WO2015070781A1 - 控制害虫的方法 - Google Patents

控制害虫的方法 Download PDF

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Publication number
WO2015070781A1
WO2015070781A1 PCT/CN2014/091023 CN2014091023W WO2015070781A1 WO 2015070781 A1 WO2015070781 A1 WO 2015070781A1 CN 2014091023 W CN2014091023 W CN 2014091023W WO 2015070781 A1 WO2015070781 A1 WO 2015070781A1
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Prior art keywords
spodoptera litura
protein
plant
seq
nucleotide sequence
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PCT/CN2014/091023
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English (en)
French (fr)
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张爱红
鲍晓明
刘海利
裴艳新
张云珠
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北京大北农科技集团股份有限公司
北京大北农科技集团股份有限公司生物技术中心
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Priority to AU2014350744A priority Critical patent/AU2014350744C1/en
Publication of WO2015070781A1 publication Critical patent/WO2015070781A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present application relates to a method of controlling pests, and more particularly to a method for controlling a Spodoptera litura-damaging plant using a CrylF protein expressed in a plant.
  • Prodenia litura belongs to the family Lepidoptera, and is a omnivorous and gluttonous pest. It is a host of pests. In addition to corn and soybeans, it can also be used to damage melon, eggplant, beans, onions, leeks, spinach and Cruciferous vegetables, food, cash crops and other nearly 100 families, more than 300 kinds of plants; Spodoptera litura is a worldwide distribution, occurring in all parts of the country, mainly in the Yangtze River Basin and the Yellow River Basin. Spodoptera litura mainly damages whole plants with larvae, and grows on the back of the cluster when they are young. After 3 years of age, the leaves are scattered, and the young stems and old larvae can feed on the fruit.
  • the annual food loss caused by Spodoptera litura is huge, and even more affects the living conditions of the local population.
  • the main control methods commonly used are: agricultural control, chemical control and physical control.
  • Agricultural control is the comprehensive coordinated management of the multi-factors of the whole farmland ecosystem, regulating crops, pests, environmental factors, and creating a farmland ecological environment that is conducive to crop growth and is not conducive to the occurrence of Spodoptera litura.
  • weeds can be removed, ploughed or irrigated after harvesting to destroy or deteriorate the site of pupation, which can help reduce the source of insects; or combined with the management of the newly hatched larvae that remove the eggs and cluster damage to reduce the source of insects.
  • agricultural control is mostly a preventive measure, its application has certain limitations and cannot be used as an emergency measure. It appears to be powerless when the Spodoptera litura breaks out.
  • Chemical control that is, pesticide control
  • chemical control methods are mainly sprayed with pharmaceuticals. However, chemical control also has its limitations. If improper use, it will lead to phytotoxicity of crops, resistance to pests, killing natural enemies, polluting the environment, destroying farmland ecosystems and threatening the safety of humans and animals. Adverse consequences.
  • Physical control mainly relies on the response of pests to various physical factors in environmental conditions, and uses various physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce pests, radiation infertility and other methods to control pests.
  • various physical factors such as light, electricity, color, temperature and humidity, and mechanical equipment to induce pests, radiation infertility and other methods to control pests.
  • a wide range of methods are mainly used to attract moths, sweet and sour traps and switchgrass 500 times of trichlorfon to trap moths; although the above methods have different degrees of control effects, they have certain difficulties in operation.
  • Cry1F insecticidal protein is one of many insecticidal proteins and is an insoluble parasporal crystal protein produced by Bacillus thuringiensis.
  • the Cry1F protein is ingested by insects into the midgut, and the protoxin is dissolved in the alkaline pH environment of the insect midgut.
  • the N- and C-termini of the protein are digested with alkaline protease to convert the protoxin into an active fragment; the active fragment binds to the receptor on the upper surface of the epithelial cell membrane of the insect and is inserted into the intestinal membrane, causing perforation of the cell membrane and destroying the inside and outside of the cell membrane. Changes in osmotic pressure and pH balance disrupt the insect's digestive process and ultimately lead to death.
  • Plants transfected with the Cry1F gene have been shown to be resistant to Lepidoptera pests such as the larvae, however, there have been no reports on the control of plant damage by Spodoptera litura by producing transgenic plants expressing the Cry1F protein.
  • the purpose of the present application is to provide a method for controlling pests, for the first time, to provide a method for controlling plant damage caused by a transgenic plant expressing a Cry1F protein, and effectively overcoming the prior art agricultural control, chemical control, physical control, etc. Technical flaws.
  • a first aspect of the present application relates to a method of controlling a pest of Spodoptera litura, wherein a Spodoptera litura pest is brought into contact with a Cry1F protein.
  • the Cry1F protein is a Cry1Fa protein.
  • the Cry1Fa protein is present in a plant cell that produces the CrylFa protein, the Spodoptera litura pest being contacted with the Cry1Fa protein by ingestion of the plant cell.
  • the Cry1Fa protein is present in a transgenic plant producing the Cry1Fa protein, the Spodoptera litura pest is contacted with the Cry1Fa protein by ingesting tissue of the transgenic plant, the twill night after contact The growth of the moth pests is inhibited and/or contacted, resulting in the death of the Spodoptera litura, thereby achieving control of the plants of Spodoptera litura.
  • the transgenic plant can be in any growth period.
  • the tissue of the transgenic plant is selected from the group consisting of leaves, stems, tassels, ears, anthers, filaments and fruits.
  • the control of the plants against Spodoptera litura is not altered by changes in planting location and/or planting time.
  • the plant is selected from the group consisting of corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, sugar beet, cabbage or eggplant, preferably the plant is selected from corn or soybean.
  • the step prior to the contacting step is the planting of a plant containing a polynucleotide encoding the Cry1Fa protein.
  • the amino acid sequence of the Cry1Fa protein has: 1) the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and 2) has at least one of SEQ ID NO: 1 or SEQ ID NO: 70% homologous and amino acid sequence having insecticidal activity against Spodoptera litura pests, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or higher, or 3) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted, deleted and/or added with one or more amino acid residues The obtained amino acid sequence having insecticidal activity against Spodoptera litura pests, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.
  • the nucleotide sequence encoding the Cry1Fa protein has: 1) the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, 2) and SEQ ID NO: 3 or SEQ ID NO: 4 has a nucleotide sequence of at least about 75% homology and encodes an amino acid sequence having insecticidal activity against Spodoptera litura pests, such as at least 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 under stringent conditions and encodes against Spodoptera litura
  • the nucleotide sequence of the pesticidal amino acid sequence of the pest 4) the amino acid sequence encoding the insecticidal activity against Spodoptera litura pests of SEQ ID NO: 3 or SEQ ID NO: 4 due to codon degeneracy Nucleotide sequence.
  • the plant further comprises at least one second nucleotide different from the nucleotide encoding the Cry1Fa 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 Cry1Ab protein, a Cry1Ac protein, a Cry1Ba protein, or a Vip3A protein.
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • a second aspect of the present application relates to the use of a Cry1F protein for controlling Spodoptera litura pests.
  • the Cry1F protein is a Cry1Fa protein.
  • the amino acid sequence of the Cry1Fa protein has: 1) the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and 2) has SEQ ID NO: 1 or SEQ ID NO: 2 At least 70% homologous An amino acid sequence having insecticidal activity against Spodoptera litura pests, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or 3) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is obtained by substitution, deletion and/or addition of one or more amino acid residues and An amino acid sequence having insecticidal activity against Spodoptera litura pests, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues.
  • the nucleotide sequence encoding the Cry1Fa protein has: 1) the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, 2) and SEQ ID NO: 3 or SEQ ID NO: 4 a nucleotide sequence having at least about 75% homology and encoding an amino acid sequence having insecticidal activity against Spodoptera litura pests, such as at least 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 under stringent conditions and encodes a pair of twill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucle
  • the Cry1F protein controls a Spodoptera frondosa pest by expressing the Cry1F protein in a plant cell and contacting the plant cell with the Cry1F protein by feeding the Spodoptera litura pest.
  • the Cry1F protein controls the Spodoptera litura pest by achieving expression of the Cry1F protein in the transgenic plant and contacting the tissue of the transgenic plant with the Cry1F protein by a Spodoptera litura pest.
  • the transgenic plant can be in any growth period.
  • the tissue of the transgenic plant is selected from the group consisting of leaves, stems, fruits, tassels, ears, anthers, and filaments.
  • the Cry1F protein controls the Spodoptera litura pests not to change due to changes in planting location and/or planting time.
  • the plant is selected from the group consisting of corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, sugar beet, cabbage or eggplant, preferably the plant is selected from corn or soybean.
  • the plant further comprises at least one second nucleotide different from the nucleotide encoding the Cry1F 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 Cry1Ab protein, a Cry1Ac protein, a Cry1Ba protein, or a Vip3A protein.
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • a third aspect of the present application relates to a plant cell, transgenic plant or transgenic plant for controlling pests of Spodoptera litura
  • a method of a part of a plant comprising introducing a coding nucleotide sequence of a Cry1F protein into a part of the plant cell, the transgenic plant or the transgenic plant, preferably introducing a coding nucleotide sequence of the Cry1F protein into the plant cell, A genome of a transgenic plant or part of a transgenic plant.
  • the portion of the transgenic plant is a propagation material or a non-propagating material.
  • the propagation material refers to the fruit, seed or callus of the plant.
  • the non-propagating material refers to a leaf, a stem, a tassel, an ear, an anther or a filament of a plant that does not have the ability to reproduce.
  • the Cry1F protein is a Cry1Fa protein.
  • the amino acid sequence of the Cry1Fa protein has: 1) the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and 2) has SEQ ID NO: 1 or SEQ ID NO: 2
  • An amino acid sequence having at least 70% homology and having insecticidal activity against Spodoptera litura pests such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or higher, or 3) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is substituted, deleted and/or added with one or more amino acid residues
  • Amino acid sequence obtained by the base and having insecticidal activity against Spodoptera litura pests such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 amino acid residues .
  • the nucleotide sequence encoding the Cry1Fa protein has: 1) the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, 2) and SEQ ID NO: 3 or SEQ ID NO: 4 a nucleotide sequence having at least about 75% homology and encoding an amino acid sequence having insecticidal activity against Spodoptera litura pests, such as at least 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, 3) hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 under stringent conditions and encodes a pair of twill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucleotwill nights a nucle
  • the plant is selected from the group consisting of corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, sugar beet, cabbage or eggplant, preferably the plant is selected from corn or soybean.
  • the method further comprises introducing at least one second nucleotide different from the nucleotide encoding the Cry1F protein into the plant cell, the transgenic plant, or a portion of the transgenic plant, preferably At least one second nucleotide different from the nucleotide encoding the Cry1F protein is introduced into the genome of the part of the plant cell, transgenic plant or transgenic plant.
  • 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 Cry1Ab protein, a Cry1Ac protein, a Cry1Ba protein, or a Vip3A protein.
  • the second nucleotide has the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6.
  • the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.
  • the coding nucleotide is introduced into the plant cell, the transgenic plant by Agrobacterium-mediated transformation, microprojection bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon mediated DNA introduction. Or a portion of the transgenic plant, preferably Agrobacterium-mediated transformation.
  • a fourth aspect of the present application relates to a part of a plant cell, a transgenic plant or a transgenic plant for controlling a Spodoptera litura pest obtained by the method of the above third aspect.
  • a fifth aspect of the present invention relates to the use of a Cry1F protein for the preparation of a plant cell, a transgenic plant or a part of a transgenic plant that controls a Spodoptera litura pest.
  • the definitions of "Cry1F protein”, "controlling Spodoptera litura pests”, “plants”, “plant cells”, “transgenic plants”, “portions of transgenic plants” and their extensions referred to in this aspect are as defined above. .
  • a sixth aspect of the present application relates to a method of cultivating a plant for controlling a pest of Spodoptera litura, comprising:
  • the plants are grown under conditions in which the artificially inoculated pests of Spodoptera litura and/or Spodoptera litura pests are naturally harmful, and harvested with reduced plant damage and/or plants other than those having no polynucleotide sequence encoding the Cry1F protein. Or plants with increased plant yield.
  • expression of a Cry1F protein in a transgenic plant can be accompanied by expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Co-expression of such more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant (the first parent) can express the Cry1F protein by genetic engineering
  • the second plant (the second parent) can express the Cry-like insecticidal protein and/or the Vip-like insecticidal protein by genetic engineering.
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • RNA interference refers to the phenomenon of highly-specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Therefore, RNAi technology can be used in this application to specifically knock out or shut down the expression of a particular gene in a target insect pest.
  • the application also relates to the following:
  • Section 1 A method of controlling a pest of Spodoptera litura, comprising: contacting a Spodoptera litura pest with a Cry1F protein.
  • the method for controlling a pest of Spodoptera litura characterized in that the Cry1F protein is a Cry1Fa protein.
  • the method for controlling a pest of Spodoptera litura characterized in that the Cry1Fa protein is present in a plant cell producing the Cry1Fa protein, and the Spodoptera litura pest ingests the plant cell by The Cry1Fa protein is contacted.
