WO1994000992A1 - Avidine et ses homologues utiles comme larvicides contre les insectes nuisibles - Google Patents

Avidine et ses homologues utiles comme larvicides contre les insectes nuisibles Download PDF

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WO1994000992A1
WO1994000992A1 PCT/US1993/006487 US9306487W WO9400992A1 WO 1994000992 A1 WO1994000992 A1 WO 1994000992A1 US 9306487 W US9306487 W US 9306487W WO 9400992 A1 WO9400992 A1 WO 9400992A1
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plant
cellε
protein
avidin
glycoprotein
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PCT/US1993/006487
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English (en)
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Thomas H. Czapla
Karl J. Kramer
Thomas D. Morgan
Brenda Oppert
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Pioneer Hi-Bred International, Inc.
United States Of America
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Priority to AU46712/93A priority Critical patent/AU4671293A/en
Publication of WO1994000992A1 publication Critical patent/WO1994000992A1/fr

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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
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • 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)
    • 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

  • This invention relates to materials and methods for killing insect larvae which are harmful to plants, and materials and methods for imparting insect resistance to plants, material harvested from the plants, and products derived from the harvested material.
  • insects are serious pests of common agricul ⁇ tural crops.
  • One method of controlling insects has been to apply insecticidal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems.
  • a more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects, fungi (milky-spore) and bacteria (Bacillus thuringiensis cv., commonly referred to as "Bt").
  • Bacillus thuringiensis cv. commonly referred to as "Bt"
  • Figure 1 illustrates the gene map of pla ⁇ mid pPHl414 which is useful as an expression cassette for protein struc ⁇ tural genes.
  • Figure 2 illustrates the gene map of plasmid pPHl412 which is also useful as an expression cassette for protein structural genes.
  • glycoprotein avidin and the protein streptavidin have potent larvicidal activity when administered enterally to the larvae of insects such as European corn borer, corn rootworm, red flour beetle, alfalfa weevil, tobacco budworm, beet armyworm, bollworm, sunflower moth, confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth.
  • this invention provides a method for killing susceptible insect larvae, including larvae selected from European corn borer, corn rootworm, red flour beetle, alfalfa weevil, tobacco budworm, beet armyworm, bollworm, sunflower moth, confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth and comprising administering enterally to the larvae a larvicidal amount of a protein or glycoprotein selected from avidin and streptavidin, and proteins and glycoproteins at least 90% homologous thereto by amino acid sequence.
  • a protein or glycoprotein selected from avidin and streptavidin, and proteins and glycoproteins at least 90% homologous thereto by amino acid sequence.
  • Avidin was originally a factor isolated from raw egg white as described by Eakin et al., J. Biol . Chem. , 136 , 801 (1940); Pennington et al. , J. Am. Chem. Soc. , 6 , 469 (1942); Fraenkel-Conrat et al., Arch. Biochem. Biophys., 39, 80, 97 (1952). Later, improved methods of purification were disclosed by Green et al . , Biochem. J. , 118, 67, 71 (1970).
  • the glycoprotein contains four essentially identical subunits having a combined molecular weight of about 66,000 daltons.
  • Each subunit is a single polypeptide chain containing 128 amino acid residues with alanine at the N- terminu ⁇ , glutamic acid at the C-terminus, and a carbohydrate moiety attached at the asparaginyl residue at position 17 through post-tran ⁇ lational processing.
  • the complete amino acid sequence has been determined and has been published. See, e.g., DeLange, H., J. Biol. Chem. , 246, 698 (1971).
  • the bacterial version, streptavidin is not glycosylated.
  • the larvicidal compound can be effectively applied to plants, harvested materials, or products consumed by the larvae by spray, dust or other formulation common to the insecticidal arts.
  • harvested plant material herein is meant any material harvested from an agricultural or horticultural crop, including without limitation grain, fruit, leaves, fibers, seeds, or other plant parts.
  • Products derived or obtained from such harvested material include flour, meal, and flakes derived from grain and products in which such materials are admixed, such as, for example, cake, pancake and biscuit mixes.
  • the larvicidal protein or glycoprotein can be incorporated into the tissues of a susceptible plant so that in the course of infesting the plant, its harvested material, or a product derived from harvested plant material, the larvae consume larvicidal amounts of the protein or glycoprotein.
  • One method of doing this is to incorporate the compound in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants.
