WO2023216141A1 - 杀虫蛋白的用途 - Google Patents

杀虫蛋白的用途 Download PDF

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WO2023216141A1
WO2023216141A1 PCT/CN2022/092220 CN2022092220W WO2023216141A1 WO 2023216141 A1 WO2023216141 A1 WO 2023216141A1 CN 2022092220 W CN2022092220 W CN 2022092220W WO 2023216141 A1 WO2023216141 A1 WO 2023216141A1
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plant
protein
pod borer
vip3aa protein
plants
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PCT/CN2022/092220
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English (en)
French (fr)
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狄少康
韩超
谢香庭
于彩虹
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北京大北农生物技术有限公司
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Priority to PCT/CN2022/092220 priority Critical patent/WO2023216141A1/zh
Priority to CN202280017829.5A priority patent/CN117396609A/zh
Publication of WO2023216141A1 publication Critical patent/WO2023216141A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

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  • the present invention relates to the use of an insecticidal protein, in particular to the use of a Vip3Aa protein to control plant damage caused by pod borer through expression in plants.
  • the pod borer (Etiella zinckenella), also known as cowpea pod borer, soybean pod borer, bean pod borer, and locust borer moth, belongs to the Lepidoptera family, and mainly damages soybeans, lentils, mung beans, cowpeas, beans, peas, locusts, tops, and bitters.
  • Legumes such as ginseng, sweet potato, and quinoa beans.
  • the larvae damage leaves, buds, flowers and bean pods. They damage the leaves or bore into the pods to feed on the young seeds. Feces often accumulate inside the pods and outside the bore holes. In mild cases, the bean grains are cut into nicks and holes, and in severe cases, the entire bean pod is bored. If the pods are empty, the affected pods will taste bitter, resulting in bud drop, flower drop, pod drop and dead shoots.
  • Cultivated soybean (Glycine max (L.) Merri) is an important economic crop grown globally as the main source of vegetable oil and vegetable protein. It is an important food and feed crop in China. Soybeans are one of the plants that the pod borer likes to feed on most. This insect is distributed worldwide and is widely distributed in my country. Except forChina, which has not been reported, it occurs in other provinces and regions, south of the Yellow River and in most places in Gansu and Qinghai. , the density is very high. Soybeans are damaged by the pod borer every year, causing serious economic losses. In order to control pod borer, the main methods commonly used include agricultural control, chemical control and biological control.
  • Agricultural prevention and control is the comprehensive and coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, and environmental factors to create a farmland ecological environment that is conducive to crop growth but not conducive to the occurrence of pod borer.
  • irrigation can be carried out several times in autumn and winter to increase the mortality of overwintering larvae.
  • irrigation can be carried out 1 to 2 times to increase the mortality of larvae entering the soil and increase soybean yield.
  • this method has had little effect and consumes a lot of water resources.
  • Chemical control that is, pesticide control, uses chemical pesticides to kill pests. It is an important part of the comprehensive management of pod borer. It is fast, convenient, simple and highly economical, especially when the pod borer occurs in large numbers. situations, it is an essential emergency measure.
  • the main chemical control method is liquid spray.
  • For chemical spray control use 21% synergistic cyanide EC, 2.5% cyhalothrin EC, 2.5% deltamethrin, 2.5% Baode EC, and 55% poison chlorine EC. From the time of budding, spraying the buds and flowers once every 10 days can control the damage. If other pests need to be controlled at the same time, spraying should be carried out comprehensively.
  • chemical control also has its limitations. Improper use can often lead to phytotoxicity in crops, pesticide resistance in pests, killing natural enemies, polluting the environment, causing damage to farmland ecosystems, and pesticide residues posing a threat to the safety of people and livestock. Adverse consequences.
  • Biological control is the use of certain beneficial organisms or biological metabolites to control pest populations in order to reduce or eliminate pests, such as selecting pesticides with low toxicity to natural enemies and adjusting pesticide application according to the differences in the occurrence periods of pests and natural enemies in the field. time, avoid applying pesticides when natural enemies occur in large numbers to protect natural enemies.
  • the control effect can reach more than 80%. Its characteristics are that it is safe for humans and livestock, causes less environmental pollution, and can achieve long-term control of certain pests; however, the effect is often unstable, and the same investment is required regardless of the severity of the pod borer occurrence.
  • Vip3Aa insecticidal protein is one of many insecticidal proteins. It is an insoluble parasporal crystal protein produced by Bacillus thuringiensis subsp. kurstaki (B.t.k.).
  • Vip3Aa insecticidal protein is one of many insecticidal proteins and is a specific protein produced by Bacillus thuringiensis. Vip3Aa protein has a poisonous effect on sensitive insects by stimulating apoptotic programmed cell death. Vip3Aa protein is hydrolyzed into four main protein products in the insect intestine, of which only one proteolytic product (66KD) is the toxic core structure of Vip3Aa protein. The Vip3Aa protein binds to the midgut epithelial cells of sensitive insects and initiates programmed cell death, causing the dissolution of the midgut epithelial cells and leading to the death of the insect. It does not cause any symptoms in non-sensitive insects and does not cause apoptosis and lysis of midgut epithelial cells.
  • Vip3Aa gene can resist Lepidoptera pests such as cutworms, cotton bollworms and Spodoptera frugiperda.
  • Lepidoptera pests such as cutworms, cotton bollworms and Spodoptera frugiperda.
  • there are no reports on the toxicity of Vip3Aa protein to pod borer and there is no report on the toxicity of Vip3Aa protein to pod borer.
  • the purpose of the present invention is to provide the use of an insecticidal protein, and for the first time provide a method for controlling pod borer by producing transgenic plants expressing Vip3Aa protein, and effectively overcome the technical shortcomings of existing agricultural control, chemical control and biological control technologies. .
  • the present invention provides a method for controlling the pod borer pest, which includes contacting the pod borer pest with at least Vip3Aa protein.
  • the Vip3Aa protein exists in at least a host cell that produces the Vip3Aa protein, and the pod borer pest comes into contact with at least the Vip3Aa protein by ingesting the host cell.
  • the Vip3Aa protein exists in at least the bacteria or transgenic plants that produce the Vip3Aa protein, and the pod borer pest comes into contact with at least the Vip3Aa protein by feeding on the bacteria or the tissue of the transgenic plant. The growth of the latter pest is inhibited and/or death is achieved, so as to control the damage caused by the pod borer to plants.
  • the transgenic plants can be at any growth stage.
  • the tissues of the transgenic plant are roots, leaves, stems, fruits and flowers.
  • the control of plant damage caused by pod borer does not change due to changes in planting location and/or planting time.
  • the plant is soybean, lentil, mung bean, cowpea, kidney bean, pea, acacia or black locust.
  • the contacting step is preceded by growing a plant containing a polynucleotide encoding the Vip3Aa protein.
  • the amino acid sequence of the Vip3Aa protein has the amino acid sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence of the Vip3Aa protein has the nucleotide sequence shown in SEQ ID NO:2.
  • the plant further includes at least a second polynucleotide different from the polynucleotide encoding the Vip3Aa protein.
  • the second polynucleotide encodes Cry insecticidal protein, Vip insecticidal protein, protease inhibitor, lectin, ⁇ -amylase or peroxidase.
  • the second polynucleotide is a dsRNA that inhibits an important gene in the target insect pest.
  • the present invention also provides the use of Vip3Aa protein to control the pest of pod borer.
  • the present invention also provides a method for producing a plant for controlling the pest of pod borer, which includes introducing a polynucleotide sequence encoding Vip3Aa protein into the genome of the plant.
  • the present invention also provides a method for producing plant seeds for controlling pod borer pests, including selfing or crossing the plant obtained by the method with a second plant, thereby producing a polynucleoside encoding the Vip3Aa protein. Acid sequence seeds.
  • the present invention also provides a method for cultivating plants for controlling pod borer pests, including:
  • Planting at least one plant seed, the genome of the plant seed includes a polynucleotide sequence encoding the Vip3Aa protein
  • the plants are grown under conditions of artificial inoculation of the pod borer pest and/or the naturally occurring harm of the pod borer pest, and harvested with reduced plant damage and/or compared with other plants that do not have a polynucleotide sequence encoding the Vip3Aa protein. or plants with increased plant yield.
  • Contact means that insects and/or pests touch, stay and/or eat plants, plant organs, plant tissues or plant cells.
  • the plants, plant organs, plant tissues or plant cells can be It means that the insecticidal protein is expressed in the body, or the surface of the plant, plant organ, plant tissue or plant cell has insecticidal protein and/or there is a microorganism that produces the insecticidal protein.
