CN101139609A - Plant-optimized polynucleotide encoding approximately 45 kda pesticidal protein - Google Patents

Abstract

The subject invention provides new, plant-optimized polynucleotide sequences that encode pesticidal toxins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, for example, that make them particularly well-suited for optimized expression in plants. Using the polynucleotide sequences described herein, the transformation of plants can be accomplished, using techniques known to those skilled in the art, in order to confer pest resistance upon said plants. In preferred embodiments, the subject invention provides plant-optimized polynucleotide sequences which encode approximately 15 kDa and approximately 45 kDa pesticidal proteins.

Description

The plant-optimized polynucleotide of the insect-killing protein of coding approximately 45 kda

The application is for dividing an application, and the applying date of its female case application is on October 21st, 1999, and application number is 99812497.4, and denomination of invention is " the plant-optimized polynucleotide of the insect-killing protein of encode about 15kDa and approximately 45 kda ".

Background of invention

The cost that insect and other insect make the crop loss in peasant every year and control these insects reaches hundreds of millions dollars.The loss of the agriculture production environment that insect pest causes comprises the raising of the minimizing of crop yield, the reduction of making amount and results cost.

Chemical insecticide provides the effective ways of Pest Control; Yet the public has begun to worry the chemical residues amount that may find in food, underground water and environment.Therefore, the synthetic chemical insecticide is subjected to careful examination just day by day, and particularly correct is its potential toxicity environmental consequences of examination.The synthetic chemical insecticide can be poisoned soil and underlying aquifer, because of runoff pollutes the surface water, and destroys non-target life form.Another shortcoming of synthetic chemical shim is, when they are applied to regional that pet, farming animals or children may be in contact with it, can cause the harm of public safety aspect.They also may work the mischief to the health of user, especially when not following correct application process.Global administrative authority is all in restriction and/or ban use of many sterilants, and particularly those are lasting and enter the synthetic chemistry sterilant of biologic chain in environment.The example of the synthetic chemistry sterilant of widespread use comprises the organochlorine class, for example DDT, mirex, kepone, lindane, aldrin, Niran, aldicarb and Dieldrin-attapulgite mixture; Organophosphorus compounds, for example Chlorpyrifos 94, thiophos, Malathion and diazinon; And amino formate.To the new restriction of the strictness of use of insecticide and eliminate from the market that some effective insecticidal agent all may limit the economy of the high insect of control cost and effective choice.

In view of with use the relevant problem of synthetic chemistry sterilant, obviously need limit the use of these agents and need to determine other control agent.Replace the synthetic chemistry sterilant, or these agents of applied in any combination and biotic pesticide, can reduce the level of environment toxic chemical.

Just day by day a kind of biotic pesticide of popularization and application be soil microorganisms bacillus thuringiensis (Bacillus thuringiensis) (B.t.).This soil microorganisms bacillus thuringiensis (B.t.) is a kind of Gram-positive, product genus bacillus.B.t. most of bacterial strains do not show insecticidal activity.Some B.t. bacterial strains produce companion cell crystalline protein inclusion bodies, and can companion cell crystalline protein inclusion body as its feature.These generally have specific insecticidal activity " delta-endotoxin " and are different from the extracellular toxin that those have nonspecific host range.When examining under a microscope, these inclusion bodies often show as the crystal of unique shape.These protein can be highly toxic to insect and be specific aspect its toxicity activity.

For many years, the gemma of use bacillus thuringiensis Ku Er Stark subspecies (B.thuringiensissubsp.kurstaki) and crystalline preparation are as the commercial sterilant of lepidoptera pest.For example, bacillus thuringiensis Ku Er Stark mutation HD-1 (B.thuringiensisvar.kurstaki HD-1) produces the deleterious crystalline delta-endotoxin of a kind of larva to many lepidopterous insects.

Before 15 years, in the document of publishing cloning and the expression (Schnepf of B.t. crystalline protein gene in intestinal bacteria (Escherichia coli) just described, H.E., H.R.Whiteley[1981] Pro.Natl.Acad.Sci.USA78:2893-2897.).U.S. Patent No. 4,448,885 and U.S. Patent No. 4,467,036 expression of B.t. crystalline protein in intestinal bacteria all disclosed.Based on the use of having produced and be given the ratification of the B.t. product of recombinant DNA.

