CN101385467B - Novel pesticidal toxins - Google Patents

Abstract

A novel pesticidal toxin that is highly active against a wide range of lepidopteran insect pests is disclosed. The DNA encoding the pesticidal toxin can be used to transform various prokaryotic and eukaryotic organisms to express the pesticidal toxin. These recombinant organisms can be used to control lepidopteran insects in various environment.

Description

New Pesticidal toxins

Technical field

The present invention relates to derive from the new Vip3 toxin of bacillus thuringiensis (Bacillus thuringiensis), its expression causes the nucleotide sequence of described toxin, prepare the method for this toxin, and utilize this toxin and corresponding nucleotide sequence to control the method for insect.

Background technology

Plant insect is the principal element of the important crop loss in the world.The Jin U.S., every year comprises that by nonmammalian insect infecting of insect just lose approximately 8,000,000,000 dollars.Except field-crop loss, insect pest, for vegetables and application person, for the producer of ornamental flower, and is also burden for homestead gardener.

At present mainly by the widespread use of chemical insecticide, control insect pest, this chemical insecticide can actively suppress insect growth, hinder the aspect of insect feed or breeding, or causes death.Therefore can reach good insect and control, but these pharmaceutical chemicalss also affect other useful insect sometimes.Caused another problem of widespread use of chemical insecticide is that resistance insect species occurs.By various resistance, process this phenomenon of having put into practice partial rcsponse, but the medicament of alternative Control pests is existed to the needs that day by day increase.Biological insect is controlled medicament, bacillus thuringiensis (Bacillusthuringiensis) strain that Pesticidal toxins resembles delta-endotoxin as expressed has also been applied to farm crop, and have satisfied result, this provides Res fungibiles or fill-in for chemical insecticide.Separated some genes of these delta-endotoxins of encoding, and confirmed that their expression in heterologous host provide the another kind of instrument of controlling for the important insect pest of economy.Especially; in transgenic plant Pesticidal toxins as the expression of bacillus thuringiensis (Bacillus thuringiensis) delta-endotoxin provide avoid for effective protection of insect pest infringement; and express the commercialization of transgenic plant of this toxin, make peasant can reduce chemical insect and control using of medicament.

Now identified the protein of other non-endotoxin gene and their codings.United States Patent (USP) 5,877,012,6,107,279,6,137,033 and 6,291,156, and Estruch etc. (1996, Proc.Natl.Acad.Sci.93:5389-5394) and Yu etc. (1997, Appl.Environ.Microbiol.63:532-536) described the new class insect-killing protein that is called Vip3, being incorporated to all these documents is here reference.The approximately 88kDa protein that produces during Vip3 encoding sequence coding genus bacillus (Bacillus) vegetative growth phase and secrete (nutritive insecticidal protein, VIP).Vip3A protein has anti-wide spectrum lepidoptera pest, include but not limited to black cutworm (BCW, Agrotis ipsilon), meadow mythimna separata (FAW, Spodoptera frugiperda), cigarette aphid noctuid (TBW, Heliothis Virescens), insecticidal activity with the real noctuid of paddy (CEW, Helicoverpa zea).Recently, have been found that and express the feed infringement that the Genes For Plant Tolerance hemipteran insect of Vip3A protein causes.Therefore Vip3A protein has shown unique insecticidal activity spectrum.Other the open WO98/18932 of patent, WO98/33991, WO98/00546, and WO99/57282 has also identified the homologue of Vip3A proteinoid now.

The continuous application of chemistry and biological control method has increased insect development to the drug-fast chance of this control method.And, by current method, only can control several specific insect pests.

Therefore, need to develop can be for peasant provides economic interests, and be environmentally acceptable new and effectively insect control medicament.In special needs target is compared with the important insect pest of the economy of wide spectrum with can effectively control existing insect is controlled to the control medicament that medicament has the insect strain that resistance maybe may develop immunity to drugs.And also expectation obtains it and uses the impact of environment is down to minimum medicament.

Summary of the invention

The present invention is by providing new gene and toxin to be devoted to solve the needs that new insect is controlled medicament, and described new gene and toxin are different from United States Patent (USP) 5,877,012,6,107,279, and 6,137,033, Estruch etc. (1996), Yu etc. (1997) and WO98/18932, WO98/33991, WO99/57282, and the disclosed gene of WO98/00546 and toxin.

The invention provides composition and the method for controlling plant insect.Especially, provide from the separated new vip3 nucleotide sequence of bacillus thuringiensis (Bacillus Thuringiensis), the basic sequence identical with it, the expression of described sequence caused the important insect pest of economy, and the insect pest that particularly infects plant has the desinsection toxicity of high special toxicity.The invention further relates to by described nucleotide sequence and express the new Pesticidal toxins producing, the composition that contains this Pesticidal toxins and preparation, they can suppress the ability of insect pest survival, Growth and reproduction, maybe can limit farm crop infringement or the loss relevant with insect.For example the present invention also relates to have in the hybrid toxins of stronger insecticidal activity or to utilize in as DNA reorganization in recombination method in preparation the method for this nucleotide sequence.The invention further relates to the method for this toxin of preparation; for example in microorganism, utilize this nucleotide sequence to control the method for insect; or in transgenic plant, utilize this nucleotide sequence that the method for the provide protection of avoiding insect damage is provided; relate to and utilize Pesticidal toxins and the composition that comprises this Pesticidal toxins and the method for preparation, the region of for example easily infecting to region or the preventive treatment insect of infestation by insect or plant to use Pesticidal toxins or composition or preparation so that the protection of avoiding insect pest infringement to be provided.

The activity of the new anti-insect of Pesticidal toxins described here is very high.For example, by Pesticidal toxins, can control the important insect pest of a large amount of economy, as lepidopteran European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), Southwest Maize snout moth's larva (Diatraea grandiosella), sugarcane borer (Diatraeasaccharalis), Mediterranean Sea Pyrausta nubilalis (Hubern). (Sesanlia nonagroides), choice refreshments dash forward noctuid (Helicoverpa punctigera) and bollworm (Helicoverpa Armigera).Described Pesticidal toxins can be used separately or combine use with other insect control strategy, to provide, has maximum insect control effect, the impact on environment is down to minimum simultaneously.

According to an aspect, the invention provides the separated nucleic acid molecule of the nucleotide sequence that comprises toxin-encoding, described toxin has anti-insect active, wherein said nucleotide sequence: (a) have at least 92% sequence identity with SEQ ID NO:1; Or nucleotides sequence dependent of dead military hero isocoding (isocoding) (b) and (a); Or (c) coding has the aminoacid sequence of at least 91% sequence identity with SEQ ID NO:3.

In an embodiment aspect this, separated nucleic acid molecule comprises the nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1.

In another embodiment aspect this, separated nucleic acid molecule comprises the nucleotide sequence that has the nucleotides sequence dependent of dead military hero isocoding of at least 92% sequence identity with same SEQ IDNO:1.

In further embodiment, separated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQID NO:3.

In another embodiment aspect this, separated nucleic acid molecule comprises to encode the nucleotide sequence of the aminoacid sequence of at least 91% sequence identity with SEQ IDNO:2.In further embodiment, the nucleotide sequence that separated nucleic acid molecule comprises aminoacid sequence shown in coding SEQ ID NO:3.

In one embodiment, separated nucleic acid molecule comprises the contained approximately 2.4kbDNA fragment of pNOV1325 (called after ATCC PTA-3868) containing in intestinal bacteria (E.coli) strain DH5 α.In another embodiment, separated nucleic acid molecule comprises contained approximately 2.4kb DNA fragmentation in the pNOV1328 (called after ATCC PTA-3869) containing in intestinal bacteria (E.coli) strain DH5 α.

According to an embodiment of the invention, the toxin of the separated anti-lepidopterous insects activity of nucleic acid molecule encoding tool.In further embodiment, lepidopterous insects is selected from: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodoptera exigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

The present invention also provides mosaic gene, and it comprises the allogeneic promoter sequence being operably connected with nucleic acid molecule of the present invention.Further, the invention provides the recombinant vectors that comprises this mosaic gene.The present invention also provides the virus that comprises this mosaic gene.The virus of this aspect of the present invention can be animal virus or plant virus.Further, the invention provides the genetically modified host cell that comprises this mosaic gene.The genetically modified host cell of this aspect of the present invention can be zooblast, bacterial cell, yeast cell or vegetable cell, preferred plant cell.Further, the invention provides the transgenic plant that comprise this vegetable cell.The transgenic plant of this aspect of the present invention can be jowar, wheat, Sunflower Receptacle, tomato, wild cabbage crop, cotton, paddy rice, soybean, beet, sugarcane, tobacco, barley, oilseed rape or corn, preferably corn.Further, the invention provides the seed from transgenic plant, described transgenic plant comprise: jowar, wheat, Sunflower Receptacle, tomato, wild cabbage crop, cotton, paddy rice, soybean, beet, sugarcane, tobacco, barley, oilseed rape and corn.In a preferred embodiment, seed comes from rotaring gene corn plant.

The present invention also provides and has further comprised coding the second nucleotide sequence of the second desinsection element or the transgenic plant of nucleotide sequence group.Particularly preferred the second nucleotide sequence is the nucleotide sequence of coding delta-endotoxin, and the nucleotide sequence of other nutritive insecticidal archon of encoding or coding are for the nucleotide sequence of nonprotein desinsection element the way of production.

According to an aspect, the invention provides the separated toxin with anti-insect active, wherein toxin comprises monoamino-acid sequence, this aminoacid sequence: (a) have at least 91% sequence identity with SEQ ID NO:2; Or (b) be to have the expression of nucleic acid molecule of the nucleotide sequence of at least 92% sequence identity to produce by comprising with SEQ ID NO:1.

In an embodiment aspect this, separated toxin comprises the aminoacid sequence that has at least 91% sequence identity with SEQ ID NO:2.

In further embodiment, separated toxin comprises the aminoacid sequence shown in SEQ ID NO:2.

In another embodiment aspect this, this separated toxin is to have the expression of nucleic acid molecule of the nucleotide sequence of at least 92% sequence identity to produce by comprising with SEQ IDNO:1.

In another embodiment, this separated toxin is that the expression by comprising the nucleic acid molecule of nucleotide sequence shown in SEQ ID NO:1 or SEQID NO:3 produces.

In another embodiment, toxin of the present invention has anti-lepidopterous insects activity.In further embodiment, toxin has the activity of anti-following insect: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

In further embodiment, described toxin is to be that intestinal bacteria (E.coli) strain of ATCC preserving number PPTA-3868 or intestinal bacteria (E.coli) strain that preservation is ATCC preserving number PPTA-3869 produce by preservation.

The present invention also provides and has comprised the endotoxic composition of the present invention of controlling insect significant quantity.

On the other hand, the invention provides the method with anti-insect toxins activity of producing, comprise that (a) obtains the genetically modified host cell that comprises mosaic gene, described mosaic gene itself comprises the allogeneic promoter sequence being operably connected with nucleic acid molecule of the present invention; (b) in transgenic cell, express this nucleic acid molecule, produced at least one and there is the toxin of anti-insect active.

Aspect further, the invention provides the method for producing anti-insecticidal transgenic plant, comprise in transgenic plant and introduce nucleic acid molecule of the present invention, wherein nucleic acid molecule can be expressed to control the significant quantity of insect in transgenic plant.According to an embodiment, insect is lepidopterous insects.In further embodiment, lepidopterous insects is selected from: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodoptera exigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

Aspect further, the invention provides the method for controlling insect, comprise toxin of the present invention from significant quantity to insect that send.According to an embodiment, insect is lepidopterous insects.In further embodiment, lepidopterous insects is selected from: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).In another embodiment, oral delivery is to insect toxins.In further embodiment, described toxin is transgenic plant oral delivery by comprising the nucleotide sequence of expressing toxin of the present invention.

The present invention also provides the hybrid toxins with anti-insect active, and wherein hybrid toxins is by the nucleic acid molecule encoding that comprises nucleotide sequence of the present invention.

In one embodiment, hybrid toxins of the present invention has anti-lepidopterous insects activity.In further embodiment, lepidopterous insects is selected from European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

In another embodiment, hybrid toxins is by nucleotide sequence coded described in SEQ ID NO:6.

