WO2015074325A1 - 新型的杀虫蛋白 - Google Patents

新型的杀虫蛋白 Download PDF

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WO2015074325A1
WO2015074325A1 PCT/CN2014/001029 CN2014001029W WO2015074325A1 WO 2015074325 A1 WO2015074325 A1 WO 2015074325A1 CN 2014001029 W CN2014001029 W CN 2014001029W WO 2015074325 A1 WO2015074325 A1 WO 2015074325A1
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Prior art keywords
polypeptide
amino acid
plant
insecticidal
nucleic acid
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PCT/CN2014/001029
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English (en)
French (fr)
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凃巨民
罗炬
陈浩
张晓波
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浙江大学
中国水稻研究所
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Priority to CN201480063955.XA priority Critical patent/CN105980546B/zh
Priority to US15/039,234 priority patent/US9879056B2/en
Publication of WO2015074325A1 publication Critical patent/WO2015074325A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • 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
    • 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)
    • 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
    • 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 invention relates to the field of plant genetic engineering and molecular breeding technology.
  • the invention relates to artificially designed and engineered pesticidal protein polypeptides or fragments thereof having insecticidal activity against insect pests.
  • the invention also provides nucleic acids, insecticidal compositions, DNA constructs, and transformed microorganisms and plants comprising the pesticidal protein polypeptides or fragments thereof. These compositions are useful in methods for controlling pests, particularly plant pests.
  • the invention also relates to the use of the nucleic acid or DNA construct to improve the resistance of a plant.
  • Rice is one of the most important food crops in the world and an important economic crop in China. Nearly half of the world's people use rice as their staple food [Khush, 2005], mainly in Asia, and rice in Europe, America, Africa and Oceania. All are planted. However, rice is also one of the most seriously endangered food crops. According to incomplete statistics, the annual loss of rice caused by rice pests such as lepidoptera has exceeded 10 million tons [Herdt, 1991; Zhu et al., 1999 ]. Although the use of pesticides can play a role in controlling pests, it also brings serious residual pollution to humans.
  • Pesticides remain in the environment, may pollute the atmosphere and water resources, destroy soil traits; and, while killing pests, many beneficial insects are also eliminated, which seriously damages the ecological balance; in addition, the long-term use of pesticides may also cause pests Producing drug resistance; therefore, breeding insect-resistant rice varieties and improving rice self-resistance is considered to be a more environmentally-friendly and effective method of control.
  • the cycle of breeding new varieties is also longer, so there is no good problem in overcoming the breeding of new varieties of resistant insects.
  • Bacillus thuringiensis is the most widely used insecticidal microorganism in the world. During the production of Bacillus thuringiensis, it is able to form and secrete some insecticidal crystal proteins (ICPs or Cry) consists of parasporal crystals, such as insects such as lepidoptera (cryI), lepidoptera and diptera (cryII), coleoptera (cryIII) and diptera (cryIV), as well as arthropods such as animal and plant nematodes. Specific toxic activity (Schnepf et al, 1998; Li Changyou et al., 2007). However, long-term single planting of Bt-transgenic plants may also cause pest resistance.
  • ICPs or Cry insecticidal crystal proteins
  • VIP Vegetative Insecticidal Protein
  • VIP is a type of extracellular toxin protein secreted by Bacillus thuringiensis in the mid-log phase of vegetative growth [Schnepf et al, 1998]. It is widely found in Bt bacteria in nature. As of 2008, a total of 8 types and 37 species were found. [Crickmore, 2008]. Previous studies have shown that these VIPs proteins are genetically relatively conserved. Under normal conditions, at least 75% of them are present in the supernatant culture medium and are thermally unstable compared to ICPs and treated at 95 °C. It loses its activity in 20 minutes [Estruch et al, 1996].
  • VIPs can be distinguished into three major categories based on their protein sequence homology, namely VIP1, VIP2 and VIP3 [Crickmore et al, 2008].
  • VIP1 and VIP2 together constitute a binary toxin that has insecticidal specificity against Coleoptera, the genus Corydalis (Warren et al, 1998);
  • VIP3 has a broad spectrum of insecticidal activity against many pests in the family Lepidoptera, genus, and species. The research on its insecticidal mechanism is also deeper [Estruch et al, 1998]. By 2008, fifty-seven Vip genes had been identified and isolated [Crickmore, 2008].
  • VIP1 and VIP2 act as binary toxins and act independently on the insecticidal mechanism [Barth et al, 2002 and 2004].
  • the Vip2 gene is located upstream of the Vip1 gene and its products can be independently Their respective functions [Barth et al, 2004], but the greatest insecticidal virulence of toxin proteins can only be achieved when both proteins are present and synergistic [Warren et al, 1998].
  • VIP2 containing the NAD binding site has ADP-ribosyltransferase activity, which can transfer ribose groups to actin, accompanied by the release of nicotinamide, which leads to the inhibition of multimerization of actin monomers, affecting cells.
  • the composition of the skeleton leads to the death of insect cells (Han et al, 1999).
  • the insecticidal mechanism of VIP3 is mainly reflected in the damage of toxins to insect midgut cells [Whalon & Wingerd, 2003]. After 8888 of VIP3A was swallowed by lepidopteran insects, it was hydrolyzed and activated by intestinal trypsin. The activated VIP3A protein could bind to 80ku and 100ku unknown receptor molecules on BBMVs of sensitive larval midgut. Ion channel perforation is formed on intestinal epithelial cells, which induces apoptosis of insect cells, nucleus lysis, and ultimately leads to insect death [Lee et al, 2003].
  • VIP3A protein can be dissolved as long as the pH is lower than 7.5, and the C' end is not excised, and the two receptors are also different from any known Cry receptor [Lee et al, 2003]; In the absence of any receptor, VIP3A can also form channels on artificial double lipid membranes (BLMs) [Warren et al, 1998; Lee et al, 2003].
  • BBMs artificial double lipid membranes
  • the cytopathological test showed that after feeding the VIP3A(a) protein of the sensitive insects such as the small tiger and the grasshopper, the midgut epithelial goblet cells and the columnar cells completely detached from the basement membrane, and the insect died [Yu, et al, 1997].
  • Vip3A The symptoms caused by Vip3A are similar to those of ICPs, but are delayed in time (Estruch et al, 1996). It can be seen that the receptor and mode of action of VIP3A are significantly different from those of ICPs. Therefore, research and utilization of VIP3A is of great significance for broadening the insecticidal spectrum, improving insecticidal virulence and preventing insect resistance.
  • the molecular mass of ICP protein is generally 130-160 ku, which is only soluble in a highly alkaline solution with pH >10. It exists in the form of protoxin, which is not toxic by itself. It is alkaline and reducing environment in the intestine after being swallowed by insect larvae.
  • the active polypeptide can be dissolved and cleaved into 65-75 ku; while VIP3A can be cleaved by trypsin to a 62 ku-sized active polypeptide under weak alkaline conditions even at pH slightly below 7.5 (Lee et al, 2003).
  • the ICP protein is a crystal protein secreted in the cell
  • the VIP protein is an extracellular secreted protein; in the secretory process, the 100 ku VIP1A protein is cleaved before the formation of the 80 ku mature toxin, The N-terminal signal peptide of the 88 ku VIP3A protein in the secretory process is often not excised due to the absence of an enzyme cleavage site [Schnepf et al, 1998].
  • the VIP protein is wider than the Cry protein and has insecticidal activity against agricultural pests that are not sensitive to ICP protein, such as snails [Estruch et al, 1996].
  • Vip3 gene capable of highly resistant to rice aphids has not been isolated in the natural strain of Bacillus thuringiensis. Therefore, artificially synthesize, engineer and innovate Vip3 gene resources against rice aphids to address the persistence of insect resistance in the use of Cry genes [McGaughey, 1985; Van, 1990; Gould, 1992; Lee, 1995; Tabashnik et al, 1997 Ni Wanchao and Guo Sandui, 1998; High et al, 2004] have far-reaching significance.
  • Vip3A gene which is highly resistant to insect pests, particularly to lepidopteran pests, and particularly has high resistance to rice lepidoptera which is not possessed by all other Vip3 genes which have been identified and isolated so far.
  • the ability of pests such as stem borer, stem borer and rice leaf roller, named Vip3ArLr1; provides a plasmid vector system for transforming Vip3A rLr1 by Agrobacterium-mediated transformation and its construction scheme, thereby providing a pest
  • a breeding method in which a plant is transformed into an insect-resistant plant, and the main main line/variety is converted into an insect-resistant main line/variety.
  • the invention relates to the following aspects:
  • a nucleic acid molecule selected from the group consisting of
  • nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 or a full length complement thereof;
  • nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof;
  • nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof;
  • nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having a deletion, substitution, insertion of one or more amino acids as compared to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof.
  • a DNA construct comprising the nucleic acid molecule of item 1.
  • the DNA construct of item 3 which further comprises a nucleic acid molecule encoding a heterologous polypeptide.
  • a host cell comprising the DNA construct of item 2 or 3, preferably a bacterial cell and a eukaryotic cell, more preferably a plant cell and a yeast cell, preferably a plant cell, Rice (Oryza sativa L.) cells are particularly preferred.
  • a transgenic plant comprising the host cell of item 4.
  • a polypeptide having insecticidal pest activity selected from the group consisting of:
  • polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof;
  • a polypeptide comprising an amino acid sequence having a deletion, substitution, insertion of one or more amino acids compared to the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof;
  • composition comprising the polypeptide of item 7.
  • composition of clause 9 wherein the composition is selected from the group consisting of powders, dusts, pellets, granules, sprays, emulsions, gels, and solutions.
  • composition of clause 9 or 10 which comprises from about 1% to about 99% by weight of said polypeptide.
  • a method for controlling a population of insect pests comprising contacting the population with a pesticidally effective amount of a polypeptide of item 7.
  • a method for killing an insect pest comprising contacting the pest with a pesticidally effective amount of the polypeptide of item 7, or feeding the pest with a pesticidally effective amount of the polypeptide of item 7.
  • a method for producing a polypeptide of item 7 having insecticidal pest activity comprising culturing a host cell of item 4 under conditions in which a nucleic acid molecule encoding a polypeptide is expressed.
  • a plant having stably incorporated a DNA construct into its genome comprising a nucleotide sequence encoding a protein having insecticidal pest activity, wherein the nucleotide sequence is selected from the group consisting of:
  • nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 2 or a full length complement thereof;
  • nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof;
  • nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof;
  • nucleic acid encoding a polypeptide comprising an amino acid sequence having a deletion, substitution, insertion of one or more amino acids compared to the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof child;
  • nucleotide sequence is operably linked to a promoter that drives expression of the coding sequence in a plant cell.
  • a method for protecting a plant from insect pests comprising introducing at least one expression vector comprising the nucleotide sequence of claim 1 into said plant or a cell thereof, said nucleotide sequence encoding Insect polypeptide.
  • a method of improving plant stress resistance comprising introducing at least one expression vector comprising a nucleotide sequence of the first item into the plant or a cell thereof, the nucleotide sequence encoding an insecticidal pest polypeptide .
  • a plant breeding method comprising introducing at least one expression vector comprising the nucleotide sequence of the first item into the plant or a cell thereof, the nucleotide sequence encoding an insecticidal pest polypeptide.
  • insect pest is preferably a lepidopteran pest, more preferably a rice aphid and a Spodoptera litura.
  • Figure 1 Structure of the prokaryotic expression plasmid vector pETVip3A6.
  • Figure 2 Structure of the Agrobacterium plasmid vector pSBVip3A6.
  • FIG. 3 PCR detection of Vip3ArLr1 in transgenic resistant callus.
  • M molecular weight marker
  • + plasmid positive control
  • - empty plasmid negative control
  • 1-10 transformed callus
  • FIG. 12GV-19, 12GV-26, 12GV-37 and 12GV-43 are Vip3ArLr1 independent transformation lines.
  • Vip3 genes cloned from the natural strain of Bacillus thuringiensis Many pests in the family Lepidoptera, genus, and species, such as Spodoptera frugiperda, Spodoptera litura, Helicoverpa armigera, and Pieris rapae, have high and specific insecticidal activity, but have never been reported to indicate these natural
  • the Vip3 gene produced by the strain has insecticidal activity against lepidopteran pests such as rice stem borer, stem borer and rice leaf roller.
  • Fang J. et al. (2007) reported that an artificial chimeric gene Vip3AcAa is resistant to corn borer.
  • Vip3 insect-resistant gene in the present invention follows the principle of uniform structure and function of proteins, and on the basis of determining the functional domains related to the existing Vip3 protein sequence, artificial design improvement is carried out, and finally, rice stem borer can be developed.
  • a gene sequence (SEQ ID No. 1 and SEQ ID No 2) that produces resistance to lepidopteran pests such as Cnaphalocrocis medinalis.
  • Embodiments of the invention relate to compositions and methods for affecting insect pests, particularly plant pests. More specifically, nucleic acids isolated according to embodiments, and fragments and variants thereof, include nucleotide sequences encoding insecticidal polypeptides (eg, proteins). The disclosed pesticidal proteins are biologically active (e.g., insecticidal) against insect pests such as, but not limited to, lepidopteran insect pests. Insect pests of interest include, but are not limited to, Chilo suppressalis, Cnaphalocrocismedialis Guenee, and Prodenia litura.
  • compositions of embodiments include nucleic acids, and fragments and variants thereof, which encode insecticidal polypeptides or fragments thereof, including expression cassettes for the nucleotide sequences of the embodiments, isolated pesticidal proteins, and pesticidal compositions.