At first, the commerce of B.t. sterilant is used lepidopteran (caterpillar) insect that is restricted to close limit.Yet, recent research person found to wide ranges many insects have specific B.t. sterilant.For example, B.t. other kind, promptly, Israel's kind (israelensis) and Mo Lixun kind (morrisoni) (a.k.a.tenebrionis, a.k.a.B.t.M-7) respectively at the commercial insect (Gaertner that is used to control Diptera and Coleoptera, F.H.[1989] " Cellular Delivery Systems forInsecticidal Proteins:Living and Non-Living Microorganisms ", referring to Controlled Delivery of Crop Protection Agents, R.M.Wilkins, editor, Taylor and Francis, New York and London, 1990, pp.245-255).

Now identified the new subspecies of B.t., and to the gene of being responsible for active delta-endotoxin proteins matter carried out separating and order-checking (H  fte, H., H.R.Whiteley[1989] Microbiological Reviews 52 (2): 242-255).H  fte and Whiteley are divided into four big classes with the B.t. crystalline protein gene.This four class is cryI (lepidopteran is specific), cryII (lepidopteran and Diptera are specific), cryIII (Coleoptera is specific) and cryIV (Diptera is specific).Reported and found that other insect was had the toxic bacterial strain of specificity (Feitelson, J.S., J.Payne, L.Kim[1992] Bio/Technology 10:271-275).For example, CryV and CryVI name have been proposed for two groups of new nematode active toxins.

The nomenclature in 1989 of H  fte and Whiteley and classification schemes based on the host range of aminoacid sequence of inferring and toxin the two.This system is suitable for summarizing 14 kinds of dissimilar toxin genes, and they are divided into five big classes.The quantity of the bacillus thuringiensis crystalline protein gene that checked order at present, is more than 50.A kind of nomenclature scheme of revising had been proposed, it is only based on amino acid identity (Crickmore etc., [1996] Society forInvertebrate Pathology, 29th Annual Meeting, IIIrd InternationalColloquium on Bacillus thuringiensis, University ofCordoba, Cordoba, Spain, September 1-6,1996, abstract)." cry " that has all kept mnemonic(al) for all toxin genes except that cytA and cytB (they remain an independent class).In the first estate, Roman number has been changed into Arabic numerals, in the tertiary gradient, removed parantheses.Have in the original title manyly to be retained, although there are some to be reclassified.

Adopt gene engineering, developing the novel method that is used for the B.t. toxin is discharged into agricultural environment, comprise and use the plant of handling and use the release vehicle (Gaertner of the microorganism cells of stabilization as the B.t. toxin with B.t. toxin gene (being used for insect-resistant) genetic engineering, F.H., L.Kim[1988] TIBTECH 6:S4-S7).Therefore, the isolating B.t. endotoxin genes commercial value that just becoming.

Various improvement have been obtained by modifying B.t. toxin and/or their gene.For example, U.S. Patent No. 5,380,831 and No.5,567,862 relate to the synthetic insect crystalline protein gene that kills that the expression of production in plant is improved.

Obstacle to the successful agricultural application of B.t. toxin comprises the formation of insect to the resistance of B.t. toxin.In addition.Some insect is that the effect of B.t. is unmanageable.The latter comprises such as the such insect of boll weevil and little low tiger and does not show obviously remarkable most several adult of susceptibility to the B.t. delta-endotoxin so far.

Therefore, the resistance control strategy in the B.t. plant technology causes great concern, and presses for new toxin gene.For example, WO97/40162 (disclosed PCT application) discloses the 15kDa that obtains from B.t. isolate PS80JJ1 and PS149B1 and the coleopteran-active protein of 45kDa.

Because deep research and resource input, continue to have issued patent about the new purposes of the patent of new B.t. isolate, toxin and gene and relevant B.t. isolate.Summary (above) referring to people such as Feitelson.U.S. Patent No. 5,589,382 disclose B.t. isolate PS80JJ1 has the nematicide activity.U.S. Patent No. 5,632,987 disclose B.t. isolate PS80JJ1 has anti-pumpkin 12 asterophyllite first activity.Yet, find new B.t. isolate and find that the new purposes of known B.t. isolate remains by rule of thumb, unpredictalbe technology.