  • the method for controlling a Spodoptera litura pest according to paragraph 3, wherein the Cry1Fa protein is present in a transgenic plant producing the Cry1Fa protein, and the Spodoptera litura pest ingests the transgenic plant The tissue is contacted with the Cry1Fa protein, and the growth of the Spodoptera litura pest is inhibited and eventually causes death to achieve control of the plant against Spodoptera litura.
  • tissue of the transgenic plant can be a leaf, a stem, a tassel, an ear, an anther, a filament or a fruit.
  • Item 7 The method of controlling Spodoptera litura pests according to paragraph 4, characterized in that the control of the plants of the Spodoptera litura is not changed by the change of the planting site.
  • Item 8 The method of controlling Spodoptera litura pests according to paragraph 4, characterized in that the control of the plants of the Spodoptera litura is not changed by the change of the planting time.
  • the method of controlling a Spodoptera litura pest according to any one of paragraphs 3 to 8, wherein the plant is derived from corn, soybean, cotton, sweet potato, alfalfa, lotus, celery, tobacco, beet, Cabbage or eggplant.
  • step prior to the contacting step is planting a plant containing a polynucleotide encoding the Cry1Fa protein.
  • nucleotide sequence of the Cry1Fa protein has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.
  • the method for controlling a pest of Spodoptera litura characterized in that the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, a lectin, Alpha-amylase or Oxidase.
  • Item 15 The method of controlling Spodoptera litura pests according to paragraph 14, wherein the second nucleotide encodes a Cry1Ab protein, a Cry1Ac protein, a Cry1Ba protein or a Vip3A protein.
  • the second nucleotide comprises the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • Item 17 The method of controlling Spodoptera litura pests according to paragraph 13, characterized in that the second nucleotide is a dsRNA which inhibits an important gene in a target insect pest.
  • Section 18 Use of a Cry1F protein to control Spodoptera litura pests.
  • FIG. 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a Cry1Fa-01 nucleotide sequence in the method for controlling pests of the present application;
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100014 containing a Cry1Fa-01 nucleotide sequence according to the method for controlling pests of the present application;
  • Figure 3 is a flow chart showing the construction of a recombinant expression vector DBN100015 containing the Cry1Fa-01 nucleotide sequence of the method for controlling pests of the present application;
  • FIG. 4 is a diagram showing the insect resistance effect of the transgenic corn plants inoculated with Spodoptera litura by the method for controlling pests of the present application;
  • Fig. 5 is a diagram showing the insect-resistant effect of the transgenic soybean plants inoculated with Spodoptera litura by the method for controlling pests of the present application.
  • Prodenia litura and Agrotis ypsilon Rottemberg belong to the family Lepidoptera, and are omnivorous pests, all of which are harmful to corn, soybean, cotton, and sweet potato. Despite this, there are at least two major differences between the two species, the Spodoptera litura and the small tiger, which are biologically distinct and distinct:
  • the distribution area is different.
  • the worldwide distribution of Spodoptera litura occurs in all parts of China, mainly in the provinces of Henan, Jiangsu, Hunan, Hubei, Zhejiang, Anhui and the Yellow River in the Yangtze River Basin, Henan, Hebei, Shandong and other provinces.
  • Spodoptera frugiperda is mainly distributed overseas, including Canada, Mexico, the United States, Argentina, Cambodia, Brazil, Chile, Colombia, Ecuador, French Guiana, Guyana, Paraguay, Peru, Suriname, Republic, Venezuela and Central America. And the Caribbean.
  • the small tigers are world-wide pests, which are also distributed throughout China, especially in the Yangtze River basin and the southeast coast where rainfall is abundant and the climate is humid. The northeast region mostly occurs in the eastern and southern humid regions.
  • the Spodoptera litura is plagued by larvae, and when it is young, the leaves of the leaves are fed back to the epidermis and mesophyll. Only the epidermis is left with clear plaque; after 3 years of age, it is dispersed into leaves and tender stems; after 4th instar, it enters overeating, biting leaves, leaving only the main vein; old larvae can eat fruits; their eating habits are mixed and endangering organs, old age When it is overeating, it is a very harmful pest.
  • the underground larvae of the genus Diptera, 1-2 instar larvae can be clustered at the top of the young leaves of the seedlings for feeding; the larvae are scattered after the third age, the larvae are agile, have a false-killing habit, are extremely sensitive to light, and are disturbed.
  • Shrinking into groups lurking between the dry and wet layers of the topsoil during the day, and unearthing from the ground to pull the seedling plants into the earth's hole or biting the unearthed seeds.
  • the young stems of the seedlings are hardened and then eaten with young leaves and leaves and growing points. When there is insufficient food or looking for a wintering place, there is a migration phenomenon; the young larvae have a high rate of seedling cutting and a large amount of food intake.
  • the larvae of Spodoptera litura are 33-50 mm in length, dark brown in the head, and varied in the chest. From khaki to black-green, the body surface is scattered with white spots, and the winter festival has a triangular half-moon. A pair of black spots, the larva is generally 6 years old; while the small tiger larvae are cylindrical, the mature larvae are 37-50mm in length, the head is brown, with dark brown irregular mesh, body gray brown to dark brown, rough surface
  • the granules of different sizes and separated from each other, the back line, the sub-back line and the valve line are dark brown, the front chest plate is dark brown, and the yellow-brown hip plate has two distinct dark brown longitudinal bands, the chest and the abdomen. The feet are yellowish brown.
  • the outer edge of the scorpion section is taupe, the end of each section of the midfoot is gray-brown, the front wing is brown, the front edge is dark brown, the outer edge is dark brown, the base is light brown, the black wavy inner double line, black ring
  • the kidney pattern is black with black edges
  • the outer middle part has a wedge-shaped black line extending to the outer horizontal line
  • the middle horizontal line is dark brown wavy
  • the double line wavy outer horizontal line is brown
  • the irregular zigzag shape is
  • the outer edge line is gray
  • the inner edge has three tines between the midribs.
  • the outer edge line is black
  • the outer horizontal line and the outer outer line are light brown.
  • the outer edge of the line is dark brown
  • the hind wings are grayish white
  • the longitudinal veins and the margin are brown
  • the back of the abdomen is gray.
  • Each female moth can lay 3-5 eggs, each with about 100-200 eggs.
  • the eggs are mostly produced in the leaf veins of the leaf back, and the dense, green crops lay more eggs and pile up.
  • the egg mass is often covered with scaly hair and easy to be found.
  • the incubation temperature of the egg is about 24 °C; the larvae have a cluster hazard habit at 14 °C for 25-20 °C, and the larvae begin to disperse after the age of 3, old age. The larvae are crouching and suspended. During the day, they are lurking in the cracks of the soil. They climb out of the food in the evening and will squat and succumb to death.
  • insects When the food is insufficient or not, the larvae can migrate to the nearby fields in a group, so there is a common name for "running insects"; the suitable soil moisture for the phlegm is about 20% of the soil moisture, and the flood season is 11-18 days.
  • Spodoptera litura is a kind of pest that is sensitive to temperature and high temperature, and the development temperature of each insect state is 28-30 °C, but at high temperature (33-40 °C), life is basically normal; The cold power is very weak, and it is basically impossible to survive under the long-term low temperature of about 0 °C in winter.
  • high temperature years and seasons are favorable for their development and reproduction, and low temperatures are likely to cause a large number of insects to die.
  • the larvae are divided into 6 years old, and the 1st and 2nd instar larvae first hide in the leather or In the heart of the plant, staying up late to eat, when the amount of food is small, the damage is not very significant; 3 days after the day to hide under the topsoil, night out of harm; 5, 6 instar larvae eaten greatly, each larva can bite overnight 4-5 strains of broken cabbage seedlings, more than 10 strains; the resistance of the larvae to the medicinal agent increased significantly after 3 years of age; from the end of March to the middle of April is the serious period of the first generation larvae; the generation from October to the second In April of the year, it happened and harmed; two to three generations in the northwest, two to three generations in the north of the Great Wall, three generations in the north of the Yellow River to the south of the Yellow River, four generations south of the Yellow River to the Yangtze River, and four to five generations south of the Yangtze River
  • the larvae After the larvae mature, they are phlegded in a soil chamber about 5 cm deep, and the pupa period is about 9-19 days.
  • the high temperature affects the development of the small tiger. It is unfavorable for reproduction, so the number is less in summer, and the suitable survival temperature is 15°C-25°C. In winter, the temperature is too low, and the mortality rate of larvae is increased. Where the terrain is low and the rainfall is abundant, the first year occurs.
  • Autumn rain soil The high humidity and weeds in the soil are conducive to adult spawning and larval feeding activities, which is a sign of the second year of large occurrence; however, excessive precipitation and excessive humidity are not conducive to larval development, and early larvae are prone to death after flooding.
  • Adults in the spawning period of the soil water content in the 15-20% area is more harmful; sandy loam soil, easy to permeable, rapid drainage, suitable for small tigers to breed, while heavy clay and sand are lighter.
  • the genome of a plant, plant tissue or plant cell as referred to in this application refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • contacting means that insects and/or pests touch, stay and/or feed on plants, plant organs, plant tissues or plant cells, which are both plants, plant organs, plant tissues or plant cells. It may be that the insecticidal protein is expressed in the body, and the surface of the plant, plant organ, plant tissue or plant cell may have a pesticidal protein and/or a microorganism having a pesticidal protein.
  • control and/or “control” as used herein refers to the contact of a Spodoptera litura pest with a Cry1F protein, which is inhibited from growing and/or causing death after contact. Further, the Spodoptera litura pests are in contact with the Cry1F protein by feeding plant tissues, and all or part of the Spodoptera litura pest growth is inhibited and/or causes death after the contact. Inhibition refers to sublethal death, that is, it has not been killed but can cause certain effects in growth, behavior, behavior, physiology, biochemistry and organization, such as slow growth and/or cessation. At the same time, the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • plants and/or plant seeds containing a polynucleotide sequence encoding a Cry1F protein for controlling Spodoptera litura pests under conditions in which artificially inoculated pests of Spodoptera litura and/or Spodoptera litura pests are naturally harmful, and non-transgenic Wild-type plants have reduced plant damage compared to specific manifestations including, but not limited to, improved stem resistance, and/or increased kernel weight, and/or increased yield, and the like.
  • the "control" and/or “control” effects of the Cry1F protein on Spodoptera litura can be independently and not attenuated and/or disappeared by other substances that can "control” and/or "control” the pests of Spodoptera litura. .
  • any tissue of a transgenic plant (containing a polynucleotide sequence encoding a Cry1F protein) is present and/or asynchronously, present and/or produced, a Cry1F protein and/or another substance that can control a pest of Spodoptera litura,
  • the presence of the other substance neither affects the "control” and/or "control” effect of the Cry1F protein on Spodoptera litura, nor does it cause the "control” and/or “control” effects to be completely
  • a substance is achieved regardless of the Cry1F protein.
  • the process of feeding on plant tissues by Spodoptera litura pests is short and difficult to observe with the naked eye.
  • Any tissue of a plant (containing a polynucleotide sequence encoding a Cry1F protein) is present in a dead Spodoptera litura pest, and/or a Spodoptera litura pest inhibiting growth growth thereon, and/or with a non-transgenic wild-type plant
  • the method and/or use of the present application is achieved by having attenuated plant damage, that is, by contacting the Cry1F protein with Spodoptera litura pests.
  • a method and/or use for controlling pests of Spodoptera litura is achieved by having attenuated plant damage, that is, by contacting the Cry1F protein with Spodoptera litura pests.
  • 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 present application can be placed under the control of regulatory sequences in a host of interest.
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the application includes 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.
  • 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 present application also includes RNA and PNA (peptide nucleic acid) having comparable functions to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present application hybridizes to the Cry1Fa gene of the present application under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1Fa gene of the present application.
  • a nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present application, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes.
  • the stringent conditions described herein may be specific hybridization with SEQ ID NO: 3 or SEQ ID NO: 4 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1 The membrane was washed once with %SDS and 1 ⁇ SSC and 0.1% SDS.
  • sequences having insect resistance and hybridizing under stringent conditions to SEQ ID NO: 3 and/or SEQ ID NO: 4 of the present application are included in the present application. These sequences are at least about 40%-50% homologous to the sequences of the present application, about 60%, 65% or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. Sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99% or greater.
  • genes and proteins described in this application include not only specific exemplary sequences, but also portions and/or fragments that retain the insecticidal activity characteristics of the proteins of the specific examples (including internal and/or end ratios compared to full length proteins). Deletions), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • variant or “variant” is meant a nucleotide sequence that encodes the same protein or an equivalent protein encoded with insecticidal activity.
  • the "equivalent protein” refers to a protein having the same or substantially the same biological activity as the Spodoptera litura pest of the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as referred to in this application refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • the length of the aforementioned sequence may vary, but is of sufficient length to ensure that the (encoding) protein is an insect toxin.
  • Genes can be modified and gene variants can be easily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. Further, for example, U.S. Patent No. 5,605,793 describes a method of using DNA reassembly to generate other molecular diversity after random fragmentation. Fragments of full-length genes can be made using commercial endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A gene encoding an active fragment can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
  • the present application can derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries.
