个核苷酸)，如序列表中SEQ ID NO:14所示。
2、合成上述核苷酸序列
所述Cry1Ab-01核苷酸序列(如序列表中SEQ ID NO:2所示)、所述Cry1Ab-02核苷酸序列(如序列表中SEQ ID NO:4所示)、所述Cry1Ac-01核苷酸序列(如序列表中SEQ ID NO:6所示)、所述Cry1Ac-02核苷酸序列(如序列表中SEQ ID NO:8所示)、所述Cry1Ab/Ac核苷酸序列(如序列表中SEQ ID NO:10所示)、所述Cry1Ie核苷酸序列(如序列表中SEQ ID NO:12所示)和所述Cry1Fa核苷酸序列(如序列表中SEQ ID NO:14所示)由南京金斯瑞生物科技有限公司合成；合成的所述Cry1Ab-01核苷酸序列(SEQ ID NO:2)的5’端连接有Spe I酶切位点，3’端连接有Kas I酶切位点；合成的所述Cry1Ab-02核苷酸序列(SEQ ID NO:4)的5’端连接有Spe I酶切位点，3’端连接有Kas I酶切位点；合成的所述Cry1Ac-01核苷酸序列(SEQ ID NO:6)的5’端连接有Sac I酶切位点，3’端连接有Kas I酶切位点；合成的所述Cry1Ac-02核苷酸序列(SEQ ID NO:8)的5’端连接有Sac I酶切位点，3’端连接有Kas I酶切位点；合成的所述Cry1Ab/Ac核苷酸序列(SEQ ID NO:10)的5’端连接有Spe I酶切位点，3’端连接有Kas I酶切位点；合成 的所述Cry1Ie核苷酸序列(SEQ ID NO:12)的5’端连接有SnaB I酶切位点，3’端连接有Kas I酶切位点；合成的所述Cry1Fa核苷酸序列(SEQ ID NO:14)的5’端连接有Asc I酶切位点，3’端连接有BamH I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基因的重组克隆载体
将合成的Cry1Ab-01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SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；Cry1Ab-01为Cry1Ab-01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Ahd I和Xho I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中插入的所述Cry1Ab-01核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即Cry1Ab-01核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Ab-02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，Cry1Ab-02为Cry1Ab-02 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Cry1Ab-02核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Ac-01核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN03-T，其中，Cry1Ac-01为Cry1Ac-01核苷酸序列(SEQ ID NO:6)。酶切和测序验证重组克隆载体DBN03-T中所述Cry1Ac-01核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Ac-02核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN04-T，其中，Cry1Ac-02为Cry1Ac-02核苷酸序列(SEQ ID NO:8)。酶切和测序验证重组克隆载体DBN04-T中所述Cry1Ac-02核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Ab/Ac核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN05-T，其中，Cry1Ab/Ac为Cry1Ab/Ac核苷酸序列(SEQ ID NO:10)。酶切和测序验证重组克隆载体DBN05-T中所述Cry1Ab/Ac核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Ie核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN06-T，其中，Cry1Ie为Cry1Ie核苷酸序列(SEQ ID NO:12)。酶切和测序验证重组克隆载体DBN06-T中所述Cry1Ie核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry1Fa核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN07-T，其中，Cry1Fa为Cry1Fa核苷酸序列(SEQ ID NO:14)。酶切和测序验证重组克隆载体DBN07-T中所述Cry1Fa核苷酸序列正确插入。
2、构建含有Cry1A基因的重组表达载体
用限制性内切酶Spe I和Kas I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Cry1Ab-01核苷酸序列片段插到表达载体DBNBC-01的Spe I和Kas I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DBN100124，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:15)；Cry1Ab-01：Cry1Ab-01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:16)；Hpt：潮霉素磷酸转移酶(SEQ ID NO:17)；LB：左边界)。