  • Embodiments further provide plants and microorganisms transformed with these novel nucleic acids, and methods involving the use of such nucleic acids, insecticidal compositions, transformed organisms, and their products in affecting insect pests.
  • the nucleic acid and nucleotide sequences of the embodiments can be used to transform any organism to produce the encoded insecticidal protein. Methods are provided for affecting or controlling plant pests using such transformed organisms. The nucleic acid and nucleotide sequences of the embodiments can also be used to transform an organelle.
  • nucleotide sequence of the embodiments is directly useful in methods for affecting pests, particularly insect pests, such as lepidopteran pests.
  • embodiments provide a new method for affecting insect pests that does not rely on the use of conventional, synthetic chemical insecticides.
  • Embodiments relate to naturally occurring, biodegradable insecticides and substrates encoding the same The discovery of the cause.
  • nucleic acids are written from left to right in the 5' to 3' direction, respectively; amino acid sequences are written from left to right in the amino to carboxyl direction.
  • the range of numbers includes a limited number of numbers.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleotides can be referred to by their commonly accepted one-letter codes. The above terms are more fully defined by reference to the specification as a whole.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer in a single or double stranded form.
  • the term "encoding" or "encoding" when used in the context of a particular nucleic acid means that the nucleic acid includes the necessary information to direct translation of the nucleotide sequence into a particular protein.
  • the information encoded by the protein is specified by the use of the codon.
  • the nucleic acid encoding the protein may comprise a non-translated sequence (eg, an intron) within the translational region of the nucleic acid, or may lack such intervening non-translated sequences (eg, as in cDNA).
  • polypeptide peptide
  • protein protein
  • amino acid residue or “amino acid” are used interchangeably herein to refer to an amino acid incorporated into a protein, polypeptide or peptide (collectively “protein”).
  • Amino acids can be naturally occurring amino acids and, unless otherwise defined, can include known analogs of natural amino acids, which can function in a manner similar to naturally occurring amino acids.
  • Polypeptides of embodiments can be produced from the nucleic acids disclosed herein or using standard molecular biology techniques.
  • a protein of an embodiment can be produced by expression of a recombinant nucleic acid of an embodiment in a suitable host cell or alternatively by a combination of ex vivo procedures.
  • affecting insect pests refers to achieving changes in insect feeding, growth, and/or behavior at any stage of development, including but not limited to: killing insects; Growth; prevention of fertility; antifeedant activity, etc.
  • insecticidal effect As used herein, the terms “insecticidal effect,” “insecticidal activity,” “insecticidal activity,” and “insect resistance” are used synonymously to refer to the activity of a organism or substance (eg, a protein), which may pass but not Measurements are limited to: pest mortality, pest weight loss, pest drive, and other behavioral and physical changes of the pest after ingestion and exposure for a suitable length of time. Thus, an organism or substance having insecticidal activity adversely affects at least one measurable parameter of pest suitability.
  • insecticidal protein is a protein that exhibits insecticidal activity by itself or in combination with other proteins.
  • insecticidal effective amount means the amount of a substance or organism having insecticidal activity when present in the environment of a pest. For each substance or organism, the pesticidally effective amount is determined empirically for each pest affected in a particular environment. Similarly, an “insecticidal effective amount” can be used to refer to an “insecticidal effective amount” when the pest is an insect pest.
  • the term "artificially engineered and engineered” means the use of recombinant DNA techniques to introduce (eg, engineer) changes in the structure of a protein based on the understanding of the mechanism of action of the protein and the amino acids to be introduced, deleted or substituted. consider.
  • the term "improved insecticidal activity” or “improved insecticidal activity” refers to an insecticidal polypeptide of an embodiment that has enhanced insecticidal activity relative to the activity of its corresponding wild-type protein, and/or The insecticidal polypeptides that are effective against a wider range of insects, and/or insecticidal polypeptides that are specific to insects, are not susceptible to toxicity to wild-type proteins.
  • the discovery of improved or enhanced insecticidal activity requires an increase in insecticidal activity of at least 10% against insect targets relative to the insecticidal activity of wild-type insecticidal polypeptides determined for the same insect, or at least 20%, 25%, 30% An increase in insecticidal activity of 35%, 40%, 45%, 50%, 60%, 70%, 100%, 150%, 200% or 300% or more.
  • improved insecticidal or insecticidal activity is provided wherein a broader or narrower range of insects is affected by the polypeptide relative to the range of insects affected by the wild-type Bt toxin.
  • a wider range of effects may be desirable when versatility is desired, and a narrower range of effects may be desirable when, for example, beneficial insects may otherwise be affected by the use or presence of toxins.
  • a polypeptide encoded by a nucleotide sequence comprising a mutation will comprise at least one amino acid change or addition, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, relative to the background sequence. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70 or 80 or more amino acid changes or additions.
  • the insecticidal activity of the polypeptide can also be improved by truncation of the full length sequence, as is known in the art.
  • the pesticidal protein of an embodiment provides a full length insecticidal polypeptide, a fragment of a full length insecticidal polypeptide, and a nucleic acid produced by mutagenesis treatment designed to introduce a particular amino acid sequence into a polypeptide of an embodiment.
  • Variant polypeptide a nucleic acid produced by mutagenesis treatment designed to introduce a particular amino acid sequence into a polypeptide of an embodiment.
  • Mutations can be placed in any background sequence, including such truncated polypeptides, so long as the polypeptide retains insecticidal activity.
  • Those skilled in the art can readily compare two or more proteins with respect to insecticidal activity using assays known in the art or described elsewhere herein. It will be understood that the polypeptides of the embodiments can be produced by expression of the nucleic acids disclosed herein or by using standard molecular biology techniques.
  • fragments refers to a portion of a nucleotide sequence of a polynucleotide of an embodiment or a portion of an amino acid sequence of a polypeptide.
  • a fragment of a nucleotide sequence may encode a protein fragment that retains the biological activity of the full length protein and thus has insecticidal activity.
  • fragments and mutants may be referred to correctly as fragments and mutants.
  • fragment when used in reference to a nucleic acid sequence of an embodiment, also encompasses sequences useful as hybridization probes. Such nucleotide sequences generally do not encode a fragment protein that retains biological activity. Thus, a fragment of a nucleotide sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full length nucleotide sequence of a protein encoding an embodiment.
  • variant is used herein to refer to a substantially similar sequence.
  • conservative variants include those sequences encoding the amino acid sequence of one of the pesticidal polypeptides of the embodiments due to the degeneracy of the genetic code.
  • Variant nucleotide sequences also include synthetically derived nucleotide sequences, for example, generated by site-directed mutagenesis, but still encoding the insecticidal proteins of the embodiments.
  • variants of a particular nucleotide sequence of an embodiment will have at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89% with that particular nucleotide sequence, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • Variants of the nucleotide sequence of an embodiment may differ from the original sequence by 1-240 nucleotides, as little as 1-100, as little as 1-10, as few as 5, 4, 3, 2 or even 1 nucleus. Glycosylate.
  • Embodiments further comprise a microorganism transformed with at least one nucleic acid of an embodiment, an expression cassette comprising a nucleic acid, or a vector comprising an expression cassette.
  • the microorganism is the one that is propagated on the plant.
  • Embodiments provide pesticidal compositions comprising the transformed microorganisms of the embodiments.
  • the transformed microorganism is typically present in the pesticidal effective amount together with a suitable carrier in a pesticidally effective amount.
  • Embodiments further comprise a pesticidal composition comprising an isolated protein in combination with a suitable carrier, in an insecticidally effective amount, alone or in combination with the transformed organism of the embodiment and/or the pesticidal protein of the embodiment.
  • Embodiments also encompass a transformed or transgenic plant comprising at least one nucleotide sequence of an embodiment.
  • the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of an embodiment operably linked to a promoter driven in a plant Expression in cells.
  • the terms "transformed plant” and "transgenic plant” refer to a plant that includes a heterologous polynucleotide within its genome.
  • heterologous polynucleotides are stably integrated within the genome of a transgenic or transformed plant, thereby allowing the polynucleotide to be delivered to successive generations.
  • the heterologous polynucleotide can be integrated into the genome either alone or as part of a recombinant expression cassette.
  • transgenic includes any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including those genetically altered as such. Organisms and those produced by the original transgenic organism by sexual or asexual reproduction. As used herein, the term “transgenic” does not encompass genomic alterations (chromosomal or extrachromosomal) by conventional plant breeding methods or by naturally occurring events, such as random allogeneic fertilization, non-recombinant viral infection, Non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • the term "plant” includes whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof. Portions of the transgenic plant are within the scope of the embodiments and include, for example, plant cells, protoplasts, tissues, callus derived from a transgenic plant previously transformed with a DNA molecule of the embodiments or a progeny thereof and thus at least partially composed of transgenic cells. , embryos and flowers, stems, fruits, leaves and roots.
  • the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant blocks, and plant cells, which are intact in a plant or part of a plant, For example, embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, shells, stems, roots, root tips, anthers, and the like.
  • embodiments do not rely on specific biological mechanisms for increasing plant resistance to plant pests, expression of the nucleotide sequence of the embodiments in plants can result in the production of insecticidal proteins of the embodiments and plant-to-plant pests. An increase in resistance.
  • the plants of the embodiments are useful in agriculture for methods for affecting insect pests.
  • proteins of the embodiments can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such operations are generally known in the art.
  • amino acid sequence variants of pesticidal proteins can be prepared by introducing mutations into synthetic nucleic acids, such as DNA molecules.
  • the nucleotide sequence of the mutagenized treatment of an embodiment may be modified such that about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more present in the primary sequence of the encoded polypeptide is altered Amino acids.
  • even more changes from the natural sequence can be introduced such that the encoded protein can have at least about 1% or 2%, or about 3%, 4%, 5%, compared to the corresponding original protein. 6%, 7%, 8%, 9%, 10% or more codons altered or otherwise modified.
  • amino acid additions and/or substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, charge, size, and the like.
  • exemplary groups of amino acid substitutions that take into account various of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and day Asparagine; and valine, leucine and isoleucine.
  • the gene and nucleotide sequences of the embodiments include naturally occurring sequences and mutant forms.
  • the proteins of the embodiments comprise artificially engineered proteins and variants thereof (e.g., truncated polypeptides) and modified (e.g., mutant) forms.
  • Such variants will continue to have the desired insecticidal activity.
  • Deletions, insertions, and substitutions of protein sequences encompassed herein are not expected to result in fundamental changes in protein characteristics. However, when it is difficult to predict the exact role of a substitution, deletion or insertion prior to doing so, those skilled in the art will recognize that the effect will be assessed by routine screening assays such as insect feeding assays.
  • Variant nucleotide sequences and proteins also comprise sequences and proteins derived from mutagenesis and recombination procedures such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to produce a new pesticidal protein having the desired properties. In this manner, a library of recombinant polynucleotides is generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • a full-length coding sequence, a sequence motif encoding a domain of interest, or an implementation Any fragment of the nucleotide sequence of the scheme can be shuffled between the nucleotide sequence of the embodiment and the corresponding portion of other known Vip nucleotide sequences to obtain a novel gene encoding a protein having improved properties of interest.
  • Embodiments further relate to plant propagation materials of transformed plants of the embodiments, including, but not limited to, seeds, tubers, bulbs, bulbs, leaves, and cuttings of roots and shoots.
  • Embodiments can be used to transform any plant species including, but not limited to, monocots and dicots.
  • compositions of the embodiments are useful in protecting plants, seeds and plant products in a variety of ways.
  • the composition can be used in a method involving placing an effective amount of the pesticidal composition in a pest environment by a procedure selected from the group consisting of spraying, dusting, spreading, or seed coating.
  • the plant propagation material (fruit, tubers, bulbs, bulbs, grains, seeds), but especially the seeds, are sold as commercial products, it is treated as usual with a protective agent coating comprising a herbicide, an insecticide, a fungicide, bactericide, nematicide, molluscicide or a mixture of several of these formulations, if desired together with further carriers, surfactants or application promoting adjuvants as conventionally employed in the formulation field, To provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coating can be applied to the seed by infiltrating the tubers or grains with a liquid formulation or by coating it with a combined wet or dry formulation.
  • other methods of application for plants are possible, for example for the treatment of buds or fruits.
  • the gene encoding the pesticidal protein of an embodiment can be introduced into a microbial host via a suitable vector and applied to the environment, or to a plant or animal.
  • the term "introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated Within the genome of a cell (eg, chromosome, plasmid, plastid, or mitochondrial DNA), it is converted to an autonomous replicon or transient expression (eg, transfected mRNA).
  • an expression cassette can be constructed comprising a nucleotide construct of interest operably linked to transcriptional and translational regulatory signals for expression of a nucleotide construct, and a nucleotide sequence homologous to a sequence in a host organism.
  • transcriptional and translational regulatory signals include, but are not limited to, promoters, initiation sites for transcription initiation, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons Sub, termination signal, etc.
  • the transformed microorganism (which includes intact organism, cell, one or more spores, one or more insecticidal proteins, one or more insecticidal components, one or more pest affected groups) a fraction, one or more mutants, live or dead cells and cellular components, including a mixture of live and dead cells and cellular components, and including disrupted cells and cellular components) or isolated pesticidal proteins
  • the accepted carrier is formulated together as one or more pesticidal compositions, for example, as suspensions, solutions, emulsions, dusting powders, dispersible granules or pellets, wettable powders and emulsifiable concentrates, aerosols or sprays, Encapsulation of infiltrated particles, adjuvants, coatable pastes, colloids and, for example, polymeric materials.