  • insecticidal proteins of the present application can be used to identify and isolate other proteins from protein mixtures.
  • antibodies may be caused by protein portions that are most constant in protein and most different from other B.t. proteins.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies raised in the present application 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.
  • amino acid changes are conventional in the art, and it is preferred that such amino acid changes are: small changes in properties, ie, 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 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 unsubstitution 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 Cry1F protein includes, but is not limited to, Cry1Fa2, Cry1Fa3, Cry1Fb3, Cry1Fb6 or Cry1Fb7 protein, or an insecticidal fragment having at least 70% homology to the amino acid sequence of the above protein and having insecticidal activity against Spodoptera litura or Functional Area.
  • amino acid sequences having a certain homology to the amino acid sequences shown in SEQ ID NO: 1 and/or 2 are also included in the present application. These sequences are typically greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%, similar to the sequence of the present application.
  • Preferred polynucleotides and proteins of the present application may also be defined according to a more specific range of identity and/or similarity.
  • Regulatory sequences as described herein include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the Vip-like and Cry-like proteins.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the intron includes, but is not limited to, maize hsp70 intron, corn ubiquitin Intron, Adh intron 1, sucrose synthase intron or rice Act1 intron.
  • 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.
  • operably linked can be such that a promoter is ligated to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • insecticidal or “insect-resistant” means toxic to crop pests, thereby achieving “control” and/or “control” of crop pests.
  • said "insecticide” or “insect-resistant” means killing crop pests. More specifically, the target insect is a Spodoptera litura pest.
  • the Cry1F protein in the present application is toxic to Spodoptera litura pests.
  • the plants of the present application particularly soybean and corn, contain exogenous DNA in their genome, the exogenous DNA comprising a nucleotide sequence encoding a Cry1F protein, and the Spodoptera litura pest is contacted with the protein by feeding plant tissue, contacting The growth of Spodoptera litura pests is inhibited and eventually leads to death. Inhibition refers to death or sub-lethal death.
  • the plants should be normal in morphology and can be cultivated under conventional methods. Raised for consumption and/or production of the product.
  • the plant substantially eliminates the need for chemical or biological insecticides that are insecticides against Spodoptera litura pests targeted by the Cry1F 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 insects in this application are mainly Spodoptera litura.
  • the Cry1F protein may have the amino acid sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 2 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 Cry1F protein of the present application 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 Cry1F protein or an expression cassette or a recombinant vector
  • the conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-launch bombardment, Direct DNA uptake into protoplast, electroporation or whisker silicon-mediated DNA introduction.
  • the present application provides a method of controlling pests, which has the following advantages:
  • the prior art mainly controls the pests of Spodoptera litura by external action, ie, external factors, such as agricultural control, chemical control and physical control; and the present application controls the twill night by producing a Cry1F protein capable of killing Spodoptera litura in plants. Moth pests are controlled by internal factors.
  • the method for controlling Spodoptera litura pests used in the prior art has an effect that is incomplete and only serves to alleviate the effect; and the transgenic plant (Cry1F protein) of the present application can cause a large number of deaths of the larvae of Spodoptera litura, and is small
  • the developmental progress of some surviving larvae was greatly inhibited. After 3 days, the larvae were still in the initial hatching state or between the initial hatching-negative control state, all of which were obviously dysplastic and had stopped development.
  • the transgenic plants were generally only affected. Minor damage.
  • Cry1Ab nucleotide sequence (2457 nucleotides) encoding the amino acid sequence of Cry1Ab insecticidal protein (818 amino acids), as shown in SEQ ID NO: 5 in the Sequence Listing; amino acid sequence encoding Vip3A insecticidal protein (789 The amino acid sequence of Vip3A (2370 nucleotides) is shown in SEQ ID NO: 6 in the Sequence Listing.
  • the Cry1Fa-01 nucleotide sequence (as shown in SEQ ID NO: 3 in the Sequence Listing), the Cry1Fa-02 nucleotide sequence (as shown in SEQ ID NO: 4 in the Sequence Listing), the Cry1Ab core
  • the nucleotide sequence (as shown in SEQ ID NO: 5 in the Sequence Listing) and the Vip3A nucleotide sequence (as shown in SEQ ID NO: 6 in the Sequence Listing) by Nanjing Kingsrayson Synthetic Technology Co., Ltd.
  • the Cry1Fa-01 nucleotide sequence (SEQ ID NO: 3) synthesized at the 5' end is also ligated with an AscI cleavage site, the Cry1Fa-01 nucleotide sequence (SEQ ID NO) The 3' end of 3) is also ligated with a BamHI restriction site; the 5' end of the synthesized Cry1Fa-02 nucleotide sequence (SEQ ID NO: 4) is also ligated with an AscI cleavage site, the Cry1Fa The 3' end of the -02 nucleotide sequence (SEQ ID NO: 4) is also ligated with a BamHI cleavage site; the 5' end of the synthesized Cry1Ab nucleotide sequence (SEQ ID NO: 5) is also ligated with NcoI At the cleavage site, the 3' end of the Cry1Ab nucleotide sequence (SEQ ID NO: 5) is further ligated with
  • the synthetic Cry1Fa-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).
  • Cry1Fa-01 is the Cry1Fa-01 nucleotide sequence (SEQ ID NO: 3); 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
  • TE 10 mM Tris-HCl, 1 mM The precipitate was dissolved by EDTA, pH 8.0; the RNA was digested in a water bath at a temperature of 37 ° C for 30 min; and stored at -20 ° C for use.
  • the positive clone was verified by sequencing, and the result showed that the Cry1Fa-01 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 3 in the sequence listing.
  • the synthesized Cry1Fa-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein Cry1Fa-02 was Cry1Fa-02. Nucleotide sequence (SEQ ID NO: 4).
  • the Cry1Fa-02 nucleotide sequence in the recombinant cloning vector DBN02-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Cry1Ab nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein the Cry1Ab was a Cry1Ab nucleotide sequence (SEQ ID NO: 5).
  • the Cry1Ab nucleotide sequence in the recombinant cloning vector DBN03-T was correctly inserted by restriction enzyme digestion and sequencing.
  • the synthesized Vip3A nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein Vip3A was a Vip3A nucleotide sequence (SEQ ID NO: 6).
  • the correct insertion of the Vip3A nucleotide sequence in the recombinant cloning vector DBN04-T was confirmed by restriction enzyme digestion and sequencing.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases AscI and BamHI, respectively, and the cut Cry1Fa-01 nucleotide sequence fragment was inserted. Between the AscI and BamHI sites of the expression vector DBNBC-01, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100014 is constructed.
  • FIG. 2 Kanamycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 7); Cry1Fa-01: Cry1Fa-01 nucleotide sequence (SEQ ID NO: 3); Nos : terminator of the nopaline synthase gene (SEQ ID NO: 8); PMI: phosphomannose isomerase gene (SEQ ID NO: 9); LB: left border).
  • the recombinant expression vector DBN100014 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ l E. coli T1 competent cells, 10 ⁇ l of plasmid DNA (recombinant expression vector DBN100014), water bath at 42 ° C for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50mg/L, pH adjusted with NaOH Incubate overnight to 7.5) at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases AscI and BamHI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the AscI and BamHI sites of the recombinant expression vector DBN100014 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown by 3, that is, the Cry1Fa-01 nucleotide sequence.
  • the Cry1Fa-02 nucleotide sequence excised by AscI and BamHI restriction recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100013.
  • the nucleotide sequence in the recombinant expression vector DBN100013 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 4 in the sequence listing, that is, the Cry1Fa-02 nucleotide sequence, and the Cry1Fa-02 nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleotide sequence excised by the recombinant cloning vectors DBN01-T and DBN03-T were respectively inserted into AscI and BamHI, NcoI and SpeI, respectively.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100075.
  • the nucleotide sequence in the recombinant expression vector DBN100075 contains the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 5 in the sequence listing, namely the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleoside.
  • the acid sequence, the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the Cry1Fa-01 nucleotide sequence and the Vip3A nucleotide sequence excised by the recombinant cloning vectors DBN01-T and DBN04-T were respectively inserted into AscI and BamHI, ScaI and SpeI, respectively.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100276.
  • the nucleotide sequence in the recombinant expression vector DBN100276 contains the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 6 in the sequence listing, namely the Cry1Fa-01 nucleotide sequence and the Vip3A nucleoside.
  • the acid sequence, the Cry1Fa-01 nucleotide sequence and the Vip3A nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-02 were digested with restriction endonucleases AscI and BamHI, respectively, and excised.
  • the Cry1Fa-01 nucleotide sequence fragment was inserted between the AscI and BamHI sites of the expression vector DBNBC-02, and the vector was constructed by a conventional restriction enzyme digestion method, and was constructed into a recombinant expression vector DBN100015.
  • FIG. 3 Kanamycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 7); Cry1Fa-01: Cry1Fa-01 nucleotide Sequence (SEQ ID NO: 3); Nos: terminator of the nopaline synthase gene (SEQ ID NO: 8); PAT: glufosinate acetyltransferase gene (SEQ ID NO: 22); LB: left border).
  • the Cry1Fa-02 nucleotide sequence excised by the AscI and BamHI recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-02 to obtain a recombinant expression vector.
  • the nucleotide sequence in the recombinant expression vector DBN100030 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 4 in the sequence listing, that is, the Cry1Fa-02 nucleotide sequence, and the Cry1Fa-02 nucleotide sequence was digested and sequenced.
  • the Ubi promoter and the Nos terminator can be ligated.
  • the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleotide sequence excised by the recombinant cloning vectors DBN01-T and DBN03-T were respectively inserted into AscI and BamHI, NcoI and SpeI, respectively.
  • the expression vector DBNBC-02 was obtained to obtain a recombinant expression vector DBN100012.
  • the nucleotide sequence in the recombinant expression vector DBN100012 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 3 and SEQ ID NO: 5 in the sequence listing, that is, the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleoside.
  • the acid sequence, the Cry1Fa-01 nucleotide sequence and the Cry1Ab nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the expression vector DBNBC-01 was obtained to obtain a recombinant expression vector DBN100031.
  • the nucleotide sequence in the recombinant expression vector DBN100031 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 3 and SEQ ID NO: 6 in the sequence listing, that is, the Cry1Fa-01 nucleotide sequence and the Vip3A nucleoside.
  • the acid sequence, the Cry1Fa-01 nucleotide sequence and the Vip3A nucleotide sequence can be ligated to the Ubi promoter and the Nos terminator.
  • the recombinant expression vectors DBN100014, DBN100013, DBN100075, DBN100276, DBN100015, DBN100030, DBN100012 and DBN100031, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and their transformation was carried out.
  • the conditions are: 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, rotation speed Incubate for 2 hours at 200 rpm, 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 The plasmids were extracted and the recombinant expression vectors DBN100014, DBN100013, DBN100012, DBN100075, DBN100015 and DBN100030 were digested with restriction endonucleases AhdI and XhoI, and the recombinant expression vectors DBN100031 and DBN100276 were treated with restriction endonucleases AhdI and EcoRV.
  • the immature embryo of the aseptically cultured maize variety Heisei 31 was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the second embodiment.
  • Recombinant expression vector DBN100014, T-DNA in DBN100013, DBN100075 and DBN100276 (including promoter sequence of maize Ubiquitin gene, Cry1Fa-01 nucleotide sequence, Cry1Fa-02 nucleotide sequence, Cry1Ab nucleotide sequence, Vip3A nucleotide sequence, PMI gene) And Nos terminator sequence) transferred into the maize genome, obtained a maize plant transformed into the Cry1Fa-01 nucleotide sequence, a maize plant transformed into the Cry1Fa-02 nucleotide sequence, and transferred into the Cry1Fa-01-Cry1Ab nucleus Maize plants with a nucleotide sequence and maize plants transfected with the Cry
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can express Cry1Fa-01 nucleotide sequence, Cry1Fa-02 nucleotide
  • the sequence, the Cry1Fa-01-Cry1Ab nucleotide sequence and/or the Cry1Fa-01-Vip3A nucleotide sequence is delivered to at least one cell of one of the young embryos (step 1: infection step), in which the immature embryo is preferred
  • infecting medium MS salt 4.3g / L, MS vitamin, casein 300mg / L, sucrose 68.5g / L, glucose 36g / L, acetosyringone ( AS) 40 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3)
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, agar 8 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selective agent (mannose) and the grown transformed callus is selected (step 4: selection step).
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 5 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene).
  • MS salt 4.3 g/L MS vitamin, casein 300 mg/L, sucrose 5 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene
  • oxyacetic acid (2,4-D) 1 mg/L
  • agar 8 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 MS rooting medium) Recycled plants.
  • 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, mannose) 5g/L, agar 8g/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, agar 8 g/L, pH 5 .8)
  • culture at 25 ° C to a height of about 10 cm and move to a greenhouse to grow to firmness. In the greenhouse, culture at 28 ° C for 16 hours a day, then at 20 Incubate for 8 hours at °C.