将重组表达载体DBN100124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DBN100124)，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Spe I和Kas I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DBN100124在Spe I和Kas I位点间的核苷酸序列为序列表中SEQ ID NO:2所示核苷酸序列，即Cry1Ab-01核苷酸序列。
按照上述构建重组表达载体DBN100124的方法，将Spe I和Kas I酶切重组克隆载体DBN02-T切下的所述Cry1Ab-02核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100743。酶切和测序验证重组表达载体DBN100743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Cry1Ab-02核苷酸序列，所述Cry1Ab-02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DBN100124的方法，将通过Sac I和Kas I酶切重组克隆载体DBN03-T所切下的所述Cry1Ac-01核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100741。酶切和测序验证重组表达载体DBN100741中的核苷酸序列含有为序列表中SEQ ID NO:6所示核苷酸序列，即Cry1Ac-01核苷酸序列，所述Cry1Ac-01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DBN100124的方法，将Sac I和Kas I、SnaB I和Kas I分别酶切重组克隆载体DBN04-T和DBN06-T切下的所述Cry1Ac-02核苷酸序列和所述Cry1Ie核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100734。酶切和测序验证重组表达载体DBN100734中的核苷酸序列含有为序列表中SEQ ID NO:8和SEQ ID NO:12所示核苷酸序列，即Cry1Ac-02核苷酸序列和Cry1Ie核苷酸序列，所述Cry1Ac-02核苷酸序列和所述Cry1Ie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DBN100124的方法，将Spe I和Kas I酶切重组克隆载体DBN05-T切下的所述Cry1Ab/Ac核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100737。酶切和测序验证重组表达载体DBN100737中的核苷酸序列含有为序列表中SEQ ID NO:10所示核苷酸序列，即Cry1Ab/Ac核苷酸序列，所述Cry1Ab/Ac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DBN100124的方法，将Asc I和BamH I酶切重组克隆载体DBN07-T切下的所述Cry1Fa核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100735。酶切和测序验证重组表达载体DBN100735中的核苷酸序列含有为序列表中SEQ ID NO:14所示核苷酸序列，即Cry1Fa核苷酸序列，所述Cry1Fa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DBN100124的方法，将Spe I和Kas I、Asc I和BamH I 分别酶切重组克隆载体DBN01-T和DBN07-T切下的所述Cry1Ab-01核苷酸序列和所述Cry1Fa核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100013。酶切和测序验证重组表达载体DBN100013中的核苷酸序列含有为序列表中SEQ ID NO:2和SEQ ID NO:14所示核苷酸序列，即Cry1Ab-01核苷酸序列和Cry1Fa核苷酸序列，所述Cry1Ab-01核苷酸序列和所述Cry1Fa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DBN100124、DBN100743、DBN100741、DBN100734、DBN100737、DBN100735和DBN100013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Ahd I和Xho I对重组表达载体DBN100124、DBN100743、DBN100741、DBN100734、DBN100737、DBN100735和DBN100013酶切后进行酶切验证，结果表明重组表达载体DBN100124、DBN100743、DBN100741、DBN100734、DBN100737、DBN100735和DBN100013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DBN100124、DBN100743、DBN100741、DBN100734、DBN100737、DBN100735和DBN100013中的T-DNA(包括玉米Ubiquitin基因的启动子序列、Cry1Ab-01核苷酸序列、Cry1Ab-02核苷酸序列、Cry1Ac-01核苷酸序列、Cry1Ac-02核苷酸序列、Cry1Ie核苷酸序列、Cry1Ab/Ac核苷酸序列、Cry1Fa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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株和转入Cry1Ab-01-Cry1Fa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Cry1Ab-01核苷酸序列、Cry1Ab-02核苷酸序列、Cry1Ac-01核苷酸序列、Cry1Ac-02-Cry1Ie核苷酸序列、Cry1Ab/Ac核苷酸序列、Cry1Fa核苷酸序列、Cry1Ab-01-Cry1Fa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150-250mg/L)，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；抗性愈伤组织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(30-50mg/L)。