Still press for so new toxin gene, they can be in plant in the mode of effective control insect and other insect, successfully express with enough levels.

The invention summary

The present invention relates to be used for the material and the method for Pest Control, especially plant insect.More particularly, the invention provides polynucleotide sequence novelty, plant-optimized of coded insect-killing protein.Polynucleotide sequence of the present invention is compared with wild-type sequence to have and is made them be particularly suitable for some modification that optimizing is expressed in plant.Utilize method known to those skilled in the art, can use polynucleotide sequence described herein to finish the conversion of plant, thereby give described plant insect resistance.In a preferred embodiment, the invention provides the plant-optimized polynucleotide sequence of the insect-killing protein of about 15kDa of coding and approximately 45 kda.

Sequence description

Sequence 1 is the polynucleotide sequence that is called the gene of 80JJ1-15-PO5, and it carried out optimization at the expression in corn.The protein of the about 15kDa of this genes encoding.This gene and protein are disclosed in WO97/40162.

Sequence 2 is the new polynucleotide sequences that are called the gene of 80JJ1-15-PO7, and it carried out optimization at the expression in corn.This is the proteinic alternative gene of the about 15kDa of coding.

Sequence 3 is the proteinic aminoacid sequences of new insecticidal activity by the genes encoding that is called 80JJ1-15-PO7.

Sequence 4 is the polynucleotide sequences that are called the gene of 80JJ1-45-PO, and it carried out optimization at the expression in corn.The protein of this genes encoding approximately 45 kda.This gene is disclosed in WO97/40162.

Sequence 5 is the new polynucleotide sequences that are called the gene of 149B1-15-PO, and it carried out optimization at the expression in Zea mays (Zea mays).The protein of the about 15kDa of this genes encoding, this protein derives from the PS149B1 that is disclosed in WO97/40162.

Sequence 6 is the new polynucleotide sequences that are called the gene of 149B1-45-PO, and it carried out optimization at the expression in Zea mays.The protein of this genes encoding approximately 45 kda, this protein derives from the PS149B1 that is disclosed in WO97/40162.

That invents is open in detail

The present invention relates to be used for the material and the method for Pest Control, especially plant insect.More particularly, the invention provides polynucleotide sequence novelty, plant-optimized of coded insect-killing protein.Polynucleotide sequence of the present invention is compared with wild-type sequence to have and is made them be particularly suitable for some modification that optimizing is expressed in plant.Utilize method known to those skilled in the art, can use polynucleotide sequence described herein to finish the conversion of plant, thereby give described plant insect resistance.In a preferred embodiment, the invention provides the plant-optimized polynucleotide sequence of the insect-killing protein of about 15kDa of coding and approximately 45 kda.

The method that the contrast of utilization such as computer or software auxiliary sequencel is so just can notice that plant-optimized gene of the present invention compares with wild type gene or compare in the difference aspect the nucleotide sequence with previously known gene.Similarly, can notice also that the present invention compares with the wild-type toxin or compare difference aspect peculiar aminoacid sequence with previously known toxin.

It will be readily apparent to one skilled in the art that if provide the sequence of gene as herein described, just can obtain gene of the present invention by several means.In preferred embodiments, gene of the present invention can make up by synthesis method, for example uses gene machine.The concrete gene that this paper exemplifies also can obtain by modifying some wild type gene that derives from some isolate (the culture collection unit that is deposited in as mentioned below) (for example, by the point mutation technology) in instruction according to the present invention.

Some culture of discussing among the application has been deposited in agricultural research institute patent culture collection center (NRRL), Northern Regional Research Center, 1815 NorthUniversity Street, Peoria, Illinois 61604, USA.The preservation strain of hereinafter listing is disclosed in as mentioned in the patent documentation of " background of invention " part discussion.