  • TaqMan was used to verify the maize plants transferred into the Cry1F gene.
  • Step 11 The maize plants transformed with the Cry1Fa-01 nucleotide sequence, the maize plants transformed with the Cry1Fa-02 nucleotide sequence, the maize plants transformed with the Cry1Fa-01-Cry1Ab nucleotide sequence, and the Cry1Fa-
  • the corn plants of the 01-Vip3A nucleotide sequence and the leaves of the wild-type maize 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 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ l;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Probe 1 CGTGCAAGAATGTCTCCTCCCGTGAAC as shown in SEQ ID NO: 12 in the Sequence Listing;
  • Probe 2 (CP2): CGTGCAAGAATGTCTCCTCCCGTGAAC as shown in SEQ ID NO: 15 in the Sequence Listing;
  • Probe 3 CTACCCGATCCGCACCGTGTCC as shown in SEQ ID NO: 18 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ l of each primer at a concentration of 1 mM, 50 ⁇ l of a probe at a concentration of 100 ⁇ M, and 860 ⁇ l of 1 ⁇ TE buffer, and stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Maize plants in the group which were transferred into the Cry1Fa-01 nucleotide sequence, maize plants transformed into the Cry1Fa-02 nucleotide sequence, maize plants transformed into the Cry1Fa-01-Cry1Ab nucleotide sequence, and transferred to Cry1Fa-01
  • the maize plants of the -Vip3A nucleotide sequence obtained transgenic maize plants containing a single copy of the Cry1F gene, the Cry1Ab gene and/or the Vip3A gene.
  • Maize sequences, wild-type maize plants, and non-transgenic maize plants identified by Taqman were tested for insect resistance to Spodoptera litura.
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (number of initial hatching / total number of insects) + 60 ⁇ (initial hatching - negative control Number of insects/total number of insects) +10 ⁇ (number of negative control insects/total number of insects)]+100 ⁇ (1-blade damage rate).
  • a total of 3 lines (S1, S2 and S3) transferred into the Cry1Fa-01 nucleotide sequence were transferred to the Cry1Fa-02 nucleotide sequence for a total of 3 lines (S4, S5 and S6) and transferred to Cry1Fa- A total of 3 strains (S7, S8 and S9) of the nucleotide sequence of 01-Cry1Ab were transferred into the Cry1Fa-01-Vip3A nucleotide sequence (S10, S11 and S12), which were identified by Taqman as One strain of non-transgenic (NGM1) and one strain of wild type (CK1) were selected; 3 strains were selected from each strain, and each plant was repeated 6 times. The results are shown in Table 1 and Figure 4.
  • Table 1 The results in Table 1 indicate that a maize plant transformed with the Cry1Fa-01 nucleotide sequence, a maize plant transformed with the Cry1Fa-02 nucleotide sequence, a maize plant transformed with the Cry1Fa-01-Cry1Ab nucleotide sequence, and transferred to Cry1Fa
  • the total score of the -01-Vip3A nucleotide sequence of maize plants was above 250 points or so, and some of them reached a maximum of 300 points; while the total number of non-transgenic maize plants and wild-type maize plants identified by Taqman was The score is generally around 15 points.
  • the results in Figure 4 indicate that maize plants transfected with the Cry1Fa-01 nucleotide sequence, maize plants transfected with the Cry1Fa-02 nucleotide sequence, and transferred to the Cry1Fa-01-Cry1Ab nucleotide were compared to wild-type maize plants.
  • the sequence of maize plants and maize plants transfected with the Cry1Fa-01-Vip3A nucleotide sequence can cause a large number of deaths of the larvae of Spodoptera litura in 3 days, and greatly inhibit the development of a small number of surviving larvae, after 3 days.
  • the larvae are still in the initial hatching state, and the maize plants that have been transferred into the Cry1Fa-01 nucleotide sequence, the maize plants that have been transferred into the Cry1Fa-02 nucleotide sequence, the maize plants that have been transferred into the Cry1Fa-01-Cry1Ab nucleotide sequence, and Maize plants transformed into the Cry1Fa-01-Vip3A nucleotide sequence were only slightly damaged, and the leaf damage rate was about 10% or less. In addition, when the duration of the biopsy reached 5 days, all the larvae died.
  • a maize plant transformed with the Cry1Fa-01 nucleotide sequence a maize plant transformed with the Cry1Fa-02 nucleotide sequence, a maize plant transformed with the Cry1Fa-01-Cry1Ab nucleotide sequence, and transformed into Cry1Fa-01- were confirmed.
  • Maize plants of the Vip3A nucleotide sequence all showed high activity against Spodoptera litura, which was sufficient to exert an adverse effect on the growth of Spodoptera litura, thereby allowing it to be controlled.
  • the cotyledonary node tissue of the aseptically cultivated soybean variety Zhonghuang 13 is co-cultured with the Agrobacterium described in the third embodiment, to reconstitute the second embodiment.
  • T-DNA in the expression vectors DBN100015, DBN100030, DBN100012 and DBN100031 (including the promoter of the maize Ubiquitin gene)
  • the sequence, Cry1Fa-01 nucleotide sequence, Cry1Fa-02 nucleotide sequence, Cry1Ab nucleotide sequence, Vip3A nucleotide sequence, PAT gene and Nos terminator sequence) were transferred into the soybean genome and obtained transfer Soybean plants of the Cry1Fa-01 nucleotide sequence, soybean plants transformed with the Cry1Fa-02 nucleotide sequence, soybean plants transformed with the Cry1Fa-01-Cry1Ab nucleotide sequence, and the Cry1Fa-01-Vip3A nucleo
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5.6.
  • the seeds were inoculated on a germination medium and cultured under the following conditions: temperature 25 ⁇ 1 ° C; photoperiod (light/dark) was 16/8 h.
  • photoperiod light/dark
  • the soybean sterile seedlings of the fresh green cotyledon nodes were taken, the hypocotyls were cut at 3-4 mm below the cotyledonary nodes, and the cotyledons were cut longitudinally to remove the top buds, lateral buds and seed roots.
  • infecting medium MS salt 2.15 g/L, B5 vitamin, sucrose 20 g/L, glucose
  • AS aceto
  • the cotyledonary node tissue is in solid medium (MS salt 4.3 g / L, B5 vitamin, sucrose 20 g / L, after the infection step, Glucose 10g / L, 2-morpholine ethanesulfonic acid (MES) 4g / L, zeatin 2mg / L, agar 8g / L, pH 5.6) culture.
  • MS salt 4.3 g / L
  • B5 vitamin sucrose 20 g / L
  • Glucose 10g / L Glucose 10g / L, 2-morpholine ethanesulfonic acid (MES) 4g / L, zeatin 2mg / L, agar 8g / L, pH 5.6
  • MES 2-morpholine ethanesulfonic acid
  • step 3 restore the medium (B5 salt 3.1g / L, B5 vitamins, 2-morpholine ethanesulfonic acid (MES) 1g / L, sucrose 30g / L, zein (ZT) 2mg/L, agar 8 At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium exists in g/L, cephalosporin 150 mg/L, glutamic acid 100 mg/L, aspartic acid 100 mg/L, pH 5.6) Adding a selection agent for plant transformants (step 3: recovery step).
  • MES 2-morpholine ethanesulfonic acid
  • ZT zein
  • the cotyledonary node-regenerated tissue pieces are cultured on a solid medium with antibiotics but no selective agent to eliminate Agrobacterium and provide recovery period for infected cells.
  • the cotyledonary node-regenerated tissue block is cultured on a medium containing a selective agent (glufosinate) and the grown transformed callus is selected (step 4: selection step).
  • the cotyledonary node-regenerated tissue block is Screening solid medium with selective agent (B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg /L, agar 8g / L, cefotaxime 150mg / L, glutamic acid 100mg / L, aspartic acid 100mg / L, glufosinate 6mg / L, pH 5.6) culture, resulting in transformed cell selectivity Growth.
  • selective agent B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg /L, agar 8g / L, cefotaxime 150mg / L, glutamic acid 100mg / L, aspart
  • the transformed cells then regenerate the plants (step 5: regeneration step), preferably, the tissue segments regenerated from the cotyledonary nodes grown on the medium containing the selection agent are Culture medium to regenerate the plants body (B5 B5 differentiation medium and rooting medium).
  • the selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)) 1mg/L, agar 8g/L, cephalosporin 150mg/L, Glutamic acid 50 mg / L, aspartic acid 50 mg / L, gibberellin 1 mg / L, auxin 1 mg / L, glufosinate 6 mg / L, pH 5.6), culture differentiation at 25 ° C.
  • B5 differentiation medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)
  • MES 2-morpholine ethanesulfonic acid
  • ZT zeatin
  • the differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin) 150 mg/L, indole-3-butyric acid (IBA) 1 mg/L), cultured in rooting culture at 25 ° C to a height of about 10 cm, and transferred to a greenhouse for cultivation to firmness. In the greenhouse, the cells were cultured at 26 ° C for 16 hours and then at 20 ° C for 8 hours.
  • B5 rooting medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin
  • IBA indole-3-butyric acid
  • Soybean plants transformed with the Cry1Fa-01 nucleotide sequence soybean plants transferred to the Cry1Fa-02 nucleotide sequence, soybean plants transferred to the Cry1Fa-01-Cry1Ab nucleotide sequence, and transferred to Cry1Fa-01-Vip3A Approximately 100 mg of the leaves of the soybean plants of the nucleotide sequence were used as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of the Cry1F gene, the Cry1Ab gene and the Vip3A gene was detected by Taqman probe fluorescent quantitative PCR.
  • wild type soybean plants were used as a control, and the detection and analysis were carried out according to the method of TaqMan to verify the maize plants transferred to the Cry1F gene in the above third embodiment.
  • the experiment was set to repeat 3 times and averaged.
  • soybean plants into the Cry1Fa-01 nucleotide sequence soybean plants transferred to the Cry1Fa-02 nucleotide sequence, soybean plants transferred to the Cry1Fa-01-Cry1Ab nucleotide sequence, and transferred to Cry1Fa-01
  • the soybean plants of the -Vip3A nucleotide sequence each obtained a transgenic soybean plant containing a single copy of the Cry1F gene, the Cry1Ab gene and/or the Vip3A gene.
  • total score 100 ⁇ mortality + [100 ⁇ mortality + 90 ⁇ (initial hatching) Number / total number of insects) +60 ⁇ (first hatching - negative control insects / Total number of insects) +10 ⁇ (negative control number of insects / total number of insects)] +100 ⁇ (1 - blade damage rate).
  • a total of 3 lines (S13, S14 and S15) transferred into the Cry1Fa-01 nucleotide sequence were transferred to the Cry1Fa-02 nucleotide sequence for a total of 3 lines (S16, S17 and S18) and transferred to Cry1Fa-
  • a total of 3 strains (S19, S20 and S21) of the nucleotide sequence of 01-Cry1Ab were transferred into the Cry1Fa-01-Vip3A nucleotide sequence (S22, S23 and S24) and identified by Taqman as One strain of non-transgenic (NGM2) and one strain of wild type (CK2) were selected; 3 strains were selected from each strain, and each plant was repeated 6 times. The results are shown in Table 2 and Figure 5.
  • the results in Table 2 indicate that soybean plants transferred to the Cry1Fa-01 nucleotide sequence, soybean plants transformed into the Cry1Fa-02 nucleotide sequence, soybean plants transferred to the Cry1Fa-01-Cry1Ab nucleotide sequence, and transferred to Cry1Fa
  • the total scores of the soybean plants of the -01-Vip3A nucleotide sequence were all above 220 points or so, and some of them reached a maximum of 300 points; and the total bioassay of non-transgenic soybean plants and wild-type soybean plants identified by Taqman The score is usually around 40 minutes.
  • the larvae After 3 days, the larvae are still in the initial hatching-negative control state, and the leaf damage rate is about 20%; the soybeans transferred into the Cry1Fa-01-Cry1Ab nucleotide sequence Plants and soybean plants transferred to the Cry1Fa-01-Vip3A nucleotide sequence have almost 100% control effect on the newly hatched larvae, and very few surviving larvae have basically stopped developing, and only a small amount of pinhole-like damage is observed on the leaves.
  • soybean plants transformed into the Cry1Fa-01 nucleotide sequence, soybean plants transformed into the Cry1Fa-02 nucleotide sequence, soybean plants transformed into the Cry1Fa-01-Cry1Ab nucleotide sequence, and transformed into Cry1Fa-01- were confirmed.
  • Soybean plants of the Vip3A nucleotide sequence all showed activity against Spodoptera litura, which was sufficient to exert an adverse effect on the growth of Spodoptera litura, thereby allowing it to be controlled.
  • the poisoning effect can produce a similar transgenic plant that can express the Cry1F protein and can be used to control the damage of Spodoptera litura.
  • the Cry1F protein in the present application includes, but is not limited to, the Cry1F 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 Cry1F protein, such as Cry1Ab protein, Cry1Ac protein, Cry1Ba protein or Vip3A protein and the like.