由此获得了转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株、转入Cry1Ab/Ac核苷酸序列的高粱植株、转入Cry1Fa核苷酸序列的高粱植株和转入Cry1Ab-01-Cry1Fa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℃下暗培养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30-50mg/L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Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株、转入Cry1Ab/Ac核苷酸序列的甘蔗植株、转入Cry1Fa核苷酸序列的甘蔗植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株；同时以野生型甘蔗植株作为对照。
第四实施例、用TaqMan验证转基因植株
分别取转入Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株和转入Cry1Ab-01-Cry1Fa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基因和Cry1Fa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基因和Cry1Fa基因拷贝数的具体方法如下：
步骤11：分别取转入Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa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引物和探针序列如下：
以下引物和探针用来检测Cry1Ab-01核苷酸序列和Cry1Ab-01-Cry1Fa核苷酸序列：
引物1：CGAACTACGACTCCCGCAC，如序列表中SEQ ID NO:18所示；
引物2：GTAGATTTCGCGGGTCAGTTG，如序列表中SEQ ID NO:19所示；
探针1：CTACCCGATCCGCACCGTGTCC，如序列表中SEQ ID NO:20所示；
以下引物和探针用来检测Cry1Ab-02核苷酸序列和Cry1Ab/Ac核苷酸序列：
引物3：TGCGTATTCAATTCAACGACATG，如序列表中SEQ ID NO:21所示；
引物4：CTTGGTAGTTCTGGACTGCGAAC，如序列表中SEQ ID NO:22所示；
探针2：CAGCGCCTTGACCACAGCTATCCC，如序列表中SEQ ID NO:23所示；
以下引物和探针用来检测Cry1Ac-01核苷酸序列：
引物5：CATTCAATTCAATGACATGAACAGC，如序列表中SEQ ID NO:24所示；
引物6：GACAAGTGCAGGTTGGCAGC，如序列表中SEQ ID NO:25所示；
探针3：TCCGCTCTTCGCCGTTCAGAATTACC，如序列表中SEQ ID NO:26所示；
以下引物和探针用来检测Cry1Ac-02核苷酸序列和Cry1Ac-02-Cry1Ie核苷酸序列：
引物7：GGTTACACTCCCATCGACATCTC，如序列表中SEQ ID NO:27所示；
引物8：CACAAGACCAAGCACGAAACC，如序列表中SEQ ID NO:28所示；
探针4：CCTTACCCAGTTCCTTCTTTCCGAGTTCG，如序列表中SEQ ID NO:29所示；
以下引物和探针用来检测Cry1Fa核苷酸序列：
引物9：CAGTCAGGAACTACAGTTGTAAGAGGG，如序列表中SEQ ID NO:30所示；
引物10：ACGCGAATGGTCCTCCACTAG，如序列表中SEQ ID NO:31所示；
探针5：CGTCGAAGAATGTCTCCTCCCGTGAAC，如序列表中SEQ ID NO:32所示；
PCR反应体系为：
Figure PCTCN2016082587-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 PCTCN2016082587-appb-000002
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，Cry1Ab-01核苷酸序列、Cry1Ab-02核苷酸序列、Cry1Ac-01核苷酸序列、Cry1Ac-02-Cry1Ie核苷酸序列、Cry1Ab/Ac核苷酸序列、Cry1Fa核苷酸序列、Cry1Ab-01-Cry1Fa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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株和转入Cry1Ab-01-Cry1Fa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，对转基因高粱植株和转基因甘蔗植株进行检测分析。