Subculture	Go into to hide registration number	Preservation day
			B.t.PS80JJ1	NRRL B-18679	July 17 nineteen ninety
Intestinal bacteria (NM522) are (PS80JJ1 14kDa ﹠ 45kDa) (pMYC2421)	NRRL B-21555	On March 28th, 1996
			Intestinal bacteria (NM522) are (PS80JJ1 14kDa ﹠ 45kDa) (pMYC2426)	NRRL B-21671	On March 26th, 1997
B.t.PS149B1	NRRL B-21553	On March 28th, 1996
			Intestinal bacteria (NM522) are (PS149B1 15kDa ﹠ 45kDa) (pMYC2429)	NRRL B-21673	On March 26th, 1997

It should be understood that can obtain the preservation thing not component part abolish the patent right that action by government authorizes and permit enforcement the present invention.

Gene and toxinIn preferred embodiments, the present invention includes at the expression in plant and carried out optimized polynucleotide sequence, wherein said sequence is selected from sequence 2, sequence 5 and sequence 6.Preferred protein shown in sequence 2 encoding sequences 3.

Polynucleotide of the present invention can be used for forming complete " gene " with coded protein or peptide in required host cell.For example, as the technician can recognize easily, sequence 2, sequence 5 and sequence 6 did not have terminator codon and show.As this area was known easily, sequence 2, sequence 5 and/or sequence 6 can suitably be placed in the purpose host under the promotor control.

As the technician recognize easily, DNA can double chain form exists.In this structure, a chain and another chain complementation, vice versa.Refer to have the chain (codon is can once read three and three Nucleotide producing a specific amino acids) of a series of codons in this area through commonly used " coding strand ", this chain can be used as open reading-frame (ORF) (ORF) and reads and form target protein matter or peptide.In order to express a kind of protein in vivo, usually a DNA chain is translated into a RNA complementary strand, it is used as proteinic template.When DNA duplicates, produce additional, DNA complementary strand in plant for example.Therefore, the present invention includes the exemplary polynucleotide represented in the appended sequence table or the application of complementary strand.The RNA and the PNA (peptide nucleic acid(PNA)) that are equivalent to the new dna molecular that specifically exemplifies on those functions are included within the present invention.

This paper has specifically exemplified some dna sequence dna of the present invention.These sequences are demonstrations of the present invention.What it should be obvious that is, the present invention not only comprises gene and the sequence that this paper specifically exemplifies, and (for example comprise its Equivalent and variant, mutant, syzygy, mosaic, block thing, fragment and littler gene), described Equivalent is compared with sequence with the concrete those disclosed gene of this paper with variant, shows identical or close feature aspect the expression toxin in plant." variant " used herein and " Equivalent " are meant such sequence, and they have expression and the Nucleotide (or amino acid) of the gained insecticidal activity displacement, disappearance (inner and/or terminal) of not remarkably influenced theme gene in plant, add or insert.Keeping the proteinic fragment of polynucleotide of insecticidal activity and " the desinsection part " of full length protein is also included within this definition.

The application standard technology can be modified gene, also can make up the version of gene easily.For example, the technology of manufacturing place sudden change is well known in the art.In addition, can use commercially available exonuclease or endonuclease according to standard method, and can use enzyme (for example Bal31) or site-directed mutagenesis from the end system of these genes cut away Nucleotide.Also can use various restriction enzymes and obtain useful gene.

It should be noted that gene of equal value has the toxin of encoding those and theme genes encoding the toxin of height amino acid identity or homology.Critical area amino acid identity at toxin will be the highest, and these critical areas cause biological activity or definite relevant with 3-d modelling (its final decision biological activity).About this point, some displacement is acceptable and is to expect, is to influence the conservative amino acid metathetical of the 3-d modelling of molecule words if these displacements are in active not crucial zone or these displacements.For example, amino acid can be divided into following a few class: nonpolar, uncharged polar, alkalescence and tart.Preservative replacement (wherein one type a amino acid is by another aminoacid replacement of same type) falls within the scope of the present invention, as long as this displacement does not significantly change the biological activity of compound.Table 1 provides the table look-up of the examples of amino acids that belongs to each type.

Table 1
		The amino acid type	Examples of amino acids
Nonpolar uncharged polar tart alkalescence	Ala，Val，Leu，Ile，Pro，Met，Phe，Trp Gly，Ser，Thr，Cys，Tyr，Asn，Gln Asp，Glu Lys，Arg，His

In some cases, also can carry out the non-conservation displacement.Key factor is that these displacements can not significantly impair the ability of expression of plants theme dna sequence dna or impair the biological activity of toxin.