  • control and/or “prevention” in the present invention refer to the fact that the pod borer pest is at least in contact with the Vip3Aa protein, and the growth of the pod borer pest is inhibited and/or causes death after contact. Furthermore, the pod borer pest comes into contact with at least the Vip3Aa protein by feeding on plant tissue. After contact, the growth of all or part of the pod borer pest is inhibited and/or causes death. Inhibition refers to sub-lethal effects, which are not lethal but can cause certain effects in growth and development, behavior, physiology, biochemistry and tissue, such as slowed growth and development and/or cessation.
  • plants and/or plant seeds containing a polynucleotide sequence encoding the Vip3Aa protein to control pod borer pests, under conditions where artificial inoculation of pod borer pests and/or pod borer pests naturally occur are the same as non-transgenic Compared with wild-type plants, plant damage is reduced, and specific manifestations include but are not limited to improved stem resistance, and/or increased grain weight, and/or increased yield, etc.
  • the "control” and/or "prevention” effect of Vip3Aa protein on pod borer can exist independently and will not be weakened and/or eliminated by the presence of other substances that can "control” and/or "prevent” pod borer pests. .
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a Vip3Aa protein
  • contains and/or produces the Vip3Aa protein and/or another substance that can control the pod borer pest contains and/or produces the Vip3Aa protein and/or another substance that can control the pod borer pest.
  • the presence of the other substance neither affects the "control” and/or "prevention” effect of Vip3Aa protein on pod borer, nor can it cause the "control” and/or “prevention” effect to be complete and/or partial.
  • the process of feeding plant tissue by the pod borer pest is short-lived and difficult to observe with the naked eye. Therefore, under conditions where the pest is artificially inoculated and/or the pod borer pest naturally causes damage, such as genetically modified Any tissue of a plant (containing a polynucleotide sequence encoding a Vip3Aa protein) contains dead pod borer pests, and/or has pod borer pests that remain there and have its growth inhibited, and/or are similar to non-transgenic wild-type plants.
  • the method and/or use of the present invention is achieved if the plant damage is reduced, that is, the method and/or use of controlling the pod borer pest is achieved by contacting the pod borer pest with at least the Vip3Aa protein.
  • the expression of Vip3Aa protein in a transgenic plant can be accompanied by the expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins.
  • This co-expression of more than one insecticidal toxin in the same transgenic plant can be achieved by genetically engineering the plant to contain and express the desired gene.
  • one plant (the first parent) can express the Vip3Aa protein through genetic engineering
  • the second plant (the second parent) can express the Cry-like insecticidal protein and/or the Vip-like insecticidal protein through genetic engineering.
  • Progeny plants expressing all the genes introduced into the first parent and the second parent are obtained by crossing the first parent and the second parent.
  • RNA interference refers to a phenomenon that is highly conserved during evolution, induced by double-stranded RNA (dsRNA), and causes efficient and specific degradation of homologous mRNA. Therefore, RNAi technology can be used in the present invention to specifically knock out or turn off the expression of specific genes in target insect pests.
  • the pod borer (Etiella zinckenella) of the present invention is an insect of the family Etiella zinckenella of the order Lepidoptera.
  • the adult body length is 10-12mm
  • the wingspan is 20-24mm
  • the body color is grayish brown.
  • the hind wings are gray-white, with brown along the outer edge; the forewings are long and narrow, gray-brown, covered with dark brown, yellow and white scales, with a white longitudinal band along the front edge, and a yellow-brown crescent-shaped horizontal band near the base 1/3 of the wing. bring.
  • the eggs are oval, with a long diameter of 0.5-0.6mm and a short diameter of about 0.4mm. They are milky white at first and turn red and yellow later.
  • the surface of the egg is filled with irregular reticular patterns.
  • Mature larvae are 14 to 18 mm long, purple-red on the back and green on the ventral surface; there are "herringbone"-shaped black spots on the front and back of the thorax, one black spot on each side, and two small black spots in the center of the trailing edge.
  • the topline, subtopline, valve line and valve lower line are obvious.
  • the pupal body is 9 to 10 mm long. It is green at first and turns yellowish brown later.
  • the ventral end is pointed and darker along the dorsal midline.
  • the antennae and wings are as long as the posterior edge of the fifth abdominal segment. There are 6 hooks at the end of the abdomen.
  • Pod borer is widely distributed in my country, with East China, Central China, South China, and Shaanxi suffering the most damage. Likes to eat legumes. The adult lifespan is 6 to 7 days. It lurks on the back of leaves during the day and is active at night. It has weak flying ability and weak phototaxis. They can mate on the same day after emerging and lay eggs the next day. Each pod usually lays only 1 egg, but rarely more than 2 eggs. Eggs are mostly laid between the fine hairs on the pods and under the sepals, and a few can be laid on petioles and other places. Each female moth can lay 80-90 eggs, and the egg period lasts 3-6 days. The larvae have 5 instars in total, and the larval period is 9 to 12 days.
  • the larvae crawl on the pod or spin silk to hang and transfer to the pod. After selecting the pod, they first spin silk on the pod to make a small white silk sac. They bore into the pod from under the silk sac and sneak into the beans to feed.
  • the first instar larvae do not turn.
  • the 2nd to 5th instar larvae have the habit of turning to pods to cause damage, and each larvae can turn to pods to cause damage 1 to 3 times.
  • the damage first occurs in the upper part of the plant, and gradually reaches the lower part. Generally, the larvae are most distributed in the upper part. After the larvae mature, they leave the pod and enter the soil, form cocoons and pupate, with soil particles sticking to the outside of the cocoons.
  • the larvae usually penetrate from the middle of the pod and eat the bean kernels inside the pod.
  • the damaged kernels may be nicked or hollowed out in severe cases.
  • the damaged kernels may also be filled with insect feces, turn brown and become moldy.
  • Lepidoptera In terms of classification system, Lepidoptera is generally divided into suborders, superfamilies, families, etc. based on morphological characteristics such as the venation sequence of adult wings, linkage patterns, and types of antennae.
  • the Botryidae family is one of the most diverse families in Lepidoptera. More than 10,000 species have been discovered around the world, and thousands of them have been recorded in China alone. Most borer insects are pests of crops, and most of them cause damage in the form of stem borers, such as stem borers and corn borers. Although the corn borer and the pod borer both belong to the family Lepidoptera, apart from similarities in classification standards, they have great differences in other morphological structures; just like strawberries and apples in plants (both belong to the order Rosaceae).
  • Insects belonging to the same family are not only very different in morphological characteristics, but also in feeding habits.
  • the corn borer also a member of the family Boreridae, mainly damages corn in the Gramineae family.
  • the pod borer likes to feed on leguminous plants.
  • the difference in feeding habits also implies that the enzymes and receptor proteins produced by the digestive system in the body are different.
  • the enzymes produced in the digestive tract are the key point for the Bt gene to work. Only enzymes or receptor proteins that can combine with specific Bt genes can make a certain Bt gene have an insect-resistant effect on the pest. More and more studies have shown that insects of the same order, different families, or even different species of the same family have different sensitivity to the same Bt protein.
  • the Vip3Aa gene shows resistance to the Chilo suppressalis and the Asian corn borer Ostrinia furnacalis.
  • the Vip3Aa gene has no resistance to the Indian grain borer Plodia interpunctella and the European corn borer Ostrinia nubilalis. Insect effect.
  • the above-mentioned pests all belong to the family Lepidoptera, but the same Bt protein shows different resistance effects to these pests.
  • the European corn borer and the Asian corn borer even belong to the same genus Ostrinia (same order, same family, same genus) in terms of classification, but their responses to the same Bt protein are completely different, which fully illustrates the relationship between Bt protein and The way enzymes and receptors interact in insects is complex and unpredictable.
  • the genome of a plant, plant tissue or plant cell mentioned in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes the nucleus, plastid and mitochondrial genome.
  • polynucleotides and/or nucleotides described in the present invention form a complete "gene" encoding a protein or polypeptide in the desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in the host of interest.
  • DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other and vice versa. As DNA replicates in plants, other complementary strands of DNA are produced. As such, the present invention includes the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands. "Coding strand” as commonly used in the art refers to the strand combined with the antisense strand. To express a protein in the body, one strand of DNA is typically transcribed into a complementary strand of mRNA, which serves as a template for translation of the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • the "sense” or “coding” strand has a series of codons (codons are three nucleotides, read three at a time to produce a specific amino acid), which can be read as an open reading frame (ORF) to form the protein or peptide of interest.
  • the present invention also includes RNA having functional equivalents to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes with the Vip3Aa 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 Vip3Aa gene of the invention. Nucleic acid molecules or fragments thereof can specifically hybridize with other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. If two nucleic acid molecules show complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to show "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are able to hybridize to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency” conditions.