The present invention also provides the composition of the hybrid toxins of the present invention that comprises insecticidal effective dose.

On the other hand, the invention provides the method for producing the hybrid toxins with anti-insect active, comprise that (a) obtains the genetically modified host cell that comprises mosaic gene, described mosaic gene itself comprises the allogeneic promoter sequence being operably connected with nucleic acid molecule of the present invention; (b) in transgenic cell, express this nucleic acid molecule, this has produced at least one and has had the hybrid toxins of anti-insect active.

Aspect further, the invention provides the method for producing anti-insecticidal transgenic plant, comprise in transgenic plant and introduce nucleic acid molecule of the present invention, wherein said nucleic acid molecule encoding hybrid toxins, and this hybrid toxins can be expressed to control the significant quantity of insect in transgenic plant.According to an embodiment, insect is lepidopterous insects.In another embodiment, lepidopterous insects is selected from: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutellaxylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotisipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothisvirescens), greedy noctuid (Spodoptera exigua), pink bollworm (Pectinophoragossypiella), amyloid plaque noctuid (Trichoplusia ni), the thin leaf roller of Sunflower Receptacle (Cochylis hospes) and Sunflower Receptacle head moth (Homoeosoma electellum).

In yet another aspect, the invention provides the method for controlling insect, comprise the hybrid toxins of the present invention of sending significant quantity to insect.According to an embodiment, insect is lepidopterous insects.In a preferred embodiment, lepidopterous insects is selected from: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), pink bollworm (Pectinophora gossypiella), amyloid plaque noctuid (Trichoplusia ni), the thin leaf roller of Sunflower Receptacle (Cochylis hospes) and Sunflower Receptacle head moth (Homoeosoma electellum).In another embodiment, hybrid toxins oral administration is delivered to insect.In further embodiment, by transgenic plant, to insect oral delivery hybrid toxins, described transgenic plant comprise the nucleotide sequence of expressing hybrid toxins of the present invention.

The present invention also provides the hybrid toxins with anti-insect active, wherein from the C-terminal region that comprises Vip3 toxin and the amino terminal region of the different Vip3 toxin that are attached thereto to the direction of carboxyl by amino, wherein C-terminal district inclusion and SEQ ID NO:2 amino acid 579-787 have the aminoacid sequence of at least 75% identity; There is at least 75% identity amino terminal region and SEQ IDNO:4 amino acid/11-578.In further embodiment, C-terminal district inclusion SEQ ID NO:2 amino acid 578-787, amino terminal region comprises SEQID NO:5 amino acid/11-579.In further embodiment, hybrid toxins comprises SEQ IDNO:7 amino acid/11-787.

According to the present invention, the hybrid toxins of this aspect has anti-lepidopterous insects activity.In further embodiment, lepidopterous insects is selected from: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), pink bollworm (Pectinophora gossypiella), amyloid plaque noctuid (Trichoplusia ni), the thin leaf roller of Sunflower Receptacle (Cochylis hospes) and Sunflower Receptacle head moth (Homoeosoma electellum).

This aspect of the present invention also comprises the nucleic acid molecule of the nucleotide sequence of the hybrid toxins that comprises this aspect of encoding.

The present invention also provides the method for controlling insect, and wherein transgenic plant further comprise the second nucleotide sequence or the nucleotide sequence group of the second desinsection key element of encoding.Particularly preferred the second nucleotide sequence is the nucleotide sequence of coding delta-endotoxin, and the nucleotide sequence of the nutritive insecticidal archon of encoding other or coding are for the nucleotide sequence of nonprotein insects key element the way of production.

Yet, the present invention provides the method for sudden change nucleic acid molecule of the present invention on the other hand, wherein said nucleic acid molecule has been cut into the big or small double-stranded random fragment storehouse of expectation, the method comprises: (a) in double-stranded random fragment storehouse, add one or more strands or double chain oligonucleotide, wherein every kind of oligonucleotide comprises the region that has the region of identity with double-stranded template polynucleotide and have heterology; (b) by the mixture sex change of the double-stranded random fragment obtaining thus and oligonucleotide, be single-chain fragment; (c) causing that single-chain fragment has under the condition of regional annealing of identity, single-chain fragment storehouse and polysaccharase that incubation obtains thus, to be formed into right annealing fragment, identity region is enough to make a member of pairing to cause copying of another member, forms thus the double-stranded polynucleotide of sudden change; (d) at least another two-wheeled circulation of repetition second and third step, the double-stranded polynucleotide that the mixture wherein obtaining in next round circulation second step comprises the sudden change obtaining from previous round third step, and next round circulation has formed the double-stranded polynucleotide of further sudden change.

By the description below the present invention and the study of non-limiting example, other aspects and advantages of the present invention will become apparent for those skilled in the art.

The brief description of sequence in sequence table

SEQ ID NO:1 is the encoding sequence of natural vip3B gene.

SEQ ID NO:2 is the aminoacid sequence of SEQ ID NO:1. coding.

SEQ ID NO:3 is the encoding sequence of the vip3B gene of corn optimization.

SEQ ID NO:4 is the encoding sequence of natural vip3A gene.

SEQ ID NO:5 is the aminoacid sequence of SEQ ID NO:4. coding.

SEQ ID NO:6 is the encoding sequence of heterozygosis vip3A-B gene.

SEQ ID NO:7 is the aminoacid sequence of SEQ ID NO:6. coding.

SEQ ID NO:8-13 is primer used in this invention.

Preservation

Microbial preservation budapest treaty according to international recognition for patented procedure object, material beneath is deposited in U.S. typical case culture center (ATCC), 10801University Blvd., Manassas, VA.Once grant patent, to remove the institute that preserved material is obtained restricted by irrevocable.

clone preserving number preservation date

16 days November calendar year 2001 of pNOV1325 ATCC No.PTA-3868

16 days November calendar year 2001 of pNOV1328 ATCC No.PTA-3869

Definition

" activity " of toxin of the present invention means the insect control medicament that toxin plays tool Orally active, toxic effect, or can destroy or stop the insect that may cause or not cause insect death to be taken food.When toxin of the present invention is delivered to insect, result is insect death normally, or insect not take, and to produce the resource that insect can obtain toxin be food.

" be associated "/" being operably connected " refer to two physics or the relevant nucleotide sequence of function.For example, if promotor or regulate DNA sequence dna and coding RNA or protein DNA sequence are operably connected or locate to such an extent as to regulates the DNA sequence dna will impact coding or the expression level of structural DNA sequence, claims promotor or adjusting DNA sequence dna and coding RNA or protein DNA sequence " relevant " so.

" mosaic gene " is recombinant nucleic acid sequence, promotor or regulate be operably connected coding mRNA or as the nucleotide sequence of protein expression of nucleotide sequence wherein, or associated, regulate like this nucleotide sequence can regulate transcribing or expressing of associated core acid sequence.As occurring in nature is found, the adjusting nucleotide sequence of mosaic gene is not to be generally operationally connected with the nucleotide sequence being associated.

" encoding sequence " is to be transcribed into RNA as mRNA, rRNA, and tRNA, snRNA, has the nucleotide sequence of adopted RNA or sense-rna.Preferably, RNA translates to produce protein subsequently in organism.

" control " insect means by there being toxic action to suppress the ability of insect pest survival, growth, feed and/or breeding, or means to limit infringement or the loss that in farm crop, insect is relevant.Although " control " insect preferably means kill insects, " control " insect also can refer to not kill insects.

" send " toxin and mean toxin and contact with insect, cause the control that has toxic action and insect.Can send toxin by many generally acknowledged methods, for example by insect, ingest oral or by Expressed in Transgenic Plant, the protein composition of making, sprayable protein composition, bait matrix, or any other art-recognized toxin delivery system and contacting with insect.

" effectively insect manipulated variable " means to survive by there being toxic action to suppress insect, growth, the infringement that in feed and/or fecundity or restriction farm crop, insect is relevant or the toxin concentration of loss.Although " effectively insect manipulated variable " preferably means kill insects, " effectively insect manipulated variable " also can refer to not kill insects.

Here " expression cassette " used means to guide and is applicable to the nucleotide sequence that in host cell, specific nucleotide sequence is expressed, and comprises the promotor being operably connected with object nucleotide sequence, the described object nucleotide sequence termination signal that is operably connected.Conventionally, it also comprises the required sequence of the correct translation of this nucleotide sequence.The expression cassette that comprises object nucleotide sequence can be chimeric, and meaning one of its composition is at least allos with respect to its at least one other composition.Expression cassette can be also natural, but obtains for heterogenous expression with recombinant forms.Yet expression cassette is allos with respect to host conventionally, that is, the specific nucleic acid sequence of expression cassette is natural appearing in host cell not, and must be introduced into by transformation event the precursor of host cell or host cell.In expression cassette the expression of nucleotide sequence can be subject to constitutive promoter or only when host cell is exposed to some specific outside stimulus things just initial inducible promoter of transcribing control.The in the situation that of multicellular organisms, as plant, promotor also can Shi Dui particular organization organ or the etap special.

" gene " is to be positioned at genome, except aforementioned nucleic acid sequence encoding, also to comprise the definite area that other mainly regulates nucleotide sequence, described adjusting nucleotide sequence be responsible for encoding part expression, transcribe and translate control.Gene also can comprise other 5 ' and 3 ' non-translated sequence and terminator sequence.The element that further can exist is, for example intron.

" goal gene " refers to any gene, when shifting it in plant time, can give plant with expectation characteristic as antibiotics resistance, virus resistance, insect-resistant, disease resistance or the resistance to other insect, herbicide tolerant, improved nutritive value, the fecundity of improved performance or change in Industrial processes." goal gene " can be also to transfer in plant to have the enzyme of commercial value or the gene of production of metabolites for plant.

" allos " nucleotide sequence is the not natural relevant nucleotide sequence of host cell being introduced into it, comprises natural acid sequence and exists with multiple copied non-natural.

" homology " nucleotide sequence is the natural nucleotide sequence being associated of host cell being introduced into it.

" homologous recombination " is the mutual exchange of the intermolecular nucleic acid fragment of homologous nucleic acid.

Here " hybrid toxins " used is the Pesticidal toxins of artificial preparation, the amino acid region that it comprises a toxin or fragment are connected from the amino acid region or the fragment that derive from different toxin, for example, Vip3B C-stub area by SEQ ID NO:2 amino acid 579 to amino acid 787 is connected with the Vip3A N-stub area of SEQ ID NO:5 amino acid/11 to amino acid 578, but is not limited to this.

" desinsection " is defined as and can controls insect, preferably kills their toxicity biological activity.

When nucleic acid sequence encoding is when having the polypeptide of same acid sequence with reference to the polypeptide of nucleic acid sequence encoding, this nucleotide sequence with reference to nucleotide sequence, belong to " isocoding ".

" separation " nucleic acid molecule or separated enzyme are artificially and the separated existence of its natural surroundings, are not therefore nucleic acid molecule or the enzyme of natural product.Separated nucleic acid molecule or enzyme can exist with purified form, or may reside in non-natural environment, for example, in recombinant host cell.

" nucleic acid molecule " or " nucleotide sequence " is can be from the list of any source separation or the linear fragment of double-stranded DNA or RNA.In the context of the invention, preferably, nucleic acid molecule is DNA fragmentation.

" plant " is any plant, particularly spermatophyte in any etap.

" vegetable cell " is structure and the physiology unit of plant, comprises protoplastis and cell walls.Vegetable cell can be separated unicellular or culturing cell form, or as a high organized unit part for plant tissue, plant organ or whole plant for example.

The plant unit that " plant cell cultures " means the various etap as, protoplastis for example, cell culture cell, the cell in plant tissue, pollen, pollen tube, ovule, blastular, the culture of zygote and embryo.

" vegetable material " refers to blade, stem, root, the part of flower or flower, fruit, pollen, ovum, zygote, seed, cutting, cell or tissue culture, or any other parts or the product of plant.

" plant organ " be plant obviously and visible structure and break up part, as root, stem, leaf, bud or embryo.