This paper is meant these molecules when mentioning " isolating " polynucleotide and/or " purifying " toxin, and at this moment they do not combine with other molecule; Described " other molecule " is present in occurring in nature jointly and comprises their application in plant originally with " these molecules " originally.Represent to comprise when therefore, mentioning " isolating " and/or " purifying " " manually " as herein described.

Recombinant hostToxin encoding gene of the present invention can be imported in various microorganisms or the plant host.In some embodiments of the present invention, can be used for preparing in the preliminary step of precursor through the microorganism transformed host, for example, this precursor will finally be used for transformed plant cells and plant in some preferred embodiments, so that they express the toxin by genes encoding of the present invention.The microorganism that transforms by this way and use belongs to scope of the present invention.Recombinant microorganism for example can be B.t., intestinal bacteria or Rhodopseudomonas (Pseudomonas).Conversion can use standard method to carry out by those skilled in the art.The required material of these conversions is disclosed in this paper or the easy acquisition of those skilled in the art.

Therefore, in some preferred embodiments, expression of gene of the present invention directly or indirectly causes the interior generation of the born of the same parents of target protein matter and keeps.When plant transformed is ingested by insect, the insect toxin of will ingesting.Consequently to the control of insect.

B.t. toxin gene can import host, plant host preferably by suitable carrier.Many purpose plants are arranged, for example corn, wheat, rice, cotton, soybean and Sunflower Receptacle.Gene of the present invention is particularly suitable for providing the stable maintenance and the expression of the gene of express polypeptide sterilant in plant transformed, and, preferably can protect sterilant to avoid environment degradable and inactivation better.

Therefore, the present invention includes recombinant host, they comprise at the expression in plant carried out optimized polynucleotide sequence, and wherein said sequence is selected from sequence 2, sequence 5 and sequence 6.For example, recombinant host can be a vegetable cell.The complete plant that comprises the theme polynucleotide also within the scope of the invention.In particularly preferred embodiments, damage has resistance to pumpkin 12 asterophyllite first can to make a kind of plant, method is that it is transformed to express coding proteinic a kind of polynucleotide of about 15kDa (for example sequence 2) and the coding proteinic second kind of polynucleotide of 45kDa (for example sequence 4).Equally, also can under the control of a kind of promotor or independently a plurality of promotor (for example ubiquitin promoter), together use sequence 5 and sequence 6.For example, in this respect, the polynucleotide of sequence 2 and sequence 6 can together use, and perhaps, can use sequence 4 and sequence 5.

Although the invention provides the specific embodiments of synthetic gene, other gene that is equivalent to the gene that this paper exemplifies on the function also can be used for transforming host, plant host preferably.Other guidance of relevant production synthetic gene can be referring to for example U.S. Patent No. 5,380,831.

It is for referencial use that this paper reference or all public publications of quoting and patent documentation are incorporated this paper in full at this, as long as they do not contradict with the clearly instruction of this specification sheets.

Below be to set forth the embodiment that implements program of the present invention.This embodiment can not be regarded as restrictive.

Embodiment 1-inserts plant with toxin gene

One aspect of the present invention is the theme polynucleotide sequence conversion plant with the coded insect-killing toxin.Through plant transformed the attack of target insect there is resistance.Gene of the present invention carried out optimization at the application in plant.

Obviously, the promoter region that can express this gene in plant needs.Therefore, (in planta) in the plant expressed, DNA of the present invention is under the control of suitable promoter region.The method that adopts this class construction to obtain to express in the plant is known in the art.A preferred promoter zone that is used to express 15kDa and 45kDa transgenosis is that the Zea mays ubiquitin promoter adds Zea mays exons 1 and Zea mays introne 1 (Christensen, people such as A.H., 1992 Plant Mol.Biol.18:675-689).For two transgenosiss, a preferred transcription terminator is potato proteinase inhibitor II (Pin II) terminator (An, people such as G., 1989 Plant Cell 1:115-22).

Can the gene of coded insect-killing toxin disclosed herein be inserted vegetable cell with the whole bag of tricks well known in the art.For example, in some preferred embodiments,, use Bio-Rad to produce basically as described in the people such as Klein (1987)

The PDS-100He particle gun obtains to contain 14kDa and the genetically modified maize plant of 44kDa by microprojectile bombardment methods.