  • the resulting sequence can be inserted into an appropriate expression cassette, and introduced into cells of a susceptible plant species, so that an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA sequence coding for one or more insecticidal plant proteins or glycoproteins selected from avidin and streptavidin and proteins and glycoprotein ⁇ having at least 90% homology thereto by amino acid sequence, in proper reading frame relative to transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA ⁇ equence under control of the plant-active regulatory sequences causes expression of the larvicidal gene product at levels which provide an insecticidal amount of the compound in the tissues of the plant which are normally infested by the larvae.
  • a dietary bait containing one or more of the selected compounds can be employed, with, optionally, an added pheromonal or other larval attractant material.
  • the plant is preferably a plant su ⁇ ceptible, or whose harvested material or products are susceptible to infesta- tion and damage by the larvae of one or more in ⁇ ect larvae selected from European corn borer, corn rootworm and alfalfa weevil, sunflower moth, bollworm, tobacco budworm, and beet armyworm or whose harvested material is subject to attack by red flour beetle, confused flour beetle, black cutworm, le ⁇ er grain borer, ⁇ awtoothed grain beetle, rice weevil and Indian meal moth.
  • in ⁇ ect larvae selected from European corn borer, corn rootworm and alfalfa weevil, sunflower moth, bollworm, tobacco budworm, and beet armyworm or whose harvested material is subject to attack by red flour beetle, confused flour beetle, black cutworm, le ⁇ er grain borer, ⁇ awtoothed grain beetle, rice weevil and Indian meal moth.
  • corn
  • Preferred plants that are to be transformed according to the methods of this invention are cereal crop ⁇ , including maize, rye, barley, wheat, sorghum, oat ⁇ , millet, rice, triticale, ⁇ unflower, alfalfa, rape ⁇ eed and ⁇ oybean, fiber crops, such as cotton, fruit crop ⁇ , ⁇ uch as melon ⁇ , and vegetable crop ⁇ , including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.
  • the DNA sequence which when expressed imparts insecti ⁇ cidal activity is a structural gene which codes for avidin, streptavidin, or a protein or glycoprotein having at least 90% homology to avidin or streptavidin.
  • tissue specific promoter can be used in any instance where it may be desirable to localize production of the compound to an infested tissue or to a tissue which is efficient in production of the protein or glycoprotein.
  • numerou ⁇ plant expression cassettes and vectors are well known in the art.
  • expression cas ⁇ ette is meant a complete set of control sequences including initiation, promoter and termination sequence ⁇ which function in a plant cell when they flank a structural gene in the proper reading frame.
  • Expression cas ⁇ ette ⁇ frequently and preferably contain an a ⁇ sortment of restric- tion sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence.
  • the plant expression ca ⁇ ette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA.
  • a preferred (empty) expression cassette into which the DNA sequence of the present invention can be inserted is the pPHl414 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., John ⁇ ton, IA.
  • Highly preferred plant expression cassettes will be designed to include one or more selectable marker genes, such as kanamycin re ⁇ istance or herbicide tolerance genes.
  • vector herein is meant a DNA sequence which is able to replicate and expres ⁇ a foreign gene in a ho ⁇ t cell.
  • the vector ha ⁇ one or more endo- nuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme.
  • Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve a ⁇ selectable markers to identify and separate transformed cells.
  • Preferred selection agents include kanamycin, chlorosulfuron, pho ⁇ phonothricin, hygro- mycin and methotrexate, and preferred markers are genes conferring resistance to these compounds.
  • a cell in which the foreign genetic material in a vector is functionally expressed has been "tran ⁇ formed" by the vector and is referred to as a "transformant" .
  • a particularly preferred vector is a pla ⁇ mid, by which i ⁇ meant a circular double-stranded DNA molecule that is not a part of the chromosome ⁇ of the cell.
  • genomic, synthetic and cDNA encoding the gene of interest may be used in this invention.
  • the vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone.
  • genetic con ⁇ truct ⁇ are made which contain the nece ⁇ ary regulatory sequences to provide for efficient expre ⁇ ion of the gene in the host cell.
  • the genetic construct will contain (a) a fir ⁇ t genetic sequence coding for the protein or glycoprotein of intere ⁇ t and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest.
  • the regulatory sequences will be selected from the group comprising of promoters and terminators.
  • the regulatory sequences may be from autologous or heterologous sources.
  • Promoters that may be used in the genetic sequence include no ⁇ , ocs, phaseolin, CaMV, FMV and other promoters isolated from plants or plant pests.
  • An efficient plant promoter that may be used is an overproducing plant promoter.
  • Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxyla ⁇ e from soybean (Berry-Lowe et al, J. Molecular and App. Gen. , 1:483-498 (1982)), and the promoter of the cholorophyll a-b binding protein. These two promoters are known to be light-induced, in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural
  • the expre ⁇ sion cassette comprising the structural gene for the compound of intere ⁇ t operably linked to the desired control sequences can be ligated into a suitable cloning vector.
  • plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used.
  • the cloning vector will typically carry a replication origin, a ⁇ well as specific genes that are capable of providing phenotypic selection markers in tran ⁇ formed ho ⁇ t cell ⁇ .
  • gene ⁇ conferring re ⁇ i ⁇ tance to antibiotic ⁇ or ⁇ elected herbicide ⁇ are u ⁇ ed.
  • an intermediate ho ⁇ t cell will be u ⁇ ed in the practice of this invention to increase the copy number of the cloning vector. ith an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cell ⁇ .
  • Ho ⁇ t cell ⁇ that can be u ⁇ ed in the practice of this invention include prokaryotes, including bacterial hosts such a ⁇ E. coli , £>. typhimurium, and Ej. marcescens.
  • Eukaryotic host ⁇ such as yeast or filamentous fungi may also be used in this invention.
  • the isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ ⁇ ing electroporation (in protoplasts) , retroviruses, microparticle bombardment, and microinjection, into cell ⁇ from monocotyledonou ⁇ or dicotyledonous plant ⁇ , in cell or ti ⁇ ue culture, to provide transformed plant cells containing as foreign DNA at lea ⁇ t one copy of the DNA ⁇ equence of the plant expre ⁇ ion cassette.
  • the monocotyledonous specie ⁇ will be ⁇ elected from maize, ⁇ orghum, wheat and rice, and the dicotyledonous specie ⁇ will be selected from soybean, sunflower, cotton, rape ⁇ eed (either edible or indu ⁇ trial), alfalfa, tobacco, and Solanaceae such a ⁇ potato and tomato.
  • U ⁇ ing known technique ⁇ protopla ⁇ ts can be regenerated and cell or ti ⁇ ue culture can be regenerated to form whole fertile plants which carry and express the desired gene for the selected protein.
  • a highly preferred embodiment of the present invention i ⁇ a tran ⁇ formed maize plant, the cell ⁇ of which contain a ⁇ foreign DNA at lea ⁇ t one copy of the DNA ⁇ equence of an expression cas ⁇ ette of this invention.
  • This invention also provides methods of imparting re ⁇ i ⁇ tance to in ⁇ ect ⁇ ⁇ elected from European corn borer, corn rootworm, red flour beetle and alfalfa weevil, tobacco budworm, beet armyworm, bollworm, ⁇ unflower moth, confused flour beetle, black cutworm, lesser grain borer, sawtoothed grain beetle, rice weevil and Indian meal moth to plants of a su ⁇ ceptible taxon, compri ⁇ ing the ⁇ tep ⁇ of: a) culturing cell ⁇ or ti ⁇ ue ⁇ from at lea ⁇ t one plant from the taxon, b) introducing into the cell ⁇ of the cell or tissue culture at lea ⁇ t one copy of an expre ⁇ ion ca ⁇ ette compris ⁇ ing a structural gene coding for a protein or glycoprotein selected from avidin, streptavidin and proteins and glycoproteins having at least 90% homology thereto by amino acid sequence, or a combination of such proteins,operably linked
  • conven ⁇ tional plant breeding method ⁇ can be u ⁇ ed to tran ⁇ fer the protein ⁇ tructural gene and associated regulatory sequence ⁇ via cro ⁇ ing and backcro ⁇ ing.
  • Such intermediate method ⁇ will comprise the further ⁇ tep ⁇ of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon; b) recovering reproductive material from the progeny of the cross; and c) growing insect-resistant plants from the reproductive material.
  • the agronomic characteristic ⁇ of the ⁇ u ⁇ ceptible taxon can be substantially preserved by expanding thi ⁇ method to include the further ⁇ teps of repetitively: a) backcrossing the insect-resistant progeny with insect-susceptible plants from the su ⁇ ceptible taxon; and b) ⁇ electing for expre ⁇ ion of in ⁇ ect re ⁇ istance (or an associated marker gene) among the progeny of the back- cross, until the desired percentage of the characteri ⁇ tic ⁇ of the susceptible taxon are present in the progeny along with the gene imparting insect resistance.
  • tax herein is meant a unit of botanical cla ⁇ ification of genu ⁇ or lower. It thu ⁇ include ⁇ genus, species, cultivar ⁇ , varieties, variant ⁇ , and other minor taxonomic groups which lack a consi ⁇ tent nomenclature.
  • the plant vector ⁇ provided herein can be incorporated into Agrobacterium tumefacien ⁇ , which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous specie ⁇ .