Here " plant tissue " used means to be organized into one group of vegetable cell of structure and function unit.Any tissue that comprises plant in plant or in culture.This term includes but not limited to whole plant, plant organ, plant seed, tissue culture and any vegetable cell group that is organized into structure and/or functional unit.The combined utilization of this term and above-mentioned any particular type plant tissue or separately application maybe this definition comprise other application and do not mean and get rid of any other type plant tissue.

" promotor " is the non-translation DNA sequence of coding region upstream, the binding site that it comprises RNA polymerase 11, and the transcribing of initiate dna.Promoter region also can comprise other element as genetic expression instrumentality.

" protoplastis " is the separated vegetable cell that there is no cell walls or only have parts of fine cell wall.

" regulatory element " refers to participate in controlling the sequence that nucleotide sequence is expressed.The promotor that regulatory element comprises be operably connected object nucleotide sequence and termination signal.Conventionally they also comprise the required sequence of the correct translation of nucleotide sequence.

" reorganization " nucleic acid is by Shuffling Method, the nucleic acid that any Shuffling Method produces as described herein.Mode by manually and alternatively circulation (physics or in fact) the two or more nucleic acid (or character string) of recombinating can produce reorganization nucleic acid.Usually, in Shuffling Method, utilize a step or multistep screening step to identify object nucleic acid; Can before or after any reconstitution steps, carry out this screening step.In some (but not all) reorganization embodiments, be desirably in before screening and carry out many wheel restructuring to increase the diversity in screening storehouse.Optionally, can be cycled to repeat all processes of restructuring and screening.Based on context, reorganization can refer to all processes of restructuring and screening, or can only refer to the restructuring part of all processes.

Basic identical: the phrase " basic identical " in two nucleic acid or protein sequence context refer to when as utilize below one of sequence comparison algorithm or range estimation and relatively and when maximum degree of correspondence is measured in comparison, have at least 60%, preferably 80%, more preferably 90%, even more preferably two or more sequences or the subsequence of 95% and most preferably at least 99% Nucleotide or amino-acid residue identity.Preferably, at the sequence area at least about 50 residue length, more preferably the region at least about 100 residues exists basic identity, most preferably at least about 150 residues, sequence is essentially identical.In particularly preferred embodiments, in whole encoding sequence length, sequence is essentially identical.And essentially identical nucleic acid or protein sequence are fulfiled essentially identical function.

For sequence comparison, conventionally, a sequence is compared with detection sequence as canonical sequence.When utilizing sequence comparison algorithm, input detects and canonical sequence enters in computer, if necessary, specifies the coordinate of subsequence and the parameter of sequence algorithm program.Then, according to selected program parameter, sequence comparison algorithm detects sequence with respect to the percentage sequence identity of canonical sequence by calculating.

For example, by Smith & Waterman, local homology's algorithm of Adv.Appl.Math.2:482 (1981), by Needleman & Wunsch, the sequence analysis algorithm of J.Mol.Biol.48:443 (1970), by Pearson & Lipman, Proc.Nat the similarity retrieval method of Acad.Sci.USA 85:2444 (1988), (GAP in Wisconsin Genetics software package is implemented in computerize by these algorithms, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575 Science Dr., Madison, WI) or by range estimation (conventionally referring to, Ausubel etc., below) can compare the best comparison of sequence.

An examples of algorithms that is suitable for measuring percentage sequence identity and sequence similarity is BLAST algorithm, and at Altschul etc., J.Mol.Biol.215:403-410 has described this algorithm in (1990).The software that can openly obtain carrying out BLAST analysis by NCBI (http://www.ncbi nlm.nih.gov/).This algorithm comprises: be tested and appraised out and search the short word that in sequence, length is W and first identify high sub-sequence to (HSP), described short word with database in the word of equal length compare meet or meet some and divide T on the occasion of thresholding.T is called adjacent words score threshold (Altschul etc., 1990).These initial neighborhood word are hit the longer HSP that goes discovery to comprise them as the clue that starts to search.Then, these words hit the extension far away as far as possible of the both direction along each sequence, until accumulation comparison score value no longer increases.For nucleotide sequence, operation parameter M (the award score value that mates in pairs residue; Always be greater than zero) and N (the point penalty value of mispairing residue; Always be less than zero) calculating accumulation score value.For aminoacid sequence, with the matrix computations accumulation score value of scoring.When accumulation comparison score value reduces X from obtained maximum value, stop hitting extension to the word of all directions, due to one or more negative score value residues comparison accumulation, or any of two sequences be when reach home, and accumulation score value reaches or lower than zero.The parameter W of BLAST algorithm, T and X have determined susceptibility and the speed of comparison.BLASTN program inner setting value (for nucleotide sequence) is word length value (W) 11, expected value (E) 10, and cutoff value 100, M=5, N=-4, and compare two chains.For aminoacid sequence, BLASTP program inner setting value is word length value (W) 3, expected value (E) 10 and BLOSUM62 score matrix (referring to, Henikoff & Henikoff, Proc.Natl.Acad.SCI.USA 89:10915 (1989)).

Except calculating percentage sequence identity, BLAST algorithm also carry out similarity between two kinds of sequences statistical analysis (referring to, Karlin & Altschul for example, Proc.Nat ' l.Acad.Sci.USA 90:5873-5787 (1993)).It is minimum and probability (P (N)) that the similarity that BLAST algorithm provides is measured, and it provides the accidental probability level that occurs coupling between two Nucleotide or aminoacid sequence.For example, if detect nucleotide sequence minimum and probability when with reference to nucleotide sequence comparison, be less than approximately 0.1, more preferably less than approximately 0.01, be most preferably less than approximately 0.001, think that so detection nucleotide sequence is similar to canonical sequence.

Two kinds of essentially identical another indexs of nucleotide sequence are the hybridization mutually under stringent condition of two kinds of molecules.Phrase " specific hybrid " refers to that under stringent condition, molecule is only combined with specific nucleotide sequence, forms duplex or hybridization when this sequence is for example present in, in complex mixture (, total cell) DNA or RNA molecule." basic combination " refers to the complementation hybridization between probe nucleic acid and target nucleic acid, and comprises less mispairing, by reducing the severity of hybridization medium, can tolerate this mispairing, to realize the expectation of target nucleic acid sequence, detects.

In nucleic acid hybridization is tested as Southern and Northern hybridization context, " stringent hybridization condition " and " strictly hybridizing rinsing condition " is sequence dependent, and is different under varying environment parameter.Longer sequence is specific hybrid under comparatively high temps.At Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, the 2nd chapter part i " Overview of principles of hybridization and the strategyof nucleic acid probe assays " Elsevier, the detailed guide that can find related nucleic acid to hybridize in New York.Usually, the hybridization that severity is strong and rinsing condition are chosen to be lower approximately 5 ℃ in definite ionic concn and the heat fusion joint temperature (Tm) under pH than particular sequence.Normally, under " stringent condition ", probe will with the hybridization of its target subsequence, and not with other sequence hybridization.

The temperature of Tm when be (under definite ionic concn and pH condition) 50% target sequence, with the probe mating completely, hybridization occurs.For specific probe, select to equal the very strict condition of Tm.For length on filter membrane in Southern or Northern trace, be the hybridization of the complementary nucleic acid of 100 above complementary residues, an example of stringent hybridization condition is at 42 ℃, has 50% methane amide of 1mg heparin, spends the night and carries out this hybridization.The example of the rinsing condition of high severity is 72 ℃, 0.15M NaCl approximately 15 minutes.The example of strict rinsing condition is at 65 ℃, 0.2xSSC rinsing 15 minutes (referring to, Sambrook, below, the description of SSC damping fluid).Conventionally, before high severity washing, carry out the rinsing of low severity, to remove background probe signals.For for example, more than for the duplex of 100 Nucleotide, the example of moderate severity rinsing is 45 ℃, 1xSSC rinsing 15 minutes.For for example, more than for the duplex of 100 Nucleotide, the example of low severity rinsing is 40 ℃, 4-6xSSC rinsing 15 minutes.For short probe (for example, approximately 10 to 50 Nucleotide), stringent condition generally includes the salt concn that is less than about 1.0M Na ion, conventionally at pH7.0 to 8.3, Na ionic concn (or other salt) is approximately 0.01 to 1.0M, and typical temperature is at least approximately 30 ℃.By adding destabilizing agent, also can obtain stringent condition as methane amide.Usually, in specific cross is measured, the just irrelevant viewed value of probe high 2 times (or higher) of signal to noise ratio shows to have detected specific hybridization.The nucleic acid of not hybridizing mutually under stringent condition, if the protein of their codings is essentially identical, they are still essentially identical so.For example, when the maximum codon degeneracy allowing with genetic code obtains nucleic acid copy, just there will be this situation.

The example that hybridization/rinsing condition arranges below, described condition can be for clone with the present invention with reference to the essentially identical homologous nucleotide sequence of nucleotide sequence: preferably with reference to nucleotide sequence with reference to nucleotides sequence, be listed in 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 2XSSC, rinsing in 0.1%SDS, more preferably at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 1XSSC, rinsing in 0.1%SDS, more preferably at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 0.5XSSC, rinsing in 0.1%SDS, preferably at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5MNaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 0.1XSSC, rinsing in 0.1%SDS, more preferably at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 65 ℃, 0.1XSSC, rinsing in 0.1%SDS.

Two nucleotide sequences or another substantially identical index of protein are that immunological cross-reaction or specific combination can occur for the protein of the first nucleic acid encoding and the protein of the second nucleic acid encoding.Therefore,, when for example wherein two protein is only different due to preservative replacement, they are normally essentially identical.

" synthesize " nucleotide sequence that refers to comprise non-existent constitutional features in native sequences.For example, G+C content and normal codon distribute and synthesize closer to artificial sequence dicotyledonous and/or monocotyledons gene.

" conversion " is the process of heterologous nucleic acids of introducing in host cell or organism, especially, transforms " mean DNA molecular stable integration and enter in object organism genome.

" conversion/genetically modified/restructuring " refer to introduce the host organisms of heterologous nucleic acids molecule, as bacterium or plant.Nucleic acid molecule can stably be integrated into host genome, or nucleic acid molecule also can be used as extrachromosomal molecule and exists.This extrachromosomal molecule can be self-replicating.The cell transforming, tissue, or plant is interpreted as the final product that not only comprises conversion process, also comprise its transgenosis filial generation " non-transformed ", " not genetically modified ", or " nonrecombinant " host refers to the wild-type organisms that does not contain heterologous nucleic acids molecule, for example bacterium or plant.

" Vip proteinoid " comprises Vip3A (a), Vip3A (b), and Vip3A (c), Vip3B, Vip3C (a), Vip3C (b), Vip3Z and their homologue, but be not limited to this.Here other member that " homologue " used means described protein or polypeptide and Vip3 proteinoid has definite relation.This definite relation comprises, but be not limited to: 1) in sequence level, have at least 70% with another member of Vip3 proteinoid, more preferably at least 80%, at least 90% identity most preferably, also kept the protein of insecticidal activity simultaneously, 2) can there is the protein of cross reaction with another member's of immunity identification Vip3 proteinoid antibody, 3) can there is cross reaction with another member's of Vip3 proteinoid acceptor, and the protein that has retained induction apoptosis ability, with 4) in sequence level, have at least 70% with another member's of Vip3 proteinoid toxicity nucleus, more preferably at least 80%, at least 90% identity most preferably, also kept the protein of insecticidal activity simultaneously.At WO98/18932, WO98/33991, discloses other Vip3 homologue in WO98/00546 and WO99/57282.

By the base of standardized abbreviations below, represent Nucleotide: VITAMIN B4 (A), cytosine(Cyt) (C), thymus pyrimidine (T) and guanine (G).Similarly, with standardized abbreviations below, represent amino acid: L-Ala (Ala; A), arginine (Arg; R), l-asparagine (Asn; N), aspartic acid (Asp; D), halfcystine (Cys; C), glutamine (Gln; Q), L-glutamic acid (Glu; E), glycine (Gly; G), Histidine (His; H), Isoleucine (He; 1), leucine (Leu; L), Methionin (Lys; K), methionine(Met) (Met; M), phenylalanine (Phe; F), proline(Pro) (Pro; P), Serine (Ser; S), Threonine (Thr; T), tryptophane (Trp; W), tyrosine (Tyr; Y) and α-amino-isovaleric acid (Val; V).