  • two nucleic acid molecules are said to be “complementary” if they can hybridize to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency” conditions.
  • Deviations from perfect complementarity are permissible as long as such departures do not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe, it is only necessary to ensure that it has sufficient sequence complementarity to form a stable double-stranded structure under the specific solvent and salt concentration used.
  • a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matching nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0 ⁇ SSC at 50°C, will be apparent to those skilled in the art. is publicly known.
  • the salt concentration in the wash step can be selected from low stringency conditions of about 2.0 ⁇ SSC, 50°C to highly stringent conditions of about 0.2 ⁇ SSC, 50°C.
  • the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for highly stringent conditions. Temperature conditions and salt concentration can both change, or one variable can remain constant while the other variable changes.
  • the stringent conditions of the present invention can be specific hybridization with SEQ ID NO:2 in 6 ⁇ SSC, 0.5% SDS solution at 65°C, and then using 2 ⁇ SSC, 0.1% SDS and 1 ⁇ SSC. , 0.1% SDS each washed the membrane once.
  • sequences that have anti-insect activity and hybridize to SEQ ID NO: 2 of the present invention under stringent conditions are included in the present invention. These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology.
  • genes and proteins described in the present invention not only include specific example sequences, but also include parts and/or fragments that retain the insecticidal activity characteristics of the specific example proteins (including the internal and/or terminal ends compared with the full-length protein). deletions), variants, mutants, substitutions (proteins with substituted amino acids), chimeras and fusion proteins.
  • the "variant” or “variation” refers to a nucleotide sequence encoding the same protein or encoding an equivalent protein with insecticidal activity.
  • the "equivalent protein” refers to a protein that has the same or substantially the same biological activity against the pod borer pest as the claimed protein.
  • fragment or “truncation” of a DNA molecule or protein sequence described in the present invention refers to a part of the original DNA or protein sequence (nucleotides or amino acids) involved or its artificially modified form (such as a sequence suitable for plant expression). ), the length of the aforementioned sequence may vary, but the length is sufficient to ensure that the (encoded) protein is an insect toxin.
  • Genes can be modified and genetic variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Another example is US Patent No. 5,605,793, which describes the use of DNA reassembly after random fragmentation to generate other molecular diversity. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used following standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically excise nucleotides from the ends of these genes. A variety of restriction enzymes can also be used to obtain genes encoding active fragments. Active fragments of these toxins can be obtained directly using proteases.
  • the invention allows for the derivation of equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries.
  • insecticidal proteins of the invention There are many ways to obtain the insecticidal proteins of the invention.
  • antibodies to the insecticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from a mixture of proteins.
  • antibodies may be caused by the portion of the protein that is most constant and most distinct from other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies to the proteins disclosed in the present invention or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. The genes encoding these proteins can then be obtained from the microorganism.
  • nucleic acid sequences can encode the same amino acid sequence. It is within the skill of those in the art to generate these alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included within the scope of the present invention.
  • substantially identical sequences refer to sequences with amino acid substitutions, deletions, additions or insertions that do not substantially affect the insecticidal activity, and also include fragments that retain insecticidal activity.
  • amino acid changes in the present invention is a routine technology in the art.
  • amino acid changes are: small characteristic changes, that is, conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, Typically a deletion of about 1-30 amino acids; a small amino- or carboxyl-terminal extension, such as an amino-terminal extension of one methionine residue; a small linking peptide, such as about 20-25 residues long.
  • conservative substitutions are substitutions that occur 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 specific activity are well known in the art and have been described, for example, by N. Neurath and R.L.
  • amino acid residues essential for the activity of a polypeptide of the invention and therefore selected not to be substituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, e.g., Cunningham and Wells , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, e.g., Cunningham and Wells , 1989, Science 244: 1081-1085).
  • the latter technique involves introducing mutations at every positively charged residue in the molecule and testing the anti-insect activity of the resulting mutant molecules to identify the amino acid residues that are 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, e.g., 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).
  • Vip3Aa protein includes but is not limited to SEQ ID NO: 1, and amino acid sequences having certain homology with the amino acid sequence shown in SEQ ID NO: 1 are also included in the present invention.
  • the similarity/identity between these sequences and the sequences of the present invention is typically greater than 60%, preferably greater than 75%, 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 in terms of more specific ranges of identity and/or similarity.
  • transgenic plants producing the Vip3Aa protein include, but are not limited to, COT102 transgenic cotton events and/or plant materials containing COT102 transgenic cotton events (as described in CN1004395507C), COT202 transgenic cotton events and/or containing COT202
  • the plant material of the transgenic cotton event (as described in CN1886513A), or the MIR162 transgenic corn event and/or the plant material comprising the MIR162 transgenic corn event (as described in CN101548011A) can implement the method of the present invention and/or Or a use, that is, a method and/or use for controlling the pod borer pest by contacting the pod borer pest with at least the Vip3Aa protein.
  • the methods and/or uses of the present invention can also be achieved by expressing the Vip3Aa protein in the above transgenic event in different plants. More specifically, the Vip3Aa protein exists in at least a transgenic plant that produces the Vip3Aa protein, and the pod borer pest comes into contact with at least the Vip3Aa protein by feeding on the tissue of the transgenic plant, and after contact, the pod borer pest Control of plant damage caused by pod borer is achieved by inhibiting pest growth and/or causing death.
  • the regulatory sequences in the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns and other regulatory sequences operably connected to the Vip3Aa protein.
  • the promoter is a promoter expressible in plants, and the "promoter expressible in plants” refers to a promoter that ensures that the coding sequence connected thereto is expressed in plant cells. Promoters expressible in plants may be constitutive promoters. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the Arabidopsis Ubi10 promoter, the corn Ubi promoter, the promoter of the rice GOS2 gene, etc.
  • a promoter expressible in a plant may be a tissue-specific promoter, that is, the promoter directs the expression of the coding sequence to a higher level in some tissues of the plant, such as in green tissues, than in other tissues of the plant (can be determined by conventional RNA assay), such as the PEP carboxylase promoter.
  • the promoter expressible in plants may be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-induced expression pattern means that when a plant undergoes trauma caused by machinery or insect gnawing, the expression of the coding sequence under the control of the promoter is significantly increased compared with that under normal growth conditions.
  • wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitor genes (pinI and pinII) and the maize protease inhibitor gene (MPI).
  • the transit peptide (also known as secretion signal sequence or guide sequence) guides the transgene product to a specific organelle or cell compartment.
  • the transit peptide can be heterologous, for example, using a protein encoding a chloroplast transporter.
  • the peptide sequences were targeted to chloroplasts, either to the endoplasmic reticulum using the 'KDEL' retention sequence, or to the vacuole using the CTPP of the barley lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (encephalomyocarditis virus 5' non-coding region); a potato virus group Y leader sequence, such as an MDMV (Maize Dwarf Mosaic Virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader of alfalfa mosaic virus coat protein mRNA (AMV RNA4); tobacco mosaic virus (TMV) leader.
  • EMCV leader sequence encephalomyocarditis virus 5' non-coding region
  • a potato virus group Y leader sequence such as an MDMV (Maize Dwarf Mosaic Virus) leader sequence
  • MDMV Maize Dwarf Mosaic Virus
  • BiP Human immunoglobulin heavy chain binding protein
  • AMV RNA4 alfalfa mosaic virus coat protein mRNA
  • TMV tobacco mosaic virus
  • the enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, figwort mosaic virus (FMV) enhancer, carnation weathered ring virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer , purple jasmine mosaic virus (MMV) enhancer, tuberose yellowing leaf curl virus (CmYLCV) enhancer, Multan cotton leaf curl virus (CLCuMV), commelina yellow mottle virus (CoYMV) and peanut chlorotic streaks leaf virus (PCLSV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathered ring virus
  • CsVMV cassava vein mosaic virus
  • MMV purple jasmine mosaic virus
  • CmYLCV tuberose yellowing leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV commelina yellow mottle virus
  • PCLSV peanut chlorotic streaks leaf virus
  • the intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron.
  • such introns include, but are not limited to, CAT-1 introns, pKANNIBAL introns, PIV2 introns, and "superubiquitin" introns.
  • 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 , the polyadenylation signal sequence derived from the protease inhibitor II (pinII) gene, the polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and the polyadenylation signal sequence derived from the ⁇ -tubulin gene.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • pinII protease inhibitor II
  • pea ssRUBISCO E9 the polyadenylation signal sequence derived from the ⁇ -tubulin gene.