  • Such formulated compositions can be prepared by such conventional methods, such as dehydration, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of cell cultures comprising
  • Such a composition as disclosed above can be obtained by adding a surfactant, an inert carrier, a preservative, a wetting agent, a feeding stimulating agent, an attractant, a capsularizing agent, a binder, an emulsifier, a dye, and UV.
  • a surfactant an inert carrier, a preservative, a wetting agent, a feeding stimulating agent, an attractant, a capsularizing agent, a binder, an emulsifier, a dye, and UV.
  • One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, defoliants (harVest aid) And fertilizers, which may be combined with carriers, surfactants or adjuvants or other components as commonly employed in the formulation field to facilitate product processing and application to specific target pests.
  • Suitable carriers and adjuvants may be solid or liquid and correspond to materials commonly employed in formulating techniques, such as natural or regenerative minerals, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • the active ingredients of the embodiments are normally applied in the form of a composition and can be applied to crop areas, plants or seeds to be treated.
  • the composition of the embodiments can be applied to cereals used in the preparation of granaries or kiln, etc. or in storage in granaries or mantles and the like.
  • the compositions of the embodiments can be applied simultaneously or sequentially with other compounds.
  • Methods of applying the active ingredients or embodiments of the agrochemical compositions of the embodiments comprising at least one pesticidal protein produced by the bacterial strain of the embodiments include, but are not limited to, leaf application, seed coating, and soil application. The number of applications and the application ratio depend on the intensity of intrusion through the corresponding pests.
  • compositions of the embodiments may be used in a suitable form for direct application or as a concentrate of the base composition, which requires dilution with a suitable amount of water or other diluent prior to application.
  • the insecticidal concentration will vary depending on the nature of the particular formulation, depending in particular on whether it is a concentrate or to be used directly.
  • the composition (including the transformed microorganisms and insecticidal proteins of the embodiments) can be applied to the environment of insect pests.
  • the pesticidal proteins and/or transformed microorganisms of the embodiments can be mixed with the cereal to protect the cereal during storage. It is generally important to obtain good control of pests in the early stages of plant growth, as this is the time when plants can be most severely damaged.
  • the composition of the embodiment may conveniently comprise another insecticide if deemed necessary.
  • the composition is applied directly to the soil in the form of granules of the composition at the time of planting, the composition having the carrier and the Bacillus strain of the embodiment or the dead cells of the transformed microorganism.
  • Another embodiment is in the form of granules of the composition comprising an agrochemical such as a herbicide, an insecticide, a fertilizer, an inert carrier, and a Bacillus strain of the embodiment or a dead cell of the transformed microorganism.
  • the compounds of the embodiments exhibit activity against insect pests, which may include economically important agronomy, forests, greenhouses, nurseries, ornamental plants, food and fiber, public and animal health, household and commercial structures, household and stored product pests .
  • the general procedure includes the dietary source of adding the test compound or organism to a closed container.
  • Insecticidal activity can be measured by, but not limited to, changes in mortality, weight loss, attractiveness, driveability, and other behavioral and physical changes after ingestion and exposure for a suitable length of time.
  • the bioassays described herein can be used for any feeding insect pests in the larval or adult stage.
  • the modified Vip3 coding sequence was constructed on the prokaryotic expression vector pET22b (Fig. 1), and the E. coli DH5 ⁇ strain was used for prokaryotic expression and inclusion body extraction. Specific steps are as follows: Take the monoclonal colonies and raise them overnight, then raise 50ml to OD600 to 0.4-0.6; take 1ml of culture solution and centrifuge for 1min at 13000rpm for 1min, then dissolve with 30ul of water; then, add IPTG to 1mM, culture for 6hr; The solution was centrifuged at 4 ° C and 8000 rpm for 10 min, and the supernatant was removed.
  • the pellet was resuspended in 10 ml of 1 ⁇ PBS, 10 ml of lysozyme was added, and treated at 25 ° C for 30 min; then the protease inhibitor PMSF was added to a final concentration of 1 mM on ice.
  • coli DNA was cut with nuclease S1, and centrifuged at 4 ° C and 5000 rpm for 10 min, after removing the supernatant, the precipitate was washed 3 times with 25 ml of inclusion body protein purification buffer to obtain inclusion bodies. Saved at -20 Refrigerator spare.
  • the insect resistance of the Vip3 protein was identified by artificial feeding.
  • the artificial feed used for larvae is configured according to the formulation invented by Han Lanzhi, Hou Maolin, Peng Yufa et al. (2009).
  • the feed was divided into squares of 2 cm in length or the artificial feed was placed in a 24-well plate without cooling, and the body protein samples to be tested were diluted to 640 ⁇ g by the target pest-determined lethal dose determined by the preliminary test. After ml, 100 ul was applied to the feed, air dried, and then 5 worms were placed in each feed block, and 3 replicates were set. Then, it was covered with a semipermeable membrane and continuously recorded for 7 days to observe its survival.
  • the plasmid constructed in this example for the Vip3ArLr1 Agrobacterium vector is pSB130actin-nos (an empty vector modified by our laboratory) and consists of a double transfer DNA (T-DNA) region.
  • the purpose of constructing the double T-DNA plasmid vector is to allow the target gene and the marker gene to have an independent integration in the recipient genome after transformation of the rice, so that the marker gene can be separated and eliminated by self-crossing in subsequent segregation generations.
  • the promoter driving the expression of the target gene is the rice actin I promoter with a size of 0.839 kb and the donor organism is rice; the terminator is a synthetic nosT with a size of 0.271 kb, which is used to terminate transcription and guide messengers.
  • Polyadenylation of RNA (mRNA) Fraley et al, 1983
  • the marker gene for plasmid self-screening is the anti-kanamycin gene kan+.
  • the marker gene for transformant selection was the hygromycin phosphotransferase gene (hph), which was 1.026 kb in size and the donor organism was E. coli. This marker gene is constructed in a T-DNA region different from the gene of interest when the vector is designed.
  • the constructed plasmid vector was named pSBVip3A6, and its structural diagram is shown in Fig. 2.
  • the Vip3ArLr1 ligated fragment was amplified from the prokaryotic expression vector of pET11a by PCR and introduced into the cleavage site of the cloning site of the Agrobacterium plasmid vector at the same time of amplification.
  • the PCR amplification primers and reaction system were used. Table 1, the PCR reaction procedure used was denatured at 94 ° C for 5 min, followed by 35 cycles of 95 ° C denaturation for 15 sec, 55 ° C annealing for 30 sec and 72 ° C extension for 2 min, and then extended at 72 ° C for 10 min and then incubated at 10 ° C for 10 min.
  • the PCR product was subjected to electrophoresis separation using a 1% agarose gel, and then the target DNA fragment was recovered according to the Axygen Gel Recovery Kit operating instructions.
  • the specific procedure is: the target DNA fragment in the agarose gel is cut under ultraviolet light, the surface liquid is exhausted with absorbent paper, and chopped into a 1.5 ml centrifuge tube, and the weight of the gel is measured on a balance.
  • the purified Vip3ArLr DNA fragment and the Agrobacterium double T-DNA plasmid (pSB130actin-nos) fragment were double digested with XbaI and SmaI restriction enzymes.
  • the enzyme digestion reaction system is shown in Table 2.
  • the PCR product was purified using Axygen's AxyPrep PCR Cleaner Kit. The procedure was as follows: Add 3 volumes of PCR-A buffer to the PCR reaction solution (if the PCR-A buffer is less than 100 ⁇ l, add to 100 ⁇ l).
  • the recovered vector fragment and the target gene were first subjected to concentration measurement, and then added to a 50 ⁇ l sterile centrifuge tube at a ratio of 1:3, and ligated overnight at 4 ° C.
  • the connection system used is shown in Table 3.
  • the strain used as a cloning receptor in the present invention is Escherichia coli strain DH5 ⁇ , and the transformation method For the heat hit.
  • the specific procedure for transformation was as follows: 10 ⁇ l of the ligation product was added to 80 ⁇ l of E. coli DH5 ⁇ competent cells, and evenly pipetted with a pipette, placed on an ice bath for 30 min, and heat-shocked at 42 ° C for 90 s, and then quickly set back to the ice bath.
  • the transparent single colonies were selected and picked up with a toothpick as a template and placed in a PCR reaction system for amplification and identification.
  • the PCR reaction system and the reaction procedure are the same as before.
  • Select the PCR-positive colony and extract the plasmid according to the plasmid purification kit of Axygen, and verify the plasmid by enzyme digestion. After the result is confirmed, take 10 ⁇ l of the plasmid and send it to Shanghai Handsome Biotechnology Co., Ltd. for further sequencing verification.
  • the transformation of Agrobacterium by the Vip3ArLr1 vector plasmid was carried out by electroporation, and the Agrobacterium strain used was EHA105.
  • the specific procedure was as follows: 0.5 ⁇ l of Vip3ArLr1 vector plasmid was added to a 1.5 ml centrifuge tube containing 60 ⁇ l of Agrobacterium EHA105 electroporation competent cells, and the head was pipetted and mixed into the electrode cup; after the electric shock, 1 ml of LB liquid was quickly added. The medium was smothered, transferred to a previous 1.5 ml centrifuge tube, and shaken on a constant temperature shaker at 28 ° C for 1 h.
  • Rice transformation was carried out according to the method steps reported by Liu Qiaoquan et al. (2004). The specific procedure is as follows: the Agrobacterium liquid stored at -80 ° C is taken out, and 200 ⁇ l of the solution is uniformly applied to the surface of the LB solid medium containing 25 mg/l of rifampicin and 50 mg/l of kanamycin at 28 ° C. Culturing overnight; then cultivating the colony from the single colony, using the same liquid medium; after that, taking 200-300 ⁇ l of fresh bacterial solution into the 20 ml LB liquid containing 25 mg/l rifampicin and 50 mg/l kanamycin Culture in medium at 28 ° C with shaking (220 rpm) 16-18h.
  • a sufficient amount of the bacterial solution was centrifuged at 4000 rpm for 15 min, and the supernatant of the LB medium was discarded.
  • Agrobacterium (resorbed gently with a pipette) was added by adding 20 ml of 0.1 M MgSO4 solution, and centrifuged at 4000 rpm for 10-15 min, discarded. Remove the MgSO4 supernatant containing antibiotics; resuspend Agrobacterium by adding 5 ml of AA-AS infection medium containing 200 ⁇ M acetosyringone (AS), and add appropriate amount of AA-AS infection medium to make the bacteria solution.
  • the OD600 value was finally adjusted between 0.8 and 1.0; after the concentration was adjusted, the bacterial solution was dispensed in a sterile 50 ml centrifuge tube, 20-25 ml/tube, and used.
  • the Japanese wild embryogenic callus which was pre-cultured for about 7 days, was transferred from the subculture dish to an empty culture dish covered with sterile filter paper, and air-dried on a clean bench for about 10-15 minutes, during which it was sterilized.
  • the spoon slowly tumbling and making it fully dry; after it is dried, it is transferred into a centrifuge tube containing the bacterial liquid, gently shaken at room temperature (not too vigorous) for 40 minutes, and the centrifuge tube is allowed to stand on the ultra-clean workbench.
  • the embryogenic callus was placed on a sterile filter paper for about 15 minutes, and then transferred to a CC-AS co-cultivation medium containing 200 ⁇ M acetosyringone covered with sterile filter paper.
  • the cells were cultured in the dark at 28 °C for 50-55 h; the embryos with no growth or uncontaminated growth of Agrobacterium were selected and transferred to N6 containing 2.0 mg/L of 2,4-D, 500 mg/l cephalosporin.
  • bacteriostatic culture was carried out in a dark room at 28 ° C for 3-4 days; the callus after bacteriostatic culture was transferred to N6 screening medium containing 500 mg/l cephalosporin and 65 mg/L hygromycin, 28 ° C Dark room culture; in the first week, the Agrobacterium contamination should be checked every day. If the pollution cannot be controlled, it needs to be replaced in time. Screen the medium and select the growth-producing callus on the fresh screening medium every half month, and adjust the concentration of cephalosporin in the medium according to the degree of Agrobacterium self-contamination. Generally, the third or the third Half round of secondary screening can be considered to halve its concentration.
  • transgenic resistant callus was weighed from each of the subcultured individual transformants, placed in a mortar that was washed and sterilized by alcohol burning, and 600 ⁇ l of 1.5 ⁇ CTAB extract was added to grind to Homogenize, transfer the homogenate to a 1.5 ml centrifuge tube, place in a 60 ° C water bath for 25 min (during it and repeatedly invert it twice), then add 400 ⁇ l of chloroform, invert several times and mix thoroughly, then centrifuge at room temperature for 6 min (13000 r / Min); Pipette 400 ⁇ l of supernatant into a new centrifuge tube, add 800 ⁇ l of absolute ethanol, mix and place at -30 ° C for more than 30 min.
  • Vip3ArLr1 in transgenic resistant callus was performed using conventional PCR techniques.
  • the detection primers used were: vip-F, 5'-GCTGTTAT GCGGCCATTGTC-3' and vip-R, 5'-GACGTCTGTCGAGAAGTTTC-3', and the amplified target fragment was about 300 bp, and the PCR reaction system is shown in Table 3- 4.