Summary of the invention

The present invention relates to it and express the nucleotide sequence that produces new toxin, relate to preparation and the application of the described toxin that can control insect pest.Nucleotide sequence derives from bacillus (Bacillus), a kind of microorganism of gram-positive formation spore.Especially, provide the new Vip3 protein that can be used as sterilant.

For object of the present invention, insect pest comprises and being selected from: Coleoptera, Diptera, Hymenoptera, lepidopteran, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera etc., particularly lepidopterous insect.

The expression of nucleotide sequence of the present invention has produced and can be used for control example as the toxin of following lepidopterous insects: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutellaxylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotisipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothisvirescens), greedy noctuid (Spodoptera exigua), the choice refreshments noctuid (Helicoverpapunctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (ManducaSexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophoragossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

In one embodiment, the present invention comprises separated nucleic acid molecule, and described nucleic acid molecule comprises the nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 sequence, and the expression of the nucleic acid molecule that this is separated has produced the activity that can control insect.When expressing SEQ ID NO:1 nucleic acid molecule in heterologous host, its control that has produced anti-following insect is active: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodoptera exigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), the activity of the control insect of pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes), show that the nucleotide sequence shown in SEQID NO:1 enough produces the activity of this control insect.In further embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 93% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 94% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 95% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 97% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 98% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence that has at least 99% sequence identity with SEQ ID NO:1.In another embodiment, this separated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:1.

In another embodiment, the present invention includes the nucleic acid molecule being contained in pNOV1325, the expression of this nucleic acid molecule has produced Pesticidal toxins, and this pNOV1325 is deposited in intestinal bacteria (E.coli) the strain DH5 α of called after ATCC preserving number PTA-3868.

In one embodiment, the present invention includes the separated nucleic acid molecule that comprises a kind of nucleotide sequence, this nucleotide sequence has produced with have the nucleotides sequence dependent of dead military hero isocoding of at least 92% sequence identity, the expression of the nucleic acid molecule that this is separated with SEQ ID NO:1 the activity of controlling insect.When expressing SEQ ID NO:3 nucleic acid molecule in heterologous host, its control that has produced anti-following insect is active: European corn borer (Ostrinia Nubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodoptera frugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpa zea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodoptera exigua), choice refreshments dash forward noctuid (Helicoverpa punctigera) and bollworm (Helicoverpa Armigera), show that the nucleotide sequence shown in SEQ ID NO:3 enough produces the activity of this control insect.In further embodiment, the present invention includes the nucleic acid molecule being contained in pNOV1328, the expression of this nucleic acid molecule has produced Pesticidal toxins, and this pNOV1328 is deposited in intestinal bacteria (E.coli) the strain DH5 α of called after ATCC preserving number PTA-3869.

In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 91% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 92% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 93% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 94% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 95% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 96% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 97% sequence identity with aminoacid sequence shown in SEQID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 98% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, this separated nucleic acid molecule encoding comprises the toxin that has the aminoacid sequence of at least 99% sequence identity with aminoacid sequence shown in SEQ ID NO:2.In another embodiment, the toxin that this separated nucleic acid molecule encoding comprises aminoacid sequence shown in SEQ ID NO:2.

The present invention also comprises the recombinant vectors that comprises nucleotide sequence of the present invention.In this carrier, preferably, this nucleotide sequence is included in expression cassette, and this expression cassette comprises for express the regulatory element of this nucleotide sequence at the host cell that can express described nucleotide sequence.This regulatory element comprises promotor and termination signal conventionally, preferably, also comprises the element that the polypeptide of nucleic acid sequence encoding of the present invention can effectively be translated.Therefore the carrier that comprises described nucleotide sequence has the ability (preferably, it is as extrachromosomal molecule) copying in particular host cell conventionally, can utilize its nucleic acid molecule of the present invention that increases in host cell.In one embodiment, for the host cell of this carrier, be microorganism, as bacterium, particularly intestinal bacteria (E.coli) or genus bacillus (Bacillus).In another embodiment, the host cell for this recombinant vectors is endophyte or epiphyte.Preferred host cell for this carrier is eukaryotic cells, as yeast cell, and vegetable cell or insect cell.Vegetable cell as maize cell be most preferred host cell.In another preferred implementation, this carrier is virus vector, and is used in particular host cell and for example in insect cell or vegetable cell, copies described nucleotide sequence.Recombinant vectors is also for nucleotide sequence of the present invention is transformed in host cell, thereby this nucleotide sequence stable integration enters in the DNA of this host cell.In one embodiment, this host cell is prokaryote.In a preferred embodiment, this host cell is eukaryotic cell, as yeast cell, and insect cell or vegetable cell.In most preferred embodiments, host cell is vegetable cell, as maize cell.

The present invention also provides the method for producing the toxin with anti-insect active, comprise that (a) obtains genetically modified host cell of the present invention, (b) in genetically modified host cell, express nucleic acid molecule of the present invention, thereby produce the toxin that at least one has anti-insect active.

The present invention further provides the method for the transgenic plant that produce anti-insect, comprised in transgenic plant and introduce nucleic acid molecule of the present invention, wherein in transgenic plant, this nucleic acid molecule can be expressed to control the significant quantity of insect.In another embodiment, insect is lepidopterous insects.In another embodiment, this lepidopterous insects is selected from: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia n í), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).

On the other hand, the invention provides the separated toxin with anti-insect active, wherein said toxin comprises: the aminoacid sequence that (a) has at least 91% sequence identity with SEQ ID NO:2; Or (b) by comprising the aminoacid sequence of nucleic acid molecule encoding that has the nucleotide sequence of at least 92% sequence identity with SEQ ID NO:1.

In one embodiment, the invention provides the separated toxin with anti-insect active, wherein said toxin comprises the aminoacid sequence that has at least 91% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 92% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 93% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 94% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 95% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 96% sequence identity with SEQ IDNO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 97% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 98% sequence identity with SEQ ID NO:2.In one embodiment, this toxin comprises the aminoacid sequence that has at least 99% sequence identity with SEQ ID NO:2.In one embodiment, toxin comprises the aminoacid sequence shown in SEQ ID NO:2.

In one embodiment, the invention provides the toxin with anti-insect active, wherein said toxin is to have the expression of nucleic acid molecule of the nucleotide sequence of at least 92% sequence identity to produce by comprising with SEQ ID NO:1.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 93% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 94% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 95% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 96% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 97% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 98% sequence identity.In another embodiment, described nucleotide sequence and SEQ ID NO:1 have at least 99% sequence identity.In another embodiment, described toxin is the expression generation of the nucleotide sequence by comprising SEQ ID NO:1 Nucleotide 1-2364 or SEQ IDNO:3 Nucleotide 1-2364.

In one embodiment, toxin of the present invention is by the nucleotide sequence that comprises approximately 2.4kb DNA fragmentation contained in the pNOV1325 as ATCC preserving number PTA-3868 preservation is expressed and produced.In another embodiment, toxin of the present invention is by the nucleotide sequence that comprises approximately 2.4kbDNA fragment contained in the pNOV1328 as ATCC preserving number PTA-3869 preservation is expressed and produced.

In another embodiment, toxin of the present invention is to be produced by intestinal bacteria (E.coli) strain of called after ATCC preserving number PTA-3868.In another embodiment, toxin of the present invention is to be produced by intestinal bacteria (E.coli) strain of called after ATCC preserving number PTA-3869.

When testing the anti-insect pest of toxin of the present invention in biological assay, toxin of the present invention has the activity of controlling insect.In one embodiment, toxin of the present invention has the activity of anti-lepidopterous insects.In further embodiment, lepidopterous insects is selected from: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), the choice refreshments noctuid (Helicoverpa punctigera) that dashes forward, bollworm (Helicoverpa Armigera), maduca sexta (Manduca Sexta), amyloid plaque noctuid (Trichoplusia ni), pink bollworm (Pectinophora gossypiella) and the thin leaf roller of Sunflower Receptacle (Cochylis hospes).In embodiment 5 and embodiment 8, further example has been set forth the insect control characteristic of Pesticidal toxins of the present invention.

The present invention also comprises the hybrid toxins with anti-insect active, and wherein said hybrid toxins is by the nucleic acid molecule encoding that comprises following nucleotide sequence, and this nucleotide sequence (a) is at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, can hybridize (at 65 ℃, 0.1XSSC, rinsing in 0.1%SDS) with SEQ ID NO:1 Nucleotide 1734-2364; Or nucleotides sequence dependent of dead military hero isocoding (b) and (a); Or (c) comprise according to the order of sequence and (a) or continuous 20 base pair nucleotide segments that (b) continuous 20 base pair nucleotide segments of nucleotide sequence are identical, the expression of wherein said nucleic acid molecule has produced the activity of controlling insect.Here concrete example is assorted house toxin nucleotide sequence coded shown in SEQ ID NO:6.When expressing SEQ ID NO:6 nucleic acid molecule in heterologous host, its control that has produced anti-following insect is active: European corn borer (OstriniaNubilalis), small cabbage moth (Plutella xylostella), autumn mythimna separata (Spodopterafrugiperda), black cutworm (Agrotis ipsilon), the real noctuid (Helicoverpazea) of paddy, cigarette aphid noctuid (Heliothis virescens), greedy noctuid (Spodopteraexigua), pink bollworm (Pectinophora gossypiella), amyloid plaque noctuid (Trichoplusia ni), the thin leaf roller of Sunflower Receptacle (Cochylis hospes) and Sunflower Receptacle head moth (Homoeosoma electellum).

The present invention also comprises the hybrid toxins with anti-insect active, it comprises Vip3 toxin C-terminal region and the amino terminal region of the different Vip3 toxin that are attached thereto by the direction from amino to carboxyl, and wherein said C-terminal district inclusion and SEQ ID NO:2 amino acid 579-787 have the aminoacid sequence of at least 75% identity; There is at least 75% identity described amino terminal region and SEQ IDNO:5 amino acid/11-578.In further embodiment, this C-terminal district inclusion SEQ ID NO:2 amino acid 579-787, this amino terminal region comprises SEQ ID NO:5 amino acid/11-578.In another embodiment, hybrid toxins comprises SEQ ID NO:6 amino acid/11-787.

The expression of nucleotide sequence in allos microorganism host

As the reagent of biological control insect, Pesticidal toxins can produce by this nucleotide sequence of heterologous host cells expressing nucleotide sequence.In first embodiment, produced and comprised bacillus thuringiensis (B.Thuringiensis) cell that nucleotide sequence of the present invention is modified.The sudden change that this modification comprises existing regulatory element or disappearance, therefore caused the expression that nucleotide sequence changes, or introduced and controlled the new regulatory element that nucleotide sequence is expressed.In another embodiment, by inserting in karyomit(e) or the introducing of extrachromosomal replication molecule by containing this nucleotide sequence, to the additional copy that adds one or more described nucleotide sequences in bacillus thuringiensis (Bacillus Thuringiensis) cell.

In another embodiment, at least one nucleotide sequence of the present invention is inserted in applicable expression cassette, this expression cassette comprises promotor and termination signal.The expression of nucleotide sequence is composing type, maybe can utilize the reaction of all kinds stimulator with initial inducible promoter of transcribing.In a preferred embodiment, the cell of expressing toxin is microorganism, as virus, and bacterium or fungi.In a preferred embodiment, virus, as contained nucleotide sequence of the present invention in Baculovirus Gene group, can be expressed a large amount of corresponding Pesticidal toxins after the eukaryotic cells that the applicable virus replication of infection and described nucleotide sequence are expressed.Consequent Pesticidal toxins is as sterilant.Alternately, the baculovirus In vivo infection insect that comprises this nucleotide sequence with genetic engineering modified one-tenth, the expression by Pesticidal toxins or the expression by virus infection and Pesticidal toxins combine to come kill insects.