  • the method for controlling pests of the present invention controls a Spodoptera litura pest by producing a Cry1F protein capable of killing Spodoptera litura in a plant; compared with the agricultural control methods, chemical control methods, and physical control methods used in the prior art.
  • the present application protects plants from the whole growth period and whole plants to prevent the damage of Spodoptera litura pests, and has no pollution and no residue, and the effect is stable, thorough, simple, convenient and economical.

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Abstract

提供了一种控制斜纹夜蛾的方法，其中斜纹夜蛾与Cry1F蛋白发生接触。该方法包括使用Cry1Fa蛋白产生转基因植物，斜纹夜蛾通过摄食该转基因植物与Cry1Fa蛋白接触，接触后斜纹夜蛾生长受到抑制和/或最终导致死亡，从而实现对斜纹夜蛾为害植物的控制。

Description

控制害虫的方法
本申请要求2013年11月15日提交的发明名称为“控制害虫的方法”的中国专利申请第201310573804.6号的优先权，其全文通过引用并入本文。
技术领域
本申请涉及一种控制害虫的方法，特别是涉及一种用在植物中表达的Cry1F蛋白来控制斜纹夜蛾为害植物的方法。
背景技术
斜纹夜蛾(Prodenia litura)属鳞翅目夜蛾科，为杂食性和暴食性害虫，为害寄主相当广泛，除玉米和大豆外，还可危害包括瓜、茄、豆、葱、韭菜、菠菜以及十字花科蔬菜、粮食、经济作物等近100科、300多种植物；斜纹夜蛾属世界性分布，国内各地都有发生，主要发生在长江流域和黄河流域。斜纹夜蛾主要以幼虫为害全株、小龄时群集叶背啃食；3龄后分散为害叶片、嫩茎、老龄幼虫可蛀食果实。
玉米和大豆是中国重要的粮食作物，每年因斜纹夜蛾造成的粮食损失巨大，更甚者影响到当地人口的生存状况。为了防治斜纹夜蛾，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斜纹夜蛾发生的农田生态环境。如清除杂草，收获后翻耕晒土或灌水，以破坏或恶化其化蛹场所，有助于减少虫源；或者结合管理随手摘除卵块和群集危害的初孵幼虫，以减少虫源。因农业防治大多为预防性措施，应用有一定的局限性，不能作为应急措施，在斜纹夜蛾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，是斜纹夜蛾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，特别是斜纹夜蛾大发生的情况下，是必不可少的应急措施，它可以在斜纹夜蛾造成危害前将其消灭。目前化学防治方法主要是药剂喷施。但化学防治也有其局限性，如使用不当往往会导致农作物发生药害、害虫产生抗药性，以及杀伤天敌、污染环境，使农田生态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。目前应用比较广泛的方法主要有点灯诱蛾、糖醋诱杀和柳枝蘸洒500倍敌百虫诱杀蛾子；上述方法虽然有不同程度的防治效果，但是在操作上具有一定的难度。
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。Cry1F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不溶性伴孢结晶蛋白。
Cry1F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表面上受体结合，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Cry1F基因的植株可以抵抗小地老虎等鳞翅目(Lepidoptera)害虫的侵害，然而，至今尚无关于通过产生表达Cry1F蛋白的转基因植株来控制斜纹夜蛾对植物为害的报道。
发明内容
本申请的目的是提供一种控制害虫的方法，首次提供了通过产生表达Cry1F蛋白的转基因植株来控制斜纹夜蛾对植物为害的方法，且有效克服现有技术农业防治、化学防治和物理防治等技术缺陷。
本申请的第一方面涉及一种控制斜纹夜蛾害虫的方法，其中斜纹夜蛾害虫与Cry1F蛋白发生接触。
在一些实施方案中，所述Cry1F蛋白为Cry1Fa蛋白。
在进一步的实施方案中，所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的植物细胞中，所述斜纹夜蛾害虫通过摄食所述植物细胞与所述Cry1Fa蛋白接触。
在进一步的实施方案中，所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的转基因植物中，所述斜纹夜蛾害虫通过摄食所述转基因植物的组织与所述Cry1Fa蛋白接触，接触后所述斜纹夜蛾害虫生长受到抑制和/或接触后导致所述斜纹夜蛾死亡，从而实现对斜纹夜蛾为害植物的控制。
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选自叶片、茎秆、雄穗、雌穗、花药、花丝和果实。
所述对斜纹夜蛾为害植物的控制不因种植地点和/或种植时间的改变而改变。
所述植物选自玉米、大豆、棉花、甘薯、芋、莲、田菁、烟草、甜菜、白菜或茄子，优选地，所述植物选自玉米或大豆。
所述接触步骤之前的步骤为种植含有编码所述Cry1Fa蛋白的多核苷酸的植物。
在一些实施方案中，所述Cry1Fa蛋白的氨基酸序列具有：1)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，2)与SEQ ID NO:1或SEQ ID NO:2具有至少70％同源性且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，或3)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸残基所获得的且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如1、2、3、4、5、6、7、8、9、10、15、20、30、50个氨基酸残基。
在一些实施方案中，所述Cry1Fa蛋白的编码核苷酸序列具有：1)SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列，2)与SEQ ID NO:3或SEQ ID NO:4具有至少大约75％同源性且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，如至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，3)在严格条件下与SEQ ID NO:3或SEQ ID NO:4杂交且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，4)由于密码子简并性而不同于SEQ ID NO:3或SEQ ID NO:4的编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列。
在一些实施方案中，所述植物还包含至少一种不同于编码所述Cry1Fa蛋白的核苷酸的第二种核苷酸。
在进一步的实施方案中，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。
在进一步的实施方案中，所述第二种核苷酸编码Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白。
在更进一步的实施方案中，所述第二种核苷酸具有SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列。
在另一些实施方案中，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
本申请的第二方面涉及一种Cry1F蛋白质控制斜纹夜蛾害虫的用途。
在一些实施方案中，所述Cry1F蛋白为Cry1Fa蛋白。
在进一步的实施方案中，所述Cry1Fa蛋白的氨基酸序列具有：1)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，2)与SEQ ID NO:1或SEQ ID NO:2具有至少70％同源 性且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，或3)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸残基所获得的且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如1、2、3、4、5、6、7、8、9、10、15、20、30、50个氨基酸残基。
在进一步的实施方案中，所述Cry1Fa蛋白的编码核苷酸序列具有：1)SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列，2)与SEQ ID NO:3或SEQ ID NO:4具有至少大约75％同源性且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，如至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，3)在严格条件下与SEQ ID NO:3或SEQ ID NO:4杂交且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，4)由于密码子简并性而不同于SEQ ID NO:3或SEQ ID NO:4的编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列。
在一些实施方案中，Cry1F蛋白质控制纹夜蛾害虫通过使Cry1F蛋白质表达于植物细胞并通过纹夜蛾害虫摄食所述植物细胞与所述Cry1F蛋白接触而实现。
在一些实施方案中，Cry1F蛋白质控制斜纹夜蛾害虫通过使Cry1F蛋白质表达于转基因植物并通过斜纹夜蛾害虫摄食所述转基因植物的组织与所述Cry1F蛋白接触而实现。
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选自叶片、茎秆、果实、雄穗、雌穗、花药和花丝。
所述Cry1F蛋白控制斜纹夜蛾害虫不因种植地点和/或种植时间的改变而改变。
所述植物选自玉米、大豆、棉花、甘薯、芋、莲、田菁、烟草、甜菜、白菜或茄子，优选地，所述植物选自玉米或大豆。
在一些实施方案中，所述植物还包含至少一种不同于编码所述Cry1F蛋白的核苷酸的第二种核苷酸。
在进一步的实施方案中，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。
在进一步的实施方案中，所述第二种核苷酸编码Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白。
在更进一步的实施方案中，所述第二种核苷酸具有SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列。
在一些实施方案中，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
本申请的第三方面涉及一种制备控制斜纹夜蛾害虫的植物细胞、转基因植物或转基因 植物的部分的方法，其包括将Cry1F蛋白的编码核苷酸序列引入所述植物细胞、转基因植物或转基因植物的部分中，优选地，将Cry1F蛋白的编码核苷酸序列引入所述植物细胞、转基因植物或转基因植物的部分的基因组中。
在一些实施方案中，转基因植物的部分是繁殖材料或非繁殖材料。
所述繁殖材料是指植物的果实、种子或愈伤组织。
所述非繁殖材料是指不具有繁殖能力的植物的叶片、茎秆、雄穗、雌穗、花药或花丝。
在一些实施方案中，所述Cry1F蛋白为Cry1Fa蛋白。
在进一步的实施方案中，所述Cry1Fa蛋白的氨基酸序列具有：1)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，2)与SEQ ID NO:1或SEQ ID NO:2具有至少70％同源性且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，或3)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸残基所获得的且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，如1、2、3、4、5、6、7、8、9、10、15、20、30、50个氨基酸残基。
在进一步的实施方案中，所述Cry1Fa蛋白的编码核苷酸序列具有：1)SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列，2)与SEQ ID NO:3或SEQ ID NO:4具有至少大约75％同源性且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，如至少75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高，3)在严格条件下与SEQ ID NO:3或SEQ ID NO:4杂交且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，4)由于密码子简并性而不同于SEQ ID NO:3或SEQ ID NO:4的编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列。
所述植物选自玉米、大豆、棉花、甘薯、芋、莲、田菁、烟草、甜菜、白菜或茄子，优选地，所述植物选自玉米或大豆。
在一些实施方案中，所述方法还包括将至少一种不同于编码所述Cry1F蛋白的核苷酸的第二种核苷酸引入所述植物细胞、转基因植物或转基因植物的部分中，优选地，将至少一种不同于编码所述Cry1F蛋白的核苷酸的第二种核苷酸引入所述植物细胞、转基因植物或转基因植物的部分的基因组中。
在一些实施方案中，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。
在进一步的实施方案中，所述第二种核苷酸编码Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白。
在更进一步的实施方案中，所述第二种核苷酸具有SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列。
在另一些实施方案中，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
在一些实施方案中，通过农杆菌介导的转化、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导入将编码核苷酸引入所述植物细胞、转基因植物或转基因植物的部分中，优选农杆菌介导的转化。
本申请的第四方面涉及上述第三方面所述的方法获得的控制斜纹夜蛾害虫的植物细胞、转基因植物或转基因植物的部分。
本申请的第五方面涉及Cry1F蛋白在制备控制斜纹夜蛾害虫的植物细胞、转基因植物或转基因植物的部分中的用途。在该方面中涉及的“Cry1F蛋白”、“控制斜纹夜蛾害虫”、“植物”、“植物细胞”、“转基因植物”、“转基因植物的部分”及其延伸内容的限定如上述方面所限定。
本申请的第六方面涉及一种培养控制斜纹夜蛾害虫的植物的方法，其包括：
种植至少一种植物种子，所述植物种子的基因组中包括编码Cry1F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斜纹夜蛾害虫和/或斜纹夜蛾害虫自然发生危害的条件下生长，收获与其他不具有编码Cry1F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。
在该方面中涉及的“Cry1F蛋白”、“植物”及其延伸内容的限定如上述方面所限定。“控制斜纹夜蛾害虫”的意思与“抗斜纹夜蛾害虫”的意思类似，具体限定如上所述。
在本申请中，Cry1F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Cry1F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。因此在本申请中可以使用RNAi技术特异性剔除或关闭目标昆虫害虫中特定基因的表达。
在某些方面中，本申请还涉及下述内容：
段1、一种控制斜纹夜蛾害虫的方法，其特征在于，包括将斜纹夜蛾害虫与Cry1F蛋白接触。
段2、根据段1所述的控制斜纹夜蛾害虫的方法，其特征在于，所述Cry1F蛋白为Cry1Fa蛋白。
段3、根据段2所述的控制斜纹夜蛾害虫的方法，其特征在于，所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的植物细胞中，所述斜纹夜蛾害虫通过摄食所述植物细胞与所述Cry1Fa蛋白接触。
段4、根据段3所述的控制斜纹夜蛾害虫的方法，其特征在于，所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的转基因植物中，所述斜纹夜蛾害虫通过摄食所述转基因植物的组织与所述Cry1Fa蛋白接触，接触后所述斜纹夜蛾害虫生长受到抑制并最终导致死亡，以实现对斜纹夜蛾为害植物的控制。
段5、根据段4所述的控制斜纹夜蛾害虫的方法，其特征在于，所述转基因植物可以处于任意生育期。
段6、根据段4所述的控制斜纹夜蛾害虫的方法，其特征在于，所述转基因植物的组织可以为叶片、茎秆、雄穗、雌穗、花药、花丝或果实。
段7、根据段4所述的控制斜纹夜蛾害虫的方法，其特征在于，所述对斜纹夜蛾为害植物的控制不因种植地点的改变而改变。
段8、根据段4所述的控制斜纹夜蛾害虫的方法，其特征在于，所述对斜纹夜蛾为害植物的控制不因种植时间的改变而改变。
