WO2010077672A2 - Lutte contre les larves de moustique par les toxines bti et tmof - Google Patents

Lutte contre les larves de moustique par les toxines bti et tmof Download PDF

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Publication number
WO2010077672A2
WO2010077672A2 PCT/US2009/067180 US2009067180W WO2010077672A2 WO 2010077672 A2 WO2010077672 A2 WO 2010077672A2 US 2009067180 W US2009067180 W US 2009067180W WO 2010077672 A2 WO2010077672 A2 WO 2010077672A2
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Prior art keywords
bti
tmof
cell
cells
seq
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PCT/US2009/067180
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WO2010077672A3 (fr
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Dov Borovsky
Arieh Zaritsky
Eitan Ben-Dov
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University Of Florida Research Foundation, Inc.
Ben-Gurion University Of The Negev
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Publication of WO2010077672A2 publication Critical patent/WO2010077672A2/fr
Publication of WO2010077672A3 publication Critical patent/WO2010077672A3/fr

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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

Definitions

  • Bti Bacillus thuringiensis Israelensis was originally isolated from naturally- infected mosquito larvae inhabiting a temporary pond in the Negev (Goldberg and Margalith, 1977). Because the ⁇ -endotoxin produced by Bti affects larval mosquitoes and black flies, but little else, Bti has become the main mosquito larvicide used throughout the world (Margalith and Ben-Dov, 2000).
  • the ⁇ -endotoxin is composed of six polypeptides (134, 128, 78, 72, 27 and 29 kDa) encoded by Cry4Aa, Cry4Ba, CrylOAa, Cryl lAa, CytlAa, and Cyt2Ba, respectively (Hofte and Whitely, 1989; Federici el al, 1990; Margelith and Ben-Dov, 2000).
  • the Bti toxin proteins are produced in significant amounts (20-30% of the dry weight) during sporulation and are contained in parasporal crystalline bodies (Federici et al., 1990).
  • the ⁇ -endotoxin crystal is composed of Cry4Aa, Cryl IAa, and CytlAa at about 15, 25, and 50%, respectively.
  • Bti toxin the larval midgut epithelium in mosquitoes and black flies, where proteases activate the protoxin at alkaline pH.
  • Bt toxins exert their effects in an impressively specific manner. Those of Bacillus thuringiensis kurstaki (Btk) are active against some lepidopterans, but not mosquitoes. In contrast, BH toxins profoundly affect mosquito larvae, but are inactive or cause negligible effects in other kinds of invertebrates and vertebrates (reviewed by Floore, 2006).
  • the Cr)' and Cyt polypeptides do not have significant sequence similarities, although both form pores in the insect gut that lead to cell lysis and death.
  • Ellar, 1987 postulates that the crystallized ⁇ -endotoxin is released from the bacterial spores as a protoxin, solubilized by the high pH with the insect's gut, and activated by gut-alkaline proteases.
  • the truncated activated toxin domain II first binds to a brush- border membrane receptor causing a conformational change allowing domain I to insert itself into the membrane and form pores (Dean et al., 1996; Flores et al, 1997).
  • the pores that are formed selectively channel K + and Na + cations. Equilibrium of these ions across the insect cell membrane results in an influx of water that leads to a colloid osmotic lysis (Knowles el al, 1989).
  • TMOF Trypsin Modulating Oostatic Factor
  • BH toxins mosquito TMOF affects a relatively narrow range of targets.
  • the mosquito ovary serves as a rich source of "oostatic hormone" (Borovsky, 1985). When the hormone is injected into decapitated and ovariectomized females, they fail to synthesize a trypsin-like enzyme required for blood digestion. Although decapitated females fail to synthesize ecdysteroids and or to develop eggs, their guts synthesize protease.
  • the hormone is not species-specific, as injection of the Ae. aegypti hormone inhibits egg development and trypsin biosynthesis in Culex quinquefasciatus, Cx. nigripalpus and Anophelas albimaniis (Borovsky, 1988). Because the target tissue of the hormone is the mosquito midgut and not the ovary or the brain, the hormone was named "Trypsin Modulating Oostatic Factor" (TMOF).
  • the hormone has been purified, sequenced and characterized by means of mass spectroscopy as an unblocked decapeptide (YDP APPPPPP) (SEQ ID NO:1) (Borovsky et al, 1990).
  • YDP APPPPPP unblocked decapeptide
  • SEQ ID NO:1 Borovsky et al, 1990.