When preparing that foreign gene inserted higher plant, can obtain many cloning vectors, they comprise a dubbing system and the mark that allows to select the cell that transformed in intestinal bacteria.For example, these carriers comprise pBR322, pUC series, M13mp series, pACYC184 etc.So, the sequence of coding B.t. toxin can be inserted carrier at suitable restriction site.The gained plasmid is used for being transformed into intestinal bacteria.Bacillus coli cells is cultivated in the appropriate nutrition substratum, then results and cracking.Plasmid is reclaimed.As analytical procedure, carry out sequential analysis, restriction analysis, electrophoresis and other biological chemistry-molecular biology method usually.After each operation, used dna sequence dna is cut and is connected on the next dna sequence dna.Each plasmid sequence can be cloned in same plasmid or other plasmid.

According to required gene is inserted the method for plant, may need other dna sequence dna.For example, if with Ti or Ri plasmid transformed plant cells, then must be but usually its right side is connected as the flanking region that is inserted into gene with left border with the right border at least of Ti or Ri plasmid T-DNA.Obtained further investigation and done fully describing with the T-DNA transformed plant cells, referring to: EP 120516; Hoekema (1985), The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, the 5th chapter; People such as Fraley, Crit.Rev.Plant Sci.4:1-46; And people (1985) EMBOJ.4:277-278 such as An.

In case the DNA that inserts is incorporated in the genome, it just more stably exists, and no longer comes out usually.It contains selective marker usually, and this selective marker is given anti-biocide of transformed plants cell or antibiotic resistance, for example, and particularly kantlex, G 418, bleomycin, Totomycin or paraxin.Correspondingly, this mark that uses separately cell that should allow to select the cell that transformed rather than not conform to the DNA of insertion.

The technology of DNA being inserted plant host cell has a lot.Those technology comprise use Agrobacterium tumefaciens (Agrobacterium tumefaciens) or rhizobiaceae (Agrobacterium rhizogenes) as transforming agent with T-DNA transform, merge, injection, biolistics (microparticle bombardment) or electroporation and other possible method.If transform with edaphic bacillus, the dna clone that is inserted into must be gone into special carrier, that is, perhaps be cloned into intermediate carrier or be cloned into binary vector.Sequence in view of the sequence homology among those and the T-DNA can be integrated into Ti or Ri plasmid with intermediate carrier by homologous recombination.Ti or Ri plasmid also comprise the necessary vir of transfer T-DNA zone.Intermediate carrier can not oneself duplicate in edaphic bacillus.Can change intermediate carrier over to Agrobacterium tumefaciens by helper plasmid (joint).Binary vector can oneself duplicate in intestinal bacteria and in edaphic bacillus.They comprise a selectable marker gene and joint or polylinker (they by right and left T-DNA fringe region frame firmly).They directly can be transformed into (people [1978] Mol.Gen.Genet.163:181-187 such as Holsters) in the edaphic bacillus.The edaphic bacillus that is used as host cell will comprise a plasmid that carries the vir zone.This vir zone is that to change T-DNA over to vegetable cell necessary.Can comprise additional T-DNA.The bacterium that so transforms is used for transformed plant cells.In order to change DNA over to vegetable cell, can advantageously plant explants be cultivated with Agrobacterium tumefaciens or rhizobiaceae.Then, can will complete plant regeneration in infection plant's material (for example, the fragment of blade, stem, root also have protoplastis or suspension cultured cells) from appropriate media, described substratum may contain and is useful on microbiotic or the biocide of selecting.Then, whether the plant that can test such acquisition exists the DNA of insertion.In the situation of injection and electroporation, plasmid there is not particular requirement.It is possible using common plasmid, for example the pUC derivative.

Grow at inside plants in a conventional way through cell transformed.They can form sexual cell and will pass to progeny plant through the proterties that transforms.Such plant can grow by normal way, and with have identical conversion gene or other genic plant hybridization.Gained hybridization individuality has corresponding phenotypic characteristic.

It should be understood that embodiment as herein described and embodiment are illustrative, according to they various modifications or variation be to those skilled in the art's prompting and be included in the application and the essence and scope of following claims within.