段9、根据段3至8任一项所述的控制斜纹夜蛾害虫的方法，其特征在于，所述植物可以来自玉米、大豆、棉花、甘薯、芋、莲、田菁、烟草、甜菜、白菜或茄子。
段10、根据段2至9任一项所述的控制斜纹夜蛾害虫的方法，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Cry1Fa蛋白的多核苷酸的植物。
段11、根据段2至10任一项所述的控制斜纹夜蛾害虫的方法，其特征在于，所述Cry1Fa蛋白的氨基酸序列具有SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列。
段12、根据段11所述的控制斜纹夜蛾害虫的方法，其特征在于，所述Cry1Fa蛋白的核苷酸序列具有SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列。
段13、根据段3至12任一项所述的控制斜纹夜蛾害虫的方法，其特征在于，所述植物还可以产生至少一种不同于所述Cry1Fa蛋白的第二种核苷酸。
段14、根据段13所述的控制斜纹夜蛾害虫的方法，其特征在于，所述第二种核苷酸可以编码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。
段15、根据段14所述的控制斜纹夜蛾害虫的方法，其特征在于，所述第二种核苷酸可以编码Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白。
段16、根据段15所述的控制斜纹夜蛾害虫的方法，其特征在于，所述第二种核苷酸包括SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列。
段17、根据段13所述的控制斜纹夜蛾害虫的方法，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。
段18、一种Cry1F蛋白质控制斜纹夜蛾害虫的用途。
附图说明
图1为本申请控制害虫的方法的含有Cry1Fa-01核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本申请控制害虫的方法的含有Cry1Fa-01核苷酸序列的重组表达载体DBN100014构建流程图；
图3为本申请控制害虫的方法的含有Cry1Fa-01核苷酸序列的重组表达载体DBN100015构建流程图；
图4为本申请控制害虫的方法的转基因玉米植株接种斜纹夜蛾的抗虫效果图；
图5为本申请控制害虫的方法的转基因大豆植株接种斜纹夜蛾的抗虫效果图。
具体实施方式
斜纹夜蛾(Prodenia litura)与小地老虎(Agrotis ypsilon Rottemberg)同属鳞翅目夜蛾科，为杂食性害虫，均为害玉米、大豆、棉花、甘薯等。尽管如此，斜纹夜蛾与小地老虎在生物学上是清晰的、截然不同的两个物种，至少存在以下主要区别：
1、分布区域不同。斜纹夜蛾属世界性分布，中国各地都有发生，主要发生在长江流域的江西、江苏、湖南、湖北、浙江、安徽和黄河流域的河南、河北、山东等省。而草地贪夜蛾主要分布在境外，包括美洲的加拿大、墨西哥、美国、阿根廷、玻利维亚、巴西、智利、哥伦比亚、厄瓜多尔、法属圭亚那、圭亚那、巴拉圭、秘鲁、苏里南、乌拉圭、委内瑞拉及整个中美洲和加勒比海地区。而小地老虎是世界性的害虫，在我国各地也均有分布，尤以雨量丰富、气候湿润的长江流域和东南沿海发生量大，东北地区多发生在东部和南部湿润地区。
2、为害习性不同。斜纹夜蛾以幼虫为害全株，小龄时群集叶背啃食下表皮及叶肉， 仅留上表皮呈透明斑；3龄后分散为害叶片、嫩茎；4龄以后进入暴食，咬食叶片，仅留主脉；老龄幼虫可蛀食果实；其食性既杂又危害各器官，老龄时形成暴食，是一种危害性很大的害虫。而小地老虎属地下害虫，1-2龄幼虫昼夜均可群集于幼苗顶心嫩叶处取食为害；3龄后分散，幼虫行动敏捷、有假死习性、对光线极为敏感、受到惊扰即卷缩成团，白天潜伏于表土的干湿层之间，夜晚出土从地面将幼苗植株咬断拖入土穴、或咬食未出土的种子，幼苗主茎硬化后改食嫩叶和叶片及生长点，食物不足或寻找越冬场所时，有迁移现象；高龄幼虫剪苗率高，取食量大。
3、形态特征不同。
1)卵形态不同：斜纹夜蛾的卵呈扁平的半球状，初产黄白色，后变为暗灰色，块状粘合在一起，上覆黄褐色绒毛；而小地老虎的卵成馒头形，具纵横隆线，初产乳白色，渐变黄色，孵化前卵一顶端具黑点。
2)幼虫形态不同：斜纹夜蛾幼虫体长33-50毫米，头部黑褐色，胸部多变，从土黄色到黑绿色都有，体表散生小白点，冬节有近似三角形的半月黑斑一对，幼虫一般6龄；而小地老虎幼虫圆筒形，老熟幼虫体长37-50mm，头部褐色，具黑褐色不规则网纹，体灰褐至暗褐色，体表粗糙、布大小不一而彼此分离的颗粒，背线、亚背线及气门线均黑褐色，前胸背板暗褐色，黄褐色臀板上具两条明显的深褐色纵带，胸足与腹足黄褐色。
3)蛹形态不同：斜纹夜蛾的蛹长15-20mm，圆筒形，红褐色，尾部有一对短刺；而小地老虎的蛹长18-24mm，赤褐有光，口器与翅芽末端相齐，均伸达第4腹节后缘，腹部第4-7节背面前缘中央深褐色，且有粗大的刻点，两侧的细小刻点延伸至气门附近，第5-7节腹面前缘也有细小刻点，腹末端具短臀棘1对。
4)成虫形态不同：斜纹夜蛾成虫体长14-20mm左右，翅展35-46mm，体暗褐色，胸部背面有白色丛毛，前翅灰褐色，花纹多，内横线和外横线白色、呈波浪状、中间有明显的白色斜阔带纹，所以称斜纹夜蛾；而小地老虎成虫体长17-23mm、翅展40-54mm，头、胸部背面暗褐色，足褐色，前足胫、跗节外缘灰褐色，中后足各节末端有灰褐色环纹，前翅褐色，前缘区黑褐色，外缘以内多暗褐色，基线浅褐色，黑色波浪形内横线双线，黑色环纹内有一圆灰斑，肾状纹黑色具黑边、其外中部有一楔形黑纹伸至外横线，中横线暗褐色波浪形，双线波浪形外横线褐色，不规则锯齿形亚外缘线灰色、其内缘在中脉间有三个尖齿，亚外缘线与外横线间在各脉上有小黑点，外缘线黑色，外横线与亚外缘线间淡褐色，亚外缘线以外黑褐色，后翅灰白色，纵脉及缘线褐色，腹部背面灰色。
4、生长习性和发生规律不同。斜纹夜蛾一年发生4代(华北)-9代(广东)，一般以老熟幼虫或蛹在田基边杂草中越冬，广州地区无真正越冬现象；在长江流域以北的地区， 该虫冬季易被冻死，越冬问题尚未定论，推测当地虫源可能从南方迁飞过去；长江流域多在7-8月大发生，黄河流域则多在8-9月大发生。成虫夜出活动，飞翔力较强，具趋光性和趋化性，对糖醋酒等发酵物尤为敏感。每只雌蛾能产卵3-5块，每块约有卵位100-200个，卵多产于叶背的叶脉分叉处，以茂密、浓绿的作物产卵较多，堆产，卵块常覆有鳞毛而易被发现，卵的孵化适温是24℃左右；幼虫在气温25℃时，历经14-20天，初孵幼虫具有群集危害习性，3龄以后则开始分散，老龄幼虫有昼伏性和假死性，白天多潜伏在土缝处，傍晚爬出取食，遇惊就会落地蜷缩作假死状。当食料不足或不当时，幼虫可成群迁移至附近田块危害，故又有“行军虫”的俗称；化蛹的适合土壤湿度是土壤含水量在20％左右，蛹期为11-18天。斜纹夜蛾是一种喜温性而又耐高温的间歇猖獗危害的害虫，各虫态的发育适温为28-30℃，但在高温下(33-40℃)，生活也基本正常；抗寒力很弱，在冬季0℃左右的长时间低温下，基本上不能生存。一般高温年份和季节有利其发育、繁殖，低温则易引致虫蛹大量死亡。该虫食性虽杂，但食料情况，包括不同的寄主，甚至同一寄主不同发育阶段或器官，以及食料的丰缺，对其生育繁殖都有明显的影响。间种、复种指数高或过度密植的田块有利其发生。天敌有寄生幼虫的小茧蜂和多角体病毒等。而小地老虎一年发生3-4代，老熟幼虫或蛹在土内越冬；早春3月上旬成虫开始出现，一般在3月中下旬和4月上中旬会出现两个发蛾盛期；成虫白天不活动，傍晚至前半夜活动最盛，喜欢吃酸、甜、酒味的发酵物和各种花蜜，并有趋光性，幼虫共分6龄，1、2龄幼虫先躲伏在杂革或植株的心叶里，昼夜取食，这时食量很小，为害也不十分显著；3龄后白天躲到表土下，夜间出来为害；5、6龄幼虫食量大增，每条幼虫一夜能咬断菜苗4-5株，多的达l0株以上；幼虫3龄后对药剂的抵抗力显著增加；3月底到4月中旬是第1代幼虫为害的严重时期；发生世代从10月到第2年4月都见发生和为害；西北地区二到三代，长城以北一般年二到三代，长城以南黄河以北年三代，黄河以南至长江沿岸年四代，长江以南年四到五代，南亚热带地区年六至七代；无论年发生代数多少，在生产上造成严重为害的均为第一代幼虫；南方越冬代成虫二月份出现，全国大部分地区羽化盛期在3月下旬至4月上、中旬，宁夏、内蒙古为4月下旬；小地老虎成虫多在下午3时至晚上10时羽化，白天潜伏于杂物及缝隙等处，黄昏后开始飞翔、觅食，3-4天后交配、产卵；卵散产于低矮叶密的杂草和幼苗上、少数产于枯叶、土缝中，近地面处落卵最多，每雌产卵800-1000粒、多达2000粒；卵期约5天左右，幼虫6龄、个别7-8龄，幼虫期在各地相差很大，但第一代约为30-40天；幼虫老熟后在深约5cm土室中化蛹，蛹期约9-19天；高温对小地老虎的发育与繁殖不利，因而夏季发生数量较少，适宜生存温度为15℃-25℃；冬季温度过低，小地老虎幼虫的死亡率增高；凡地势低湿，雨量充沛的地方，发生较多；头年秋雨多、土 壤湿度大、杂草丛生有利于成虫产卵和幼虫取食活动，是第二年大发生的预兆；但降水过多，湿度过大，不利于幼虫发育，初龄幼虫淹水后很易死亡；成虫产卵盛期土壤含水量在15-20％的地区为害较重；沙壤土，易透水、排水迅速，适于小地老虎繁殖，而重黏土和沙土则发生较轻。
综合上述，可确定斜纹夜蛾与小地老虎是两种害虫，且亲缘关系较远，无法交配产生后代。
本申请中所述的植物、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。
本申请中所述的“接触”，是指昆虫和/或害虫触碰、停留和/或取食植物、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。
本申请的术语“控制”和/或“防治”是指斜纹夜蛾害虫与Cry1F蛋白接触，接触后斜纹夜蛾害虫生长受到抑制和/或导致死亡。进一步地，斜纹夜蛾害虫通过摄食植物组织与Cry1F蛋白接触，接触后全部或部分斜纹夜蛾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Cry1F蛋白的多核苷酸序列的控制斜纹夜蛾害虫的植物和/或植物种子，在人工接种斜纹夜蛾害虫和/或斜纹夜蛾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。Cry1F蛋白对斜纹夜蛾的“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”斜纹夜蛾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Cry1F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，Cry1F蛋白和/或可控制斜纹夜蛾害虫的另一种物质，则所述另一种物质的存在既不影响Cry1F蛋白对斜纹夜蛾的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全由所述另一种物质实现，而与Cry1F蛋白无关。通常情况下，在大田，斜纹夜蛾害虫摄食植物组织的过程短暂且很难用肉眼观察到，因此，在人工接种斜纹夜蛾害虫和/或斜纹夜蛾害虫自然发生危害的条件下，如转基因植物(含有编码Cry1F蛋白的多核苷酸序列)的任何组织存在死亡的斜纹夜蛾害虫、和/或在其上停留生长受到抑制的斜纹夜蛾害虫、和/或与非转基因的野生型植株相比具有减弱的植物损伤，即为实现了本申请的方法和/或用途，即通过斜纹夜蛾害虫与Cry1F蛋白接触以实现 控制斜纹夜蛾害虫的方法和/或用途。
本申请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，可以将本申请的多核苷酸和/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，本申请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。本申请还包括与示例的DNA有相当功能的RNA和PNA(肽核酸)。
本申请中核酸分子或其片段在严格条件下与本申请Cry1Fa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申请Cry1Fa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。本申请中，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”。本申请中，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。
本申请中，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，本申请所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:3或SEQ ID NO:4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申请SEQ ID NO:3和/或SEQ ID NO:4杂交的序列包括在本申请中。这些序列与本申请序列至少大约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。
本申请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斜纹夜蛾害虫的生物活性的蛋白。
本申请中所述的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。
本申请可以从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。有多种方法获取本申请的杀虫蛋白。例如，可以使用本申请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申请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序列包括在本申请的范围内。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。
本申请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。对于由本申请的多肽，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)。
在本申请中，Cry1F蛋白包括但不限于Cry1Fa2、Cry1Fa3、Cry1Fb3、Cry1Fb6或Cry1Fb7蛋白，或者与上述蛋白的氨基酸序列具有至少70％同源性且对斜纹夜蛾具有杀虫活性的杀虫片段或功能区域。
因此，与序列1和/或2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申请中。这些序列与本申请序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的相同性和/或类似性范围定义本申请的优选的多核苷酸和蛋白质。例如与本申请示例的序列有49％、 50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
本申请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Vip类蛋白和Cry类蛋白的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(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。
本申请中所述“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。在本申请中所述“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)。
本申请中所述的“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，目标昆虫是斜纹夜蛾害虫。
本申请中Cry1F蛋白对斜纹夜蛾害虫具有毒性。本申请中的植物，特别是大豆和玉米，在其基因组中含有外源DNA，所述外源DNA包含编码Cry1F蛋白的核苷酸序列，斜纹夜蛾害虫通过摄食植物组织与该蛋白接触，接触后斜纹夜蛾害虫生长受到抑制并最终导致死亡。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Cry1F蛋白所靶向的斜纹夜蛾害虫的杀虫剂)。
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。本申请中目标昆虫主要为斜纹夜蛾。
本申请中，所述Cry1F蛋白可以具有序列表中SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。除了包含Cry1F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，包含编码本申请Cry1F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。
本申请中，将外源DNA导入植物，如将编码所述Cry1F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。
本申请提供了一种控制害虫的方法，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斜纹夜蛾害虫的为害，如农业防治、化学防治和物理防治；而本申请是通过植物体内产生能够杀死斜纹夜蛾的Cry1F蛋白来控制斜纹夜蛾害虫的，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斜纹夜蛾害虫的为害起到了一定作用，但同时也对人、畜和农田生态系统带来了污染、破坏和残留；使用本申请控制斜纹夜蛾害虫的方法，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斜纹夜蛾害虫的方法都是阶段性的，而本申请是对植物进行全生育期的保护，转基因植物(Cry1F蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受斜纹夜蛾的侵害。
4、全植株防治。现有技术使用的控制斜纹夜蛾害虫的方法大多是局部性的，如叶面喷施；而本申请是对整个植株进行保护，如转基因植物(Cry1F蛋白)的叶片、茎秆、雄穗、 雌穗、花药、花丝、果实等都是可以抵抗斜纹夜蛾侵害的。