  • Various synthetic peptide analogues possess TMOF activity (Borovsky et al, 1990; 1991; 1993; Borovsky and Meola, 2004).
  • NMR analyses (Curto et al, 1993) confirm computer-modeling suggestions that the polyproline portion of TMOF in solution is a left-handed alpha helix (Borovsky et al , 1990, 1993).
  • Mosquito-derived TMOF inhibits the synthesis of serine proteases in the cat flea, (Ctenocephalides felis), stable fly (Stomoxys calcitrans), house fly (Musce domestica), and the midge (Culicoides variipennis) (Borovsky et al, 1990; 1993).
  • This hormone does not inhibit trypsin biosynthesis in flesh flies; the TMOF of these insects, in turn, does not inhibit trypsin biosynthesis in mosquitoes (Borovsky et al., 1990; 1992; 1993; Deloof et al, 1995; Bylenians et al, 1994).
  • Mosquitoes are presently controlled with a variety of chemical agents, including oil, growth regulators (methoprene), biochemicals (Bti), and organophosphates. All of these control agents suffer from significant problems including resistance, detrimental environmental and health effects, and lack of sufficient bioavailability.
  • TMOF by terminating the biosynthesis of trypsin like enzymes, prevents insect digestion, thereby starving the target insect to death even though satiated whereas activated Bti toxins can be immediately effective by passing the activation process that takes place in the larval gut.
  • Both Bti and TMOF polypeptides are nontoxic to non-target organisms, biodegradable and, being surrounded by the cell wall of yeast cells, have a longer half life sufficient for effective insect control with no anticipated long-term persistence. Both TMOF and Bti polypeptides have Environmental Protection Agency (EPA) approval.
  • EPA Environmental Protection Agency
  • the subject invention concerns materials and methods for controlling pests.
  • the pests are mosquito larvae.
  • methods of the invention comprise treating or contacting a pest with an effective amount of a composition comprising a Bti toxin and a TMOF toxin.
  • TMOF enhances the effect of Bti toxins, such that the toxins can be very effective at concentrations that are 5- to 10-fold lower than the concentrations that currently are used, thereby making treatment of mosquito habitat, such as marshes, less expensive.
  • the Bti and TMOF toxins are provided in cells that are consumed by the pest.
  • the cells can be transformed with polynucleotides encoding BH toxins and/or TMOF.
  • the cells are yeast cells.
  • the cell expresses one or more of the Bti toxins Cry4Aa, Cry4Ba, Cry 1 IAa, and/or Cytl Aa.
  • the BU toxins are expressed in activated form.
  • the subject invention also concerns compositions comprising one or more BU toxins and TMOF.
  • the one or more Bti toxin and TMOF is provided as a fusion protein wherein the toxin(s) and TMOF are operably linked together.
  • a composition of the invention comprises one or more polynucleotide encoding one or more Bti toxins and encoding TMOF peptide. In one embodiment, a composition comprises one or more polynucleotide encoding one or more Bti toxins and separate polynucleotides encoding TMOF peptide.
  • the subject invention also concerns a cell comprising one or more Bti toxins and TMOF peptide, and/or polynucleotides encoding the one or more Bti toxins and TMOF peptide.
  • SEQ ID NO:1 is the amino acid sequence of a TMOF peptide that can be used according to the present invention.
  • SEQ ID NO: 2 is an amino acid sequence of a Cry4Aa polypeptide that can be used according to the present invention.
  • SEQ ID NO:3 is an amino acid sequence of a Cry4Ba polypeptide that can be used according to the present invention.
  • SEQ ID N0:4 is an amino acid sequence of a Cry 10Aa polypeptide that can be used according to the present invention.
  • SEQ ID NO:5 is an amino acid sequence of a Cryl IAa polypeptide that can be used according to the present invention.
  • SEQ ID NO:6 is an amino acid sequence of a Cytl Aa polypeptide that can be used according to the present invention.
  • SEQ ID NO:7 is an amino acid sequence of a Cyt2Ba polypeptide that can be used according to the present invention. DETAILED DESCRIPTION OF THE INVENTION
  • the subject invention concerns materials and methods for controlling pests.
  • the pests are blood-sucking insects or larvae thereof.
  • the pests are mosquito larvae.
  • Mosquitos contemplated within the scope of the invention include, but are not limited to, Aedes aegypti, Culex species, and Anopheles species.
  • methods of the invention comprise treating or contacting a pest with an effective amount of a compound or composition comprising one or more Bti toxins and one or more TMOF peptides.