Bacterial cell is also the host who expresses for nucleotide sequence of the present invention.In a preferred embodiment, the non-pathogenic symbiotic bacterium (usually said endophyte) that utilization can be lived within plant tissue and breed or can be the non-pathogenic symbiotic bacterium (usually said epiphyte) of phyllosphere or rhizosphere colony.This bacterium comprises Agrobacterium (Agrobacterium), Alkaligenes (Alcaligenes), nitrogen-fixing root nodule Pseudomonas (Azospirillum), Azotobacter (Azotobacter), bacillus (Bacillus), rod shape Bacillaceae (Clavibacter), enterobacter (Enterobacter), erwinia (Erwinia), Flavobacterium (Flavobacter), Klebsiella (Klebsiella), Rhodopseudomonas (Pseudomonas), rhizobium (Rhizobium), serratia (Serratia), the bacterium of streptomyces (Streptomyces) and xanthomonas (Xanthomonas).Symbiosis fungi, the host that also may express as nucleotide sequence of the present invention as trichoderma (Trichoderma) and the mould genus of glue (Gliocladium) is for same object.

These gene manipulation techniqueses be specific for the different hosts that can supply to utilize, and are known in the art.For example, expression vector pKK223-3 and pKK223-2 can be for transcribing or translate the heterologous gene of fusion form after expression tac or trc promotor in intestinal bacteria (E.coli).For the expression of coding many ORFs (open reading frame) operon, the simplest method is to transcribe amalgamation mode at carrier, as inserted this operon in pKK223-3, makes it possible to utilize the homology ribosome bind site of heterologous gene.Gram-positive species as genus bacillus (Bacillus) in the technology of overexpression be also known in the art, it also can use (Quax etc. in the context of the invention, In:Industrial Microorganisms:Basic and Applied Molecular Genetics, the editors such as Baltz, AmericanSociety for Microbiology, Washington (1993)).Alternative overexpression system depends on for example yeast vector, comprise Pichia (Pichia), yeast belong (Saccharomyces) and kluyveromyces spp (Kluyveromyces) (Sreekrishna, In:Industrial microorganisms:basic andapplied molecular genetics, Baltz, Hegeman and Skatrud edit, American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology L2:173-177 (1994); Van den Berg etc., Biotechnology 8:135-139 (1990)).

Plant Transformation

In embodiment particularly preferably, in higher organism style, express at least one Pesticidal toxins of the present invention in as plant.The transgenic plant of in this case, expressing the toxin of significant quantity are protected the infringement of itself avoiding insect pest.When insect starts take this transgenic plant as when food, its toxin of expressing of also having ingested.This further stings food plant tissue by prevention insect, or even injures or kill insects.Nucleotide sequence of the present invention can be inserted in expression cassette, then preferably, expression cassette stable integration is in described Plant Genome.In another preferred implementation, described nucleotide sequence is included in the self-replacation virus of non-virulent.The plant transforming according to the present invention can be monocotyledons or dicotyledons, include but not limited to corn, wheat, barley, rye, sweet potato, beans, pea, witloof, lettuce, wild cabbage, Cauliflower, asparagus broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, hemp, zucchini, apple, pears, Wen Quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, Chinese sorghum, sugarcane, beet, Sunflower Receptacle, Semen Brassicae campestris, trifolium, tobacco, Radix Dauci Sativae, cotton, clover, paddy rice, potato, eggplant, cucumber, Arabidopis thaliana and xylophyta are as coniferale and Deciduous tree.

Once the nucleotide sequence of expectation be transformed and is entered in specified plant species, just can breed it or with traditional breeding method, it is shifted to other kind that enters same species in these species, in commercial variety.

Preferably, in transgenic plant, express nucleotide sequence of the present invention, therefore in transgenic plant, caused the biosynthesizing of corresponding toxin.By this way, can produce and there are the transgenic plant that strengthen anti-insect-resistant.In order to express nucleotide sequence of the present invention in transgenic plant, nucleotide sequence of the present invention may need to modify and optimize.Although deriving from many cases that the gene of microorganism just need not modify can high-caliber expression in plant, the microorganism nucleotide sequence with the codon showing no favouritism in plant may cause low-level expression in transgenic plant.All organisms have specific codon usage bias, and this is known in the art, can keep nucleotide sequence coded amino acid whose its codon that simultaneously changes of the present invention to meet plant-preference.And, from having at least about 35%, preferably more than approximately 45%, more preferably more than 50%, most preferably more than the encoding sequence of about 60%GC content, can realize best high-caliber expression in plant.Owing to existing, may make information remove stable ATTTA motif and the AATAAA motif that can cause inappropriate polyadenylation, have the microorganism nucleotides sequence of low GC content to be listed in plant and may to express low-levelly.Although can express fully preferred gene order in monocotyledons and dicotyledons species, can modification sequence to adapt to special codon preference and the GC content preference of monocotyledons or dicotyledons, because these preferences have been proved to be different (Murray etc., Nucl.Acids Res.17:477-498 (1989)).In addition, can screen nucleotide sequence to find the unconventional splice site that causes information brachymemma.Utilize Patent Application Publication EP 0 385 962, the method described in EP 0 359 4721 and WO93/07278, uses side-directed mutagenesis well known in the art, and PCR and synthetic gene build and can carry out needing in above-mentioned these nucleotide sequences the institute carrying out to change.

In one embodiment of the present invention, according to being incorporated herein by reference disclosed method in the United States Patent (USP) 5,625,136 of document here, can prepare vip3B gene.In the method, utilized the preferred codon of corn, amino acid whose single cipher in the corn of the most usually encoding.The preferred codon of corn of specific amino acids can derive from, for example the known sequence of corn.At Murray etc., in Nucleic Acids Research 17:477-498 (1989), instructed the corn codon of 28 kinds of genes of maize plant to use, the disclosure that is incorporated to this piece of document is here reference.The composition sequence with corn optimal codon of preparation is as shown in SEQ ID NO:2.

Nucleotide sequence can be optimized by this way to express in any plant.All or any part of generally acknowledged gene order can be optimized or be synthetic.That is, also can utilize sequence synthetic or that part is optimized.

Effectively initial for what translate, the sequence that may need to modify contiguous initial methionine.For example, by comprise known in plant effectively sequence can modify them.Joshi has proposed the applicable consensus sequence (NAR 15:6643-6653 (1987)) of plant, and Clonetech has proposed the sub-consensus sequence of further translation initiation (1993/1994 catalogue, 210 pages).These consensus sequences are applicable to using together with nucleotide sequence of the present invention.To comprising described nucleotide sequence, mix this sequence, until and comprise ATG (simultaneously keeping second amino acid not modified), or until and comprise the GTC (likely modifying genetically modified second amino acid) after ATG.

Can using as mentioned above as its native sequences or as the new vip3 toxin gene of the present invention who optimizes composition sequence operationally with the various promotors of expressing in plant, comprise composing type, induction type, sequential regulates, grow to regulate, Chemical Regulation, tissue preferably and tissue-specific promoter be separated by and close, to prepare recombinant DNA molecules, i.e. mosaic gene.The selection of promotor will be along with expression time and space requirement and is changed, and depends on target species.Therefore, preferably at blade, stem or cane, fringe, inflorescence (for example, spike, panicle, cob etc.), root, and/or in seedling, express nucleotide sequence of the present invention.Yet, in many cases, need to provide the protection of the anti-insect pest more than a type, be therefore desirably in many tissues and express.Although proved that the many promotors that derive from dicotyledons are operational in monocotyledons, vice versa, but ideally, select dicotyledons promotor for the expression of dicotyledons, monocotyledonous promotor is for the expression of monocotyledons.Yet, do not limit the origin of selected promotor, as long as promotor can drive the expression of described nucleotide sequence just enough in expectation cell.

Preferred constitutive promoter comprises CaMV35S and 19S promotor (Fraley etc., the U.S. Patent number 5,352,605 that on October 4th, 1994 announces).Preferred promotor derives from any of several actin genes of expressing in most cell types in addition.Can easily modify the described promoter expression cassettes such as McElroy (Mol.Gen.Genet.231:150-160 (1991)) for the expression of new toxin gene, this expression casette is particularly suitable for using in monocotyledons host.

Another preferred constitutive promoter derives from ubiquitin, and it is the known another kind of gene product accumulating in many cell types.From several species, such as Sunflower Receptacle (Binet etc., 1991.Plant Science79:87-94), corn (Christensen etc., 1989.Plant Molec.Biol.12:619-632) and Arabidopis thaliana (1993.PlantMolec.Biol.21:895-906 such as Norris) cloned ubiquitin promoter, can be in transgenic plant.The corn ubiquitin having developed in transgenosis monocotyledons system starts (UbiP), and announces in patent the carrier that discloses its sequence and transformed for monocotyledons in EP 0 342 926.Ubiquitin promoter is suitable for transgenic plant, particularly in monocotyledons, expresses this new toxin gene.

Be used in plant, tissue specificity or the tissue preference promotor of particularly in corn, expressing the new toxin gene of the present invention are can be at root, pith (pith), the promotor that in blade or pollen, guiding is expressed.In WO93/07278, disclose this promotor, integral body is incorporated to it for reference here.Other in the present invention available tissue-specific promoter comprises United States Patent (USP) 6,040, disclosed cotton rubisco promotor in 504; United States Patent (USP) 5,604, disclosed paddy rice sucrose synthase promotor in 121; With the bent virus of disclosed Night-Blooming jessamine yellow leaf roll (cestrum yellow leaf curlingvirus) promotor in WO01/73087, being incorporated to all these documents is reference.At United States Patent (USP) 5,614, disclose in 395 for instructing chemical induction type (the cestrum yellow leaf curling virus) promotor of the new Toxin gene expression of plant, to be incorporated to this piece of document be reference to integral body here.

Also can under regulating, Chemical Regulation type promotor express nucleotide sequence of the present invention.This can make only when processing farm crop with induction chemical reagent, could synthesize Vip3 toxin.The optimization technique of genetic expression chemical induction has been described in detail in detail in disclosed application EP 0 332 104 (applicant Ciba-Geigy) and United States Patent (USP) 5,614,395.The preferred promoter of chemical induction is tobacco PR-la promotor.

Preferred promotor kind is wound-induced type promotor.A large amount of promotors in wound site and the expression of phytopathogen sites of infection have been described.Ideally, this promotor should only have activity in sites of infection part, thereby Pesticidal toxins is only needing synthetic insecticide toxin to accumulate in killing the cell of Invasive insect species insect.Preferred this promotor comprises Stanford etc., Mol.Gen.Genet.215:200-208 (1989), Xu etc., Plant Molec.Biol.22:573-588 (1993), Logemann etc., Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec.Biol.22:783-792 (1993), Firek etc., Plant Molec.Biol.22:129-142 (1993), and Warner etc., the promotor that PlantJ.3:191-201 (1993) is described.

Preferred tissue specific expression pattern comprises chlorenchyma specificity, root-specific, stem specificity and flower specific.The promotor that is suitable for expressing in chlorenchyma comprises that many adjustings participate in the promotor of photosynthesis gene, and so many promotors have been cloned out from monocotyledons and dicotyledons.Preferred promotor is the corn PEPC promotor (Hudspeth & Grula, Plant Molec.Biol.12:579-589 (1989)) that derives from phosphoric acid enol carboxylase gene.The preferred promoter that root-specific is expressed is de Framond (FEBS290:103-106 (1991); EP 0 452 269 to Ciba-Geigy) described promotor.Preferred stem specificity promoter is United States Patent (USP) 5,625, the promotor described in 136 (authorizing Ciba-Geigy), the expression of this promoters driven corn trpA gene.

Other preferred implementation is with wound-induced mode or pathogenic infection induction mode, to express the transgenic plant of described nucleotide sequence.

Except being applicable to the selection of promotor, the structure that in plant, Pesticidal toxins is expressed need to be connected to the applicable transcription terminator in heterologous nucleotide sequence downstream.Can obtain several such terminators, and they are (for example derive from the tml of CaMV, derive from the E9 of rbcS) known in the art.Any known available terminator working in plant can be used in the context of the invention.

Also can in expression cassette of the present invention, introduce other sequence in a large number.These sequences comprise the verified sequence that strengthens expression, for example, for example, as intron sequences (deriving from Adhl and bronzel) and virus leader sequence (deriving from TMV, MCMV and AMV).