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；本申请是使所述Cry1F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，且本申请转基因植物(Cry1F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。现有技术使用的物理防治方法在农业生产操作上具有一定的难度；本申请只需种植能够表达Cry1F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斜纹夜蛾害虫的方法，其效果是不彻底的，只起到减轻作用；而本申请转基因植物(Cry1F蛋白)可以造成初孵斜纹夜蛾幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态或介于初孵-阴性对照状态之间，都是明显的发育不良，且已停止发育，转基因植物大体上只受到轻微损伤。
下面通过具体实施例进一步说明本申请控制害虫的方法的技术方案。
实施例
第一实施例、Cry1Fa基因的获得和合成
1、获得Cry1Fa核苷酸序列
Cry1Fa-01杀虫蛋白质的氨基酸序列(605个氨基酸)，如序列表中SEQ ID NO:1所示；编码相应于所述Cry1Fa-01杀虫蛋白质的氨基酸序列(605个氨基酸)的Cry1Fa-01核苷酸序列(1818个核苷酸)，如序列表中SEQ ID NO:3所示；Cry1Fa-02杀虫蛋白质的氨基酸序列(1148个氨基酸)，如序列表中SEQ ID NO:2所示；编码相应于所述Cry1Fa-02杀虫蛋白质的氨基酸序列(1148个氨基酸)的Cry1Fa-02核苷酸序列(3447个核苷酸)，如序列表中SEQ ID NO:4所示。
2、获得Cry1Ab和Vip3A核苷酸序列
编码Cry1Ab杀虫蛋白质的氨基酸序列(818个氨基酸)的Cry1Ab核苷酸序列(2457个核苷酸)，如序列表中SEQ ID NO:5所示；编码Vip3A杀虫蛋白质的氨基酸序列(789个氨基酸)的Vip3A核苷酸序列(2370个核苷酸)，如序列表中SEQ ID NO:6所示。
3、合成上述核苷酸序列
所述Cry1Fa-01核苷酸序列(如序列表中SEQ ID NO:3所示)、所述Cry1Fa-02核苷酸序列(如序列表中SEQ ID NO:4所示)、所述Cry1Ab核苷酸序列(如序列表中SEQ ID NO:5所示)和所述Vip3A核苷酸序列(如序列表中SEQ ID NO:6所示)由南京金斯瑞生 物科技有限公司合成；合成的所述Cry1Fa-01核苷酸序列(SEQ ID NO:3)的5’端还连接有AscI酶切位点，所述Cry1Fa-01核苷酸序列(SEQ ID NO:3)的3’端还连接有BamHI酶切位点；合成的所述Cry1Fa-02核苷酸序列(SEQ ID NO:4)的5’端还连接有AscI酶切位点，所述Cry1Fa-02核苷酸序列(SEQ ID NO:4)的3’端还连接有BamHI酶切位点；合成的所述Cry1Ab核苷酸序列(SEQ ID NO:5)的5’端还连接有NcoI酶切位点，所述Cry1Ab核苷酸序列(SEQ ID NO:5)的3’端还连接有SpeI酶切位点；合成的所述Vip3A核苷酸序列(SEQ ID NO:6)的5’端还连接有ScaI酶切位点，所述Vip3A核苷酸序列(SEQ ID NO:6)的3’端还连接有SpeI酶切位点。
第二实施例、重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有Cry1F基因的重组克隆载体
将合成的Cry1Fa-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；Cry1Fa-01为Cry1Fa-01核苷酸序列(SEQ ID NO:3)；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，10mMEDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入150μl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(4M醋酸钾，2M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AscI和BamHI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Cry1Fa-01核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即Cry1Fa-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Cry1Fa-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，Cry1Fa-02为Cry1Fa-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Cry1Fa-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，Cry1Ab为Cry1Ab核苷酸序列(SEQ ID NO:5)。酶切和测序验证重组克隆载体DBN03-T中所述Cry1Ab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Vip3A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，Vip3A为Vip3A核苷酸序列(SEQ ID NO:6)。酶切和测序验证重组克隆载体DBN04-T中所述Vip3A核苷酸序列正确插入。
2、构建含有Cry1F基因的重组表达载体
用限制性内切酶AscI和BamHI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Cry1Fa-01核苷酸序列片段插到表达载体DBNBC-01的AscI和BamHI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DBN100014，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:7)；Cry1Fa-01：Cry1Fa-01核苷酸序列(SEQ ID NO:3)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:9)；LB：左边界)。
将重组表达载体DBN100014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DBN100014)，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AscI和BamHI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DBN100014在AscI和BamHI位点间的核苷酸序列为序列表中SEQ ID NO:3所示核苷酸序列，即Cry1Fa-01核苷酸序列。
按照上述构建重组表达载体DBN100014的方法，将AscI和BamHI酶切重组克隆载体DBN02-T切下的所述Cry1Fa-02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:4所示核苷酸序列，即Cry1Fa-02核苷酸序列，所述Cry1Fa-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DBN100014的方法，将AscI和BamHI、NcoI和SpeI分别酶切重组克隆载体DBN01-T和DBN03-T切下的所述Cry1Fa-01核苷酸序列和Cry1Ab核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100075。酶切和测序验证重组表达载体DBN100075中的核苷酸序列含有为序列表中SEQ ID NO:3和SEQ ID NO:5所示核苷酸序列，即Cry1Fa-01核苷酸序列和Cry1Ab核苷酸序列，所述Cry1Fa-01核苷酸序列和所述Cry1Ab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DBN100014的方法，将AscI和BamHI、ScaI和SpeI分别酶切重组克隆载体DBN01-T和DBN04-T切下的所述Cry1Fa-01核苷酸序列和Vip3A核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100276。酶切和测序验证重组表达载体DBN100276中的核苷酸序列含有为序列表中SEQ ID NO:3和SEQ ID NO:6所示核苷酸序列，即Cry1Fa-01核苷酸序列和Vip3A核苷酸序列，所述Cry1Fa-01核苷酸序列和所述Vip3A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DBN100014的方法，用限制性内切酶AscI和BamHI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-02(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的Cry1Fa-01核苷酸序列片段插到表达载体DBNBC-02的AscI和BamHI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DBN100015，其构建流程如图3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:7)；Cry1Fa-01：Cry1Fa-01核苷酸序列(SEQ ID NO:3)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:22)；LB：左边界)。
按照上述构建重组表达载体DBN100015的方法，将AscI和BamHI酶切重组克隆载体DBN02-T切下的所述Cry1Fa-02核苷酸序列插入表达载体DBNBC-02，得到重组表达载 体DBN100030。酶切和测序验证重组表达载体DBN100030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Cry1Fa-02核苷酸序列，所述Cry1Fa-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DBN100015的方法，将AscI和BamHI、NcoI和SpeI分别酶切重组克隆载体DBN01-T和DBN03-T切下的所述Cry1Fa-01核苷酸序列和Cry1Ab核苷酸序列插入表达载体DBNBC-02，得到重组表达载体DBN100012。酶切和测序验证重组表达载体DBN100012中的核苷酸序列含有为序列表中SEQ ID NO:3和SEQ ID NO:5所示核苷酸序列，即Cry1Fa-01核苷酸序列和Cry1Ab核苷酸序列，所述Cry1Fa-01核苷酸序列和所述Cry1Ab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DBN100015的方法，将AscI和BamHI、ScaI和SpeI分别酶切重组克隆载体DBN01-T和DBN04-T切下的所述Cry1Fa-01核苷酸序列和Vip3A核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100031。酶切和测序验证重组表达载体DBN100031中的核苷酸序列含有为序列表中SEQ ID NO:3和SEQ ID NO:6所示核苷酸序列，即Cry1Fa-01核苷酸序列和Vip3A核苷酸序列，所述Cry1Fa-01核苷酸序列和所述Vip3A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DBN100014、DBN100013、DBN100075、DBN100276、DBN100015、DBN100030、DBN100012和DBN100031用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和100mg/L的卡那霉素(Kanamycin)的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶AhdI和XhoI对重组表达载体DBN100014、DBN100013、DBN100012、DBN100075、DBN100015和DBN100030进行酶切验证，用限制性内切酶AhdI和EcoRV对重组表达载体DBN100031和DBN100276进行酶切验证，结果表明重组表达载体DBN100014、DBN100013、DBN100075、DBN100276、DBN100015、DBN100030、DBN100012和DBN100031结构完全正确。
第三实施例、转入Cry1F基因的玉米植株的获得及验证
1、获得转入Cry1F基因的玉米植株
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DBN100014、 DBN100013、DBN100075和DBN100276中的T-DNA(包括玉米Ubiquitin基因的启动子序列、Cry1Fa-01核苷酸序列、Cry1Fa-02核苷酸序列、Cry1Ab核苷酸序列、Vip3A核苷酸序列、PMI基因和Nos终止子序列)转入到玉米染色体组中，获得了转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A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Cry1Fa-01核苷酸序列、Cry1Fa-02核苷酸序列、Cry1Fa-01-Cry1Ab核苷酸序列和/或Cry1Fa-01-Vip3A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、琼脂8g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(甘露糖)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5g/L、甘露糖12.5g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/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、甘露糖5g/L、琼脂8g/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、琼脂8g/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、用TaqMan验证转入Cry1F基因的玉米植株
分别取转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A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Cry1F基因、Cry1Ab基因和Vip3A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Cry1F基因、Cry1Ab基因和Vip3A基因拷贝数的具体方法如下：
步骤11、分别取转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株、转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μl；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型玉米植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测Cry1Fa-01核苷酸序列：
引物1(CF1)：CAGTCAGGAACTACAGTTGTAAGAGGG如序列表中SEQ ID NO:10所示；
引物2(CR1)：ACGCGAATGGTCCTCCACTAG如序列表中SEQ ID NO:11所示；
探针1(CP1)：CGTCGAAGAATGTCTCCTCCCGTGAAC如序列表中SEQ ID NO:12所示；
以下引物和探针用来检测Cry1Fa-02核苷酸序列：
引物3(CF2)：CAGTCAGGAACTACAGTTGTAAGAGGG如序列表中SEQ ID NO:13所示；
引物4(CR2)：ACGCGAATGGTCCTCCACTAG如序列表中SEQ ID NO:14所示；
探针2(CP2)：CGTCGAAGAATGTCTCCTCCCGTGAAC如序列表中SEQ ID NO:15所示；
以下引物和探针用来检测Cry1Ab核苷酸序列：
引物5(CF3)：CGAACTACGACTCCCGCAC如序列表中SEQ ID NO:16所示；
引物6(CR3)：GTAGATTTCGCGGGTCAGTTG如序列表中SEQ ID NO:17所示；
探针3(CP3)：CTACCCGATCCGCACCGTGTCC如序列表中SEQ ID NO:18所示；
以下引物和探针用来检测Vip3A核苷酸序列：
引物7(VF1)：ATTCTCGAAATCTCCCCTAGCG如序列表中SEQ ID NO:19所示；
引物8(VR1)：GCTGCCAGTGGATGTCCAG如序列表中SEQ ID NO:20所示；
探针4(VP1)：CTCCTGAGCCCCGAGCTGATTAACACC如序列表中SEQ ID NO:21所示；
PCR反应体系为：
Figure PCTCN2014091023-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 PCTCN2014091023-appb-000002
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，Cry1Fa-01核苷酸序列、Cry1Fa-02核苷酸序列、Cry1Fa-01-Cry1Ab核苷酸序列和Cry1Fa-01-Vip3A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Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株均获得了含有单拷贝Cry1F基因、Cry1Ab基因和/或Vip3A基因的转基因玉米植株。