  • the composition is applied to a body of water (e.g. , a lake or pond) or to an aquatic environment (e.g., standing water, an aquarium, etc.).
  • the Bti and TMOF are provided in cells or viral particles, e.g., in cells that are consumed by the pest.
  • the cells can be transformed with polynucleotides encoding Bti toxins and/or TMOF.
  • the cells are bacterial (e.g., E. coli), yeast, and/or algal cells.
  • the cell or viral particle comprises or expresses one or more of the Bti toxins Cry4Aa, Cry4Ba, Cryl IAa, and/or CytlAa, and/or one or more TMOF peptides.
  • a Cry4Aa Bti toxin comprises the amino acid sequence shown in SEQ ID N0:2, or a biologically active fragment or variant thereof.
  • a Cry4Ba Bti toxin comprises the amino acid sequence shown in SEQ ID N0:3, or a biologically active fragment or variant thereof.
  • a Cry 10Aa Bti toxin comprises the amino acid sequence shown in SEQ ID NO:4, or a biologically active fragment or variant thereof.
  • a Cryl IAa Bti toxin comprises the amino acid sequence shown in SEQ ID NO:5, or a biologically active fragment or variant thereof.
  • a CytlAa Bti toxin comprises the amino acid sequence shown in SEQ ID NO:6, or a biologically active fragment or variant thereof.
  • a Cyt2Ba Bti toxin comprises the amino acid sequence shown in SEQ ID NO:7, or a biologically active fragment or variant thereof.
  • the Bti toxins are expressed in activated form.
  • an effective amount of the cells are provided in a natural environment where the pest is located, e.g., a pond or marsh.
  • compositions comprising one or more Bti toxins and TMOF.
  • the one or more Bti toxin and TMOF is provided as a fusion protein wherein the toxin(s) and TMOF are operably linked together.
  • the toxin and TMOF can be directly linked, wherein the terminal amino acid of one protein is followed by the first amino acid in the sequence of another protein.
  • the proteins of a fusion protein are separated by a spacer or linker amino acid sequence.
  • the fusion protein comprises an enzyme- specific cleavage site, e.g. , a trypsin cleavage site, between the one or more Bti toxins and TMOF peptides.
  • proteins are connected through an arginine amino acid.
  • a composition of the invention comprises one or more polynucleotides encoding one or more Bti toxins and/or encoding TMOF peptide.
  • a composition comprises a first polynucleotide encoding one or more BH toxins and a second polynucleotide encoding TMOF peptide.
  • polynucleotides in the composition are provided in the form of an expression construct.
  • the expression construct provides for expression in bacterial, yeast, or algal cells.
  • the codons of the polynucleotides can be optimized for expression in a particular cell, for example yeast cells.
  • the Bti toxin(s) encoded by the one or more polynucleotides is an activated Bti toxin.
  • the subject invention also comprises a cell or viral particle comprising one or more Bti toxins and/or one or more TMOF peptides, and/or polynucleotides encoding the one or more Bti toxins and/or the one or more TMOF peptides.
  • the Bti toxin(s) encoded by the one or more polynucleotides is an activated Bti toxin.
  • the polynucleotides can comprise sequences encoding both Bti toxin(s) and TMOF peptides.
  • the cell or viral particle can comprise a first polynucleotide encoding a Bti toxin(s) and a second polynucleotide encoding a TMOF peptide.
  • the polynucleotides are stably incorporated into the genome of the cell.
  • the cell is a cell that is consumed by the pest, e.g., a bacterial, yeast, or algal cell.
  • the cell is a yeast cell.
  • the bacterial cell is a Bacillus species.
  • the cell is an E. coli cell.
  • the cell is an algal cell, such as Chlorella.
  • the cells can be transformed or transgenic cells.
  • the cells are inactivated or killed.
  • a viral particle of the invention is a baculovirus or an entomopoxvirus.
  • the subject invention also comprises compositions that comprise a first cell comprising a polynucleotide encoding one or more Bti toxin(s) and a second cell comprising a polynucleotide encoding a TMOF peptide.
  • the first and second cells can be the same or different species.
  • the first cells are bacterial cells and the second cells are yeast cells, or vice versa.
  • the first cells comprise bacterial or yeast cells and the second cells comprise algal cells, or vice versa.
  • the subject invention also concerns synthetic capsules which comprise one or more Bti toxins and/or one or more TMOF peptides.