  • Effectively linked refers to the connection of nucleic acid sequences such that one sequence can provide the function required for the connected sequence.
  • the "effective connection” in the present invention can be connecting a promoter to a sequence of interest, so that the transcription of the sequence of interest is controlled and regulated by the promoter.
  • "effectively linked” means that the promoter is connected to the sequence in such a way that the resulting transcript is efficiently translated. If the connection between the promoter and the coding sequence is a transcript fusion and expression of the encoded protein is desired, the connection is made so that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • connection between the promoter and the coding sequence is a translational fusion and expression of the encoded protein is desired, make such a connection such that the first translation initiation codon contained in the 5' untranslated sequence is fused with the promoter are connected in such a manner that the relationship between the resulting translation product and the translated open reading frame encoding the desired protein is in frame.
  • Nucleic acid sequences that can be "operably linked" include, but are not limited to: sequences that provide gene expression functions (i.e., gene expression elements, such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polypeptides, etc.
  • sequences that provide DNA transfer and/or integration functions i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites
  • sequences that provide selection Sequences that provide sexual function i.e., antibiotic resistance markers, biosynthetic genes
  • sequences that provide scoreable marker functions sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences) and sequences that provide Replication functional sequences (i.e. bacterial origin of replication, autonomous replication sequences, centromere sequences).
  • “Pesticide” or “pest-resistant” mentioned in the present invention refers to being toxic to crop pests, thereby achieving “control” and/or “prevention” of crop pests.
  • the "pesticide” or “pest-resistant” refers to killing crop pests. More specifically, the target insect is the pod borer.
  • the Vip3Aa protein in the present invention is toxic to the pod borer pest.
  • the plants of the present invention especially soybeans, contain exogenous DNA in their genome, and the exogenous DNA includes a polynucleotide sequence encoding the Vip3Aa protein.
  • the pod borer pest comes into contact with this protein by feeding on plant tissue.
  • Canker borer pest growth is inhibited and/or mortality results. Inhibition refers to lethal or sublethal.
  • the plants should be morphologically normal and cultureable under conventional methods for product consumption and/or production.
  • the plant can substantially eliminate the need for chemical or biological pesticides (such as those against the pod borer pest targeted by the Vip3Aa protein).
  • ICPs insecticidal crystal proteins
  • the target insect in the present invention is mainly the pod borer.
  • the Vip3Aa protein may have the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
  • other elements may also be included, such as proteins encoding selectable markers.
  • the expression cassette comprising the polynucleotide sequence encoding the Vip3Aa protein of the present invention can also be expressed in plants together with at least one protein encoding a herbicide resistance gene, which includes, but is not limited to, glufosinate.
  • Phosphate resistance genes such as bar gene, pat gene
  • bendichlor resistance genes such as pmph gene
  • glyphosate resistance genes such as EPSPS gene
  • bromoxynil (bromoxynil) resistance genes sulfonylurea Resistance genes, resistance genes to the herbicide quat, resistance genes to cyanamide, or resistance genes to glutamine synthetase inhibitors (such as PPT), thereby obtaining high insecticidal activity and herbicidal properties agent-resistant transgenic plants.
  • exogenous DNA is introduced into plants, such as introducing the gene or expression cassette or recombinant vector encoding the Vip3Aa protein into plant cells.
  • Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-emission bombardment, Direct DNA uptake into protoplasts, electroporation, or whisker silicon-mediated DNA introduction.
  • the invention provides a use of insecticidal protein, which has the following advantages:
  • the existing technology mainly controls the damage of pod borer pests through external effects, that is, external factors, such as agricultural control, chemical control and biological control; while the present invention controls pod borer through the production of Vip3Aa protein in plants that can kill pod borer. Borer pests are controlled through internal factors.
  • the transgenic plants (Vip3Aa protein) can be used from germination and growth to flowering and fruiting. Avoid pod borer infestations.
  • the effect is stable. Whether agricultural control methods or physical control methods used in the prior art require the use of environmental conditions to control pests, there are many variable factors; the present invention allows the Vip3Aa protein to be expressed in plants, effectively overcoming environmental conditions. Instable defects, and the control effect of the transgenic plant (Vip3Aa protein) of the present invention is stable and consistent in different locations, different times, and different genetic backgrounds.
  • the present invention only needs to plant transgenic plants capable of expressing Vip3Aa protein without taking other measures, thus saving a lot of manpower, material resources and financial resources.
  • Figure 1 is a flow chart for the construction of the recombinant cloning vector DBN01-T containing the polynucleotide sequence mVip3Aa of the Vip3Aa protein for use in the insecticidal protein of the present invention
  • Figure 2 is a construction flow chart of the recombinant expression vector DBN10702 containing the polynucleotide sequence mVip3Aa of the Vip3Aa protein for use in the insecticidal protein of the present invention
  • FIG. 3 shows the use of the insecticidal protein of the present invention on the pod borer under natural insect-infectious conditions
  • Figure 4 is a diagram of the pod damage of the transgenic soybean plant using the insecticidal protein of the present invention when it is naturally susceptible to the pod borer.
  • Vip3Aa insecticidal protein (789 amino acids), as shown in SEQ ID NO: 1 in the sequence listing; mVip3Aa polynucleotide sequence (2370 nucleotides) encoding the amino acid sequence corresponding to the Vip3Aa insecticidal protein , as shown in SEQ ID NO:2 in the sequence list.
  • the mVip3Aa polynucleotide sequence (shown as SEQ ID NO: 2 in the sequence listing) was synthesized by Nanjing Genscript Biotechnology Co., Ltd.
  • the synthesized mVip3Aa polynucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operating steps were carried out according to the instructions of the Promega product pGEM-T vector to obtain the recombinant cloning vector DBN01-T.
  • FIG. 1 where 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; T7 is the T7 RNA polymerase promoter; mVip3Aa is the mVip3Aa polynucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) using heat shock method, white colonies were picked, and cultured in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, adjust pH to 7.5 with NaOH, and culture overnight at 37°C.
  • the plasmid was extracted by alkaline method and stored at -20°C for later use.
  • the positive clones were sequenced and verified.
  • the results showed that the mVip3Aa polynucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. , that is, the mVip3Aa polynucleotide sequence is correctly inserted.
  • the recombinant expression vector DBN10702 was constructed, and its construction process is shown in Figure 2 (Kan: Ka Namycin gene; RB: right border; prAtAct2: ACT2 promoter of Arabidopsis thaliana (SEQ ID NO:3); mVip3Aa: mVip3Aa polynucleotide sequence (SEQ ID NO:2); tNos: nopaline synthase gene Terminator (SEQ ID NO:4); pr35S: Cauliflower mosaic virus 35S promoter (SEQ ID NO:5); PAT: phosphinothricin acetyltransferase gene (SEQ ID NO:6); t35S: Cauliflower mosaic virus 35S terminator (SEQ ID NO:7); LB: left border).
  • the heat shock method to transform the recombinant expression vector DBN10702 into Escherichia coli T1 competent cells, pick white colonies, and culture them in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, kanamycin 50mg/L, adjust pH to 7.5 with NaOH) and culture overnight at 37°C.
  • the plasmid was extracted by alkaline method. The extracted plasmid was digested with restriction enzymes and identified, and the positive clones were sequenced and identified. The results showed that the nucleotide sequence in the recombinant expression vector DBN10702 is the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. , that is, the mVip3Aa polynucleotide sequence.
  • the correctly constructed recombinant expression vector DBN10702 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) using the liquid nitrogen method.
  • the transformation conditions were: 100 ⁇ l Agrobacterium LBA4404, 3 ⁇ l plasmid DNA (recombinant expression Carrier); place in liquid nitrogen for 10 minutes, 37°C warm water bath for 10 minutes; inoculate the transformed Agrobacterium LBA4404 into an LB test tube, culture it for 2 hours at a temperature of 28°C and a rotation speed of 200 rpm, and apply it on a solution containing 50 mg/L Add rifampicin and 100 mg/L kanamycin to the LB plate until a positive single clone grows.
  • the third embodiment obtaining transgenic soybean plants
  • the cotyledon node tissue of the aseptically cultured soybean variety Jack is co-cultured with the Agrobacterium described in 3 in the second embodiment to recombinantly express the soybean constructed in 2 in the second embodiment.
  • the T-DNA of vector DBN10702 (including mVip3Aa polynucleotide sequence and PAT gene) was transferred into the soybean genome, and soybean plants transformed with the mVip3Aa polynucleotide sequence were obtained, while wild-type soybean plants were used as controls.