Likely preferably the expression of nucleotide sequence of the present invention is targeted to different cellular localizations in plant.In some cases, possible expected location is in cytosol, and in other cases, possible preferred orientation is in some subcellular organelles.The Subcellular Localization of the enzyme that transgenosis is coded can adopt technology well known in the art.Conventionally, operate coding derives from the DNA of target peptide of the gene product of known targeted cells device, makes it to be fused to the upstream of nucleotide sequence.Become known for the many this target sequence of chloroplast(id), and proved their functions in allos builds.The expression of nucleotide sequence of the present invention also can be targeted in the endoplasmic reticulum or vacuole of host cell.The technology that realizes above-mentioned these objects is well known in the art.

The carrier that is suitable for Plant Transformation has been described beyond this specification sheets.Conversion for Agrobacterium (Agrobacterium) mediation, binary vector or be applicable to the carrier of at least one T-DNA border sequence, and for direct transgenosis, any carrier is all applicable to, and the preferred linear DNA that only contains object construct.The in the situation that of direct gene transfer, can utilize conversion or the cotransformation (Schocher etc., Biotechnology 4:1093-1096 (1986)) of unique DNA kind.Transfer for direct gene transfer and Agrobacterium (Agrobacterium) mediation, conventionally (but not being essential) adopts selective marker, this selective marker can provide the resistance to microbiotic (kantlex, Totomycin, or methotrexate) or weedicide (basta).Yet the selection of selective marker is not vital to the present invention.

In another preferred implementation, can directly nucleotide sequence of the present invention be transformed and be entered in plastom.The major advantage of plastid transformation is that plastid does not need essence modification just can express bacterial gene conventionally, and plastid can be expressed a plurality of open reading frame under single promotor control.At United States Patent (USP) 5 451 513, in 5 545 817 and 5 545 818, in PCT application number WO95/16783 and McBride etc., (1994) Proc.Nati.Acad.Sci.USA91, has at large described plastid transformation technology in 7301-7305.The basic fundamental that chloroplast(id) transforms comprises for example utilizes biolistics or protoplast transformation (for example conversion of calcium chloride or PEG mediation) that goal gene is introduced to applicable target tissue together with being arranged in clone's plastid DNA region of selective marker flank.1 to 1.5kb flank region, is called targeting sequencing, can promote and the homologous recombination of plastom, therefore allows displacement and the modification of plastom(e) (plastome) specific region.At first, can utilize and provide the chloroplast(id) 16S rRNA of spectinomycin and/or streptomycin resistance and the point mutation of rps12 gene as the selective marker (Svab transforming, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc.Nati.Acad.Sci.USA 87,8526-8530; Staub, J.M., and Maliga, P. (1992) Plant Cell4,39-45).This bombards the frequency of approximately 1 time with approximately every 100 target blades and produces stable homogeneity transformant.The cloning site existing between these marks can be used to produce plastid targeting vector for importing foreign gene (Staub, J.M., and Maliga, P. (1993) EMBO J.12,601-606).By using dominant selectable marker, rRNA as recessive in the bacterium aadA gene substitution of the spectinomycin detoxifying enzymes of encoding (cletoxifying enzyme) enzyme aminoglycoside-3 '-adenylyl transferase or r-protein antibiotics resistance gene can make transformation frequency significantly increase (Svab, Z. and Maliga, P. (1993) Proc.Natl.Acad.Sci.USA90,913-917).In the past, this mark had been successfully used to the plastom ((Goldschmidt-Clermont) M. (1991) Nucl.Acids Res.19:4083-4089) that high frequency transforms Chlamydomonasreinhardtii.Other selective marker that can be used for plastid transformation is known in the art, and comprises within the scope of the present invention.Conventionally, after conversion, need approximately 15 to 20 cell fission to circulate to reach homogeneity state.The plastid by homologous recombination, gene being inserted in the annular plastom of all thousands of copies existing in each vegetable cell is expressed the advantage of having utilized copy number to be much higher than nuclear expression gene, makes expression level can easily surpass 10% of total solvable plant protein.In a preferred embodiment, nucleotide sequence of the present invention is inserted in plastid targeting vector, and conversion enters in the plant host plastom of expectation.Obtained the plant that belongs to homogeneity for the plastom that contains nucleotide sequence of the present invention, preferably, this plant has the ability of expressing this nucleotide sequence high-levelly.

Control the associating of insect key element

Can combine Bt delta-endotoxin or other desinsection will usually use Pesticidal toxins of the present invention to increase insect target scope.And, the desinsection key element of Pesticidal toxins of the present invention and Bt delta-endotoxin or other different qualities combine use, for prevention and/or the processing of insect-resistant, there is specific purposes.According to activity profile and the sequence similarity of the various desinsection crystallization of protein of bacillus thuringiensis (Bacillus thuringiensis), it is classified.Then, Hofte and Whiteley, the classification that Microbiol.Rev.53:242-255 (1989) proposes is divided into 4 large classes by known desinsection crystallization of protein.Conventionally, by activity profile, define described large class, Cry1 protein has anti-lepidopteran-active, and Cry2 protein has anti-lepidopteran and Diptera is active, and Cry3 protein has anti-coleopteran-active, and it is active that Cry4 protein has anti-Diptera.

In each large class, according to sequence similarity by delta-endotoxin component.Cry1 protein produces with toxin protein before 130-140kDa conventionally, and proteolysis cuts the active toxin that produces about 60-70kDa after this front toxin.The active part of delta-endotoxin is present in the NH of full-length molecule ₂-terminal portions.Then, Hofte and Whiteley (above) are divided into 6 groups by known Cry1 protein, 1Aa, 1Ab, 1Ac, 1B, 1C and 1D.From then on, also identified and be categorized as CrylEa, CrylFa, Cry9A, the protein of Cry9C and Cry9B and other class.

The insecticidal activity spectrum of each delta-endotoxin of bacillus thuringiensis (Bacillus thuringiensis) trends towards very narrow, and a kind of delta-endotoxin is anti-several insects only.Specificity be participate in to produce the efficiency of each step of active toxin protein and its subsequently with the result of the interactional ability of insect gastrointestinal epithelial cells.In a preferred implementation, in transgenic plant, the expression of nucleic acid molecule of the present invention is accompanied by the expression of one or more Bt delta-endotoxins.Particularly preferred Bt delta-endotoxin is at United States Patent (USP) 5,625, and disclosed Bt delta-endotoxin in 136 is incorporated to it here for reference.

Know delta-endotoxin proteins matter toxin (protoxin) the form expression in fact in the past of many bacillus thuringiensiss (Bacillus thuringiensis).These front toxin dissolve in insect gut alkaline environment, and the proteolysis by proteolytic enzyme is converted into poisonous core fragment (Hofte and Whiteley, Microbiol.Rev.53:242-255 (1989)).For the delta-endotoxin proteins matter of Cry1 class, poisonous core fragment is positioned at front toxin N-terminal in half.Within the scope of the present invention, in plant conversion carrier, can utilize the gene of poisonous core fragment of clipped form of toxin form before coding total length or new toxin protein so that the insecticidal properties to host plant to be provided.

Other desinsection key element comprises proteinase inhibitor (Serine and halfcystine type), lectin, α-amylase, peroxidase and rCO.In the present invention, also can utilize other Vip encoding sequence, as United States Patent (USP) 5,849, disclosed vip1A (a) and vip2A (a) in 870, be incorporated to it here for reference.

Can be by genetic engineering modified plant to contain and to express the coexpression that all essential genes are realized the plurality of desinsection key element in same transgenic plant.Alternately, can genetic engineering modified plant parent 1 to express gene of the present invention.Can genetic engineering modified the second strain plant be that parent 2 is to express supplementary control insect key element.By hybrid strain 1 and parent 2, can obtain and express the progeny plant of introducing all genes in parent 1 and parent 2.

The present invention also comprises the variant of disclosed nucleic acid molecule.The Protocols in Molecular Biology of knowing by application, for example, PCR and hybridization technique as outlined below, can identify and/or variant sequence that separating natural exists.

Variant vip3 nucleotide sequence comprises the nucleotide sequence in synthetic source, as, for example, by the nucleotide sequence that utilizes site-directed mutagenesis to produce, or the nucleotide sequence producing by the exchange of total territory, but these nucleotide sequences have still shown insecticidal activity.The method that mutagenesis and nucleotide sequence change is known in the art.Referring to, Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492 for example; Kunkel etc. (1987) Methods inEnzymol.154:367-382; U.S. Patent number 4,873,192; Walker and Gaastra edit (1983) Techniques in Molecular Biology (MacMillan) Publishing Company, New York) and the reference wherein quoted.Conventionally, nucleotide sequence of the present invention is its corresponding shows at least 80% with reference to vip3 nucleotides sequence, and preferably 85%, 90%, 95%, reach 98% or more sequence identity, and have insecticidal activity.

Variant Vip3 nucleotide sequence also comprises and derives from mutagenesis and recombination method as the sequence of DNA reorganization.In this way, can for example, by one or more different vip3 sequences of the present invention (vip3B and vip3A-B, but be not limited to this) be binned in together, or with other vip3 or correlated series (non-vip3A (SEQ ID NO:5) for example, but be not limited to this) recombinate mutually, to produce coding, there is the new vip3 nucleic acid molecule of the Vip3 toxin of desired characteristic.In this way, can produce from the vip3 polynucleotide storehouse of Serial relation restructuring vip3 polynucleotide library, described serial correlation vip3 polynucleotide include the sequence area of basic sequence identity, and can be in vitro or homologous recombination in body.Strategy for this DNA reorganization is known in the art.Referring to, Stemmer (1994) Proc.Natl.Acad.Sci.USA91:10747-10751 for example; Stemmer (1994) Nature370:389-391; Crameri etc., (1997) Nature Biotech.15:436-438; Moore etc., (1997) J.Mol.Biol.272:336-347; Zhang etc., (1997) Proc.Natl.Acad.Sci.USA94:4504-4509; Crameri etc., (1998) Nature391:288-291; International Patent Application WO 99/57128 and U.S. Patent number 5,605,793,5,837,458 and 6,335,179.

Can be by the screening method combined utilization of mutafacient system disclosed herein and format high throughput, to detect the insecticidal activity of the mutagenesis Vip3 polypeptide of cloning in host cell.Can from host cell, reclaim the active Vip3 polypeptide of coding (for example, secretion property and by antibody test; Or in insect biological assay, have an insecticidal activity) mutagenized dna molecule, and check order fast by standard technology method.These methods can Fast Measurement object Vip3 polypeptide in the importance of single amino acids residue, can be applied to the polypeptide of unknown structure.

Can, to screening by the restructuring vip3 gene library that DNA Shuffling Method produces, to identify to be presented at for the protection of Genes For Plant Tolerance insect aspect, there is the restructuring vip3 gene that improves characteristic.What can utilize the improved vip3 pest resistance genes characteristic of DNA reorganization acquisition is the effect that has the anti-target insect of increase, the target insect scope increasing, the possibility of insect development resistance reduces, the expression level increasing, the resistance that proteasome degradation is had to increase, the environmental stability of increase and the toxicity reduction to host plant.By utilizing applicable screening strategy, can or obtain successively the vip3 gene that a plurality of characteristics are optimized simultaneously.

Utilize DNA reorganization can obtain the vip3 pest resistance genes of toxin-encoding, described toxin has shown the anti-target insect effect strengthening.Once complete reorganization, can screen the reorganization vip3 gene library obtaining thus, to identify the vip3 gene that shows the insecticidal activity strengthening.A method of carrying out this screening is that the protein coding region clone of the vip3 gene through reorganization is entered in expression vector, described expression vector is suitable at selected host cell, and for example intestinal bacteria (E.coli) or bacillus thuringiensis (Bacillus thuringiensis) are without expressing said gene in crystal strain.Those skilled in the art will recognize that the merits and demerits of utilizing these two kinds of expression systems.For example in producing secretion property Vip3 protein, will more expect to use bacillus thuringiensis (Bacillus thuringiensis).If expectation, can carry out preliminary screening to clone, for example, by immunoassay, to identify the clone of the Vip3 protein that produces correct size.Then in functional screening, detect the positive colony in preliminary screening, with identification code, have the reorganization vip3 gene of toxin of the enhanced activity of expectation.