  • the PCR reaction procedure used was denaturation at 94 ° C for 5 min, followed by 35 cycles of denaturation at 95 ° C for 15 sec, annealing at 55 ° C for 30 sec and 72 ° C for 30 sec, followed by extension at 72 ° C for 10 min and incubation at 10 ° C.
  • the obtained PCR amplification product was separated by 0.8% agarose gel electrophoresis, and photographed and stored.
  • Vip3ArLr1 positive callus was carried out according to the method reported by Yang R et al (2011) et al.
  • the specific procedure is as follows: the resistant callus positive for the target gene Vip3ArLr1 is transferred to N6 differentiation medium (N6 basic medium + 2 mg/l Kinetin + 1 mg/l NAA + 4% Gelrite), and pre-conditioned in a dark room at 28 ° C Differentiation for 7-9 days, and then transferred to fresh differentiation medium, green seedling differentiation in 25 ° C light room (generally, 7-14 days after differentiation of green spots can be seen, after 3 weeks can be differentiated into green seedlings).
  • N6 differentiation medium N6 basic medium + 2 mg/l Kinetin + 1 mg/l NAA + 4% Gelrite
  • the obtained green seedlings are washed and adhered to the medium on the root system, and directly (when the root buds are simultaneously differentiated) or after rooting through the rooting medium (the buds are differentiated), they are transferred to the Yoshida medium for transient culture, and are grown. After being in good condition and stable, transplant it to the greenhouse until it is ripe.
  • the positive transgenic plant Vip3ArLr1 protein was detected by Shanghai Youlong Company's Vip test strip. Protein extraction and test strip detection in rice leaves were performed as described in the product data sheet.
  • the specific steps are as follows: Take the leaves of transgenic rice with a length of about 2-3 cm into a mortar, add 1 ml of distilled water or commercially available purified water, and after thorough grinding, transfer to a 1.5 ml centrifuge tube with a pipette and centrifuge for 30 s at 12000 rpm. After that, the supernatant was transferred to another 1.5 ml centrifuge tube. Then put the test strip, and observe the color development of the test strip after 5 minutes.
  • the plant material used for the identification of the insects is the independent transformation line which is positive for the detection of DNA and protein.
  • the identification method of the insects adopts the natural insect and artificial insect identification method, and the indicators for identification of the insects have the heart rate, the white ear rate, and the leaf curl. Rate and average corrected mortality.
  • the identification of natural insects is mainly carried out for rice leaf roller; the artificial insect identification method is carried out for rice stem borer and rice leaf roller.
  • the artificial insect identification method includes the in vitro leaf method, the isolated stem method, the single insect resistance identification method, and the Daejeon artificial insect collection method.
  • In vitro leaf method In the middle stage of rice tillering, 3 pieces of leaves from different tillers and 1 leaf of 3 different lengths were randomly selected from the rice plants of the tested lines, and 3 strains/test strains were repeatedly identified. material. Thereafter, small filter paper impregnated with 0.1 g/L benzoxazole preservative solution was pressed on both ends of the blade, and transferred into a small flat bottom glass test tube having a length of 9.5 cm and an inner diameter of 1.5 cm, and then each tube was separately Connected to the first instar larvae of 12 heads of rice stem borer or rice leaf roller, inserted into the cotton wool at the mouth of the tube, placed in a light-breeding condition at 28 ° C, and added 2 to 3 fresh test leaves to the tube after 4 days, and checked after 7 days.
  • corrected mortality% (the test specimen material mortality rate of the test insecticide-the average mortality of the susceptible insects)/(1) Mortality) ⁇ 100.
  • the resistance of the test materials to the target pests namely Chilo suppressalis and/or Rice leaf roller, was evaluated according to the in vitro leaf resistance evaluation criteria listed in Table 4.
  • In vitro stalk method 2 pieces of the main tiller rice seedlings were collected for each test plant material, and the rice seedlings were dried, and 2 rice seedlings were cut into 2 pieces of 5 cm stalks containing nodules and leaf sheaths, followed by Press the small filter paper impregnated with 0.1g/L benzoxazole preservative solution on both ends of the stem and move it It was placed in a small flat bottom glass test tube having a length of 9.5 cm and an inner diameter of 1.5 cm. Then, each tube was connected to 12 first-instar larvae, and the tube was stuffed with absorbent cotton.
  • Daejeon artificial insect inoculation mainly in the two stages of tillering and booting.
  • the former is the second instar larvae at about 15 cm in height, and the latter is the first hatching larvae, 15 heads/plants, 10-30 strains/lines, 7-3 days before heading, repeated three times.
  • the heart rate and white ear rate were investigated, and the effect of the transgenic field on insect resistance was judged.
  • the mean corrected mortality rate was above 66%, significantly higher than the mean corrected mortality on the parental and susceptible control lines (Tables 6 and 7); the four independent transformation lines of the inoculated mites in Vip3ArLr1
  • the rate of heart failure caused by the hazard on the plants was also significantly lower than that caused by the hazard on the control gene Vip3Aa1 transformed line (Table 8).
  • Table 8 Identification of resistant resistance of artificially infected insects to stem borer of Vip3ArLr1 and control Vip3Aa1 transformed plants (2013).
  • Tables 9 to 11 show the results of the identification of the resistance of natural outbreaks and artificial insects of Cnaphalocrocis medinalis by four independent transformed lines and their corresponding sister lines.
  • the natural leaf curl rate and the average corrected death of artificial insects In addition to the transformation line 12GV-43 and its corresponding sister line 13GV-38 reaching 12.5% and not more than 20%, respectively, the other three independent transformation lines and their corresponding sister lines are within 2% and 100% respectively.