实验结果表明，Cry1Ab-01核苷酸序列、Cry1Ab-02核苷酸序列、Cry1Ac-01核苷酸序列、Cry1Ac-02-Cry1Ie核苷酸序列、Cry1Ab/Ac核苷酸序列、Cry1Fa核苷酸序列、Cry1Ab-01-Cry1Fa核苷酸序列均己分别整合到所检测的高粱植株和甘蔗植株的染色体组中，而且转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株、转入Cry1Ab/Ac核苷酸序列的高粱植株、转入Cry1Fa核苷酸序列的高粱植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株、转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株、转入Cry1Ab/Ac核苷酸序列的 甘蔗植株、转入Cry1Fa核苷酸序列的甘蔗植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株；转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株、转入Cry1Ab/Ac核苷酸序列的高粱植株、转入Cry1Fa核苷酸序列的高粱植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株；转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株、转入Cry1Ab/Ac核苷酸序列的甘蔗植株、转入Cry1Fa核苷酸序列的甘蔗植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株；相应的野生型玉米植株、高粱植株和甘蔗植株，以及经Taqman鉴定为非转基因的玉米植株、高粱植株和甘蔗植株对高粱条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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa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头高粱条螟(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，根据高粱条螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：抗性总分＝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Cry1Ab-01核苷酸序列的共3个转化事件株系(S1、S2和S3)，转入Cry1Ab-02核苷酸序列的共3个转化事件株系(S4、S5和S6)，转入Cry1Ab-01-Cry1Fa核苷酸序列的共3个转化事件株系(S7、S8和S9)，转入Cry1Ac-01核苷酸序列的共3个转化事件株系(S10、S11和S12)，转入Cry1Ac-02-Cry1Ie核苷酸序列的共3个转化事件株系(S13、S14和S15)，转入Cry1Ab/Ac核苷酸序列的共3个转化事件株系(S16、S17和S18)， 转入Cry1Fa核苷酸序列的共3个转化事件株系(S19、S20和S21)，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高粱条螟的抗虫实验结果
Figure PCTCN2016082587-appb-000003
表1的结果表明：转入Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株和转入Cry1Ab/Ac核苷酸序列的玉米植株对高粱条螟均具有较好的杀虫效果，高梁条螟平均死亡率基本上可达80％以上，部分甚至达到100％。其抗性总分也基本上均在270分以上，部分甚至达到299分(满分300分)；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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株和转入Cry1Ab/Ac核苷酸序列的玉米植株可以造成高粱条螟初孵幼虫的大量死亡，少数存活的幼虫也基本上停止发育，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Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株和转入Cry1Ab/Ac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高粱条螟的取食痕迹，其叶片损伤率均在5％以下。
对于转入Cry1Fa核苷酸序列的玉米植株对高粱条螟则没有表现出明显的防治效果，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，均未表现出明显差异。
由此证明转入Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株和转入Cry1Ab/Ac核苷酸序列的玉米植株都显示出高抗高粱条螟的活性，这种活性足以对高粱条螟的生长产生不良效应从而使其在田间得以控制。同时通过控制高粱条螟的钻蛀为害，也有可能降低玉米上病害的发生，极大地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Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株、转入Cry1Ab/Ac核苷酸序列的甘蔗植株、转入Cry1Fa核苷酸序列的甘蔗植株、转入Cry1Ab-01-Cry1Fa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头高粱条螟(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，根据高粱条螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：抗性总分＝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Cry1Ab-01核苷酸序列的共3个转化事件株系(S22、S23和S24)，转入Cry1Ab-02核苷酸序列的共3个转化事件株系(S25、S26和S27)，转入Cry1Ab-01-Cry1Fa核苷酸 序列的共3个转化事件株系(S28、S29和S30)，转入Cry1Ac-01核苷酸序列的共3个转化事件株系(S31、S32和S33)，转入Cry1Ac-02-Cry1Ie核苷酸序列的共3个转化事件株系(S34、S35和S36)，转入Cry1Ab/Ac核苷酸序列的共3个转化事件株系(S37、S38和S39)，转入Cry1Fa核苷酸序列的共3个转化事件株系(S40、S41和S42)，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所示。
表2  转基因甘蔗植株接种高粱条螟的抗虫实验结果
Figure PCTCN2016082587-appb-000004