Sequence table

<110>Mycogen Corporation

＜120〉desinsection of about 15kDa of coding and approximately 45 kda

Proteinic plant-optimized polynucleotide

<130>MA723/4X

<140>

<141>

<150>60/105,408

<151>1998-10-23

<150>60/105,359

<151>1998-10-23

<160>6

<170>PatentIn Ver.2.1

<210>1

<211>357

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic bacillus thuringiensis toxin gene

<400>1

atgtccgccc gcgaggtgca catcgagatc aacaacaaga cccgccacac cctccagctc 60

gaggacaaga ccaagctctc cggcggcagg tggcgcacct ccccgaccaa cgtggcccgc 120

gacaccatca agacgttcgt ggcggagtcc cacggcttca tgaccggcgt cgagggcatc 180

atctacttct ccgtgaacgg cgacgccgag atctccctcc acttcgacaa cccgtacatc 240

ggctccaaca agtgcgacgg ctcctccgac aagcccgagt acgaggtgat cacccagtcc 300

ggctccggcg acaagtccca cgtgacctac accatccaga ccgtgtccct ccgcctc 357

<210>2

<211>357

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic bacillus thuringiensis toxin gene

<400>2

atgtccgccc gcgaggtgca catcgagatc aacaacaaga cccgccacac cctccagctc 60

gaggacaaga ccaagctctc cggcggcagg tggcgcacct ccccgaccaa cgtggcccgc 120

gacaccatca agacgttcgt ggcggagtcc cacggcttca tgaccggcgt cgagggcatc 180

atctacttct ccgtgaacgg cgacgccgag atctccctcc acttcgacaa cccgtacatc 240

ggctccaaca agtccgacgg ctcctccgac aagcccgagt acgaggtgat cacccagtcc 300

ggctccggcg acaagtccca cgtgacctac accatccaga ccgtgtccct ccgcctc 357

<210>3

<211>119

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: by the toxin of synthetic bacillus thuringiensis genes encoding

<400>3

Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His

1 5 10 15

Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg

20 25 30

Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala

35 40 45

Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser

50 55 60

Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile

65 70 75 80

Gly Ser Asn Lys Ser Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val

85 90 95

Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile

100 105 110

Gln Thr Val Ser Leu Arg Leu

115

<210>4

<211>1155

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic bacillus thuringiensis toxin gene

<400>4

atgctcgaca ccaacaaggt gtacgagatc tccaacctcg ccaacggcct ctacacctcc 60

acctacctct ccctcgacga ctccggcgtg tccctcatgt ccaagaagga cgaggacatc 120

gacgactaca acctcaagtg gttcctcttc ccgatcgaca acaaccagta catcatcacc 180

tcctacggcg ccaacaactg caaggtgtgg aacgtgaaga acgacaagat caacgtgtcc 240

acctactcct ccaccaactc cgtgcagaag tggcagatca aggccaagga ctcctcctac 300

atcatccagt ccgacaacgg caaggtgctc accgcgggcg tgggccagtc cctcggcatc 360

gtgcgcctca ccgacgagtt cccggagaac tccaaccagc aatggaacct caccccggtg 420

cagaccatcc agctcccgca gaagccgaag atcgacgaga agctcaagga ccacccggag 480

tactccgaga ccggcaacat caacccgaag accaccccgc agctcatggg ctggaccctc 540

gtgccgtgca tcatggtgaa cgactccaag atcgacaaga acacccagat caagaccacc 600

ccgtactaca tcttcaagaa atacaagtac tggaacctcg ccaagggctc caacgtgtcc 660

ctcctcccgc accagaagcg cagctacgac tacgagtggg gcaccgagaa gaaccagaag 720

accaccatca tcaacaccgt gggcctgcag atcaacatcg actcggggat gaagttcgag 780

gtgccggagg tgggcggcgg caccgaggac atcaagaccc agctcaccga ggagctgaag 840

gtggagtact ccaccgagac caagatcatg accaagtacc aggagcactc cgagatcgac 900

aacccgacca accagccgat gaactccatc ggcctcctca tctacacctc cctcgagctg 960

taccgctaca acggcaccga gatcaagatc atggacatcg agacctccga ccacgacacc 1020

tacaccctca cctcctaccc gaaccacaag gaggcgctgc tgctgctgac caaccactcc 1080

tacgaggagg tggaggagat caccaagatc ccgaagcaca ccctcatcaa gctcaagaag 1140

cactacttca agaag 1155

<210>5

<211>369

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic bacillus thuringiensis toxin gene