  • the difference from the parental and susceptible control strains is extremely significant.
  • the transgenic Vip3ArLr1 gene resistant rice new germplasm was successfully created. Further using the germplasm as a parent, the traditional backcross breeding program and modern molecular marker-assisted selection can also be used to select the derived lines, and finally select a good transgenic insect-resistant new line.
  • Barth H Blocker D, Aktories K.

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Abstract

提供了经人工设计和改造的杀虫蛋白或其片段，其属于Vip3A类杀虫蛋白，所述蛋白或其片段具有针对昆虫害虫，尤其是鳞翅目害虫的杀虫活性。还提供了编码所述杀虫蛋白或其片段的核酸、杀虫组合物、DNA构建体以及包括所述核酸的经转化的微生物和植物，及用于控制害虫特别是植物害虫的方法。

Description

新型的杀虫蛋白 技术领域
本发明涉及植物基因工程和分子育种技术领域。具体而言，本发明涉及经人工设计和改造的杀虫蛋白多肽或其片段，所述多肽或其片段具有针对昆虫害虫的杀虫活性。本发明还提供了编码所述杀虫蛋白多肽或其片段的核酸、杀虫组合物、DNA构建体以及包括所述核酸的转化的微生物和植物。这些组合物在用于控制害虫特别是植物害虫的方法中有用。本发明还涉及借助所述核酸或DNA构建体来改良植物抗逆性的应用。
背景技术
水稻是世界上最为重要的粮食作物之一，也是我国的重要经济作物，全球有近一半的人以稻米为主食[Khush，2005]，主要分布在亚洲，水稻在欧洲、美洲、非洲和大洋洲也均有种植。然而，水稻也是受虫害危害最为严重的粮食作物之一，据不完全统计，全球每年因鳞翅目等水稻害虫的危害造成稻米的损失已超过千万吨[Herdt，1991；朱桢等，1999]。农药的使用在防治虫害上虽能起到作用，但也给人类带来了严重的残毒污染。农药在环境中残留，可能污染大气和水资源，破坏土壤性状；而且，在杀死害虫的同时，不少益虫也被消灭，这严重破坏了生态平衡；另外，农药的长期使用还可能引起害虫产生抗药性；因此选育抗虫水稻品种、提高水稻自身抗性被认为是一种更为环保有效的防治方法。但由于水稻品种本身抗虫资源贫乏，常规育种选育新品种的周期也较长，因此一直没有较好的克服抗虫水稻新品种选育这个难题。近年来随着细胞生物学和分子生物学的飞速发展，科学家们成功地利用生物基因工程技术将外源抗虫基因通过转化整合到水稻基因组，使水稻自身产生抗虫蛋白而达到抗虫的效果，并能够稳定遗传[Tu et al，1998和2000；唐微和杨宙等，2007]。该技术打破了物种界限，实现了基因的直接选择和有效聚合，大大提高了育种效率。
苏云金芽孢杆菌(Bacillus thuringiensis)是目前世界上应用面积最广、研究最为深入的杀虫微生物。在苏云金芽孢杆菌产胞期间，能够形成并分泌一些由杀虫晶体蛋白(insecticidal crystal proteins，ICPs或 Cry)组成的伴孢晶体，对鳞翅目(cryI)、鳞翅目和双翅目(cryII)、鞘翅目(cryIII)以及双翅目(cryIV)等昆虫，以及动植物线虫等节肢动物都有特异性的毒杀活性(Schnepf et al，1998；李长友等，2007)。但是长期单一的种植转Bt基因植物，也可能导致害虫产生抗性。近几十年来人们已经陆续发现不同的害虫对ICPs及转ICPs基因植物表现出不同水平的抗性[McGaugher et al，1985；Van，1990；Gould，1992；Lee，1995；Tabashnik et al，1997；倪万潮和郭三堆，1998；High et al，2004；Griffitts JS and Aroian RV，2005]。
近年来，人们发现Bt在营养生长期可以分泌一类新型杀虫蛋白即苏云金芽孢杆菌营养期杀虫蛋白(Vegetative Insecticidal Protein，简称VIP)，它不形成晶体，在氨基酸序列的进化上与ICPs没有任何同源性，杀虫机理与ICPs也不相同，也不存在结构相似性[Estruch et al，1996；Yu et al，1997；Estruch et al，1998；Lee et al，2003；Estela et al，2004；Rang et al，2005]，对鳞翅目和鞘翅目等多种农业害虫具有一定的杀虫活性[Estruch et al，1996；Warren et al，1998]，对某些害虫的杀虫活性达到纳克级水平[刘荣梅等，2004]；另外，对小地老虎等对ICPs不敏感的害虫也具有毒性[Estruch et al，1996；Yu et al，1997]。这对于治理对ICPs不敏感或产生抗性的农业害虫提供了一种新的选择。
VIP是苏云金芽孢杆菌在营养生长对数中期开始分泌的一类胞外毒素蛋白[Schnepf et al，1998]，在自然界中广泛存在于Bt菌中，截止到2008年，共分离发现8类37种[Crickmore，2008]。已有的研究表明，这些VIPs蛋白在遗传上相对保守，在一般条件下，它们至少有75％存在于上清培养液中，且相比较于ICPs具有热不稳定性，在95℃条件下处理20min便会失去活性[Estruch et al，1996]。
在命名系统上，VIPs可根据其蛋白质序列同源性区分为以下三个大类，即VIP1、VIP2和VIP3[Crickmore et al，2008]。VIP1和VIP2共同构成二元毒素对鞘翅目萤叶甲科昆虫具有杀虫特异性[Warren et al，1998]；VIP3对鳞翅目科、属、种中的许多害虫具有较广谱的杀虫活性，其杀虫机理研究的也比较深入[Estruch et al，1998]。到2008年，已有五十七个Vip基因被鉴定分离出来[Crickmore，2008]。
VIP1和VIP2作为二元毒素，在杀虫机理上是独自起作用的[Barth et al，2002和2004]。Vip2基因位于Vip1基因的上游，其产物能独立行 使它们各自的功能[Barth et al，2004]，但只有当两种蛋白同时存在并协同作用时，才能发挥毒素蛋白最大的杀虫毒力[Warren et al，1998]。研究表明，VIP1能与昆虫幼虫中肠上皮细胞上的受体特异性结合，并在细胞膜上形成通道，为VIP2进入靶标昆虫细胞的细胞质提供途径[Barth et al，2004]。含有NAD结合位点的VIP2则具有ADP-核糖基转移酶活性，可以将核糖基转移到肌动蛋白上，同时伴随烟酰胺的释放，导致肌动蛋白单体的多聚化受阻，影响了细胞骨架的构成，从而导致昆虫细胞死亡(Han et al，1999)。
VIP3的杀虫机理主要体现在毒素对昆虫中肠细胞的破坏[Whalon&Wingerd，2003]。全长88ku的VIP3A被鳞翅目昆虫吞食后，在其中肠胰蛋白酶作用下水解活化，活化的VIP3A蛋白能与敏感幼虫中肠的BBMVs上的80ku和100ku的未知受体分子结合，并在中肠上皮细胞上形成离子通道型穿孔，诱发昆虫细胞凋亡，细胞核溶解，最终导致昆虫死亡[Lee et al，2003]。并且VIP3A蛋白只要当pH低于7.5时即可溶解，其C′端也不被切除，同时，这2种受体也不同于已知的任何Cry受体[Lee et al，2003]；另外，在没有任何受体的情况下，VIP3A也能在人工双脂质膜(BLMs)上形成通道[Warren et al，1998；Lee et al，2003]。细胞病理学试验表明：饲喂小地老虎和草地贪夜蛾等敏感昆虫VIP3A(a)蛋白72h后，中肠上皮杯状细胞和柱状细胞与基膜完全脱落，昆虫死亡[Yu，et al，1997]。Vip3A引起的症状与ICPs的相似，但在时间上推迟了(Estruch et al，1996)。由此可见，VIP3A的受体和作用方式与ICPs明显不同，因此研究和利用VIP3A，对于拓宽杀虫谱、提高杀虫毒力、防止昆虫产生抗性具有重要意义。
ICP蛋白的分子质量一般为130～160ku，仅溶于pH＞10的高碱性溶液，以原毒素的形式存在，本身不具有毒性，被昆虫幼虫吞食后在其中肠的碱性和还原性环境下能够溶解并剪切成65～75ku的活性多肽；而VIP3A在弱碱性条件下甚至pH略低于7.5时也可被胰蛋白酶剪切成62ku大小的活性多肽(Lee et al，2003)。另外，ICP蛋白是分泌于胞内的晶体蛋白，VIP蛋白则是胞外分泌蛋白；在分泌过程中，全长为100ku的VIP1A蛋白在形成80ku的成熟毒素之前其N′端信号肽会被切除，而全长为88ku的VIP3A蛋白在分泌过程中N′端信号肽则往往因无酶切位点存在一般不被切除[Schnepf et al，1998]。在杀虫谱方面， VIP蛋白比Cry蛋白宽，对于小地老虎等对ICP蛋白不敏感的农业害虫也具有杀虫活性[Estruch et al，1996]。
目前在苏云金芽孢杆菌的自然菌株中尚未分离到能高抗水稻螟虫的Vip3基因。因此，人工合成、改造和创新抗水稻螟虫的Vip3基因资源对于解决Cry基因使用所面临的抗虫持久性问题[McGaughey，1985；Van，1990；Gould，1992；Lee，1995；Tabashnik et al，1997；倪万潮和郭三堆，1998；High et al，2004]具有深远意义。
发明概述
为解决上述问题，本申请的发明人研制出一个高抗昆虫害虫特别是抗鳞翅目害虫的Vip3A基因，尤其是具备迄今业已鉴定分离的所有其它Vip3基因所不具备的高抗水稻鳞翅目害虫如二化螟、三化螟和稻纵卷叶螟等的能力，命名为Vip3ArLr1；提供了借助农杆菌介导法转化Vip3A rLr1的质粒载体系统及其构建方案，由此提供了将感虫植物转化成为抗虫植物、将普通主栽品系/品种转化为抗虫的主栽品系/品种的育种方法。具体而言，本发明涉及下述方面：
1.一种核酸分子，其选自：
a)包括SEQ ID NO：2的核苷酸序列的核酸分子或其全长互补体；
b)编码包括SEQ ID NO：1的氨基酸序列或其片段的多肽的核酸分子；
c)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽的核酸分子；和
d)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽的核酸分子。
2.一种DNA构建体，其包括第1项的核酸分子。
3.第3项的DNA构建体，其进一步包括编码异源多肽的核酸分子。
4.一种宿主细胞，其包含第2或3项的DNA构建体，所述宿主细胞优选细菌细胞和真核生物细胞，更优选植物细胞和酵母细胞，所述植物细胞优选禾本科植物细胞，特别优选水稻(Oryza sativa L.)细胞。
5.一种转基因植物，其包括第4项的宿主细胞。
6.第5项的植物的转化的种子，其中所述种子包括第2或3项的 DNA构建体。
7.一种具有杀昆虫害虫活性的多肽，其选自：
a)包括SEQ ID NO：1的氨基酸序列或其片段的多肽；
b)包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽；和
c)包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽；或
d)由SEQ ID NO：1所示的氨基酸序列编码的多肽。
8.第7项的多肽，其进一步包括异源氨基酸序列。
9.一种组合物，其包括第7项的多肽。
10.第9项的组合物，其中所述组合物选自粉末、粉屑、小丸、颗粒、喷雾剂、乳剂、胶体和溶液。
11.第9或10项的组合物，其包括约1重量％-约99重量％的所述多肽。
12.一种用于控制昆虫害虫群体的方法，其包括使所述群体与杀虫有效量的第7项的多肽接触。
13.一种用于杀死昆虫害虫的方法，其包括使所述害虫与杀虫有效量的第7项的多肽接触，或给所述害虫喂食杀虫有效量的第7项的多肽。
14.一种用于产生具有杀昆虫害虫活性的第7项的多肽的方法，其包括在其中表达编码多肽的核酸分子的条件下，培养第4项的宿主细胞。
15.一种已将DNA构建体稳定掺入到其基因组内的植物，所述DNA构建体包括编码具有杀昆虫害虫活性的蛋白质的核苷酸序列，其中所述核苷酸序列选自：
a)包括SEQ ID NO：2的核苷酸序列的核酸分子或其全长互补体；
b)编码包括SEQ ID NO：1的氨基酸序列或其片段的多肽的核酸分子；
c)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽的核酸分子；和
d)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽的核酸分 子；
其中所述核苷酸序列与启动子可操作地连接，所述启动子驱动编码序列在植物细胞中的表达。
16.一种用于保护植物不受昆虫害虫的方法，其包括将包括第项1的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀虫多肽。
17.第16项的方法，其中所述植物产生具有针对昆虫害虫的杀虫活性的杀虫多肽。
18.一种改良植物抗逆性的方法，其包括将包括第1项的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀昆虫害虫多肽。
19.一种植物育种方法，其包括将包括第1项的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀昆虫害虫多肽。
20.根据第15-19项任一项的方法，其中所述植物优选禾本科植物，特别优选水稻。
21.根据第12-19项任一项的方法，其中所述昆虫害虫优选鳞翅目害虫，更优选水稻螟虫和斜纹夜蛾。
附图说明
图1.原核表达质粒载体pETVip3A6结构图。
图2.农杆菌质粒载体pSBVip3A6结构图。
图3.转基因抗性愈伤中Vip3ArLr1的PCR检测。图中，M：分子量标记；+：质粒阳性对照；-：空质粒阴性对照；1-10：转化的愈伤；箭头所指为目标带。
图4.Vip3ArLr1蛋白检测结果。图中，12GV-19、12GV-26、12GV-37和12GV-43为Vip3ArLr1独立转化系。