  • soybean germination medium B5 salt 3.1g/L, B5 vitamins, sucrose 20g/L, agar 8g/L, pH 5.6
  • inoculate the seeds on the germination medium under the following conditions: temperature 25 ⁇ 1°C; photoperiod (light/dark) 16/8h.
  • 4-6 days after germination take the bright green sterile soybean seedlings with enlarged cotyledon nodes, cut off the hypocotyls 3-4mm below the cotyledon nodes, cut the cotyledons lengthwise, and remove the terminal buds, lateral buds and seed roots.
  • infection medium MS salt 2.15g/L
  • B5 vitamins sucrose 20g/L, glucose 10g/L, Acetosyringone (AS) 40 mg/L, 2-morpholinoethanesulfonic acid (MES) 4 g/L, zeatin (ZT) 2 mg/L, pH 5.3
  • the cotyledon node tissue and Agrobacterium are co-cultured for a period of time (3 days) (step 2: co-culture step).
  • the cotyledon node tissue is incubated in solid culture medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, MES 4g/L, ZT 2mg/L, agar 8g/L after the infection step , pH5.6).
  • solid culture medium MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, MES 4g/L, ZT 2mg/L, agar 8g/L after the infection step , pH5.6.
  • recovery medium B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, ZT 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamine acid 100mg/L, aspartic acid 100mg/L, pH 5.6
  • antibiotics there is at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250mg/L)
  • no selection agent for plant transformants Step 3: Recovery steps.
  • the regenerated tissue pieces from the cotyledonary nodes are cultured on solid media with antibiotics but no selective agents to eliminate Agrobacterium and provide a recovery period for infected cells.
  • the regenerated tissue pieces from the cotyledonary nodes are cultured on a medium containing a selection agent (glufosinate) and the growing transformed calli are selected (step 4: selection step).
  • a selection agent glufosinate
  • the cotyledon node regenerated tissue pieces are cultured in a screening solid medium with selective agents (B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, glufosinate 6mg/L, pH5.6), leading to the selection of transformed cells sexual growth.
  • selective agents B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, ce
  • the transformed cells are regenerated into plants (step 5: regeneration step), preferably, the regenerated tissue pieces of the cotyledon nodes grown on medium containing a selective agent are grown on solid medium (B5 differentiation medium and B5 rooting medium) to regenerate plants.
  • step 5 regeneration step
  • the regenerated tissue pieces of the cotyledon nodes grown on medium containing a selective agent are grown on solid medium (B5 differentiation medium and B5 rooting medium) to regenerate plants.
  • the resistant tissue blocks obtained by screening were transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, ZT 1mg/L, agar 8g/L, cephalosporin 150mg /L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, glufosinate 6mg/L, pH 5.6), culture and differentiate at 25°C.
  • B5 differentiation medium B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, ZT 1mg/L, agar 8g/L, cephalosporin 150mg /L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, glufosinate 6mg/L, pH 5.6
  • the differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3- Butyric acid (IBA) 1mg/L), in root culture, culture at 25°C to a height of about 10cm, then move to the greenhouse and culture until firm. In the greenhouse, culture at 26°C for 16 hours and then at 20°C for 8 hours every day.
  • B5 rooting medium B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3- Butyric acid (IBA) 1mg/L
  • the fourth embodiment using TaqMan to verify transgenic plants
  • Step 11 Take 100 mg of each leaf of the soybean plant transferred with the mVip3Aa polynucleotide sequence and the leaves of the wild-type soybean plant, grind them into a homogenate using liquid nitrogen in a mortar, and take 3 replicates for each sample;
  • Step 12 Use Qiagen’s DNeasy Plant Mini Kit to extract genomic DNA from the above samples.
  • Qiagen refer to its product instructions
  • Step 13 Use NanoDrop 2000 (Thermo Scientific) to determine the genomic DNA concentration of the above sample;
  • Step 14 Adjust the genomic DNA concentration of the above sample to the same concentration value, and the concentration value range is 80-100ng/ ⁇ L;
  • Step 15 Use the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample. Use the identified sample with known copy number as the standard, and use the wild-type soybean plant sample as the control. Each sample is repeated 3 times, and the average is taken. value; the fluorescence quantitative PCR primer and probe sequences are:
  • Primer 1 gagggtgttgtggctggtattg as shown in SEQ ID NO:8 in the sequence list;
  • Primer 2 tctcaactgtccaatcgtaagcg as shown in SEQ ID NO:9 in the sequence list;
  • Probe 1 cttacgctgggcctggaaggctag as shown in SEQ ID NO:10 in the sequence list;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ L of each primer at a 1 mM concentration, 50 ⁇ L of the probe at a 100 ⁇ M concentration, and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in amber tubes at 4°C.
  • soybean plants transformed with the mVip3Aa polynucleotide sequence and the soybean plants identified as non-transgenic by Taqman were tested for insect resistance against pod borer.
  • transgenic soybean plants to pod borer were evaluated in the field under naturally susceptible conditions. Two lines (S1, S2) transformed into the mVip3Aa polynucleotide sequence and one line identified as non-transgenic (NGM) by Taqman were selected. Planted in the planting base of Shandong province, each strain and control (NGM) were set up with 3 replicates. The experiment adopted a random block design, and each replicate was sown in 2 rows (5 meters).
  • pod-boring pests were found to harm soybeans at the planting base in Shandong Province. They were identified as pod borers. See Figure 3 for details.
  • the resistance level is evaluated based on the pod borer rate.
  • the specific evaluation criteria are shown in Table 1:
  • Table 1 show that under naturally occurring conditions, compared with NGM, soybean plants introduced with the mVip3Aa polynucleotide sequence have a good inhibitory effect on pod borer, the resistance level is high, and can effectively prevent and control pod borer. Feeding damage of soybean seeds by borers.
  • soybean plants transformed with the mVip3Aa polynucleotide sequence display high activity against pod borer, which is sufficient to have adverse effects on the growth of pod borer and thereby enable it to be controlled in the field.
  • the purpose of the insecticidal protein of the present invention is to control the pod borer pest by producing Vip3Aa protein in the plant that can kill the pod borer; it is comparable to the agricultural control methods, chemical control methods and biological control methods used in the prior art.
  • the present invention protects plants during the whole growth period and the whole plant from the pests of pod borer, and has no pollution and no residue. The effect is stable, thorough, simple, convenient and economical.