表2的结果表明：转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株和转入Cry1Ab/Ac核 苷酸序列的甘蔗植株对高粱条螟均具有较好的杀虫效果，高粱条螟的平均死亡率基本上均在80％以上，部分甚至达到100％，其抗性总分也基本上均在270分以上，部分甚至达到299分(满分300分)；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均在60分以下。
与野生型甘蔗植株相比，转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株和转入Cry1Ab/Ac核苷酸序列的甘蔗植株可以造成初孵幼虫的大量死亡，且少量存活幼虫也基本上停止发育，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Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株和转入Cry1Ab/Ac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高粱条螟的取食痕迹，其叶片损伤率均在10％以下。
对于转入Cry1Fa核苷酸序列的甘蔗植株对高粱条螟则没有表现出明显的防治效果，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，均未表现出明显差异。
由此证明转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘蔗植株和转入Cry1Ab/Ac核苷酸序列的甘蔗植株都显示出高抗高粱条螟的活性，这种活性足以对高粱条螟的生长产生不良效应从而使其在田间得以控制。同时通过控制高粱条螟的钻蛀为害，也有可能降低甘蔗上病害的发生，极大地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Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株、转入Cry1Ab/Ac核苷酸序列的高粱植株、转入Cry1Fa核苷酸序列的高粱植株、转入Cry1Ab-01-Cry1Fa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头高粱条螟(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，根据高粱条螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：抗性总分＝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Cry1Ab-01核苷酸序列的共3个转化事件株系(S43、S44和S45)，转入Cry1Ab-02核苷酸序列的共3个转化事件株系(S46、S47和S48)，转入Cry1Ab-01-Cry1Fa核苷酸序列的共3个转化事件株系(S49、S50和S51)，转入Cry1Ac-01核苷酸序列的共3个转化事件株系(S52、S53和S54)，转入Cry1Ac-02-Cry1Ie核苷酸序列的共3个转化事件株系(S55、S56和S57)，转入Cry1Ab/Ac核苷酸序列的共3个转化事件株系(S58、S59和S60)，转入Cry1Fa核苷酸序列的共3个转化事件株系(S61、S62和S63)，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所示。
表3  转基因高粱植株接种高粱条螟的抗虫实验结果
Figure PCTCN2016082587-appb-000005
表3的结果表明：转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸 序列的高粱植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株和转入Cry1Ab/Ac核苷酸序列的高粱植株对高粱条螟均具有较好的杀虫效果，高粱条螟的平均死亡率基本上均在80％以上，部分甚至达到100％，其抗性总分均在270分以上，部分甚至达到299分(满分300分)；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。
与野生型高粱植株相比，转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株和转入Cry1Ab/Ac核苷酸序列的高粱植株可以造成高粱条螟初孵幼虫的大量死亡，且少量存活幼虫也基本上停止发育，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Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株和转入Cry1Ab/Ac核苷酸序列的高粱植株大体上只受到轻微损伤，甚至肉眼都无法辨别出高粱条螟的取食痕迹，说明这些基因为高粱植株提供了很好的保护。
对于转入Cry1Fa核苷酸序列的高粱植株对高粱条螟则没有表现出明显的防治效果，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，均未表现出明显差异。
由此证明转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株和转入Cry1Ab/Ac核苷酸序列的高粱植株都显示出高抗高粱条螟的活性，这种活性足以对高粱条螟的生长产生不良效应从而使其在田间得以控制。同时通过控制高粱条螟的钻蛀为害，也有可能降低高粱上病害的发生，极大地提高高粱的产量及品质。