发明详述
为解决上述问题，本发明人在大量试验结果的基础上，完成了本发明。以下结合附图及实施例数据和表格对本发明进行具体说明。
截至目前为止，从苏云金杆菌天然菌株中所克隆的Vip3基因对对 鳞翅目科、属、种中的许多害虫如草地贪夜蛾、斜纹夜蛾、棉铃虫和菜青虫包括地老虎等具有高度的和专一的杀虫活性，但从未有报道表明这些天然菌株产生的Vip3基因对水稻二化螟、三化螟和稻纵卷叶螟等鳞翅目害虫具有杀虫活性。Fang J.等(2007)曾报道一个人工嵌合基因Vip3AcAa对玉米螟产生抗性，首次证实Vip3基因间的5′-端和3′-端功能域相互拼接可以产生新的抗性。Perlak FJ.等(1990)和Tu J.等(1998)先后报道由Cry1Ab和Cry1Ac形成的杂种基因可以综合Cry1Ab的高效杀虫活性和Cry1Ac的高度昆虫种属专一性，从而形成更优异的Bt杀虫蛋白基因。
本发明中的Vip3抗虫基因的研制遵循蛋白质结构与功能统一原则，在确定了现有Vip3蛋白质序列相关功能域的基础上，对其实施人工设计改良，并最终研制出可对水稻二化螟和稻纵卷叶螟等鳞翅目害虫产生抗性的基因序列(SEQ ID No1和SEQ ID No2)。
本发明的实施方案涉及用于影响昆虫害虫特别是植物害虫的组合物和方法。更具体而言，根据实施方案分离的核酸及其片段和变体包括编码杀虫多肽(例如蛋白质)的核苷酸序列。所公开的杀虫蛋白质针对昆虫害虫例如但不限于鳞翅目的昆虫害虫是生物学活性的(例如杀虫的)。目的昆虫害虫包括但不限于：水稻二化螟(Chilo suppressalis)、稻纵卷叶螟(Cnaphalocrocismedialis Guenee)、棉花斜纹夜蛾(Prodenia litura)。
实施方案的组合物包括核酸及其片段和变体，其编码杀虫多肽或其片段，包括实施方案的核苷酸序列的表达盒，分离的杀虫蛋白质和杀虫组合物。实施方案进一步提供了用这些新型核酸转化的植物和微生物，和涉及此种核酸、杀虫组合物、转化的生物及其产物在影响昆虫害虫中的使用的方法。
实施方案的核酸和核苷酸序列可以用于转化任何生物，以严生所编码的杀虫蛋白质。提供了涉及使用此种转化的生物来影响或控制植物害虫的方法。实施方案的核酸和核苷酸序列还可以用于转化细胞器。
实施方案的核苷酸序列在用于影响害虫的方法中直接有用，所述害虫特别是昆虫害虫，例如鳞翅目的害虫。因此，实施方案提供了用于影响昆虫害虫的新方法，其不依赖于传统的、合成化学杀昆虫剂的使用。实施方案涉及天然存在的、生物可降解的杀虫剂和编码其的基 因的发现。
在下文说明书中，广泛使用了许多术语。提供了下述定义以促进实施方案的理解。
单位和符号可以以其公认形式表示。除非另有说明，否则分别地，核酸以5′到3′方向从左往右书写；氨基酸序列以氨基到羧基方向从左往右书写。数字范围包括限定范围的数字。氨基酸在本文中可以通过其通常已知的三字母符号或通过IUPAC-IUB生物化学命名委员会(Biochemical Nomenclature Commission)推荐的单字母符号提及。同样地，核苷酸可以通过其通常公认的单字母编码提及。上述术语通过参考就整体而言的说明书更全面地定义。
如本文使用的，“核酸”包括提及单或双链形式的脱氧核糖核苷酸或核糖核苷酸聚合物。
如本文使用的，术语“编码”或“编码的”当在特定核酸背景中使用时，意指核酸包括指导核苷酸序列翻译成特定蛋白质的必需信息。蛋白质由其编码的信息由密码子的使用指定。编码蛋白质的核酸可以包括在核酸的翻译区内的非翻译序列(例如内含子)，或可以缺乏此种间插非翻译序列(例如如在cDNA中)。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，以指氨基酸残基的聚合物。
术语“残基”或“氨基酸残基”或“氨基酸”在本文中可互换使用，以指掺入蛋白质、多肽或肽(总起来说“蛋白质”)内的氨基酸。氨基酸可以是天然存在的氨基酸，并且除非另有限定，否则可以包含天然氨基酸的已知类似物，其可以以与天然存在的氨基酸相似的方式起作用。
实施方案的多肽可以由本文公开的核酸或使用标准分子生物学技术产生。例如，实施方案的蛋白质可以通过实施方案的重组核酸在合适宿主细胞中的表达或可替代地通过先体外后体内程序的组合产生。
说明书自始至终，单词“包括”或其变形应理解为暗示包括所述元件、部分或步骤，或元件、部分或步骤组，但不排除任何其他元件、部分或步骤，或元件、部分或步骤组。
如本文使用的，术语“影响昆虫害虫”指在任何发育阶段时实现昆虫摄食、生长和/或行为中的改变，包括但不限于：杀死昆虫；延缓 生长；阻止生殖能力；拒食剂活性等。
如本文使用的，术语“杀虫效果”、“杀虫活性”、“杀昆虫活性”和“抗虫性”同义使用，以指生物或物质(例如蛋白质)的活性，这可以通过但不限于下述进行测量：在摄食和暴露合适时间长度后的害虫死亡率、害虫重量减轻、害虫驱性以及害虫的其他行为和身体改变。因此，具有杀虫活性的生物或物质不利地影响害虫适合度的至少一个可测量参数。例如，“杀虫蛋白质”是自身或与其他蛋白质组合展示出杀虫活性的蛋白质。
如本文使用的，术语“杀虫有效量”意味着当存在于害虫的环境中时，具有杀虫活性的物质或生物的量。对于每种物质或生物，杀虫有效量对于特定环境中受影响的每种害虫以经验为根据进行测定。类似地，“杀昆虫有效量”可以用于指当害虫是昆虫害虫时的“杀虫有效量”。
如本文使用的，术语“人工设计和改造的”意味着利用重组DNA技术，以引入(例如工程改造)蛋白质结构中的变化，这基于蛋白质作用机制的理解和待引入、缺失或取代的氨基酸的考虑。
如本文使用的，术语“改善的杀昆虫活性”或“改善的杀虫活性”指实施方案的杀昆虫多肽，相对于其相应野生型蛋白质的活性，其具有增强的杀昆虫活性，和/或针对更广泛范围的昆虫有效的杀昆虫多肽，和/或对于昆虫具有特异性的杀昆虫多肽，所述昆虫对野生型蛋白质的毒性不易感。改善或增强的杀虫活性的发现要求相对于针对相同昆虫测定的野生型杀昆虫多肽的杀虫活性，显示针对昆虫靶至少10％的杀虫活性增加，或至少20％、25％、30％、35％、40％、45％、50％、60％、70％、100％、150％、200％或300％或更大的杀虫活性增加。
例如，提供了改善的杀虫或杀昆虫活性，其中相对于受野生型Bt毒素影响的昆虫范围，更广泛或更狭窄范围的昆虫受多肽影响。当需要通用性时，更广泛范围的影响可能是希望的，而当例如有益昆虫可能以其他方式受毒素的使用或存在影响时，更狭窄范围的影响可能是希望的。
相对于背景序列，由包括突变的核苷酸序列编码的多肽将包括至少一个氨基酸改变或添加，或2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、 28、29、30、32、35、38、40、45、47、50、60、70或80个或更多个氨基酸改变或添加。多肽的杀虫活性还可以通过对全长序列的平截加以改善，如本领域已知的。
在特定实施方案中，实施方案的杀虫蛋白质提供了全长杀昆虫多肽、全长杀昆虫多肽的片段、和通过设计为将特定氨基酸序列引入实施方案的多肽内的诱变处理的核酸产生的变体多肽。
突变可以置于任何背景序列内，包括此种截短的多肽，只要多肽保留杀虫活性。使用本领域已知或本文其他地方描述的测定，本领域技术人员可以关于杀虫活性容易地比较2种或更多种蛋白质。应当理解实施方案的多肽可以通过本文公开的核酸的表达或通过使用标准分子生物学技术产生。
核苷酸和由其编码的氨基酸序列及多肽的片段和变体也由实施方案包含。如本文使用的，术语“片段”指实施方案的多核苷酸的核苷酸序列的部分或多肽的氨基酸序列的部分。核苷酸序列的片段可以编码保留全长蛋白质的生物学活性且因此具有杀虫活性的蛋白质片段。因此，认识到实施方案的一些多核苷酸和氨基酸序列可以被正确地称为片段和突变体。
应当理解术语“片段”当其用于指实施方案的核酸序列时，还包含作为杂交探针有用的序列。这类核苷酸序列一般不编码保留生物学活性的片段蛋白质。因此，核苷酸序列的片段可以范围为至少约20个核苷酸、约50个核苷酸、约100个核苷酸、和最高达编码实施方案的蛋白质的全长核苷酸序列。
术语“变体”在本文中用于指基本上相似的序列。对于核苷酸序列，保守变体包括由于遗传密码的简并性，编码实施方案的杀虫多肽之一的氨基酸序列的那些序列。
变体核苷酸序列还包括合成衍生的核苷酸序列，例如通过使用位点定向诱变生成，但仍编码实施方案的杀虫蛋白。一般地，实施方案的特定核苷酸序列的变体将与那种特定核苷酸序列具有至少约70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性。实施方案的核苷酸序列的变体可以与原始序列相差至1-240个核苷酸，少至1-100，少至1-10，少至5、4、3、2或甚至1个核苷酸。
实施方案进一步包含用实施方案的至少一种核酸、包括核酸的表达盒、或包括表达盒的载体转化的微生物。在一些实施方案中，微生物是在植物上繁殖的那种。实施方案提供了包括实施方案的转化的微生物的杀虫组合物。在此种实施方案中，转化的微生物一般连同合适载体一起，以杀虫有效量存在于杀虫组合物中。实施方案还包含杀虫组合物，其包括连同合适的载体一起，以杀昆虫有效量，单独或与实施方案的转化的生物和/或实施方案的杀虫蛋白质组合的实施方案的分离的蛋白质。
实施方案还包含包括实施方案的至少一种核苷酸序列的转化的或转基因植物。在一些实施方案中，植物用核苷酸构建体稳定转化，所述核苷酸构建体包括与启动子可操作地连接的实施方案的至少一种核苷酸序列，所述启动子驱动在植物细胞中的表达。如本文使用的，术语“转化的植物”和“转基因植物”指在其基因组内包括异源多核苷酸的植物。一般地，异源多核苷酸在转基因或转化的植物的基因组内稳定整合，从而使得多核苷酸传递给连续世代。异源多核苷酸可以单独或作为重组表达盒的部分整合到基因组内。
应当理解如本文使用的，术语“转基因的”包括任何细胞、细胞系、愈伤组织、组织、植物部分或植物，其基因型已通过异源核酸的存在加以改变，包括最初如此改变的那些转基因生物以及通过有性杂交或无性繁殖由最初转基因生物产生的那些。如本文使用的，术语“转基因的”不包含通过常规植物育种方法或通过天然存在的事件的基因组改变(染色体或染色体外的)，所述天然存在的事件例如随机异体受精、非重组病毒感染、非重组细菌转化、非重组转座或自发突变。
如本文使用的，术语“植物”包括全植物、植物器官(例如叶、茎、根等)、种子、植物细胞及其后代。转基因植物的部分在实施方案的范围内，并且包括例如源于先前用实施方案的DNA分子转化的转基因植物或其后代且因此至少部分由转基因细胞组成的植物细胞、原生质体、组织、愈伤组织、胚以及花、茎、果实、叶和根。
如本文使用的，术语植物包括植物细胞、植物原生质体、植物可以由其再生的植物细胞组织培养物、植物愈伤组织、植物块和植物细胞，其在植物或植物的部分中是完整的，例如胚、花粉、胚珠、种子、叶、花、枝、果实、仁、穗、穗轴、外壳、茎、根、根尖、花药等。
虽然实施方案不依赖于特定生物学机制用于增加植物对植物害虫的抗性，但实施方案的核苷酸序列在植物中的表达可以导致实施方案的杀虫蛋白质的产生和植物对植物害虫的抗性中的增加。实施方案的植物在农业中在用于影响昆虫害虫的方法中有用。
因此，实施方案的蛋白质可以以各种方式加以改变，包括氨基酸取代、缺失、平截和插入。用于此种操作的方法是本领域一般已知的。例如，杀虫蛋白质的氨基酸序列变体可以通过将突变引入合成核酸(例如DNA分子)内进行制备。
实施方案的诱变处理的核苷酸序列可以如此修饰，以便改变所编码的多肽的一级序列中存在的约1、2、3、4、5、6、8、10、12个或更多个氨基酸。可替代地，来自自然序列的甚至更多改变可以这样引入，从而使得与相应原始蛋白质相比较，所编码的蛋白质可以具有至少约1％或2％，或约3％、4％、5％、6％、7％、8％、9％、10％或更多改变或以其他方式修饰的密码子。
本领域技术人员可认识到氨基酸添加和/或取代一般基于氨基酸侧链取代基的相对相似性，例如其疏水性、电荷、大小等。考虑各种前述特征的示例性氨基酸取代组是本领域技术人员众所周知的，并且包括：精氨酸和赖氨酸；谷氨酸和天冬氨酸；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。
因此，实施方案的基因和核苷酸序列包括天然存在的序列和突变体形式。同样地，实施方案的蛋白质包含人工改造的蛋白质和其变异(例如截短的多肽)及修饰(例如突变体)形式。此种变体将继续具有所需杀虫活性。本文包含的蛋白质序列的缺失、插入和取代不期望产生蛋白质特征中的根本改变。然而，当在这样做之前难以预测取代、缺失或插入的确切作用时，本领域技术人员将认识到作用将通过常规筛选测定例如昆虫摄食测定进行评价。
变体核苷酸序列和蛋白质还包含衍生自诱变和重组程序例如DNA改组的序列和蛋白质。用此种程序，一个或多个不同编码序列可以进行操作，以产生具有所需性质的新杀虫蛋白质。以这种方式，由包括序列区域的相关序列多核苷酸群体生成重组多核苷酸的文库，所述序列区域具有基本序列同一性，并且可以在体外或体内同源重组。例如，使用这种方法，全长编码序列、编码目的结构域的序列基序、或实施 方案的核苷酸序列的任何片段可以在实施方案的核苷酸序列和其他已知Vip核苷酸序列的相应部分之间改组，以获得编码具有改善的目的性质的蛋白质的新基因。
实施方案进一步涉及实施方案的转化的植物的植物繁殖材料，包括但不限于种子、块茎、球茎、鳞茎、叶以及根和枝条的插条。
实施方案可以用于转化任何植物物种，包括但不限于单子叶植物和双子叶植物。
实施方案的组合物以多种方式在保护植物、种子和植物产物中有用。例如，组合物可以在涉及将有效量的杀虫组合物置于害虫环境中的方法中使用，其通过选自喷雾、撒粉、撒播或种子涂敷的程序实现。
在植物繁殖材料(果实、块茎、鳞茎、球茎、谷粒、种子)但尤其是种子作为商业产物销售前，它照例用保护剂涂料进行处理，所述保护剂涂料包括除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂或这些制剂中的几种的混合物，若需要则连同在配制领域中照例采用的进一步载体、表面活性剂或施用促进佐剂一起，以提供针对由细菌、真菌或动物害虫引起的损害的保护。为了处理种子，通过用液体制剂浸渗块茎或谷粒或通过用组合的湿或干制剂涂敷其，保护剂涂料可以应用于种子。此外，在特定情况下，对于植物的其他应用方法是可能的，例如针对芽或果实的处理。
编码实施方案的杀虫蛋白质的基因可以经由合适载体引入微生物宿主内，并且将所述宿主应用于环境、或植物或动物。在将核酸插入细胞内的背景中的术语“引入的”意指“转染”或“转化”或“转导”，并且包括提及核酸掺入真核或原核细胞内，其中核酸可以掺入细胞的基因组(例如染色体、质粒、质体或线粒体DNA)内，转变成自主复制子或瞬时表达(例如转染的mRNA)。