第四实施例、转基因玉米植株的抗虫效果检测
将转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株、转入Cry1Fa-01-Vip3A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对斜纹夜蛾进行抗虫效果检测。
分别取转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株、转入Cry1Fa-01-Vip3A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(V3-V4期)的新鲜叶片(心叶)，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将玉米叶片去除叶脉，同时剪成约1cm×4cm的长条状，取2片剪后的长条状叶片放入圆形塑料培养皿底部的滤纸上，所述滤纸用蒸馏水润湿，每个培养皿中放10头人工饲养的斜纹夜蛾(初孵幼虫)，虫试培养皿加盖后放入底部放有湿纱布的方盒中，在温度26-28℃、相对湿度70％-80％、光周期(光/暗)16：8的条件下放置3天后，根据斜纹夜蛾幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分：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Cry1Fa-01核苷酸序列的共3个株系(S1、S2和S3)，转入Cry1Fa-02核苷酸序列的共3个株系(S4、S5和S6)，转入Cry1Fa-01-Cry1Ab核苷酸序列的共3个株系(S7、S8和S9)，转入Cry1Fa-01-Vip3A核苷酸序列的共3个株系(S10、S11和S12)，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4所示。
表1、转基因玉米植株接种斜纹夜蛾的抗虫实验结果
Figure PCTCN2014091023-appb-000003
Figure PCTCN2014091023-appb-000004
表1的结果表明：转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株的生测总分均在250分以上或左右，部分可达到满分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生测总分一般在15分左右。
图4的结果表明：与野生型玉米植株相比，转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株可以在3天内造成初孵斜纹夜蛾幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，且转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株大体上只受到轻微损伤，其叶片损伤率均在10％左右或以下。此外当生测持续时间到达5天时，幼虫全部死亡。
由此证明转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株和转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株都显示出高抗斜纹夜蛾的活性，这种活性足以对斜纹夜蛾的生长产生不良效应从而使其得以控制。
第五实施例、转入Cry1F基因的大豆植株的获得及验证
1、获得转入Cry1F基因的大豆植株
按照常规采用的农杆菌侵染法，将无菌培养的大豆品种中黄13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构建的重组表达载体DBN100015、DBN100030、DBN100012和DBN100031中的T-DNA(包括玉米Ubiquitin基因的启动子 序列、Cry1Fa-01核苷酸序列、Cry1Fa-02核苷酸序列、Cry1Ab核苷酸序列、Vip3A核苷酸序列、PAT基因和Nos终止子序列)转入到大豆染色体组中，获得了转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A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3-4毫米处切去下胚轴，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，其中农杆菌能够将所述介导植物生育力的构建体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(OD660＝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、2-吗啉乙磺酸(MES)4g/L、玉米素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、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(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)，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、2-吗啉乙磺酸(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、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(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、2-吗啉乙磺酸(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16小时，再于20℃下培养8小时。
2、用TaqMan验证转入Cry1F基因的大豆植株
分别取转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A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Cry1F基因、Cry1Ab基因和Vip3A基因的拷贝数。同时以野生型大豆植株作为对照，按照上述第三实施例中2用TaqMan验证转入Cry1F基因的玉米植株的方法进行检测分析。实验设3次重复，取平均值。
实验结果表明，Cry1Fa-01核苷酸序列、Cry1Fa-02核苷酸序列、Cry1Fa-01-Cry1Ab核苷酸序列和Cry1Fa-01-Vip3A核苷酸序列均己整合到所检测的大豆植株的染色体组中，而且转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株均获得了含有单拷贝Cry1F基因、Cry1Ab基因和/或Vip3A基因的转基因大豆植株。
第六实施例、转基因大豆植株的抗虫效果检测
将转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株、转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株、野生型大豆植株和经Taqman鉴定为非转基因的大豆植株对斜纹夜蛾进行抗虫效果检测。
分别取转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株、转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株、野生型大豆植株和经Taqman鉴定为非转基因的大豆植株(三叶期)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，同时剪成约2cm×2cm的正方形，取1片剪后的正方形叶片放入圆形塑料培养皿底部的滤纸上，所述滤纸用蒸馏水润湿，每个培养皿中放10头人工饲养的斜纹夜蛾(初孵幼虫)，虫试培养皿加盖后放入底部放有湿纱布的方盒中，在温度26-28℃、相对湿度70％-80％、光周期(光/暗)16：8的条件下放置3天后，根据斜纹夜蛾幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分：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Cry1Fa-01核苷酸序列的共3个株系(S13、S14和S15)，转入Cry1Fa-02核苷酸序列的共3个株系(S16、S17和S18)，转入Cry1Fa-01-Cry1Ab核苷酸序列的共3个株系(S19、S20和S21)，转入Cry1Fa-01-Vip3A核苷酸序列的共3个株系(S22、S23和S24)，经Taqman鉴定为非转基因的(NGM2)共1个株系，野生型的(CK2)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表2和图5所示。
表2、转基因大豆植株接种斜纹夜蛾的抗虫实验结果
Figure PCTCN2014091023-appb-000005
表2的结果表明：转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株的生测总分均在220分以上或左右，部分可达满分300分；而经Taqman鉴定为非转基因的大豆植株和野生型大豆植株的生测总分一般在40分左右。
图5的结果表明：与野生型大豆植株相比，转入Cry1Fa-01核苷酸序列的大豆植株和转入Cry1Fa-02核苷酸序列的大豆植株可以造成初孵斜纹夜蛾幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵-阴性对照状态，其叶片损伤率在20％左右；转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株对初孵幼虫的防治效果几乎为百分之百，极个别存活幼虫也基本上停止发育，其叶片上仅为极少量针孔状轻微损伤。
由此证明转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株都显示出抗斜纹夜蛾的活性，这种活性足以对斜纹夜蛾的生长产生不良效应从而使其得以控制。
上述实验结果还表明转入Cry1Fa-01核苷酸序列的玉米植株、转入Cry1Fa-02核苷酸序列的玉米植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的玉米植株、转入Cry1Fa-01-Vip3A核苷酸序列的玉米植株、转入Cry1Fa-01核苷酸序列的大豆植株、转入Cry1Fa-02核苷酸序列的大豆植株、转入Cry1Fa-01-Cry1Ab核苷酸序列的大豆植株和转入Cry1Fa-01-Vip3A核苷酸序列的大豆植株对斜纹夜蛾的防治显然是因为植物本身可产生Cry1F蛋白，所以，本领域技术人员熟知的，根据Cry1F蛋白对斜纹夜蛾的相同毒杀作用，可产生类似的可表达Cry1F蛋白的转基因植株能够用于防治斜纹夜蛾的为害。本申请中Cry1F蛋白包括但不限于具体实施方式中所给出氨基酸序列的Cry1F蛋白，同时转基因植株还可以产生至少一种不同于Cry1F蛋白的第二种杀虫蛋白质，如Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白等。
综上所述，本申请控制害虫的方法通过植物体内产生能够杀死斜纹夜蛾的Cry1F蛋白来控制斜纹夜蛾害虫；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，本申请对植物进行全生育期、全植株的保护以防治斜纹夜蛾害虫的侵害，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申请的技术方案而非限制，尽管参照较佳实施例对本申请进行了详细说明，本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的精神和范围。

Claims (19)

  1. 一种控制斜纹夜蛾害虫的方法，其中斜纹夜蛾害虫与Cry1F蛋白发生接触。
  2. 根据权利要求1所述的控制斜纹夜蛾害虫的方法，其中所述Cry1F蛋白为Cry1Fa蛋白。
  3. 根据权利要求2所述的控制斜纹夜蛾害虫的方法，其中所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的植物细胞中，所述斜纹夜蛾害虫通过摄食所述植物细胞与所述Cry1Fa蛋白接触。
  4. 根据权利要求3所述的控制斜纹夜蛾害虫的方法，其中所述Cry1Fa蛋白存在于产生所述Cry1Fa蛋白的转基因植物中，所述斜纹夜蛾害虫通过摄食所述转基因植物的组织与所述Cry1Fa蛋白接触，接触后所述斜纹夜蛾害虫生长受到抑制和/或接触后导致所述斜纹夜蛾死亡，从而实现对斜纹夜蛾为害植物的控制。
  5. 根据权利要求4所述的控制斜纹夜蛾害虫的方法，其中所述转基因植物可以处于任意生育期。
  6. 根据权利要求4所述的控制斜纹夜蛾害虫的方法，其中所述转基因植物的组织选自叶片、茎秆、雄穗、雌穗、花药、花丝和果实。
  7. 根据权利要求4所述的控制斜纹夜蛾害虫的方法，其中所述对斜纹夜蛾为害植物的控制不因种植地点和/或种植时间的改变而改变。
  8. 根据权利要求3至7任一项所述的控制斜纹夜蛾害虫的方法，其中所述植物选自玉米、大豆、棉花、甘薯、芋、莲、田菁、烟草、甜菜、白菜或茄子，优选地，所述植物选自玉米或大豆。
  9. 根据权利要求2至8任一项所述的控制斜纹夜蛾害虫的方法，其中所述接触步骤之前的步骤为种植含有编码所述Cry1Fa蛋白的多核苷酸的植物。
  10. 根据权利要求2至9任一项所述的控制斜纹夜蛾害虫的方法，其中所述Cry1Fa蛋白的氨基酸序列具有：1)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，2)与SEQ ID NO:1或SEQ ID NO:2具有至少70％同源性且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列，或3)SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列经取代、缺失和/或添加一个或多个氨基酸残基所获得的且对斜纹夜蛾害虫具有杀虫活性的氨基酸序列。
  11. 根据权利要求10所述的控制斜纹夜蛾害虫的方法，其中所述Cry1Fa蛋白的编码核苷酸序列具有：1)SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列，2)与SEQ ID NO:3或SEQ ID NO:4具有至少大约75％同源性且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序 列的核苷酸序列，3)在严格条件下与SEQ ID NO:3或SEQ ID NO:4杂交且编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列，4)由于密码子简并性而不同于SEQ ID NO:3或SEQ ID NO:4的编码对斜纹夜蛾害虫具有杀虫活性的氨基酸序列的核苷酸序列。
  12. 根据权利要求3至11任一项所述的控制斜纹夜蛾害虫的方法，其中所述植物还包含至少一种不同于编码所述Cry1Fa蛋白的核苷酸的第二种核苷酸。
  13. 根据权利要求12所述的控制斜纹夜蛾害虫的方法，其中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。
  14. 根据权利要求13所述的控制斜纹夜蛾害虫的方法，其中所述第二种核苷酸编码Cry1Ab蛋白、Cry1Ac蛋白、Cry1Ba蛋白或Vip3A蛋白。
  15. 根据权利要求14所述的控制斜纹夜蛾害虫的方法，其中所述第二种核苷酸具有SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列。
  16. 根据权利要求12所述的控制斜纹夜蛾害虫的方法，其中所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。
  17. 一种制备控制斜纹夜蛾害虫的植物细胞、转基因植物或转基因植物的部分的方法，其包括将Cry1F蛋白的编码核苷酸序列引入所述植物细胞、转基因植物或转基因植物的部分中，优选地，将Cry1F蛋白的编码核苷酸序列引入所述植物细胞、转基因植物或转基因植物的部分的基因组中。
  18. Cry1F蛋白在制备控制斜纹夜蛾害虫的植物细胞、转基因植物或转基因植物的部分中的用途。
  19. 一种培养控制斜纹夜蛾害虫的植物的方法，其包括：
    种植至少一种植物种子，所述植物种子的基因组中包括编码Cry1F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斜纹夜蛾害虫和/或斜纹夜蛾害虫自然发生危害的条件下生长，收获与其他不具有编码Cry1F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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ENTOORI, K. ET AL.: "Chimeric CrylX Gene Imparts Resistance to Spodoptera Litura and Helicoverpa Armigera in the Transgenic Groundnut", EURASIAN JOURNAL OF BIOSCIENCES, vol. 2, no. 7, 30 September 2008 (2008-09-30), pages 53 - 65 *

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AR098428A1 (es) 2016-05-26
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AU2014350744A1 (en) 2016-06-02
AU2014350744B2 (en) 2016-12-22
CN103719137A (zh) 2014-04-16
AU2014350744A8 (en) 2016-06-16

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