Complete insect assay method can be for detection of toxicity.In these assay methods, at insect bait, for example, in artitificial food or plant tissue, place the Vip3 toxin of reorganization vip3 genetic expression, and this target insect will consume this Vip3 toxin.Can in further biological assay, detect and cause that target insect growth suppresses or dead these clone to measure effect.The reorganization vip3 gene that coding has a toxin that strengthens effect can be accredited as the EC of reduction ₅₀(reducing the essential toxin concentration of insect growth 50%) and/or LC ₅₀the reorganization vip3 gene of (causing the toxin concentration that 50% mortality ratio is essential).

External test method also can be for screening reorganization vip3 gene library.This assay method generally includes application to the cultivation insect cell of Vip3 toxin sensitivity and/or expresses cell, described Vip3 toxin or the result natural or allogeneic gene expression of Vip3 toxoreceptor.Can utilize other external test method, the mensuration of morphological changes of cell for example, can be used for measuring dyestuff and the mark of necrocytosis, or the mensuration that discharges of cell ATP ase.An applicable external test example that utilizes the insect cell of cultivating to carry out Vip3 toxicity test is Sf9 (Spodoptera frugiperda) cell.Sf9 is extremely sensitive to Vip3 toxin.When mixing Vip3 toxin and Sf9 cell, it is penetrating to small molecules height that cytolemma becomes.When adding dyestuff as trypan blue to cell suspending liquid, those cell dyeings that killed by Vip3 toxin are for blue.Therefore, can measure by image analysis the cytotoxicity of Vip3 toxin.

Another external test method comprises the application of Vip3 toxoreceptor.At United States Patent (USP) 6,291, so a kind of acceptor is disclosed in 156, here and its be reference.Can accept fixedly Vip3 receptor protein of surface (for example 96 orifice plates or nitrocellulose membrane, but be not limited to this), and be exposed to and comprise the clone who reorganizes vip3 gene.Therefore, can be according to the reorganization vip3 gene of the functional toxin of binding affinity identification code to Vip3 acceptor.And, utilize methods known in the art (referring to, for example Clem and Miller, 1194, Mol.Cel.Biol.14:5212-522), the gene transformation of coding Vip3 acceptor can be entered to non-Vip3 sensitive cell line for example in Schneider 2 (S2) drosophila cell system.Then, the S2 cell of conversion is exposed to the clone who comprises reorganization vip3 gene.Therefore, can be according to the reorganization vip3 gene of the functional toxin of induction identification code of necrocytosis.

Embodiment

With reference to detailed below embodiment, present invention will be further described.These embodiment are provided only for purposes of example, and unless stated otherwise, these embodiment do not mean that it is restrictive.Here standard recombinant dna used and molecule clone technology are also known in the art, and Ansubel (editor) Current Protocols in Molecular Biology, JohnWiley and Sons .Inc. (1994); T.Maniatis, E.F.Fritsch and J.Sambrook, Molecular Cloning:A Laboratory Manual, Cold SpringHarbor laboratory, Cold Spring Harbor, NY (1998); And T.J.Silhavy, M.L.Berman, and L.W.Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) has described these technology.

Embodiment 1: the structure of bacillus thuringiensis (Bacillus thuringiensis) strain AB1183 cosmid library

By processing and be resuspended in 100mM Tris pH8 with 2mg/ml N,O-Diacetylmuramidase at 37 ℃, the cell of the fresh growth in 10mMEDTA 30 minutes, from the separated total DNA of bacillus thuringiensis (Bacillus thuringiensis) of called after AB1183.At 1%SDS, 50mM EDTA, adds proteolytic enzyme to final concentration 100 μ g/ml, at 55 ℃ of incubations in 1M urea.Add isopyknic benzene phenol-chloroform-primary isoamyl alcohol.Biased sample is 5 minutes gently, centrifugal with 3K.Repeat this step 2 time.Then with 0.7 volume Virahol mixing water, centrifugal.With 70% ethanol, wash DNA precipitation 3 times, resuspended gently in 0.5XTE.At 37 ℃, in 100 μ l volumes, the Sau3A/ μ g DNA of Yong0.3 unit processes 12 μ g DNA.Interval sampling in every 2 minutes, continues 10 minutes.Then add 1/10 volume 10XTE, 65 ℃ of heated sample 30 minutes with inactivator.Sample is carried out to electrophoresis to measure the part of 40kb scope, and in connection, utilize this sample.

Utilize BamHI cloning site, as described in supplier, prepare SuperCos cosmid vector (Stratagene, La Jolla, CA).In 5 μ l volumes, the SuperCos with 2:1 ratio being prepared by 100 η g/ml and the prior AB1183 DNA with Sau3A digestion spend the night 6 ℃ of connections.As described in supplier, with Gigapack XL III (Stratagene), pack and connect mixture.As described in supplier, make the phage-infect of packing enter XL-1MR intestinal bacteria (E.coli) cells (Stratagene).Spread plate cosmid library on the L-agar that has 50 μ g/ml, 37 ℃ of incubations 16 hours.Select and cultivate 1200 bacterium colonies for detection of anti-insect active.

Embodiment 2: clay clone's biological assay

The insecticidal activity of anti-cigarette aphid noctuid (Heliothisvirescens) newborn larvae of 1200 bacterium colonies of screening embodiment 1.On artitificial food, by surface contamination method, carry out biological assay.After 7 days, biological assay is marked.Find that 8 clones have insecticidal activity to cigarette aphid noctuid (Heliothis virescens).

Embodiment 3: insecticidal activity clay clone's analysis

In order to identify vip3 homologous sequence, utilize Carozzi etc. (1991, Appl.Env.Microbiol.57:3057-3061) method, uses the primer that comes from vip3A gene (SEQ IDNO:4) 5 ' initiation area in the enterprising performing PCR analysis of 8 cigarette aphid noctuids (Heliothis virescens) positive colony.Primer for this analysis is

Forward: 5 '-GTGATCTAACCCTAGACG-3 ' (SEQ ID NO:8)

It is reverse: 5 '-GCTTTAGTTCCATTCACTCC-3 ' (SEQ ID NO:9).

A clone has produced the DNA band of the expection size of vip3 class genes involved.The 3.8kb EcoRI fragment subclone that comes from this clone is entered to pBluescript (Stratagene), transform and enter intestinal bacteria (E.coli).With pcr analysis, confirmed that these intestinal bacteria (E.coli) clone comprises the same source coding sequence of vip3.This vip3 is named as vip3B with source coding sequence.The intestinal bacteria that comprise vip3B encoding sequence (E.coli) clone's bioassay results has proved that Vip3B toxin and cigarette aphid noctuid (Heliothisvirescens) are active relevant.The plasmid and this clone that are included in this clone are named as pCIB9400.

Embodiment 4: the Cloning and sequencing of total length vip3B gene

With BglII and EcoRV, cut pCIB9400 to remove the flanking sequence of the about 800bp of vip3B encoding sequence 3 ' end near.With Klenow polysaccharase (New England Biolabs, Beverly, Mass.), fill the end of the fragment obtaining thus, then use T4 ligase enzyme (NewEngland Biolabs, Beverly, Mass.) to link together.Transforming this connection mixture enters in intestinal bacteria (E.coli) DH5 α cell.With standard base (SB) cleavage method isolated plasmid dna from single bacterium colony, carry out several restrictive diges-tion to guarantee to have deleted the flanking sequence of this 800bp.The plasmid called after pNOV1325 obtaining thus, and be deposited in the intestinal bacteria (E.coli) of ATCC preserving number PTA-3868.

With dideoxy-chain terminating method, check order, with Applied Biosystems Inc. mode 3 700 automated DNA sequenators (Foster City, CA), complete order-checking.Sequencher4.05 assembling sequence with Gene CodesCorporation (Ann Arbor, Michigan).

The 2364bp encoding sequence (SEQ ID NO:1) of 787 aminoacid proteins (SEQ IDNO:2) of the about 88KDa molecular weight of encoding has been identified in sequential analysis.Vip3B nucleotide sequence and vip3A gene nucleotide series have 86% identity.Vip3B protein amino acid sequence and Vip3A protein amino acid sequence have 88% identity.

The biological assay of the Vip3B protein of expressing in embodiment 5:pNOV1325 and pCIB9400

To the black cutworm bait (BioServ, Frenchtown, NJ) of impouring fusing in 50mm culture dish, and it is solidified.Top10 (Invitrogen) intestinal bacteria (E.coli) cell that contains pNOV1325 or pNOV9400 (every kind all contains vip3B encoding sequence) with transfer pipet transferase 12 00 μ l is to bait surface.With bacterium ring, smear solution equably so that suspension covers the surface of whole bait.Make surperficial complete drying.With superfine point brush, on bait, place the first instar larvae of the lepidopteran species that following table lists.Each species of separate detection.Within 3 days and 5 days after larva is infected bait, record larval mortality.With comprising, there is no the sample of the Top10 of carrier intestinal bacteria (E.coli) cell as negative control.For comparison purposes, also can in identical biological assay, detect Vip3A protein.For this embodiment, the known activity spectrum data of the Vip3B data that relatively this embodiment obtains and Vip3A.

Shown in table 1 is bioassay results.Data shown in table 1 are records of the 5th day from infecting.In intestinal bacteria (E.coli) negative control, observe seldom active or there is no an activity.Result shows that Vip3B toxin has the more activity of wide spectrum than Vip3A toxin, because Vip3B has the activity of anti-European corn borer (Ostrinia Nubilalis) and small cabbage moth (Plutellaxylostella).Result also shows that Vip3B has higher specific activity to the real noctuid of paddy (Helicoverpazea) than Vip3A toxin.

Table 1

^athe insect that observes survival has serious feed and growth-inhibiting.

^b"+" represents the insect species to Vip3A sensitivity, and "-" represents Vip3A to have little or no the insect species of susceptibility.

Embodiment 6: the structure of the vip3B encoding sequence that corn is optimized

According to United States Patent (USP) 5,625, disclosed method is prepared synthetic corn and is optimized vip3B encoding sequence in 136.In the method, utilized the codon of corn preference, this amino acid whose single cipher in the corn of the most often encoding.The corn preference codon of specific amino acids can be known by inference from the known sequence of corn.At Murray etc., in Nucleic Acids Research 17:477-498 (1989), found the codon usage of 28 genes of maize plant.Synthetic vip3B encoding sequence (SEQ ID NO:3) clone is entered to pET101/D-Topo expression vector.The carrier that name obtains is thus pNOV1328, and its conversion is entered in intestinal bacteria (E.coli) DH5 α cell, with ATCC preserving number PTA-3869 preservation.

Embodiment 7: the generation of the rotaring gene corn plant that comprises vip3B gene

Select vip3B (the SEQ ID NO:3) encoding sequence that synthetic corn is optimized to enter maize plant for transforming.The expression cassette that comprises synthetic vip3B encoding sequence is transferred to the applicable carrier for the corn conversion of Agrobacterium (Agrobacterium) mediation.Build 3 carriers for this embodiment: the carrier that (a) comprises two vip3B expression cassettes, the first expression cassette comprises MTL:vip3B, the second expression cassette comprises PEPC:vip3B, (b) comprises the carrier of CMP:vip3B, (c) comprises the carrier of UbiP:vip3B.With all carriers in this embodiment, also comprise phosphomannose isomerase (PMI) gene (Negrotto etc., (2000) Plant Cell Reports 19:(798-803) for transgenic lines screening.

All 3 carriers are transformed and entered in corn respectively.Substantially as Negrotto etc., carry out the conversion of prematurity maize described in (2000) Plant Cell Reports 19:798-803.For this embodiment, all nutrient media componentses are all as above described in Negrotto etc.Yet, can replace various nutrient media components known in the art.