许多方法可用于在允许基因稳定维持和表达的条件下将表达杀虫蛋白质的基因引入微生物宿主内。例如，可以构建表达盒，其包括与用于表达核苷酸构建体的转录和翻译调节信号可操作地连接的目的核苷酸构建体，和与宿主生物中的序列同源的核苷酸序列，由此将发生整合，和/或在宿主中起作用的复制系统，由此将发生整合或稳定维持。转录和翻译调节信号包括但不限于启动子、转录起始开始位点、操纵基因、激活剂、增强子、其他调节元件、核糖体结合位点、起始密码 子、终止信号等。
在实施方案中，转化的微生物(其包括完整生物、细胞、一种或多种孢子、一种或多种杀虫蛋白质、一种或多种杀虫组分、一种或多种害虫影响组分、一种或多种突变体、活或死细胞和细胞组分，包括活和死细胞和细胞组分的混合物，并且包括破碎的细胞和细胞组分)或分离的杀虫蛋白质可以与可接受的载体一起配制成一种或多种杀虫组合物，其是例如悬浮液、溶液、乳剂、撒粉粉末、可分散颗粒或小丸、可湿性粉末和可乳化浓缩物、气溶胶或喷雾剂、浸渗颗粒、佐剂、可包被糊剂、胶体以及例如聚合物物质中的胶囊化。此种配制的组合物可以通过此种常规方法进行制备，例如包含多肽的细胞培养物的脱水、冻干、匀浆、提取、过滤、离心、沉降或浓缩。
上文公开的此种组合物可以通过添加下述获得：表面活性剂、惰性载体、防腐剂、湿润剂、摄食刺激剂、引诱剂、被囊化剂、粘合剂、乳化剂、染料、UV保护剂、缓冲剂、流动剂或肥料、微量营养物供体或影响植物生长的其他制剂。一种或多种农用化学品包括但不限于除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂、杀螨剂、植物生长调节剂、落叶剂(harVest aid)和肥料，可以与在配制领域中照例采用的载体、表面活性剂或佐剂或其他组分组合，以促进产物处理和应用于特定靶害虫。合适的载体和佐剂可以是固体或液体，并且对应于在配制技术中通常采用的物质，例如自然或再生矿物质、溶剂、分散剂、湿润剂、增粘剂、粘合剂或肥料。实施方案的活性成分正常以组合物的形式应用，并且可以应用于待处理的作物区域、植物或种子。例如，实施方案的组合物可以应用于在用于准备粮仓或地窑等或在粮仓或地窖等中的贮存过程中的谷物。实施方案的组合物可以与其他化合物同时或连续应用。应用实施方案的活性成分或实施方案的农用化学品组合物(其包含通过实施方案的细菌菌株产生的至少一种杀虫蛋白质)的方法包括但不限于叶应用、种子涂敷和土壤应用。应用数目和应用比率依赖于通过相应害虫的侵扰强度。
实施方案的组合物可以以合适形式用于直接应用或作为基本组合物的浓缩物，这在应用前需要用合适量的水或其他稀释剂稀释。杀虫浓度将依赖于特定制剂的性质而改变，特别地依赖于它是浓缩物还是待直接使用。
在害虫已开始出现时或在害虫出现前作为防护措施，通过例如喷雾、雾化、撒粉、播散、涂敷或倾注、引入土壤内或上、引入灌溉水内、通过种子处理或一般应用或撒粉，组合物(包括实施方案的转化的微生物和杀虫蛋白质)可以应用于昆虫害虫的环境。例如，实施方案的杀虫蛋白质和/或转化的微生物可以与谷物混合，以保护在贮存过程中的谷物。一般重要的是获得在植物生长的早期阶段中害虫的良好控制，因为这是植物可以被最严重损害的时间。实施方案的组合物可以方便地包含另一种杀昆虫剂，如果认为这是必要的话。在一个实施方案中，组合物在种植时以组合物的颗粒形式直接应用于土壤，所述组合物具有载体和实施方案的芽孢杆菌属菌株或转化的微生物的死细胞。另一个实施方案是组合物的颗粒形式，所述组合物包括农用化学品例如除草剂、杀昆虫剂、肥料、惰性载体、和实施方案的芽孢杆菌属菌株或转化的微生物的死细胞。
本领域技术人员将认识到并非所有化合物在针对所有害虫中是同等有效的。实施方案的化合物展示出针对昆虫害虫的活性，这可以包括经济上重要的农艺学、森林、温室、苗圃、观赏植物、食物和纤维、公共和动物健康、家用和商业结构、家庭和贮存产物害虫。
广泛多样的生物测定技术是本领域技术人员已知的。一般程序包括将实验化合物或生物添加到封闭容器中的饮食来源。杀虫活性可以通过但不限于下述进行测量：在摄食和暴露合适时间长度后的死亡率中的改变、重量减轻、吸引力、驱性以及其他行为和身体改变。本文描述的生物测定可以对于幼虫或成虫期中的任何摄食昆虫害虫使用。
实施例
本发明的以下实施例所使用的分子生物学技术和方法均为目前比较成熟的技术。其中大部分技术是本实验室常用的核心技术，另外该技术在Current Protocols in Molecular Biology和Molecular Cloning：A Labortory Manual等文献中均有详细记载。下述实施例为了举例说明而不是限制而呈现。
实施例1：原核表达和包含体抽提
将改良型Vip3编码序列构建到原核表达载体pET22b上(图1)，利用大肠杆菌DH5α菌株进行原核表达和包含体抽提。具体步骤如下： 取单克隆菌落小养过夜，再大养50ml至OD600达0.4-0.6；取1ml培养液于13000rpm条件下离心收集1min，再用30ul水溶解；之后，加入IPTG至1mM，培养6hr；再将培养液于4℃和8000rpm条件下，离心10min，去上清；用10ml 1×PBS重悬沉淀，加入溶菌酶10mg，25℃处理30min；再加入蛋白酶抑制剂PMSF至终浓度为1mM，于冰上超声破碎(200W，30S超声，30S间隔，30个循环)；之后，加入TritonX-100，至终浓度为1％，室温混合30min；接着，于4℃和12000rpm条件下离心10min；之后，将离心收集的沉淀用包涵体蛋白纯化缓冲液洗涤一次，再用1/50菌液的包涵体蛋白纯化缓冲液悬浮沉淀，按1mg/ml加入溶菌酶，于25℃水浴30min；之后，加入PMSF至1mM，800W超声破碎10min；接着，用核酸酶S1剪切大肠杆菌DNA，并于4℃和5000rpm条件下离心10min，去上清后，用25ml包涵体蛋白纯化缓冲液洗涤沉淀3次，所得包含体保存于-20℃冰箱备用。
实施例2：Vip3蛋白的抗虫性鉴定
Vip3蛋白的抗虫性鉴定按人工饲喂法进行。养虫所用的人工饲料参照韩兰芝、侯茂林、彭于发等(2009)发明的配方进行配置。接种时，将饲料分割成边长2cm正方形或者将配成的人工饲料未冷却时放入24孔板中，同时将各待测包含体蛋白样按预备试验确定的靶标害虫致死剂量稀释到640μg/ml，之后，取100ul涂在饲料上，风干，然后每个饲料块放5头虫，并设置3个重复。然后，用半透膜覆盖，连续记录7天，观察其生存状况。试验结果表明：新的编码序列表达的Vip3蛋白对二化螟呈现良好的杀虫效果，在3次重复饲喂该蛋白的总计45头3龄二化螟幼虫中有33头死亡，12头成活，统计后的平均死亡率达0.73±0.11；而空质粒载体抽提液、蛋白稀释缓冲液和清水对照重复饲喂的二化螟幼虫死亡率仅为0.20±0.00、0.15±0.07和0.20±0.00。类似的实验结果也在斜纹夜蛾的饲喂中观察到，且其抗虫性表现更佳，饲喂的斜纹夜蛾幼虫死亡率达到0.95±0.12。该结果因此证实研制的新的Vip3蛋白不仅针对水稻二化螟产生了抗性，而且和其它许多天然的Vip3蛋白一样对棉花斜纹夜蛾也具有高度抗性。这是迄今为止第一例自然条件下不存在的源自设计改良的对水稻二化螟具有抗虫活性的Vip3新基因。
进一步的序列比对分析表明，人工设计合成的新的VIP3A蛋白与 已知的VIP3A类杀虫蛋白的序列一致性最高达93％，因此，在分类上可将其基因归为Vip3A类。另外，考虑到该蛋白的设计改良导致了对水稻鳞翅目害虫如二化螟抗性的产生，为此，我们将该生产该蛋白的基因正式定名为Vip3ArLr1(Vip3A class and rice Lepidoptera resistance)。
实施例3：Vip3ArLr1农杆菌质粒载体的构建和大肠杆菌转化
本实施例用于Vip3ArLr1农杆菌载体构建的质粒为pSB130actin-nos(由本实验室改造的空载体)，由双转移DNA(T-DNA)区组成。构建双T-DNA质粒载体的目的在于，转化水稻后使目的基因和标记基因在受体基因组中有机会发生独立整合，以便于标记基因能在随后的分离世代中通过自交方式分离和剔除。驱动目的基因表达的启动子为水稻肌动蛋白actinI启动子，大小为0.839kb，其供体生物为水稻；而终止子则为人工合成的nosT，大小为0.271kb，用于终止转录和指导信使RNA(mRNA)的多聚腺苷酸化(Fraley et al，1983)；用于质粒自身筛选的标记基因为抗卡那霉素基因kan+。
用于转化体筛选的标记基因为潮霉素磷酸转移酶基因(hph)，大小为1.026kb，供体生物是大肠杆菌。该标记基因在载体设计时被构建在与目的基因不同的T-DNA区。构建的质粒载体命名为pSBVip3A6，其结构图见图2。
Vip3ArLr1质粒载体构建的具体步骤如下：
3.1 Vip3ArLr1连接片段的制备
利用PCR技术从pET11a原核表达载体中扩增获得Vip3ArLr1连接片段，在扩增的同时引入下一步连接到农杆菌质粒载体多克隆位点的酶切位点，所用的PCR扩增引物和反应体系见表1，所用的PCR反应程序为94℃变性5min，接着35个循环的95℃变性15sec，55℃退火30sec和72℃延伸2min，再经72℃延伸10min后于10℃温育保存。
表1 PCR反应体系
Figure PCTCN2014001029-appb-000001
扩增完毕后，利用1％的琼脂糖凝胶对PCR产物进行电泳分离，之后，按Axygen公司的凝胶回收试剂盒操作说明回收目标DNA片段。具体程序是：在紫外灯下将琼脂糖凝胶中的目标DNA片段切割下来，用吸水纸将其表面液体吸尽，并切碎装入1.5ml离心管中，置于天平测量凝胶的重量，该重量作为一个凝胶体积(如100mg＝100μl)；然后向离心管中加入3倍凝胶体积的DE-A缓冲液，置于72℃恒温振荡仪于250rpm条件下振荡15min；待凝胶融化后再加入1.5倍凝胶体积的DE-B缓冲液，混匀；将上述混合液转入DNA制备管(置于2ml离心管中)，于12500rpm离心1min，弃上清；加500μl洗涤缓冲液W1，于12500rpm离心1min，弃上清；加700μl洗涤缓冲液W2，12500rpm离心1min，弃上清，该步骤重复一次；再于12500rpm空离1min；向DNA制备膜中央加入60℃预热的去离子水25μl，离心收集DNA，-30℃保存。
3.2 Vip3ArLr1纯化DNA片段和农杆菌双T-DNA质粒(pSB130actin-nos)片段的双酶切
应用XbaI和SmaI限制性内切酶对纯化后的Vip3ArLr DNA片段和农杆菌双T-DNA质粒(pSB130actin-nos)片段进行双酶切。酶切反应体系见表2。
表2 酶切反应体系
Figure PCTCN2014001029-appb-000002
双酶切后，使用Axygen公司AxyPrep PCR清洁试剂盒纯化PCR产物，操作步骤如下：在PCR反应液中，加3倍体积的PCR-A缓冲液(若PCR-A缓冲液不足100μl，加至100μl)；混匀后，转移到制备管中，将制备管置于2ml离心管中，12000rpm离心1min，弃上清；将制备管置回2ml离心管，加700μl洗涤缓冲液W1(使用前须确认在洗涤缓冲液W1中已按试剂瓶上的指定体积加入了无水乙醇)，12000rpm离心1min，弃上清；将制备管置回2ml离心管，加400μl洗涤缓冲液W2，12000rpm离心1min(此为可选步骤，同时注意从离心机中取出2ml离心管时，不要让管底的洗涤缓冲液W2接触到制备管)；将制备管置于洁净的1.5ml离心管(试剂盒内提供)中，在制备管膜中央加25-30μl去离子水，室温静置1min，12000rpm离心1min，以洗脱DNA并进入下一步骤。
3.3 Vip3ArLr1 DNA片段与农杆菌双T-DNA质粒(pSB130actin-nos)片段的连接
首先对回收的载体片段及目的基因进行浓度测定，然后按1∶3的比率加入到50μl的无菌离心管中，并在4℃条件下连接过夜，所用的连接体系见表3。
表3 连接体系
Figure PCTCN2014001029-appb-000003
3.4大肠杆菌转化
本发明中用作克隆受体的菌株为大肠杆菌菌株DH5α，转化方法 为热击法。转化的具体程序是：吸取10μl连接产物加入到80μl的大肠杆菌DH5α感受态细胞中，并用移液枪吹打均匀，于冰浴上放置30min，42℃热击90s后，迅速置回冰浴上静置2min；再向每管加入1ml的LB液体培养基，于37℃恒温振荡仪振荡(250rpm)1h；待菌液复苏后，离心并吸除800μl上清液，将剩下的200μl上清液与菌块混匀，均匀的涂布于LB固体筛选培养基(50mg/l的卡那霉素)表面，37℃过夜培养。
3.5阳性菌落鉴定
挑选透明单菌落并用牙签沾取作为模板，置于PCR反应体系中进行扩增鉴定。PCR反应体系与反应程序均同前。挑选PCR鉴定为阳性的菌落摇菌，按Axygen公司的质粒纯化试剂盒提取质粒，并对质粒进行酶切验证，待结果肯定后再取10μl质粒送上海英俊生物技术公司作进一步的测序验证，一旦结果肯定即表明Vip3ArLr1载体质粒构建完成。
实施例4：农杆菌介导的遗传转化
4.1 Vip3ArLr1载体质粒的农杆菌转化
Vip3ArLr1载体质粒对农杆菌的转化应用电击转化法进行，所用的农杆菌菌株为EHA105。具体程序是：取0.5μlVip3ArLr1载体质粒，加入到含有60μl农杆菌EHA105电击感受态细胞的1.5ml离心管中，待枪头吸打混匀后移入电极杯中；电击后，迅速加入1ml的LB液体培养基，吸打混匀后移入先前的1.5ml离心管中，于28℃恒温振荡仪上振荡培养1h；菌液复苏后，吸取100μl菌液，均匀涂布于LB固体筛选(含50mg/l的卡那霉素、25mg/l的利福平)培养基表面，28℃培养2天；菌落PCR验证阳性克隆后，对阳性克隆摇菌保存菌液(50％的甘油浓度，-80℃保存)，备用。
4.2水稻转化
水稻转化按刘巧泉等人(2004)报道的方法步骤进行。其具体程序是：取出保存于-80℃农杆菌菌液，从中吸取200μl均匀涂布于到含有25mg/l利福平和50mg/l卡那霉素的LB固体培养基表面，于28℃条件下培养过夜；再从中挑单菌落扩大培养，所用液体培养基同前；之后，从中吸取200-300μl的新鲜菌液接入到20ml含有25mg/l利福平和50mg/l卡那霉素的LB液体培养基中，28℃振荡(220rpm)培养 16-18h。取足量的菌液于4000rpm下离心15min，弃去LB培养基上清液；加入20ml 0.1M MgSO4溶液重新悬浮农杆菌(用移液枪轻轻吹打)，于4000rpm下离心10-15min，弃去含有抗生素的MgSO4上清液；再加入5ml含有200μM乙酰丁香酮(Acetosyringone，AS)的AA-AS侵染培养基重新悬浮农杆菌，再加入适量的AA-AS侵染培养基，使菌液的OD600值最终调整在0.8-1.0之间；浓度调整后，用无菌的50ml离心管分装菌液，20-25ml/管，待用。
