CN1303432A - Novel insecticidal toxins from i(xenorhabdus nematophilus) and nucleic acid sequences coding therefor - Google Patents

Novel insecticidal toxins from i(xenorhabdus nematophilus) and nucleic acid sequences coding therefor Download PDF

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CN1303432A
CN1303432A CN99806688A CN99806688A CN1303432A CN 1303432 A CN1303432 A CN 1303432A CN 99806688 A CN99806688 A CN 99806688A CN 99806688 A CN99806688 A CN 99806688A CN 1303432 A CN1303432 A CN 1303432A
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CN100368544C (en
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V·C·卡拉默
M·K·摩根
A·R·安德森
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Abstract

Novel nucleic acid sequences isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii, and Photorhabdus luminescens, whose expression results in novel insecticidal toxins, are disclosed herein. The invention also discloses compositions and formulations containing the insecticidal toxins that are capable of controlling insect pests. The invention is further drawn to methods of making the toxins and to methods of using the nucleotide sequences, for example in microorganisms to control insect pests or in transgenic plants to confer insect resistance.

Description

New for the Pesticidal toxins of Xenorhabdus nematophilus and the nucleotide sequence of this toxin of encoding
The present invention relates to new toxin from Xenorhabdus nematophilus (Xenorhabdus nematophilus), Podbielniak Xenorhabdus (Xenorhabdus poinarii) and luminous smooth rod bacterium (Photorhabdus luminescens), it expresses the nucleotide sequence that produces described toxin, and the method that produces and use toxin and corresponding nucleic acids sequence control insect.
Insect is the major cause of crop production reduction.Only the loss that is caused by various insects every year in the U.S. is approximately 7,700,000,000 dollars.Except the loss that farm crop suffer, insect also is vegetable grower, orchard worker and flower-grower's a burden, and they also are the things that gardener and the owner of family are disliked.
Insect is mainly used by the high strength of chemical insecticide and is prevented and treated, and chemical insecticide stop insect feed or breeding, or kill insects works by suppressing insect growth.Good insect control therefore can be obtained, but these chemicals also influence other useful insect sometimes.Another problem that the widespread use of chemical insecticide produces is the appearance of resistant insects mutation.Different resistance operating strategies can partly relax this phenomenon, but the demand of alternative pest control agent still presents ascendant trend, the biological pest control agent, the Su Yun golden tooth spore bacillus Pesticidal toxins such as the delta-endotoxin of expressing for example, produced gratifying result in application facet, substituting or replenishing chemical insecticide is provided.Recently, encode that the gene of these delta-endotoxins is separated and their expression in heterologous host demonstrate the instrument that it provides the economic important pests of another kind of control.Especially; the expression of Pesticidal toxins in transgenic plant, for example Su Yun golden tooth spore bacillus delta-endotoxin provides the effectively anti-protection of selecting insect; and the transgenic plant of expressing these toxin are become commercialized, allow the peasant to reduce the application of chemical insecticide.Yet even in this case, still there is a kind of possibility of development in resistance, and only has a fraction of specific insect to prevent and treat.So the needs that long-term expectation does not still still have to satisfy are new and can effectively bring acceptable insect control agent in economic interests and the environmental protection to the peasant.
The present invention has satisfied long-standing demand to new insect control agent.Particularly at economic important pests and effectively control already present insect control agent is had the demand of control agent of the insect of resistance.In addition, it is desired will reducing to minimum sterilant to the burden of environment.
In the research of new insect control agent, interested in especially certain kind of the nematode that belongs to from Heterorhabdus and Steinemema, because their insecticidal properties.The larva of their kill insects and with the offspring of dead larva as food.In fact, their insecticidal activity depends on the symbiotic bacterium that lives in the nematode.These symbiotic bacteriums are that polished rod shape Pseudomonas and the Xenorhabdus under the Steinernema situation under the Heterorhabdus situation belongs to.
The present invention relates to separate nucleotide sequence from Xenorhabdus nematophilus, and to its similar basically nucleotide sequence, their expression produced to economic important pests particularly plant insect more highly toxic Pesticidal toxins is arranged.The present invention further provides and come from this nucleotide sequence and express the Pesticidal toxins that produces, and contained and can suppress the insect viability, growth or breeding, or reduce the composition and the preparation of the loss relevant of farm crop with insect.The present invention further provides and a kind ofly produced the method for toxin and utilize the Nucleotide for example pest control or the method for in transgenic plant, giving pest resistance in microorganism; and use toxin and contain the composition of toxin and the method for preparation is for example used this toxin, composition and preparation are to the insect infection district or preventive treatment insect area of liability or protect or to the resistance of insect giving with plant.