At 28 ℃, at YEP (yeast extract (5g/L), peptone (10g/L), NaCl (5g/L), 15g/L agar, pH6.8) cultivates Agrobacterium (Agrobacterium) strain LBA4404 (pSB1) 2-4 days that comprises Plant Transformation plasmid on solid medium.In the LS-inf substratum that has supplemented 100 μ M As (Negrotto etc., (2000) Plant Cell Rep 19:798-803), suspend about 0.8 * 10 ⁹individual Agrobacterium (Agrobacterium).Pre-induction bacterium 30-60 minute on this substratum.

From the fringe of 8-12 age in days, cut A188 immature embryo, put into liquid LS-inf+100 μ M As.Also can utilize the immature embryo form of other corn idioplasm.With fresh infection substratum rinse embryo once.Then add Agrobacterium (Agrobacterium) solution, vortex embryo 30 seconds, coexists itself and bacterium 5 minutes.Then, embryo is transferred in LSA substratum with scutel side direction upward, cultivated in the dark 2 to 3 days.Subsequently, by the mode of 20 to 25 embryos of each culture dish, embryo is transferred in the LSDc substratum that has supplemented cefotaxime (250mg/l) and Silver Nitrate (1.6mg/l), in 28 ℃ of dark, cultivated approximately 10 days.

The immature embryo that produces embryo callus is transferred in LSD1M0.5S substratum.On this substratum, screen culture approximately 6 weeks, simultaneously at approximately 3 weeks culturing steps that go down to posterity.Shift the callus of survival to having supplemented in the Reg1 substratum of seminose.After (hour dark management of 16 little time/8) cultivates in light, shift chlorenchyma and adjust on the Reg2 substratum of thing to not growing, about 1-2 week of incubation.Plantlet is transferred in the Magenta GA-7 box (Magenta Corp, Chicago I11.) that contains Reg3 substratum, under light, grown.About 2-3 is after week, detects the existence of PMI gene and vip3B gene in plant by PCR.The plant of PCR test positive is transferred to greenhouse, detects the resistance to lepidoptera pest.

Embodiment 8: express the analysis of the rotaring gene corn plant of Vip3B

The biological assay of insect

When transplanting plant from Magenta GA-7 box to soil time, from plant, sample.Sampling comprises cuts two small pieces blades (approximately 2-4cm is long), places every blade in little culture dish.Negative control or the vip3B gene that comes from same conversion test be the transgenic plant of PCR feminine gender, or (to detecting plant and the having similar size) non-transgenic plant of growing under similar growth conditions with transgenic plant.

By place 10 the first instar larvaes on each blade, with European corn borer (Ostrinia Nubilalis) or autumn mythimna separata (Spodoptera frugiperda), inoculate the leaf sample of each plant.Then, seal closely culture dish.Also can utilize other applicable insect pest.

After inoculation about 3-4 days, collect data.Calculate the percentage mortality ratio of larva.Can determine the visual damage level of blade simultaneously.It is senior that feeding infringement is classified as, and middle rank is rudimentary or do not have, and numerical value is respectively 3,2, and 1 or 0.

It shown in table 2, is the bioassay results of transgenic plant.Result shows that the rotaring gene corn plant that comprises vip3B gene and express Vip3B protein has insecticidal activity to European corn borer (OstriniaNubilalis) and autumn mythimna separata (Spodoptera frugiperda).

Table 2 is expressed the effect of the rotaring gene corn plant of Vip3B

Event	Promotor: vip3B construct	%FAW mortality ratio/plant a	%ECB mortality ratio/plant b
				118A	MTL:vip3B/PEPC:vip3B	100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100	90，100，100，90，100，100，80，100 100，100，100，90，90，90，100，100 90，100，70，80，70，100，100，100，70，100，80，80
121A	MTL:vip3B/PEPC:vip3B	90，100，100，100，100，100，100，100，100， 100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100	70，80，70，90，80，80，90，80，80，90，100，100，80，90，70，90，80，100，80， 100，70，80，70，70，90，100，100，90 90，90，90
				142C	MTL:vip3B/PEPC:vip3B	100，100，100，100，100，100，100，100，100	80，100，90，80，90，70，70，90，90
145B	MTL:vip3B/PEPC:vip3B	100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100，100	80，70，90，100，70，70，100，80，80，70，100，100，100，100，70，100，90， 90，90
				89A	CMP:vip3B	100，100，100，100，100，100，100，100	100，100，100，100，100，100，100，100
190A	CMP:vip3B	100，100，100，100，100，100，100，100，100	100，80，90，80，90，100，80，100，80

^afAW=autumn mythimna separata

^aeCB=European corn borer

ELISA measures

With ELISA, measure the level of Vip3B protein in various transgenic corns tissues.According to United States Patent (USP) 5,625, disclosed method has been carried out elisa assay in 136.Shown in table 3 is the result of elisa assay.

The level of Vip3B protein in table 3 transgenic corns

Embodiment 9: heterozygosis Vip3 toxin

Vip3B is virose to European corn borer (Ostrinia Nubilalis) and small cabbage moth (Plutellaxylostella), and relevant Vip toxin Vip3A has little or no activity.Vip3B and Vip3A main difference are their C-stub areas of aminoacid sequence separately, and particularly SEQ ID NO:2 amino acid 579 is to amino acid 787 regions.In order to prove that this C-stub area of Vip3B is the active part that anti-European corn borer (Ostrinia Nubilalis) and small cabbage moth (Plutella xylostella) are enough provided in Vip3B toxin, built a kind of hybrid toxins, wherein comprise by SEQ ID NO:2 amino acid 579 starts, amino acid 787 stops Vip3B C-stub area and by from amino to carboxyl aspect, connect by SEQ ID NO:5 amino acid/11, the Vip3A N-stub area that stops of amino acid 578.Naming this hybrid toxins is Vip3A-B (SEQ ID NO:7)

With primer below, utilize two-step pcr to build the nucleic acid molecule of coding Vip3A-B hybrid toxins:

VIP3A-N：5’-ATGACCAAGAATAATACTAAATTAAGCAC-3’ (SEQ ID NO：10)

VIPfus4：5’-TCCTTATGAACATATAAAGCTTTAGTTCCATT-3’ (SEQ ID NO：11)

VIP3B-C:5’-GGCGAATTCTCACTTAATCGAAAAATTCCGGAAATTTAT-3’(SEQ ID NO：12)

VIPfus3：5’-AATGGAACTAAAGCTTTATATGTTCATAAGGA-3’ (SEQ ID NO：13)

In the first step PCR, with primer Vip3A-N (SEQ ID NO:10) and Vipfus4 (SEQ ID NO:11), produce the fragment of the about 1.7kb of vip3A gene 5 ' end of coding N-stub area, with primer VipB-C (SEQ ID NO:12) and Vipfus3 (SEQ ID NO:13), produce the fragment of the about 0.7kb of vip3B gene 3 ' end of coding C-stub area.In second step PCR, combine these two fragments as the template of primer V ip3A-N (SEQ ID NO:10) and Vip3B-C (SEQ ID NO:12) to produce the heterozygosis vip3A-vip3B gene of about 2.4kb, called after vip3A-B (SEQ ID NO:6).

By the method for embodiment 5 general introduction, detect anti-autumn mythimna separata and European corn borer insecticidal activity of intestinal bacteria (E.coli) clone who expresses assorted house Vip3A-B toxin.The result of some biological assays shows, it is active with European corn borer that the C-stub area of Vip3B is enough to give assorted house toxin.

Embodiment 10: the vitro recombination of the vip3 gene of reorganizing by DNA

By one of pcr amplification vip3 gene of the present invention, SEQ ID NO:1 for example, 3, or 6.Basic as Stemmer etc., described in PNAS 91:10747-10751 (1994), process by DnaseI and digest the DNA fragmentation obtaining thus, from reaction mixture, remove PCR primer.As described in (1994) such as Stemmer, there is no the PCR reaction of primer, there is subsequently the PCR reaction of primer.The DNA fragmentation clone who obtains is thus entered to pTRC99a (Pharmacia, catalog number (Cat.No.): 27-5007-01), the condition of utilizing Biorad gene pulse producer and preparation business, is transformed and is entered intestinal bacteria (E.coli) strain SASX38 by electroporation.The bacterium that incubated overnight transforms on substratum, screening insecticidal activity.

In similar reaction, make to comprise one of vip3 gene described herein (SEQ ID NO:1,3,6 or its mutant) pcr amplified dna fragment and the pcr amplified dna fragment (or its mutant) that comprises at least one other vip3 gene as herein described there is vitro recombination, recovery as described below obtains thus has the variant that improves insecticidal properties.

In order to increase the diversity of reorganization vip3 gene library, utilize synthetic oligonucleotide reorganization to reorganize one or more vip3 genes (being called primary gene).Synthetic a large amount of (for example, 2,5,10,20,50,75, or 100 or more) oligonucleotide corresponding at least one diversity region.Can be directly to these oligonucleotide reorganization, or one or more restructuring that can Yu Gai nucleic acid family.

Can obtain oligonucleotide sequence from being called other vip3 gene of secondary gene.Secondary gene and primary gene have homology to a certain degree.There are several selections for the method for the synthetic secondary Gene Partial of oligonucleotide.For example, can select at random the part of secondary gene.DNA Shuffling Method will select those can be incorporated into the oligonucleotide in reorganization gene.

As long as selected part is applicable to synthetic, they can be any length.Also can be according to the homology design oligonucleotides between primary and secondary gene.Homology is to a certain degree necessary for the exchange that must occur between DNA fragmentation during reorganizing.Meanwhile, strong heterogeneity also needs for the diversity of reorganization gene library.And, according to protein sequence knowledge and functional relationship, can select for the synthetic secondary gene specific part of oligonucleotide.

The C-end structure territory that the invention discloses Vip3B is partly relevant with the activity profile of Vip3 toxin.When utilizing DNA shuffling technology, while modifying insecticidal spectrum by the present invention, the C-stub area that can select secondary gene nucleotide series as target region for the synthesis of the oligonucleotide of applying in oligonucleotide Shuffling Method.

Because the insecticidal activity of Vip3 protein depends on N-stub area at least in part, can select the N-stub area of secondary gene to carry out oligonucleotide reorganization, to increase insecticidal activity.

Embodiment 11: format high throughput screening insecticidal activity

The insecticidal activity of reorganization vip3 gene library in screening escherichia coli (E.coli) or bacillus thuringiensis (Bacillusthuringiensis).With Q-bot (Beckman), choose bacterium colony, be placed in the growth medium in standard 96 hole dimension boards overnight growth.Then, with 96 hole gauge lattice, each clone is taped against on insect bait surface, makes surface drying.Alternatively, to each hole, add transformant group to increase the clone's number detecting in initial screening circulation.For example, every hole sizer selects 100 clones, utilizes 10000 holes to carry out 10 ⁶individual clone's screening.

To each hole, add several newborn target insect larvaes, cigarette aphid noctuid (Heliothisvirescens) for example, the real noctuid (Helicoverpa zea) of paddy or autumn mythimna separata (Spodopterafrugiperda).With air-permeable envelope, wrap up this plate, so that larva is retained in placed hole.After 5 days, each hole assessment is consumed to amount and/or the insect mortality of bait.Select to show that the clone who has little or no in the hole that consumes bait and/or observe high insect mortality is for further analysis.Should be able to find that there is several clones of the anti-target insect active of enhancing.

All publications of mentioning in specification sheets of the present invention and patent application have shown one skilled in the art's of the present invention state of the art.Here separately and be incorporated to particularly all publications and patent application is reference.

Should be appreciated that embodiment described here and embodiment are only example objects, those skilled in the art can understand various modifications or change on its basis, and such content is all included in this application spirit and scope and within the scope of claims.

Sequence table

<110>Miles，Paul

Kramer，Vance

shen，Zhicheng

shotkoski，Frank

Warren，Greg

<120> new type disinsection toxin

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Claims (5)

1. for detect the primer pair of the existence of the vip3B gene shown in SEQ ID NO:3 by PCR.

2. primer pair, it is SEQ ID NO:12 and SEQ ID NO:13.

3. for passing through the primer pair of the vip3 gene shown in pcr amplification SEQ ID NO:1 or 3 or 6.

4. primer pair, it is SEQ ID NO:8 and SEQ ID NO:9.

5. by PCR, detect the method for the existence of the vip3B gene shown in SEQ ID NO:3.