上述实验结果还表明转入Cry1Ab-01核苷酸序列的玉米植株、转入Cry1Ab-02核苷酸序列的玉米植株、转入Cry1Ac-01核苷酸序列的玉米植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的玉米植株、转入Cry1Ab/Ac核苷酸序列的玉米植株、转入Cry1Fa核苷酸序列的玉米植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的玉米植株；转入Cry1Ab-01核苷酸序列的高粱植株、转入Cry1Ab-02核苷酸序列的高粱植株、转入Cry1Ac-01核苷酸序列的高粱植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的高粱植株、转入Cry1Ab/Ac核苷酸序列的高粱植株、转入Cry1Fa核苷酸序列的高粱植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的高粱植株；转入Cry1Ab-01核苷酸序列的甘蔗植株、转入Cry1Ab-02核苷酸序列的甘蔗植株、转入Cry1Ac-01核苷酸序列的甘蔗植株、转入Cry1Ac-02-Cry1Ie核苷酸序列的甘 蔗植株、转入Cry1Ab/Ac核苷酸序列的甘蔗植株、转入Cry1Fa核苷酸序列的甘蔗植株和转入Cry1Ab-01-Cry1Fa核苷酸序列的甘蔗植株对高粱条螟的控制/防治显然是因为植物本身可产生Cry1A蛋白，因此，如本领域技术人员所熟知的，根据Cry1A蛋白对高粱条螟的相同毒杀作用，可产生类似的可表达Cry1A蛋白的转基因植株能够用于控制/防治高粱条螟的危害。本发明中Cry1A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蛋白，同时转基因植株还可以产生至少一种不同于Cry1A蛋白的第二种杀虫蛋白质，如Cry类蛋白或Vip3A蛋白等。
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高粱条螟的Cry1A蛋白来控制高粱条螟害虫；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高粱条螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。
应理解，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 (18)

  1. 一种控制高粱条螟害虫的方法，其特征在于，包括将高粱条螟害虫至少与Cry1A蛋白接触。
  2. 根据权利要求1所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1A蛋白存在于至少产生所述Cry1A蛋白的宿主细胞中，所述高粱条螟害虫通过摄食所述宿主细胞至少与所述Cry1A蛋白接触。
  3. 根据权利要求2所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1A蛋白存在于至少产生所述Cry1A蛋白的细菌或转基因植物中，所述高粱条螟害虫通过摄食所述细菌或转基因植物的组织至少与所述Cry1A蛋白接触，接触后所述高粱条螟害虫生长受到抑制和/或导致死亡，以实现对高粱条螟危害植物的控制。
  4. 根据权利要求3所述的控制高粱条螟害虫的方法，其特征在于，所述植物为玉米、高粱、甘蔗、粟、麻或薏米。
  5. 根据权利要求2至4任一项所述的控制高粱条螟害虫的方法，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蛋白的多核苷酸的植物。
  6. 根据权利要求1至5任一项所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1A蛋白为Cry1Ab蛋白、Cry1Ac蛋白或Cry1Ab/Ac蛋白。
  7. 根据权利要求6所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1A蛋白的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7或SEQ ID NO:9所示的氨基酸序列。
  8. 根据权利要求7所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1A蛋白的核苷酸序列具有SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8或SEQ ID NO:10所示的核苷酸序列。
  9. 根据权利要求2至8任一项所述的控制高粱条螟害虫的方法，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蛋白的核苷酸的第二种核苷酸。
  10. 根据权利要求9所述的控制高粱条螟害虫的方法，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。
  11. 根据权利要求10所述的控制高粱条螟害虫的方法，其特征在于，所述第二种核苷酸编码Cry1Fa蛋白或Cry1Ie蛋白。
  12. 根据权利要求11所述的控制高粱条螟害虫的方法，其特征在于，所述Cry1Ie蛋白的氨基酸序列具有SEQ ID NO:11所示的氨基酸序列，所述Cry1Fa蛋白的氨基酸序列具有SEQ ID NO:13所示的氨基酸序列。
  13. 根据权利要求12所述的控制高粱条螟害虫的方法，其特征在于，所述第二种核 苷酸具有SEQ ID NO:12或SEQ ID NO:14所示的核苷酸序列。
  14. 根据权利要求9所述的控制高粱条螟害虫的方法，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。
  15. 一种Cry1A蛋白质控制高粱条螟害虫的用途。
  16. 一种产生控制高粱条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蛋白的多核苷酸序列。
  17. 一种产生控制高粱条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16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16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蛋白的多核苷酸序列的植物繁殖体。
  18. 一种培养控制高粱条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高粱条螟害虫和/或高粱条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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