  • this invention provides a method for imparting insect resi ⁇ tance in Agrobacterium tumefaciens-susceptible dicotyledonous plants in which the expre ⁇ sion cassette is introduced into the cell ⁇ by infect ⁇ ing the cells with Agrobacterium tumefaciens, a pla ⁇ mid of which ha ⁇ been modified to include a plant expression cassette of this invention.
  • the ⁇ e compound ⁇ can exert their larvicidal activity again ⁇ t in ⁇ ect pests of harvested material, including stored grain, ⁇ uch a ⁇ the red flour beetle (Tribolium ca ⁇ tanaeum) . Thu ⁇ , the ⁇ e insects can also be target ⁇ for the compositions and methods of thi ⁇ invention.
  • the invention al ⁇ o provide ⁇ a method ⁇ and compo ⁇ ition ⁇ for killing larvae of red flour beetle and confu ⁇ ed flour beetle, black cutworm, le ⁇ er grain borer, ⁇ awtoothed grain beetle, rice weevil and Indian meal moth and other ⁇ u ⁇ ceptible in ⁇ ect pests of harvested materials and products obtained from harvested materials, compri ⁇ ing applying, to the grain or cau ⁇ ing to be expre ⁇ sed in the grain a protein or glycoprotein selected from avidin, streptavidin, or a protein or glycoprotein having at least 90% homology thereto by amino acid sequence.
  • the following description further exemplifies the compositions of this invention and the methods of making and using them.
  • thi ⁇ te ⁇ t This was accomplished by making up a standard artificial diet at 90% of the original water and adding a solution of the test protein to thi ⁇ mixture. Concentrations of the protein in thi ⁇ diet are recorded as mg or ⁇ g of protein per ml of diet.
  • a modification of thi ⁇ te ⁇ t involves preparing the original diet, then preparing a 1% solution of the test protein that is applied (75 ⁇ l ) to the diet surface.
  • Thi ⁇ is referred to herein a ⁇ a "Topical bioassay," but it should be understood that the test compound is applied topically to the insect's diet, not to the in ⁇ ect it ⁇ elf. Weight and mortality are recorded after ⁇ even days. Specific a ⁇ ay ⁇ and variations are described in the individual examples.
  • Example 1 EUROPEAN CORN BORER Avidin and streptavidin were very effective against European corn borer with high mortality occurring during a 7-day topical bioassay. The re ⁇ ult ⁇ are ⁇ hown in Table 1.
  • Example 2 SOUTHERN CORN ROOTWORM Avidin and streptavidin have not had a ⁇ dramatic effect an on ⁇ outhern corn rootworm larvae. Avidin ⁇ how ⁇ an increa ⁇ e in mortality at 10 mg/ml, but no effect is ⁇ een at 1 mg/ml Re ⁇ ult ⁇ are ⁇ hown in Table 3.
  • BW bollworms
  • TW tobacco budworms
  • BCW black cutworms
  • SM sunflower moths
  • BAW beet armyworms
  • nucleotide sequence data which establishes an open reading frame (i.e., the correct triplet code for translation which should have only one "stop" signal at the very end of the gene.) It is also nece ⁇ ary to have an indication of where to look for the protea ⁇ e cleavage junction between the protein and the replica ⁇ e which precede ⁇ it in the sequence. This can be determined from the peptide sequence of the N-terminal portion of the protein or by comparing the protein sequence with that of other ho ologou ⁇ proteins. This can generally be accomplished and the nece ⁇ ary information obtained without sequencing the entire gene.
  • the remainder of the gene can be cloned u ⁇ ing restriction enzymes that flank the protein coding region or, more preferably, by cloning the preci ⁇ e protein coding region by oligonucleotide- directed amplification of DNA (polymera ⁇ e chain reaction or PCR) .
  • the gene ha ⁇ Once the gene ha ⁇ been i ⁇ olated, it can be cloned into a bacterial expre ⁇ sion vector with linkers added to create all three reading frames (using 8mer, lOmer, and 12mers each of which contain an ATG tran ⁇ lational ⁇ tart ⁇ ite).
  • the resulting vector ⁇ containing the fragments of intere ⁇ t, can be in ⁇ erted into, for example, BRL's Maximum Efficiency DH5 F' IQ transformation competent E_ ⁇ coli cell ⁇ . All three tran ⁇ formation ⁇ , one for each linker, are then ⁇ creened via miniprep ⁇ for the pre ⁇ ence and orientation of in ⁇ ert. Appropriate clone ⁇ are then chosen to test for expre ⁇ ion of the protein gene.
  • Clone ⁇ containing the properly oriented in ⁇ ert ⁇ are grown in culture medium conducive to the induction of the gene (LB medium with added IPTG) .