  • the capsules can comprise, for example, a microcontainer such as a microsphere having an outer shell which can degrade when exposed to the conditions of the larvae gut. Materials suitable for selective degrading under such conditions are numerous and commonly known in the art. These microcontainers can be deposited in pest larvae habitats whereby they will be passively ingested during the course of normal feeding.
  • TMOF includes the specific peptide YDP APPPPPP (SEQ ID NO:1), as well as salts, fragments, and analogs thereof that possess TMOF toxin activity.
  • fragments and analogs of TMOF contemplated within the scope of the subject invention include, but are not limited to, those described in U.S. Patent Nos. 5,011,909; 5,130,253; 5,358,934; 5,792,750; 5,629,196; 5,439,821; 6,562,590; and 6,566,129, and published application WO 94/13698 (which also disclose polynucleotides encoding TMOF fragments and analogs).
  • TMOF fragments include, but are not limited to, those having the amino acid sequence YDPAP; YDPAPP; YDPAPPP; YDPAPPPP, and YDPAPPPPP. Any TMOF peptide fragment or analog (and any polynucleotide encoding the same) that exhibits TMOF toxin activity is contemplated for use in the subject invention.
  • a TMOF peptide of the invention comprises one or more D -conformation amino acids.
  • the subject TMOF peptides can have an LD50 against mosquito larvae of less than
  • the peptides have an LD 50 of less than 2.0 ⁇ moles/ml, and, in another embodiment, the peptides have an LD 50 of less than 1.0 ⁇ moles/ml.
  • * 'LD 5 o refers to a lethal dose of a peptide able to cause 50% mortality of larvae maintained on a diet of 1 mg/ml autoclaved yeast (Borovsky and Mahmood, 1995).
  • Another aspect of the subject invention pertains to methods of controlling pests comprising preparing a host to produce a pesticidal polypeptide wherein the host is edible by the target pest and administering the host to the target pest.
  • Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.
  • regulatory elements include promoters, transcription termination sequences, translation termination sequences. enhancers, and polyadenylation elements.
  • expression construct refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.
  • operably linked refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner.
  • operably linked components are in contiguous relation.
  • An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding one or more BU toxin and/or TMOF peptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention.
  • a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity.
  • a transcription start site is typically included in the expression construct. Promoters that can be used for expression constructs in yeast cells include, for example, CMV immediate early promoter; simian virus 40 early enhancer/promoter; yeast alcohol dehydrogenase promoter (Adh 1); yeast INO4 promoter; and alcohol oxidase promoters (AOXl, A0X2).
  • promoters suitable for use with an expression construct of the invention in yeast cells include, but are not limited to, 3- phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, metallothionein promoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.
  • Constitutive promoters such as the ubiquitin, actin, or NOS promoter
  • developmentally- regulated promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • inducible promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • an expression construct of the invention can comprise promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, lambda P L promoter, ⁇ -lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7 promoter, or tac promoter (de Boer et al, 1983).
  • promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, lambda P L promoter, ⁇ -lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7 promoter, or tac promoter (de Boer et al, 1983).
  • Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements.
  • Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination.
  • a signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment.
  • Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct.
  • Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element.
  • CMV cytomegalovirus
  • Intron- mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent.
  • DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal.
  • the expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, /. e. , a transposon.
  • Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA.
  • the subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.
  • the use of the compounds and compositions of the subject invention to control pests can be accomplished readily by those skilled in the art having the benefit of the instant disclosure.
  • the Bti toxins and TMOF peptides may be provided in a cell or encapsulated, incorporated in a granular form, solubilized in water or other appropriate solvent, powdered, and included into any appropriate formulation for direct application to the pest or to a pest-inhabited locus.
  • Formulated bait granules containing an attractant and the pesticidal Bti toxins and/or TMOF peptides of the present invention, or recombinant microbes comprising BU toxin and/or TMOF encoding polynucleotide sequences can be applied to a pest- inhabited locus, such as to the soil or water. Treatments may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like).
  • inert materials such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like).
  • the formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants.
  • Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates or the like.
  • the ingredients may include rheological agents, surfactants, emulsifiers, dispersants or polymers.
  • the formulations can be applied to the environment of the pest by spraying, dusting, sprinkling or the like.
  • the pesticidal concentration of the Bti toxin and/or TMOF peptide will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly.
  • the Bti toxin and/or TMOF peptide will be present in at least about 0.0001% by weight and may be 100% by weight.