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Abstract

一种杀虫蛋白的用途，所述杀虫蛋白可用于控制豆荚斑螟害虫，所述控制豆荚斑螟害虫的方法包括：将豆荚斑螟害虫至少与Vip3Aa蛋白接触。所述方法通过植物体内产生能够杀死豆荚斑螟的Vip3Aa蛋白来控制豆荚斑螟害虫；与现有技术使用的农业防治方法、化学防治方法和生物防治方法相比，所述方法对植物进行全生育期、全植株的保护以防治豆荚斑螟害虫的侵害，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Vip3Aa蛋白质通过在植物中表达来控制豆荚斑螟为害植物的用途。
背景技术
豆荚斑螟(Etiella zinckenella)别名豇豆荚螟、大豆荚螟、豆荚螟、槐螟蛾，属鳞翅目螟蛾科，主要危害大豆、扁豆、绿豆、豇豆、菜豆、豌豆、刺槐、毛条、苦参、苕子、藜豆等豆科植物。幼虫为害叶、蕾、花及豆荚，卷叶为害或蛀入荚内取食幼嫩籽粒，荚内及蛀孔外常堆积粪便，轻者把豆粒蛀成缺刻、孔洞，重则把整个豆荚蛀空，受害豆荚味苦，造成落蕾、落花、落荚和枯梢。
栽培大豆(Glycine max(L.)Merri)，是一种全球种植的作为植物油和植物蛋白主要来源的重要经济作物，是中国重要的食用和饲用作物。大豆是豆荚斑螟最喜取食的植物之一，该虫为世界性分布，在我国分布面广，除西藏未见报道外，其余各省区均有发生，黄河以南以及甘肃、青海多数地方，密度均很高。大豆每年因豆荚斑螟为害，造成严重经济损失。为了防治豆荚斑螟，人们通常采用的主要方法有农业防治、化学防治和生物防治。
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豆荚斑螟发生的农田生态环境，如利用灌溉灭虫，在水源方便的地区，可在秋、冬灌水数次，提高越冬幼虫的死亡率，在夏大豆开花结荚期，灌水1～2次，可增加入土幼虫的死亡率，增加大豆产量。但该法收效甚微，且对水资源的耗用极大。
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是豆荚斑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，特别是豆荚斑螟大发生的情况下，是必不可少的应急措施。目前化学防治方法主要是药液喷雾。药液喷雾选用21％增效氰·马乳油、2.5％三氟氯氰菊酯乳油、2.5％溴氰菊酯、2.5％保得乳油、55％毒·氯乳油等药剂喷雾防治。从现蕾开始，每隔10d喷蕾、花1次，可控制为害，如需兼治其他害虫，则应全面喷药。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。
生物防治是利用某些有益生物或生物代谢产物来控制害虫种群数量，以达到降低或消灭害虫的目的，如选择对天敌毒性低的农药，并根据害虫和天敌田间发生期的差异，调整施药时间，避开在天敌大量发生时施药以保护天敌。其次，于产卵始盛期释放赤眼蜂(Trichogrammatidae)，防治效果可达80％以上。其特点是对人、畜安全，对环境污染少，对某些害虫可达到长期控制的目的；但是效果常不稳定，并且不论豆荚斑螟发生轻重均需同样投资进行。
为了解决农业防治、化学防治和生物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。Vip3Aa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，B.t.k.)产生的不溶性伴孢结晶蛋白。
Vip3Aa杀虫蛋白是众多杀虫蛋白中的一种，是由苏云金芽孢杆菌产生的特异性蛋白质。Vip3Aa蛋白通过激发凋亡类型的细胞程序性死亡对敏感性昆虫具有毒杀效应。Vip3Aa蛋白在昆虫肠道内被水解为4种主要蛋白产物，其中只有一种蛋白水解产物(66KD)为Vip3Aa蛋白的毒性核心结构。Vip3Aa蛋白结合敏感昆虫的中肠上皮细胞，启动细胞程序性死亡，造成中肠上皮细胞的溶解导致昆虫死亡。对非敏感昆虫不产生任何病症，不会导致中肠上皮细胞的凋亡和溶解。
已证明转Vip3Aa基因的植株可以抵抗小地老虎、棉铃虫和草地贪夜蛾等鳞翅目(Lepidoptera)害虫的侵害，然而，至今尚无关于Vip3Aa蛋白对豆荚斑螟有毒性的报道，也无关于通过产生表达Vip3Aa蛋白的转基因植株来控制豆荚斑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Vip3Aa蛋白的转基因植株来控制豆荚斑螟的方法，且有效克服现有技术农业防治、化学防治和生物防治等技术缺陷。
为实现上述目的，本发明提供了一种控制豆荚斑螟害虫的方法，包括将豆荚斑螟害虫至少与Vip3Aa蛋白接触。
进一步地，所述Vip3Aa蛋白存在于至少产生所述Vip3Aa蛋白的宿主细胞中，所述豆荚斑螟害虫通过摄食所述宿主细胞至少与所述Vip3Aa蛋白接触。
更进一步地，所述Vip3Aa蛋白存在于至少产生所述Vip3Aa蛋白的细菌或转基因植物中，所述豆荚斑螟害虫通过摄食所述细菌或所述转基因植物的 组织至少与所述Vip3Aa蛋白接触，接触后所述豆荚斑螟害虫生长受到抑制和/或导致死亡，以实现对豆荚斑螟危害植物的控制。
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、花。
所述对豆荚斑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。
所述植物为大豆、扁豆、绿豆、豇豆、菜豆、豌豆、洋槐或刺槐。
所述接触步骤之前的步骤为种植含有编码所述Vip3Aa蛋白的多核苷酸的植物。
优选地，所述Vip3Aa蛋白氨基酸序列具有SEQ ID NO:1所示的氨基酸序列。所述Vip3Aa蛋白的核苷酸序列具有SEQ ID NO:2所示的核苷酸序列。
在上述技术方案的基础上，所述植物还包括至少一种不同于编码所述Vip3Aa蛋白的多核苷酸的第二种多核苷酸。
进一步地，所述第二种多核苷酸编码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。
可选择地，所述第二种多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Vip3Aa蛋白质控制豆荚斑螟害虫的用途。
为实现上述目的，本发明还提供了一种产生控制豆荚斑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Vip3Aa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豆荚斑螟害虫的植物种子的方法，包括将由所述方法获得的植株自交或与第二植株杂交，从而产生含有编码Vip3Aa蛋白的多核苷酸序列的种子。
为实现上述目的，本发明还提供了一种培养控制豆荚斑螟害虫的植物的方法，包括：
种植至少一粒植物种子，所述植物种子的基因组中包括编码Vip3Aa蛋白的多核苷酸序列；
使所述植物种子长成植株；
使所述植株在人工接种豆荚斑螟害虫和/或豆荚斑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Vip3Aa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“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”是指豆荚斑螟害虫至少与Vip3Aa蛋白接触，接触后豆荚斑螟害虫生长受到抑制和/或导致死亡。进一步地，豆荚斑螟害虫通过摄食植物组织至少与Vip3Aa蛋白接触，接触后全部或部分豆荚斑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Vip3Aa蛋白的多核苷酸序列的控制豆荚斑螟害虫的植物和/或植物种子，在人工接种豆荚斑螟害虫和/或豆荚斑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。Vip3Aa蛋白对豆荚斑螟的“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”豆荚斑螟害虫的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码Vip3Aa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，Vip3Aa蛋白和/或可控制豆荚斑螟害虫的另一种物质，则所述另一种物质的存在既不影响Vip3Aa蛋白对豆荚斑螟的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全和/或部分由所述另一种物质实现，而与Vip3Aa蛋白无关。通常情况下，在大田，豆荚斑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，在人工接种豆荚斑螟害虫和/或豆荚斑螟害虫自然发生危害的条件下，如转基因植物(含有编码Vip3Aa蛋白的多核苷酸序列)的任何组织存在死亡的豆荚斑螟害虫、和/或在其上停留生长受到抑制的豆荚斑螟害虫、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，即通过豆荚斑螟害虫至少与Vip3Aa蛋白接触以实现控制豆荚斑螟害虫的方法和/或用途。
在本发明中，Vip3Aa蛋白在一种转基因植物中的表达可以伴随着一个或多个Cry类杀虫蛋白质和/或Vip类杀虫蛋白质的表达。这种超过一种的杀虫毒素在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达Vip3Aa蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达Cry类杀虫蛋白质和/或Vip类杀虫蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。因此在本发明中可以使用RNAi技术特异性剔除或关闭目标昆 虫害虫中特定基因的表达。
本发明所述豆荚斑螟(Etiella zinckenella)为鳞翅目螟蛾科昆虫。成虫体长10～12mm，翅展20-24mm，体色灰褐色。后翅灰白色，沿外缘褐色；前翅狭长，灰褐色，覆有深褐色、黄色及白色鳞片，沿前缘有1条白色纵带，近翅基1/3处有1条黄褐色月牙形横带。卵椭圆形，长径0.5～0.6mm，短径约0.4mm，初产乳白色，后转红黄色，卵表面弥补不规则网状纹。老熟幼虫体长14～18mm，背面紫红色，腹面绿色；前胸背板上有“人”字形黑斑，两侧各有1个黑斑，后缘中央有2个小黑斑。背线、亚背线、气门线和气门下线明显。蛹体长9～10mm，初化蛹为绿色，以后呈黄褐色，腹端尖细，沿背中线颜色较深，触角和翅长达第5腹节后缘，腹部末端有钩刺6个。
豆荚斑螟在我国分布广泛，以华东、华中、华南、陕西受害最重。喜食豆科植物。成虫寿命6～7d，白天潜伏在叶背，夜晚活动，飞翔力不强，趋光性弱。羽化后当日即能交尾，隔天就可产卵。每荚一般只产1粒卵，少数2粒以上。卵多产在荚上的细毛间和萼片下面，少数可产在叶柄等处。每头雌蛾可产卵80～90粒，卵期3～6d。幼虫共5龄，幼虫期9～12d。幼虫孵化后在荚上爬行或吐丝悬垂转荚，选荚后先在荚上吐丝作一小白丝囊，从丝囊下蛀入荚内，潜入豆粒中取食，1龄幼虫不转荚，2～5龄幼虫有转荚为害习性，每一幼虫可转荚为害1～3次。先在植株上部为害，渐至下部，一般以上部幼虫分布最多。幼虫老熟后离荚入土，结茧化蛹，茧外粘有土粒。幼虫一般从荚中部蛀入，在豆荚内蛀食豆粒，被害粒轻则成缺刻，重则被蛀空，被害籽粒还充满虫粪，变褐以致霉烂。
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。尽管玉米螟和豆荚斑螟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。但因人们较少接触昆虫，尤其是较少接触农业害虫，对于昆虫形态上的差异较少关注，而使得人们以为昆虫的形态大同小异。而事实上，豆荚斑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。