  • the cells are lysed and bacterial proteins are subjected to electrophoresis in SDS polyacrylamide gel ⁇ and then transferred to nitrocellulose.
  • the resulting protein blots are easily screened for presence of protein using rabbit polyclonal and mouse monoclonal anti-protein antibody.
  • a plant expression cassette employing the regulatory sequences developed by Beach, et al., and containing the protein gene, is constructed.
  • the restriction map of the preferred plasmid, de ⁇ ignated pPHl414, is illu ⁇ trated in Figure 1.
  • Thi ⁇ pla ⁇ mid contain ⁇ an enhanced 35S promoter spanning nucleotide ⁇ - 421 to +2 of Cauliflower Mo ⁇ aic Viru ⁇ with the region from - 421 to - 90 duplicated in tandem, a 79 bp Hindlll Sail fragment from pJIIlOl ⁇ panning the 5' leader ⁇ equence of Tobacco Mosaic Virus, a 579 bp fragment spanning the first intron from maize AdHl-S, and a 281 bp fragment spanning the polyadenylation site from the nopaline synthase gene in pTiT37.
  • pPHl412 pla ⁇ mid ⁇ hown in Figure 2 Another construct which can be used a ⁇ an expre ⁇ ion ca ⁇ ette is the pPHl412 pla ⁇ mid ⁇ hown in Figure 2. It differs from pPHl414 in that it lacks the AdH intron segment. However, like pPHl414, it i ⁇ constructed to have numerous restriction site ⁇ between the 0' ⁇ egment and the NOS ⁇ egment, which ⁇ ite ⁇ can be conveniently u ⁇ ed for ⁇ plicing any de ⁇ ired protein structural gene into position.
  • This vector can be cotransformed with a similar plasmid containing a selectable marker for antibiotic re ⁇ i ⁇ tance into Black Mexican Sweet corn protopla ⁇ t ⁇ by electroporation.
  • the ⁇ e protopla ⁇ t ⁇ can then be induced to regenerate cell walls and develop into callus by conventional techniques.
  • this callu ⁇ can then be subjected to antibiotic selection to select for transformed colonies, and these colonies can be tested for expre ⁇ ion of protein with anti ⁇ era for the appropriate protein u ⁇ ing known method ⁇ .
  • the efficiency of protection can be mea ⁇ ured by infe ⁇ ting callus (or ⁇ uspension culture ⁇ derived from callu ⁇ ) with the target in ⁇ ect and mea ⁇ uring ⁇ urvival percentage ⁇ .
  • the protein gene can be introduced into embryogenic maize callu ⁇ by method ⁇ ⁇ imilar to tho ⁇ e used for Black Mexican Sweet. Embryogenic callu ⁇ can be regenerated to whole fertile plant ⁇ .
  • the in ⁇ ect resistance imparted by the endogenous production of the protein is a simply inherited, dominant trait and can, if desired, be introduced into other plant varietie ⁇ of the ⁇ pecie ⁇ by simple crossing or backcro ⁇ ing.

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'avidine et la streptavidine se sont révélées des larvicides contre plusieurs insectes nuisibles communs qui sévicent sur les cultures agricoles et dans les grains stockés. Dans un mode préférentiel de réalisation, la résistance des plantes à de tels insectes est développée en insérant dans les cellules d'une plante un gène dont l'expression provoque la production d'une ou plusieurs de ces glycoprotéines en quantités larvicides.