<400>5

atgtccgccc gcgaggtgca catcgacgtg aacaacaaga ccggccacac cctccagctg 60

gaggacaaga ccaagctcga cggcggcagg tggcgcacct ccccgaccaa cgtggccaac 120

gaccagatca agaccttcgt ggccgaatcc aacggcttca tgaccggcac cgagggcacc 180

atctactact ccatcaacgg cgaggccgag atcagcctct acttcgacaa cccgttcgcc 240

ggctccaaca aatacgacgg ccactccaac aagtcccagt acgagatcat cacccagggc 300

ggctccggca accagtccca cgtgacctac accatccaga ccacctcctc ccgctacggc 360

cacaagtcc 369

<210>6

<211>1149

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic bacillus thuringiensis toxin gene

<400>6

atgctcgaca ccaacaaggt gtacgagatc agcaaccacg ccaacggcct ctacgccgcc 60

acctacctct ccctcgacga ctccggcgtg tccctcatga acaagaacga cgacgacatc 120

gacgactaca acctcaagtg gttcctcttc ccgatcgacg acgaccagta catcatcacc 180

tcctacgccg ccaacaactg caaggtgtgg aacgtgaaca acgacaagat caacgtgtcc 240

acctactcct ccaccaactc catccagaag tggcagatca aggccaacgg ctcctcctac 300

gtgatccagt ccgacaacgg caaggtgctc accgccggca ccggccaggc cctcggcctc 360

atccgcctca ccgacgagtc ctccaacaac ccgaaccagc aatggaacct gacgtccgtg 420

cagaccatcc agctcccgca gaagccgatc atcgacacca agctcaagga ctacccgaag 480

tactccccga ccggcaacat cgacaacggc acctccccgc agctcatggg ctggaccctc 540

gtgccgtgca tcatggtgaa cgacccgaac atcgacaaga acacccagat caagaccacc 600

ccgtactaca tcctcaagaa gtaccagtac tggcagaggg ccgtgggctc caacgtcgcg 660

ctccgcccgc acgagaagaa gtcctacacc tacgagtggg gcaccgagat cgaccagaag 720

accaccatca tcaacaccct cggcttccag atcaacatcg acagcggcat gaagttcgac 780

atcccggagg tgggcggcgg taccgacgag atcaagaccc agctcaacga ggagctcaag 840

atcgagtact cccacgagac gaagatcatg gagaagtacc aggagcagtc cgagatcgac 900

aacccgaccg accagtccat gaactccatc ggcttcctca ccatcacctc cctggagctc 960

taccgctaca acggctccga gatccgcatc atgcagatcc agacctccga caacgacacc 1020

tacaacgtga cctcctaccc gaaccaccag caggccctgc tgctgctgac caaccactcc 1080

tacgaggagg tggaggagat caccaacatc ccgaagtcca ccctcaagaa gctcaagaag 1140

tactacttc 1149

Claims (11)

1. polynucleotide, it has the nucleotide sequence of sequence 6.

2. recombinant host cell, its expresses the polynucleotide according to claim 1.

3. according to the host cell of claim 2, wherein said host cell is a vegetable cell.

4. according to the host cell of claim 2, wherein said host is a maize cell.

5. method of producing the recombinant host cell of claim 2.

6. according to the method for claim 5, wherein said host cell is a vegetable cell.

7. according to the method for claim 5, wherein said host cell is a maize cell.

8. the method for a controlling plant insect, wherein said method comprise described insect are contacted with first kind of protein by the polynucleotide encoding of claim 1 that wherein said first kind of protein is to be produced by the recombinant host cell of expressing described polynucleotide.

9. method according to Claim 8, wherein said host cell is a vegetable cell.

10. method according to Claim 8, wherein said host cell is the Zea mays cell.

11. according to the method for claim 10, wherein said method further comprises described insect is contacted with about second kind of protein of 15kDa.