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。例如同为螟蛾科的玉米螟主要为害禾本科的玉米。而豆荚斑螟喜取食豆科植物。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和亚洲玉米螟Ostrinia furnacalis都表现出了抗虫活性，但是Vip3Aa基因对于同属螟蛾科的印度谷螟Plodia interpunctella和欧洲玉米螟Ostrinia nubilalis却没有抗虫效果。上述几种害虫均属于鳞翅目螟蛾科，但同种Bt蛋白对这几种螟蛾科害虫表现出不同的抗性效果。尤其是欧洲玉米螟和亚洲玉米螟在分类上甚至同属于螟蛾科Ostrinia属(同目同科同属)，但是其对同种Bt蛋白的反应却是截然不同的，更加充分说明了Bt蛋白与昆虫体内酶和受体的相互作用方式是复杂且难以预料的。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA。
本发明中核酸分子或其片段在严格条件下与本发明Vip3Aa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Vip3Aa基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够 以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:2发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
因此，具有抗虫活性并在严格条件下与本发明SEQ ID NO:2杂交的序列包括在本发明中。这些序列与本发明序列至少大约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％或更大的序列同源性。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的杀虫活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“变异”是指编码同一蛋白或编码有杀虫活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与权利要求的蛋白具有相同或基本相同的抗豆荚斑螟害虫的生物活性的蛋白。
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸或氨基酸)的一部分或其人工改造形式(例如适合植物表达的序列)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为昆虫毒素。
使用标准技术可以修饰基因和容易的构建基因变异体。例如，本领域熟知制造点突变的技术。又例如美国专利号5605793描述了在随机断裂后使用DNA重装配产生其它分子多样性的方法。可以使用商业化核酸内切酶制造全长基因的片段，并且可以按照标准程序使用核酸外切酶。例如，可以使用酶诸如Bal31或定点诱变从这些基因的末端系统地切除核苷酸。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和与其它B.t.蛋白最不同的蛋白部分引起的。然后可以通过免疫沉淀、酶联免疫吸附测定(ELISA)或western印迹方法使用这些抗体专一地鉴定有特征活性的等价蛋白。可使用本领域标准程序容易的制备本发明中公开的蛋白或等价蛋白或这类蛋白的片段的抗体。然后可以从微生物中获得编码这些蛋白的基因。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或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。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的抗虫活性，从而确定对该分子活性而言重要的 氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol 224:899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。
在本发明中，Vip3Aa蛋白包括但不限于SEQ ID NO:1，与SEQ ID NO:1所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于90％，甚至更优选的大于95％，并且可以大于99％。也可以根据更特定的相同性和/或类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60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
在本发明中，产生所述Vip3Aa蛋白的转基因植物包括但不限于COT102转基因棉花事件和/或包含COT102转基因棉花事件的植物材料(如在CN1004395507C所描述的)、COT202转基因棉花事件和/或包含COT202转基因棉花事件的植物材料(如在CN1886513A所描述的)、或者MIR162转基因玉米事件和/或包含MIR162转基因玉米事件的植物材料(如在CN101548011A所描述的)，其均可以实现本发明的方法和/或用途，即通过豆荚斑螟害虫至少与Vip3Aa蛋白接触以实现控制豆荚斑螟害虫的方法和/或用途。本领域技术人员所理解的，使上述转基因事件中的Vip3Aa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Vip3Aa蛋白存在于至少产生所述Vip3Aa蛋白的转基因植物中，所述豆荚斑螟害虫通过摄食所述转基因植物的组织至少与所述Vip3Aa蛋白接触，接触后所述豆荚斑螟害虫生长受到抑制和/或导致死亡，以实现对豆荚斑螟危害植物的控制。
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Vip3Aa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、拟南芥Ubi10启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测 定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pinⅠ和pinⅡ)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达 时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明中所述的“杀虫”或“抗虫”是指对农作物害虫是有毒的，从而实现“控制”和/或“防治”农作物害虫。优选地，所述“杀虫”或“抗虫”是指杀死农作物害虫。更具体地，目标昆虫是豆荚斑螟。
本发明中Vip3Aa蛋白对豆荚斑螟害虫具有毒性。本发明中的植物，特别是大豆，在其基因组中含有外源DNA，所述外源DNA包含编码Vip3Aa蛋白的多核苷酸序列，豆荚斑螟害虫通过摄食植物组织与该蛋白接触，接触后豆荚斑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Vip3Aa蛋白所靶向的豆荚斑螟害虫的杀虫剂)。
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豆荚斑螟。
本发明中，所述Vip3Aa蛋白可以具有序列表中SEQ ID NO:1所示的氨基酸序列。除了包含Vip3Aa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Vip3Aa蛋白的多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，草铵膦抗性基因(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Vip3Aa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豆荚斑螟害虫的危害，如农业防治、化学防治和生物防治；而本发明是通过植物体内产生能够杀死豆荚斑螟的Vip3Aa蛋白来控制豆荚斑螟害虫的，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豆荚斑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；使用本发明控制豆荚斑螟害虫的方法，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豆荚斑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(Vip3Aa蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受豆荚斑螟的侵害。
4、全植株防治。现有技术使用的控制豆荚斑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(Vip3Aa蛋白)的根、叶片、茎秆、果实、雄穗、雌穗、花药或花丝等都是可以抵抗豆荚斑螟侵害的。
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Vip3Aa蛋白在植物体内进行表达，有效地克服了环境条件不稳定的缺陷，且本发明转基因植物(Vip3Aa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。本发明只需种植能够表达Vip3Aa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豆荚斑螟害虫的方法，其效果是不彻底的，只起到减轻作用；而本发明转基因植物(Vip3Aa蛋白)可以造成豆荚斑螟害虫的大量死亡，而转基因植物大体上只受到轻微损伤。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明杀虫蛋白的用途的含有Vip3Aa蛋白的多核苷酸序列mVip3Aa的重组克隆载体DBN01-T构建流程图；
图2为本发明杀虫蛋白的用途的含有Vip3Aa蛋白的多核苷酸序列mVip3Aa的重组表达载体DBN10702构建流程图；
图3为本发明杀虫蛋白的用途的自然感虫条件下的豆荚斑螟；
图4为本发明杀虫蛋白的用途的转基因大豆植株的豆荚斑螟自然感虫情况下的豆荚损伤图。
具体实施方式
下面通过具体实施例进一步说明本发明杀虫蛋白的用途的技术方案。
第一实施例、基因的获得和合成
1、获得核苷酸序列
Vip3Aa杀虫蛋白质的氨基酸序列(789个氨基酸)，如序列表中SEQ ID NO:1所示；编码相应于所述Vip3Aa杀虫蛋白质的氨基酸序列的mVip3Aa多核苷酸序列(2370个核苷酸)，如序列表中SEQ ID NO:2所示。
2、合成上述核苷酸序列
所述mVip3Aa多核苷酸序列(如序列表中SEQ ID NO:2所示)由南京金斯瑞生物科技有限公司合成。
第二实施例、重组表达载体的构建和重组表达载体转化农杆菌
1、构建含有mVip3Aa基因的重组克隆载体
将合成的mVip3Aa多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；mVip3Aa为mVip3Aa多核苷酸序列(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),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,于温度-20℃保存备用。
提取的质粒经酶切鉴定后，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mVip3Aa多核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即mVip3Aa多核苷酸序列正确插入。
2、构建含有mVip3Aa基因的大豆重组表达载体