PCT/US1993/006487 1992-07-10 1993-07-09 Avidine et ses homologues utiles comme larvicides contre les insectes nuisibles WO1994000992A1 (fr)

Priority Applications (1)

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AU46712/93A AU4671293A (en) 1992-07-10 1993-07-09 Avidin and homologues as larvicides against insect pests

Applications Claiming Priority (2)

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US91186492A 1992-07-10 1992-07-10
US07/911,864 1992-07-10

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WO1994000992A1 true WO1994000992A1 (fr) 1994-01-20

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WO1996034954A2 (fr) * 1995-05-01 1996-11-07 Trustees Of Boston University Systemes de confinement liant de la biotine
WO1996040949A1 (fr) * 1995-06-07 1996-12-19 Pioneer Hi-Bred International, Inc. Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine
WO1997017455A2 (fr) * 1995-11-06 1997-05-15 John Howard Production commerciale d'avidine dans des plantes
WO1998039461A1 (fr) * 1997-03-20 1998-09-11 Prodigene Inc. Methode de production et d'extraction commerciales de proteines a partir de graines
US5962769A (en) * 1995-06-07 1999-10-05 Pioneer Hi-Bred International, Inc. Induction of male sterility in plants by expression of high levels of avidin
WO2000004049A1 (fr) * 1998-07-15 2000-01-27 The Horticulture And Food Research Institute Of New Zealand Limited Polypeptides chimeres permettant l'expression de proteines nocives de plantes
WO2001088166A2 (fr) * 2000-05-17 2001-11-22 Prodigene, Inc. Methodes de lutte contre l'infestation des plantes transgeniques par les insectes a l'aide du gene de la proteine de fixation a la biotine
US6504085B1 (en) 1997-03-07 2003-01-07 Prodigene, Inc. Methods of commercial production and extraction of protein from seed
US7071384B2 (en) 1997-03-07 2006-07-04 Genencor International, Inc. Methods for commercial production of heterologous laccase in plant tissue and extraction of the laccase from plant seed
WO2007134122A2 (fr) 2006-05-09 2007-11-22 The Curators Of The University Of Missouri Plateformes végétales de chromosomes artificiels au moyen d'un troncage du télomère
EP1865059A1 (fr) 2001-03-12 2007-12-12 Japan Tobacco, Inc. Nouvelle protéine, son codage génétique et son procédé d'utilisation
US7718852B2 (en) 2007-04-12 2010-05-18 Dow Agrosciences Llc Canola cultivar DN040241
US7723579B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040244
US7723582B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN041100
US7723580B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040844
US7723577B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040847
US7723578B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040839
US7723581B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040845
US7728195B2 (en) 2007-04-12 2010-06-01 Dow Agrosciences Llc Canola cultivar DN040856
US7964774B2 (en) 2008-05-14 2011-06-21 Monsanto Technology Llc Plants and seeds of spring canola variety SCV384196
US7968764B2 (en) 2005-05-02 2011-06-28 Purdue Research Foundation Methods for increasing the yield of fermentable sugars from plant stover
US8071865B2 (en) 2009-04-15 2011-12-06 Monsanto Technology Llc Plants and seeds of corn variety CV589782
US8071864B2 (en) 2009-04-15 2011-12-06 Monsanto Technology Llc Plants and seeds of corn variety CV897903
US8124143B2 (en) 2007-06-21 2012-02-28 Suntava, Llc Anthocyanin pigment/dye compositions through corn extraction
US8304616B2 (en) 2009-04-07 2012-11-06 University Of Georgia Research Foundation, Inc. Soybean variety G00-3209
US8362332B2 (en) 2009-04-15 2013-01-29 Monsanto Technology Llc Plants and seeds of corn variety CV165560
US8378177B2 (en) 2010-02-03 2013-02-19 Dow Agrosciences, Llc Canola cultivar DN051493
US8445746B2 (en) 2007-02-23 2013-05-21 University Of Georgia Research Foundation, Inc. Compositions and methods for identifying genetic sequences with toxin resistance in plants
US8530726B2 (en) 2010-12-30 2013-09-10 Agrigenetics, Inc. Canola cultivar G030994
US8558064B2 (en) 2010-12-30 2013-10-15 Agrigenetics, Inc. Canola cultivar CL31613
US8558065B2 (en) 2010-12-30 2013-10-15 Agrigenetics, Inc. Canola cultivar G31064
US8563811B2 (en) 2010-12-30 2013-10-22 Agrigenetics, Inc. Canola cultivar DN040845A
US8563810B2 (en) 2010-12-30 2013-10-22 Agrigenetics, Inc. Canola cultivar DN040244A