  • the dry formulations will have from about 0.0001-95% by weight of the Bti toxin and/or TMOF peptide while the liquid formulations will generally be from about 0.0001-60% by weight of the solids in the liquid phase.
  • the formulations that contain cells will generally have from about 1 to about 10 10 cells/mg. These formulations can be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
  • the transformant cells or viral particles can be applied to the natural habitat of the pest. In some cases, the transformant cells will grow in the pest upon ingestion, while continuing to produce the pesticidal polypeptide(s).
  • the cells or compositions of the invention can be applied by a wide variety of methods known in the art, including pouring, spraying, soaking, injection into the soil, seed coating, seedling coating or spraying or the like.
  • pest control may be attained at or below the surface by adjusting the specific gravity of a microbe or cell of the invention. This can be accomplished by, for example, varying the lipid content of the transformant microorganism strain or cell. It is known that many indigenous aquatic algae float due to their lipid content and/or air vacuoles. A variation in lipid content will allow the transformant strain to be distributed at desired depths below the water surface.
  • the cells, viral particles, and compositions of the invention may also be provided in tablets, pellets, briquettes, bricks, blocks and the like which are formulated to float, maintain a specified depth, or sink as desired.
  • the formulations are formulated to float on the surface of an aqueous medium; in another embodiment they are formulated to maintain a depth of 0 to 2 feet in an aqueous medium; in yet another embodiment the formulations are formulated to sink in an aqueous environment.
  • the cells of the invention may be maintained in a nutrient medium which maintains selectivity and results in a low rate of proliferation.
  • a nutrient medium which maintains selectivity and results in a low rate of proliferation.
  • Various media may be used, such as yeast extract or L-broth.
  • the non-proliferating concentrate may be introduced into an appropriate selective nutrient medium, grown to high concentration, generally from about 10 5 to 10 cells/ml and may then be employed for introduction into the environment of the pest.
  • Larval starvation caused by TMOF which occurs later on after cessation of trypsin biosynthesis, will slow down repair of sublethal damaged gut epithelium caused by ⁇ -endotoxin (Khawaled et al, 1988), thereby enhancing gut absorption of TMOF into the hemo lymph.
  • Table 1 starved Ae. aegypli larvae are 10-30 fold more sensitive to Bti than well-fed larvae.
  • E. coli cells BL21
  • pVE4-ADRC Khasdan el al, 2001
  • the transformed E. coli expressed 3 toxins that will be expressed in our yeast cells (Cry4Aa, Cry 1 IAa, and Cytl Aa).
  • Minimal amount of cells 1.6 x 10 7 cells/mL
  • Yeast cells that did not produce TMOF did not cause mortality to the larvae during the 6-day feeding period, similar to control results (Table 2).
  • E coli cells transformed with pVE4-ADRC expressing Cry4Aa, Cry 11Aa, CytlAa, and p20 were fed for 6 days to groups of first instar larval Ae. aegypt (20 larvae per group in 160 mL water) in the presence of different concentrations of recombinant P. pastoris cells expressing TMOF. Larval survival was followed and is expressed as mean ⁇ S.E.M. The amount of TMOF in recombinant P. pastoris cells was determined by ELISA. a Significant difference from control by student's t-test p ⁇ 0.008. b >c not significant difference p ⁇ .6. d Pichia-TMOF (75-298 nM) does not significantly kill mosquito larvae in 6 days.
  • the subject invention concerns the use of both Bti and TMOF toxins to effectively control mosquito larvae.
  • Bti toxin has to be activated in order to be effective
  • yeast cells can express TMOF and activated Bti.
  • the use of one or more of activated Cry4Aa, Cry4Ba, Cryl IAa, CytlAa, and TMOF can be expressed in P. pastoris cells to effectively control mosquito larvae.
  • the activated BH toxins can immediately bind to the gut membrane of the larvae, whereas TMOF will starve the larvae and make them more susceptible to lower concentrations of the Bti toxins (see Table 1).
  • the activated Bti toxins and TMOF can be expressed as fusion proteins with a trypsin cleavage site between TMOF and the Bti toxins, similar to what has been done with GFP-TMOF fusion protein (see U.S. patents 6,562,590 and 6,566,129).
  • the yeast cell wall protects the activated Bti toxins from exposure to sunlight that inactivates aromatic amino acids in proteins. Since yeast cells are preferred food by mosquito larvae it will ensure effective control of mosquito larvae.
  • the yeast cells can be transformed, for example, with pPICZ B plasmid (Invitrogen, CA USA) by homologous recombination.