将预培养7天左右的日本晴胚性愈伤从继代培养皿中转移至覆有无菌滤纸的空培养皿中，在超净工作台上风干10-15min左右，期间用灭菌过的小勺缓缓翻滚愈伤使之充分干燥；待其干燥后，移入盛有菌液的离心管，在室温下轻轻摇晃(不可太剧烈)40min，将该离心管于超净工作台上静置10min；弃去菌液，将胚性愈伤置于无菌滤纸上干燥15min左右，然后，将其转移至表面以无菌滤纸覆盖的含有200μM乙酰丁香酮的CC-AS共培养培养基上，于28℃条件下暗培养50-55h；挑选表面农杆菌未大量生长或未污染的胚性愈伤，移至含有2.0mg/L的2，4-D，500mg/l头孢霉素的N6抑菌培养基上，28℃暗室中抑菌培养3-4d；再将抑菌培养后的愈伤移至含有500mg/l头孢霉素和65mg/L潮霉素的N6筛选培养基上，28℃暗室培养；在最初的一周内，每天都要检查农杆菌污染情况，若污染控制不住，需及时更换筛选培养基，每隔半月挑选生长状态良好的愈伤于新鲜筛选培养基上继代，并根据农杆菌自身污染的程度来调整培养基中头孢霉素的浓度，一般情况下至第三或第四轮继代筛选时可考虑将其浓度减半。
依照上述程序先后进行了3次重复转化，结果从101个用于转化的愈伤中，共获得35个独立转化体。
4.3转基因抗性愈伤的PCR鉴定
4.3.1抗性愈伤基因组DNA的抽提
从继代培养的每个独立转化体上称取0.2-0.3g的转基因抗性愈伤，置于洗净并经酒精燃烧灭菌的研钵中，加入600μl的1.5×CTAB抽提液研磨至匀浆，转移匀浆至1.5ml离心管中，置于60℃水浴25min(期间拿出来反复颠倒2次)，之后加入400μl的氯仿，上下颠倒数次充分混匀后室温下离心6min(13000r/min)；吸取400μl上清到新的离心管，加入800μl的的无水乙醇，混匀后于-30℃中放置30min以上， 13000rpm和室温下离心5min后弃去上清，再用75％乙醇浸洗DNA沉淀，弃去乙醇并置于室温晾干，之后用100μl无菌水溶解过夜。
4.3.2转基因抗性愈伤中Vip3ArLr1的PCR检测
转基因抗性愈伤中Vip3ArLr1的分子检测是利用常规PCR技术进行的。所用的检测引物为：vip-F，5′-GCTGTTAT GCGGCCATTGTC-3′和vip-R，5′-GACGTCTGTCGAGAAGTTTC-3′，扩增出的目的片段大小为300bp左右，其PCR反应体系见表3-4，所用的PCR反应程序为94℃变性5min，接着35个循环的95℃变性15sec，55℃退火30sec和72℃延伸30sec，再经72℃延伸10min后于10℃温育保存。所得PCR扩增产物经0.8％琼脂糖凝胶电泳分离，拍照后保存。
检测结果显示：筛选至第3-4的转基因抗性愈伤中有30-70％对Vip3ArLr1呈阳性(图3)，表明目的基因已经整合到受体细胞中。
4.4 Vip3ArLr1阳性愈伤的分化
Vip3ArLr1阳性愈伤的分化按Yang R et al(2011)等人报道的方法进行。其具体程序是：将目的基因Vip3ArLr1呈阳性的抗性愈伤转移至N6分化培养基(N6基本培养基+2mg/l Kinetin+1mg/l NAA+4％Gelrite)上，于28℃暗室中预分化7-9天，再转接到新鲜的分化培养基上，于25℃光室中进行绿苗的分化(一般7-14天后可见分化绿点，3周后可分化成绿苗)。所获得的绿苗，洗净粘附于根系上的培养基后，直接(根芽同时分化型)或经生根培养基壮根后(芽先分化型)移入Yoshida培养液中过渡培养，待其生长状态良好与稳定后，再移栽到温室，直至成熟。
实施例5：阳性转基因植株Vip3ArLr1蛋白检测
阳性转基因植株Vip3ArLr1蛋白用上海佑隆公司的Vip试纸条进行检测分析。水稻叶片中的蛋白质抽提和试纸检测按产品说明书中描述的步骤进行。
具体步骤如下：取长2-3cm左右的转基因水稻叶片置于研钵中，加入1ml的蒸馏水或市售纯净水，经充分研磨后，用移液枪移入1.5ml离心管中，经12000rpm离心30s后，再将上清液转移至另一1.5ml离心管。然后放入试纸条，5min后观察试纸条的显色情况。
结果显示阳性转基因植株的蛋白样本中均有目标条带(图4)，但检测信号的强弱随转化体的不同而有变化，这一方面说明Vip3ArLr1 基因在受体基因组中能正常表达，另一方面也显示不同转化体(如图4中12GV-26和12GV-43)的Vip3ArLr1蛋白表达水平可能因其插入位点的不同或拷贝数目的差异而有显著差异。
实施例6：Vip3ArLr1转基因系的抗虫性鉴定
接虫鉴定所用的植物材料是前述DNA和蛋白质检测呈阳性的独立转化系，接虫鉴定方法采用自然发虫和人工接虫鉴定法，接虫鉴定的指标有枯心率、白穗率、卷叶率和平均校正死亡率。自然发虫鉴定主要是针对稻纵卷叶螟进行；人工接虫鉴定法是针对二化螟和稻纵卷叶螟进行。人工接虫鉴定法包括离体叶片法、离体茎秆法、单株抗虫性鉴定法、大田人工接虫法。
离体叶片法：在水稻分蘖中期，随机从各供试株系材料的稻株上，剪取3～4cm长的来自不同分蘖倒一叶的叶片3片，重复鉴定3株/供试株系材料。之后，在叶片两端压上用0.1g/L苯骈眯唑保鲜液浸渍过的小滤纸片，并将其移入长度为9.5cm、内径为1.5cm的小平底玻璃试管内，然后每管分别接入12头二化螟或稻纵卷叶螟一龄幼虫，管口塞入脱脂棉，置于28℃光照条件下保温饲养，4天后向管内添加新鲜供试叶片2～3片，7天后检查、记录和统计供试幼虫(简称试虫，下同)存活情况，并依此计算各供试株系材料的试虫死亡率，之后，参照感虫对照的平均试虫死亡率计算各供试株系材料上的试虫校正死亡率，其算式是：校正死亡率％＝(供试株系材料试虫死亡率一感虫对照试虫平均死亡率)/(1一感虫对照试虫平均死亡率)×100。再根据表4所列离体叶片法抗虫性鉴定分级标准评价供试材料对靶标害虫即二化螟和/或稻纵卷叶螟的抗性。
表4.离体叶片法抗虫性鉴定分级标准：
Figure PCTCN2014001029-appb-000004
离体茎秆法：每供试株系材料取分蘖盛期的主分蘖稻苗2根，把稻苗擦干，将2根稻苗截取成包含节和叶鞘的5cm茎秆2根，随后在茎秆两端压上用0.1g/L苯骈眯唑保鲜液浸渍过的小滤纸片，并将其移 入长度为9.5cm、内径为1.5cm的小平底玻璃试管内。然后，每试管接入12头二化螟一龄幼虫，管口塞入脱脂棉。3天后在茎秆两端增加用0.1g/L苯骈眯唑保鲜液浸渍过的小滤纸片确保茎秆湿润。7天后剥检、记录幼虫存活数，必要时称量每个玻璃管的活虫体质量。评价标准同离体叶片法。
单株抗虫性鉴定法：将带2个分蘖的供试稻株移栽于塑料桶中，每株接入10头二化螟一龄幼虫，并置于塑料网罩(直径12cm，高70cm)内，2周后移去网罩，各供试材料重复鉴定20株。接虫后第15天和第30天调查各供试株系材料之稻株受害后的枯心表型，并按下式计算供试株系材料的校正枯心率％：供试株系材料枯心率％＝(供试株系材料枯心率一感虫对照枯心率)/(1一感虫对照枯心率)×100。
大田人工接虫法：主要在分蘖期和孕穗期两个时期进行。前者是在苗高约15cm左右时接二化螟二龄幼虫，后者是在抽穗前7-10天接二化螟初孵幼虫，15头/株，10-30株/系，重复三次，接虫15-21天后调查枯心率和白穗率，并籍此判断所转基因的大田抗虫效果。
试验结果证实Vip3ArLr1的4个独立转化系及其相应姐妹系无论是对人工接虫的二化螟还是对自然发生的稻纵卷叶螟均显示高度抗性。如表4-2至表4-5所示，2012年人工接种的二化螟二龄幼虫在Vip3ArLr1的4个独立转化系上危害的白穗率都极显著地低于在对照基因Vip3Aa1转化系上危害造成的白穗率，其中，前3个转化系的白穗率为0，后1个转化系的白穗率为6.64％(表5)。重复鉴定于2013年用前述独立转化系的姐妹系的叶片、茎秆和植株进行，并增加了亲本和感虫对照品系。所接种的二化螟除在独立转化系的姐妹系13GV-38叶片及茎秆上的平均校正死亡率低于或略高于日本晴对照和Vip3Aa1的转化系以外，在其余三个转化系上的平均校正死亡率都达到66％以上，极显著地高于在亲本和感虫对照品系上的平均校正死亡率(表6和表7)；所接种的二化螟在Vip3ArLr1的4个独立转化系植株上危害造成的枯心率也都极显著地低于在对照基因Vip3Aa1转化系上危害造成的枯心率(表8)。
表5.Vip3ArLr1和对照Vip3Aa1转化系对人工接虫的二化螟的抗性鉴定(2012年)。
Figure PCTCN2014001029-appb-000005
表6.Vip3ArLr1和对照Vip3Aa1转化系叶片对人工接虫的二化螟的抗性鉴定(2013年)。
Figure PCTCN2014001029-appb-000006
表7.Vip3ArLr1和对照Vip3Aa1转化系茎秆对人工接虫的二化螟的抗性鉴定(2013年)。
Figure PCTCN2014001029-appb-000007
表8.Vip3ArLr1和对照Vip3Aa1转化系植株对人工接虫的二化螟的抗性鉴定(2013年)。
Figure PCTCN2014001029-appb-000008
表9至表11是4个独立转化系及其相应姐妹系对自然爆发和人工接虫的稻纵卷叶螟的抗性鉴定结果，其自然爆发的卷叶率和人工接虫的平均校正死亡率除转化系12GV-43及其相应姐妹系13GV-38分别达到12.5％和不大于20％以外，其余3个独立转化系及其相应姐妹系的这两项指标分别为2％以内和100％，与亲本和感虫对照品系的差异极显著。至此，转Vip3ArLr1基因抗虫水稻新种质创制成功。进一步以该种质为亲本，利用传统的回交育种程序加上现代的分子标记辅助选择还可进行其衍生系的选育，并最终选育出优良的转基因抗虫新品系。
表9.Vip3ArLr1和对照Vip3Aa1转化系对自然发生的稻纵卷叶螟的抗性鉴定(2012年)。
Figure PCTCN2014001029-appb-000009
表10.Vip3ArLr1和对照Vip3Aa1转化系对自然发生的稻纵卷叶螟的抗性鉴定(2013年)。
Figure PCTCN2014001029-appb-000010
表11.Vip3ArLr1和对照Vip3Aa1转化系叶片对人工接虫的稻纵卷叶螟的抗性鉴定(2013年)。
Figure PCTCN2014001029-appb-000011
最后，还需注意的是，以上列举的仅是本发明的若干个具体实施例。客观上，本发明并不仅仅只限于以上实施例，还可以有相当一部分变化。因此，本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变化，均应当认为是本发明的保护范围。
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Claims (19)

  1. 一种核酸分子，其选自：
    a)包括SEQ ID NO：2的核苷酸序列的核酸分子或其全长互补体；
    b)编码包括SEQ ID NO：1的氨基酸序列或其片段的多肽的核酸分子；
    c)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽的核酸分子；和
    d)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽的核酸分子。
  2. 一种DNA构建体，其包括权利要求1的核酸分子。
  3. 权利要求3的DNA构建体，其进一步包括编码异源多肽的核酸分子。
  4. 一种宿主细胞，其包含权利要求2或3的DNA构建体，所述宿主细胞优选细菌细胞和真核生物细胞，更优选植物细胞和酵母细胞，所述植物细胞优选禾本科植物细胞，特别优选水稻(Oryza sativa L.)细胞。
  5. 一种具有杀昆虫害虫活性的多肽，其选自：
    a)包括SEQ ID NO：1的氨基酸序列或其片段的多肽；
    b)包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽；和
    c)包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽；或
    d)由SEQ ID NO：1所示的氨基酸序列编码的多肽。
  6. 权利要求5的多肽，其进一步包括异源氨基酸序列。
  7. 一种组合物，其包括权利要求5的多肽。
  8. 权利要求7的组合物，其中所述组合物选自粉末、粉屑、小丸、颗粒、喷雾剂、乳剂、胶体和溶液。
  9. 权利要求7或8的组合物，其包括约1重量％-约99重量％的所述多肽。
  10. 一种用于控制昆虫害虫群体的方法，其包括使所述群体与杀虫 有效量的权利要求5的多肽接触。
  11. 一种用于杀死昆虫害虫的方法，其包括使所述害虫与杀虫有效量的权利要求5的多肽接触，或给所述害虫喂食杀虫有效量的权利要求5的多肽。
  12. 一种用于产生具有杀昆虫害虫活性的权利要求7的多肽的方法，其包括在其中表达编码多肽的核酸分子的条件下，培养权利要求4的宿主细胞。
  13. 一种已将DNA构建体稳定掺入到其基因组内的植物，所述DNA构建体包括编码具有杀昆虫害虫活性的蛋白质的核苷酸序列，其中所述核苷酸序列选自：
    a)包括SEQ ID NO：2的核苷酸序列的核酸分子或其全长互补体；
    b)编码包括SEQ ID NO：1的氨基酸序列或其片段的多肽的核酸分子；
    c)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段具有至少90％同一性的氨基酸序列的多肽的核酸分子；和
    d)编码包括与SEQ ID NO：1中所示的氨基酸序列或其片段相比具有缺失、替换、插入一个或多个氨基酸的氨基酸序列的多肽的核酸分子；
    其中所述核苷酸序列与启动子可操作地连接，所述启动子驱动编码序列在植物细胞中的表达。
  14. 一种用于保护植物不受昆虫害虫的方法，其包括将包括权利要求1的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀虫多肽。
  15. 权利要求14的方法，其中所述植物产生具有针对昆虫害虫的杀虫活性的杀虫多肽。
  16. 一种改良植物抗逆性的方法，其包括将包括权利要求1的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀昆虫害虫多肽。
  17. 一种植物育种方法，其包括将包括权利要求1的核苷酸序列的至少一种表达载体引入所述植物或其细胞内，所述核苷酸序列编码杀昆虫害虫多肽。
  18. 根据权利要求13-17任一项的方法，其中所述植物优选禾本科 植物，特别优选水稻。
  19. 根据权利要求10-17任一项的方法，其中所述昆虫害虫优选鳞翅目害虫，更优选水稻螟虫和斜纹夜蛾。
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