US8686222B2 (en) 2009-10-16 2014-04-01 Dow Agrosciences, Llc. Use of dendrimer nanotechnology for delivery of biomolecules into plant cells
US8835720B2 (en) 2012-04-26 2014-09-16 Monsanto Technology Llc Plants and seeds of spring canola variety SCV967592
US8859857B2 (en) 2012-04-26 2014-10-14 Monsanto Technology Llc Plants and seeds of spring canola variety SCV259778
US8878009B2 (en) 2012-04-26 2014-11-04 Monsanto Technology, LLP Plants and seeds of spring canola variety SCV318181
US8901379B2 (en) 2007-04-12 2014-12-02 Dow Agrosciences, Llc. Canola cultivars having high yield and stabilized fatty acid profiles
US8945653B2 (en) 2007-06-21 2015-02-03 Suntava, Llc Extracted whole corn kernels and improved processed and processable corn produced thereby
US9204601B1 (en) 2011-11-21 2015-12-08 Agrigenetics, Inc. Canola inbred CL60855R
US9204602B1 (en) 2011-11-21 2015-12-08 Agrigenetics, Inc. Canola inbred CL77606R
US9210857B1 (en) 2011-11-21 2015-12-15 Agrigenetics, Inc. Canola inbred CL102407R
US9414556B1 (en) 2012-11-29 2016-08-16 Agrigenetics, Inc. Canola inbred restorer line G98014R
US9414557B1 (en) 2013-02-21 2016-08-16 Agrigenetics, Inc. Canola inbred restorer line CE185952R
US9426953B1 (en) 2013-02-21 2016-08-30 Agrigenetics, Inc. Canola hybrid cultivar CE216910H
US9447430B1 (en) 2012-11-29 2016-09-20 Agrigenetics, Inc. Canola inbred line G2X0023AB
US9445564B1 (en) 2012-11-29 2016-09-20 Agrigenetics, Inc. Canola inbred line DN051465A
US9538716B1 (en) 2013-02-21 2017-01-10 Agrigenetics, Inc. Canola inbred restorer line CE185942R
US9554534B1 (en) 2014-02-28 2017-01-31 Agrigenetics, Inc. Canola inbred line CL1992625A
US9596816B1 (en) 2014-02-28 2017-03-21 Agrigenetics, Inc. Canola inbred restorer line CL215695R
US9603322B2 (en) 2010-12-30 2017-03-28 Agrigenetics, Inc. Canola cultivars having high yield and stabilized fatty acid profiles
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US9968050B2 (en) 2015-04-15 2018-05-15 Agrigenetics, Inc. Canola inbred restorer line G175274R
US9968047B2 (en) 2015-03-24 2018-05-15 Agrigenetics, Inc. Canola hybrid cultivar CL2562968H
US9968051B2 (en) 2015-04-15 2018-05-15 Agrigenetics, Inc. Canola hybrid cultivar G2537376H
US9974262B2 (en) 2015-04-15 2018-05-22 Agrigenetics, Inc. Canola inbred restorer line CL134904R
US9986702B1 (en) 2014-12-05 2018-06-05 Agrigenetics, Inc. Canola inbred restorer line G1934899R
US10165751B2 (en) 2014-12-05 2019-01-01 Agrigenetics, Inc. Canola inbred line G30853A
US10244716B2 (en) 2016-09-07 2019-04-02 Agrigenetics, Inc. Canola hybrid cultivar CL3701975H
US10306852B2 (en) 2015-04-15 2019-06-04 Agrigenetics, Inc. Canola inbred line G1992650A
US10314270B2 (en) 2015-04-01 2019-06-11 Agrigenetics, Inc. Canola hybrid cultivar G3697124H
US10420296B2 (en) 2016-09-07 2019-09-24 Agrigenetics, Inc. Canola inbred restorer line G263532R
US10426110B2 (en) 2016-09-07 2019-10-01 Agrigenetics, Inc. Canola inbred restorer line CL2503899R
US10463004B2 (en) 2016-09-07 2019-11-05 Agrigenetics, Inc. Canola inbred line G1466454A/B
US10588281B2 (en) 2016-09-07 2020-03-17 Agrigenetics, Inc. Canola hybrid cultivar G5428584H
US11180751B2 (en) 2015-06-18 2021-11-23 The Broad Institute, Inc. CRISPR enzymes and systems
US11591601B2 (en) 2017-05-05 2023-02-28 The Broad Institute, Inc. Methods for identification and modification of lncRNA associated with target genotypes and phenotypes

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WO1996034954A2 (fr) * 1995-05-01 1996-11-07 Trustees Of Boston University Systemes de confinement liant de la biotine
WO1996034954A3 (fr) * 1995-05-01 1997-01-16 Univ Boston Systemes de confinement liant de la biotine
US5679533A (en) * 1995-05-01 1997-10-21 Trustees Of Boston University Biotin-binding containment systems
US5681745A (en) * 1995-05-01 1997-10-28 Trustees Of Boston University Biotin-binding containment systems
WO1996040949A1 (fr) * 1995-06-07 1996-12-19 Pioneer Hi-Bred International, Inc. Induction de la sterilite male dans des plantes par expression de niveaux eleves d'avidine
US5962769A (en) * 1995-06-07 1999-10-05 Pioneer Hi-Bred International, Inc. Induction of male sterility in plants by expression of high levels of avidin
WO1997017455A2 (fr) * 1995-11-06 1997-05-15 John Howard Production commerciale d'avidine dans des plantes
WO1997017455A3 (fr) * 1995-11-06 1997-09-12 John Howard Production commerciale d'avidine dans des plantes