  • Polynucleotides encoding Bti activated toxin and TMOF toxin can be provided as synthetic genes that are codon optimized for expression in P. pastoris cells. This ensures high levels of synthesis of Bti toxin and TMOF toxin.
  • the yeast cells can be formulated by standard procedures and applied to an environment (e.g., marsh) where mosquito larvae are found by manual or aerial applications. Since yeast cells harboring TMOF are stable at room temperature for one year or longer and Bti toxins expressed in bacterial cells are known to be stable for long periods of time, the materials will be stable in the environment allowing monthly treatments against mosquito larvae.

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Abstract

La présente invention porte sur des matières et des procédés pour lutter contre des nuisibles tels que les larves de moustique. Dans un mode de réalisation, les procédés de l'invention comprennent la mise en contact d'un nuisible avec une quantité efficace d'une toxine Bti et d'un facteur oostatique de modulation de trypsine (TMOF). Le Bti et le TMOF peuvent être introduits dans des cellules qui sont consommées par le nuisible. La présente invention porte également sur des compositions comprenant une ou plusieurs toxines Bti et TMOF.
PCT/US2009/067180 2008-12-08 2009-12-08 Lutte contre les larves de moustique par les toxines bti et tmof WO2010077672A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
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WO2012105824A1 (fr) * 2011-01-31 2012-08-09 Entogenex Industries Sdn Bhd Bioinsecticide et son procédé de production
EP2744920A4 (fr) * 2011-08-19 2015-06-03 Synthetic Genomics Inc Procédé intégré pour l'identification à haut rendement de nouvelles compositions de pesticides et ses utilisations
WO2016179086A3 (fr) * 2015-05-01 2016-12-15 Board Of Regents, The University Of Texas System Souches de chlamydomonas avec des protéines cry exprimées par les chloroplastes pour la lutte biologique contre les moustiques qui transmettent une maladie
WO2017199078A3 (fr) * 2016-05-17 2018-02-01 Universidad Nacional Autonoma De Mexico Mutants cyt1a de bacillus thuringiensis
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WO2018187342A1 (fr) * 2017-04-04 2018-10-11 Baylor University Toxines moustiquicides ciblées
TWI728264B (zh) * 2018-09-10 2021-05-21 高雄醫學大學 控制蚊媒傳染疾病傳播之微生物製劑

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WO2012105824A1 (fr) * 2011-01-31 2012-08-09 Entogenex Industries Sdn Bhd Bioinsecticide et son procédé de production
EP2744920A4 (fr) * 2011-08-19 2015-06-03 Synthetic Genomics Inc Procédé intégré pour l'identification à haut rendement de nouvelles compositions de pesticides et ses utilisations
WO2016179086A3 (fr) * 2015-05-01 2016-12-15 Board Of Regents, The University Of Texas System Souches de chlamydomonas avec des protéines cry exprimées par les chloroplastes pour la lutte biologique contre les moustiques qui transmettent une maladie
US10793611B2 (en) 2016-05-17 2020-10-06 Universidad Nacional Autónoma de México Bacillus thuringiensis CYT1A mutants
WO2017199078A3 (fr) * 2016-05-17 2018-02-01 Universidad Nacional Autonoma De Mexico Mutants cyt1a de bacillus thuringiensis
US20180086796A1 (en) * 2016-07-08 2018-03-29 Brian A. Federici USE OF Cyt1Aa AS BROAD SPECTRUM TARGETING DOMAIN FOR INSECTICIDAL PROTEINS WITH BASIC RESEARCH AND BIOTECHNOLOGY APPLICATIONS
US10472397B2 (en) * 2016-07-08 2019-11-12 The Regents Of The University Of California Use of Cyt1Aa as broad spectrum targeting domain for insecticidal proteins with basic research and biotechnology applications
WO2018187342A1 (fr) * 2017-04-04 2018-10-11 Baylor University Toxines moustiquicides ciblées
CN110475867A (zh) * 2017-04-04 2019-11-19 贝勒大学 靶向型杀蚊毒素
JP2020515628A (ja) * 2017-04-04 2020-05-28 ベイラー ユニバーシティ 標的化された殺蚊毒素
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US11479583B2 (en) 2017-04-04 2022-10-25 Baylor University Targeted mosquitocidal toxins
TWI728264B (zh) * 2018-09-10 2021-05-21 高雄醫學大學 控制蚊媒傳染疾病傳播之微生物製劑

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