用限制性内切酶分别酶切重组克隆载体DBN01-T和表达载体DBNBC-03(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的mVip3Aa多核苷酸序列片段插到表达载体DBNBC-03的限制性内切酶酶切位点之间，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DBN10702，其构建流程如图2所示(Kan：卡那霉素基因；RB：右边界；prAtAct2：拟南芥的ACT2启动子(SEQ ID NO:3)；mVip3Aa：mVip3Aa多核苷酸序列(SEQ ID NO:2)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:4)；pr35S：花椰菜花叶病毒35S启动子(SEQ ID NO:5)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:6)；t35S：花椰菜花叶病毒35S终止子(SEQ ID NO:7)；LB：左边界)。将重组表达载体DBN10702用热激方法转化大肠杆菌T1感受态细胞，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DBN10702中的核苷酸序列为序列表中SEQ ID NO:2所示核苷酸序列，即mVip3Aa多核苷酸序列。
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DBN10702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，用限制性内切酶对重组表达载体DBN10702酶切后进行验证，结果表明重组表达载体DBN10702结构完全正确。
第三实施例、获得转基因大豆植株
按照常规采用的农杆菌侵染法，将无菌培养的大豆品种Jack的子叶节组织与第二实施例中3所述的农杆菌共培养，以将第二实施例中2构建的大豆重组表达载体DBN10702的T-DNA(包括mVip3Aa多核苷酸序列和PAT基因)转入到大豆染色体组中，获得了转入mVip3Aa多核苷酸序列的大豆植株，同时以野生型大豆植株作为对照。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子在大豆萌发培养基(B5盐3.1g/L、B5维他命、蔗糖20g/L、琼脂8g/L，pH5.6)中进行萌发，将种子接种于萌发培养基上，按以下条件培养：温度25±1℃； 光周期(光/暗)为16/8h。萌发4-6天后取鲜绿的子叶节处膨大的大豆无菌苗，在子叶节下3-4mm处切去下胚轴，纵向切开子叶，去顶芽、侧芽和种子根。用解剖刀的刀背在子叶节处进行创伤，用农杆菌悬浮液接触创伤过的子叶节组织，其中农杆菌能够将载体T-DNA序列传递至创伤过的子叶节组织(步骤1：侵染步骤)在此步骤中，子叶节组织优选地浸入农杆菌悬浮液(OD 660＝0.5-0.8)，侵染培养基(MS盐2.15g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)40mg/L、2-吗啉乙磺酸(MES)4g/L、玉米素(ZT)2mg/L，pH5.3)中以启动接种。子叶节组织与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，子叶节组织在侵染步骤后在固体培养基(MS盐4.3g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、MES 4g/L、ZT 2mg/L、琼脂8g/L，pH5.6)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(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、ZT 2mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸100mg/L、天冬氨酸100mg/L，pH5.6)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，子叶节再生的组织块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，子叶节再生的组织块在含选择剂(草丁膦)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，子叶节再生的组织块在有选择剂的筛选固体培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、6-苄基腺嘌呤(6-BAP)1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸100mg/L、天冬氨酸100mg/L、草丁膦6mg/L，pH5.6)上培养，导致转化的细胞选择性生长。然后，转化的细胞再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的子叶节再生的组织块在固体培养基(B5分化培养基和B5生根培养基)上培养以再生植物。
筛选得到的抗性组织块转移到所述B5分化培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、ZT 1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸50mg/L、天冬氨酸50mg/L、赤霉素1mg/L、生长素1mg/L、草丁膦6mg/L，pH5.6)上，25℃下培养分化。分化出来的小苗转移到所述B5生根培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、琼脂8g/L、头孢霉素150mg/L、吲哚-3-丁酸(IBA)1mg/L)，在生根培养上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于26℃下培养16h，再于20℃下培养8h。
第四实施例、用TaqMan验证转基因植株
取转入mVip3Aa多核苷酸序列的大豆植株的叶片约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PAT基因的拷贝数以确定mVip3Aa基因的拷贝数。同时以野生型大豆植株作为对照，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PAT基因拷贝数的具体方法如下：
步骤11、分别取转入mVip3Aa多核苷酸序列的大豆植株和野生型大豆植株的叶片各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，以野生型大豆植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测PAT基因：
引物1：gagggtgttgtggctggtattg如序列表中SEQ ID NO:8所示；
引物2：tctcaactgtccaatcgtaagcg如序列表中SEQ ID NO:9所示；
探针1：cttacgctgggccctggaaggctag如序列表中SEQ ID NO:10所示；
PCR反应体系为：
Figure PCTCN2022092220-appb-000001
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2022092220-appb-000002
Figure PCTCN2022092220-appb-000003
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，mVip3Aa多核苷酸序列己整合到所检测的大豆植株的染色体组中，而且转入mVip3Aa多核苷酸序列的大豆植株获得了单拷贝的转基因大豆植株。
第五实施例、转基因大豆植株的抗虫效果检测
将转入mVip3Aa多核苷酸序列的大豆植株和经Taqman鉴定为非转基因的大豆植株对豆荚斑螟进行抗虫效果检测。
在田间进行自然感虫条件下评估转基因大豆植株对豆荚斑螟的抗性。选取转入mVip3Aa多核苷酸序列的2个株系(S1、S2)和经Taqman鉴定为非转基因的(NGM)1个株系。在山东省种植基地种植，每个株系及对照(NGM)各设3个重复，试验采用随机区组设计，每个重复播种2行(5米)。
R6期在山东省种植基地发现蛀荚类害虫危害大豆，经鉴定为豆荚斑螟，具体见图3。
根据蛀荚率评估抗性水平，具体评价标准如表1所示：
表1、抗性水平评估标准
Figure PCTCN2022092220-appb-000004
在大豆材料R6～R8期，开始调查豆荚中被豆荚斑螟幼虫取食的情况，每行调查5株材料，采取均匀取样，每米范围内随机选择1株长势正常，结荚饱满的植株。随机取100个荚，记录有虫食破损的荚数目，得蛀荚率 (％)，重复3次。结果如表2和图4所示。
表2、转基因大豆植株豆荚斑螟自然感虫的实验结果
Figure PCTCN2022092220-appb-000005
表1的结果表明：在自然发生条件下，与NGM相比，转入mVip3Aa多核苷酸序列的大豆植株对豆荚斑螟具有良好的抑制效果，抗性水平为高抗，可以有效防控豆荚斑螟对大豆籽粒的取食危害。
由此证明转入mVip3Aa多核苷酸序列的大豆植株显示出高抗豆荚斑螟的活性，这种活性足以对豆荚斑螟的生长产生不良效应从而使其在田间得以控制。
上述实验结果还表明：转入mVip3Aa多核苷酸序列的大豆植株对豆荚斑螟的控制/防治显然是因为植物本身可产生Vip3Aa蛋白。所以，本领域技术人员熟知的，根据Vip3Aa蛋白对豆荚斑螟的毒杀作用，本发明中转入Vip3Aa蛋白的植株还可以产生至少一种不同于Vip3Aa蛋白的第二种杀虫蛋白质，如Cry类蛋白等。
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豆荚斑螟的Vip3Aa蛋白来控制豆荚斑螟害虫；与现有技术使用的农业防治方法、化学防治方法和生物防治方法相比，本发明对植物进行全生育期、全植株的保护以防治豆荚斑螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (10)

  1. 一种控制豆荚斑螟害虫的方法，其特征在于，包括将豆荚斑螟害虫至少与Vip3Aa蛋白接触；
    优选地，所述Vip3Aa蛋白存在于至少产生所述Vip3Aa蛋白的宿主细胞中，所述豆荚斑螟害虫通过摄食所述宿主细胞至少与所述Vip3Aa蛋白接触；
    更优选地，所述Vip3Aa蛋白存在于至少产生所述Vip3Aa蛋白的细菌或转基因植物中，所述豆荚斑螟害虫通过摄食所述细菌或所述转基因植物的组织至少与所述Vip3Aa蛋白接触，接触后所述豆荚斑螟害虫生长受到抑制和/或导致死亡，以实现对豆荚斑螟危害植物的控制。
  2. 根据权利要求1所述的控制豆荚斑螟害虫的方法，其特征在于，所述转基因植物为大豆、扁豆、绿豆、豇豆、菜豆、豌豆、洋槐或刺槐；
    优选地，所述转基因植物的组织为叶片、果实、花。
  3. 根据权利要求1或2所述的控制豆荚斑螟害虫的方法，其特征在于，所述Vip3Aa蛋白具有SEQ ID NO:1所示的氨基酸序列；
    优选地，所述Vip3Aa蛋白具有SEQ ID NO:2所示的核苷酸序列。
  4. 根据权利要求1至3任一项所述的控制豆荚斑螟害虫的方法，其特征在于，所述转基因植物还包括至少一种不同于编码所述Vip3Aa蛋白的多核苷酸的第二种多核苷酸。
  5. 根据权利要求4所述的控制豆荚斑螟害虫的方法，其特征在于，所述第二种多核苷酸编码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。
  6. 根据权利要求4所述的控制豆荚斑螟害虫的方法，其特征在于，所述第二种多核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
  7. 一种Vip3Aa蛋白质控制豆荚斑螟害虫的用途。
  8. 一种产生控制豆荚斑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Vip3Aa蛋白的多核苷酸序列。
  9. 一种产生控制豆荚斑螟害虫的植物种子的方法，其特征在于，包括将由权利要求8所述方法获得的植株自交或与第二植株杂交，从而产生含有编码Vip3Aa蛋白的多核苷酸序列的种子。
  10. 一种培养控制豆荚斑螟害虫的植物的方法，其特征在于，包括：
    种植至少一粒植物种子，所述植物种子的基因组中包括编码Vip3Aa蛋白的多核苷酸序列；
    使所述植物种子长成植株；
    使所述植株在人工接种豆荚斑螟害虫和/或豆荚斑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Vip3Aa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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