US5767379A (en) * 1995-11-06 1998-06-16 John Howard Commercial production of avidin in plants
US6504085B1 (en) 1997-03-07 2003-01-07 Prodigene, Inc. Methods of commercial production and extraction of protein from seed
US7179961B2 (en) 1997-03-07 2007-02-20 Prodi Gene, Inc Methods of commercial production and extraction of protein from seed
US7071384B2 (en) 1997-03-07 2006-07-04 Genencor International, Inc. Methods for commercial production of heterologous laccase in plant tissue and extraction of the laccase from plant seed
WO1998039461A1 (fr) * 1997-03-20 1998-09-11 Prodigene Inc. Methode de production et d'extraction commerciales de proteines a partir de graines
WO2000004049A1 (fr) * 1998-07-15 2000-01-27 The Horticulture And Food Research Institute Of New Zealand Limited Polypeptides chimeres permettant l'expression de proteines nocives de plantes
US6972350B1 (en) * 1998-07-15 2005-12-06 The Horticulture And Food Research Institute Of New Zealand Pest-resistant plants comprising a construct encoding a vacuole targeting sequence and avidin or streptavidin
WO2001088166A3 (fr) * 2000-05-17 2002-06-27 Prodigene Inc Methodes de lutte contre l'infestation des plantes transgeniques par les insectes a l'aide du gene de la proteine de fixation a la biotine
WO2001088166A2 (fr) * 2000-05-17 2001-11-22 Prodigene, Inc. Methodes de lutte contre l'infestation des plantes transgeniques par les insectes a l'aide du gene de la proteine de fixation a la biotine
US7776333B2 (en) 2001-03-12 2010-08-17 Japan Tobacco Inc. Protein, a genes encoding therefor and a method of using the same
EP1865059A1 (fr) 2001-03-12 2007-12-12 Japan Tobacco, Inc. Nouvelle protéine, son codage génétique et son procédé d'utilisation
US7713531B2 (en) 2001-03-12 2010-05-11 Japan Tobacco, Inc. Protein, a gene encoding therefor and a method of using the same
US7989610B2 (en) 2001-03-12 2011-08-02 Japan Tobacco Inc Protein, a gene encoding therefor and a method of using the same
US7855282B2 (en) 2001-03-12 2010-12-21 Japan Tobacco Inc. Protein, a gene encoding therefor and a method of using the same
US7968764B2 (en) 2005-05-02 2011-06-28 Purdue Research Foundation Methods for increasing the yield of fermentable sugars from plant stover
US8921648B2 (en) 2005-05-02 2014-12-30 Purdue Research Foundation Methods for increasing the yield of fermentable sugars from plant stover
WO2007134122A2 (fr) 2006-05-09 2007-11-22 The Curators Of The University Of Missouri Plateformes végétales de chromosomes artificiels au moyen d'un troncage du télomère
US8445746B2 (en) 2007-02-23 2013-05-21 University Of Georgia Research Foundation, Inc. Compositions and methods for identifying genetic sequences with toxin resistance in plants
US7718852B2 (en) 2007-04-12 2010-05-18 Dow Agrosciences Llc Canola cultivar DN040241
US7728195B2 (en) 2007-04-12 2010-06-01 Dow Agrosciences Llc Canola cultivar DN040856
US7723581B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040845
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US7723577B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040847
US7723580B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040844
US7723582B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN041100
US8901379B2 (en) 2007-04-12 2014-12-02 Dow Agrosciences, Llc. Canola cultivars having high yield and stabilized fatty acid profiles
US7723579B2 (en) 2007-04-12 2010-05-25 Dow Agrosciences Llc Canola cultivar DN040244
US8491670B2 (en) 2007-06-21 2013-07-23 Suntava, Llc Anthocyanin pigment/dye compositions and method of providing composition through extraction from corn
US8124143B2 (en) 2007-06-21 2012-02-28 Suntava, Llc Anthocyanin pigment/dye compositions through corn extraction
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US7964774B2 (en) 2008-05-14 2011-06-21 Monsanto Technology Llc Plants and seeds of spring canola variety SCV384196
US8304616B2 (en) 2009-04-07 2012-11-06 University Of Georgia Research Foundation, Inc. Soybean variety G00-3209
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US8686222B2 (en) 2009-10-16 2014-04-01 Dow Agrosciences, Llc. Use of dendrimer nanotechnology for delivery of biomolecules into plant cells
US8378177B2 (en) 2010-02-03 2013-02-19 Dow Agrosciences, Llc Canola cultivar DN051493
US8558064B2 (en) 2010-12-30 2013-10-15 Agrigenetics, Inc. Canola cultivar CL31613
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