New toxin has very high insecticidal action to small cabbage moth (Plutella xylostella) (diamondback moth (diamonback moth)), and small cabbage moth is important economically insect.Toxin can be used to the strategy of various insects control, produces the maximum efficiency that environment is had minimum influence.
On the one hand, the invention provides a kind of isolating nucleic acid molecule, comprise: (a) one basically similar in appearance to the nucleotide sequence of the nucleotide sequence that is selected from the group that contains following sequence: the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; Or (b) nucleotide sequence with the nucleotide sequence isocoding of (a); The expression of wherein said nucleic acid molecule produces at least a active toxin of anti-insect that has.In the embodiment in this regard, nucleotide sequence with basically similar in appearance to the Nucleotide 569-979 of SEQ IDNO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and the Nucleotide isocoding of SEQ ID NO:14.Preferred nucleotide sequence is basically similar in appearance to the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and the Nucleotide of SEQ ID NO:14.Preferred nucleotide sequence coded being selected from contained SEQID NO:2, and 3,5,7,9,11,13, and 15 aminoacid sequence.Most preferred, nucleotide sequence contains the 569-979 of SEQ ID NO:1 Nucleotide, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, or SEQ ID NO:14.In another embodiment, nucleotide sequence contains the about 3.0 kb dna fragmentations that are included among the pClB9369 (NRRL B-21883).
According to an embodiment preferred, has the activity of anti-small cabbage moth from the toxin of the expression of nucleic acid molecule of the present invention.
On the other hand, the invention provides an isolated nucleic acid molecule, it contains one 20,25,30,35,40,45, or 50 (preferred 20) base pair nucleotide segment is equal to the difference successive 20,25 of the nucleotide sequence that is selected from the group that contains following sequence on sequence, 30,35,40,45, or 50 (preferred 20) base pair nucleotide segment: the 1045-2334 Nucleotide of the 569-979 Nucleotide SEQ ID NO:1 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, wherein above-mentioned nucleic acid molecule express and produce at least a active toxin of anti-insect that has.
The present invention also provides a mosaic gene, and it comprises the allogeneic promoter sequence that effectively is connected in nucleic acid molecule of the present invention.In addition, the invention provides the recombinant vectors that contains this mosaic gene.Further, the invention provides a host cell that contains this mosaic gene.According to this aspect of the invention, host cell can be a bacterial cell, a yeast cell, perhaps vegetable cell, preferably a vegetable cell.Further, the invention provides the plant that contains this vegetable cell.Preferred plant is a corn.
On the other hand, the invention provides the toxin of expressing generation by dna molecular of the present invention.According to embodiment preferred, toxin of the present invention has the activity of anti-small cabbage moth.
In one embodiment, toxin is intestinal bacteria (E.coli) the bacterial strain generation of B-21883 by called after NRRL preserving number.
In another embodiment, toxin of the present invention contains to be selected from and comprises SEQ ID NO:2, and 3,5,7,9,11,13, and the aminoacid sequence of 15 group.
The present invention also provides a kind of composition, and it contains the toxin of the present invention of insecticidal effective dose.On the other hand, the invention provides a kind of generation has the method for the toxin of anti-insect active, comprise: (a) obtain to contain the host cell of a mosaic gene, mosaic gene itself contains one section allogeneic promoter sequence that effectively is connected with nucleic acid molecule of the present invention; And (b) nucleic acid molecule is expressed in cell, and it produces the toxin of at least a effective anti-insect.
Further, the invention provides a kind of method that produces anti-insect plant, comprise importing nucleic acid molecule of the present invention in plant, nucleic acid molecule wherein in plant so that the significant quantity of control insect is expressed.According to embodiment preferred, insect is a small cabbage moth.
Further, the invention provides a kind of method of preventing and treating insect, comprise toxin of the present invention to the insect effective dosage.In a preferred embodiment, insect is a small cabbage moth.Preferably, toxin is administered orally in insect.
Another aspect of the present invention, the method of mutagenesis nucleic acid molecule of the present invention is provided, nucleic acid molecule wherein is cut into the colony of the double-stranded random fragment of required size, comprise: (a) add one or more lists of double-stranded random fragment group or double chain oligonucleotide, oligonucleotide wherein respectively contains the regional and allos zone with double-stranded template polynucleotide identity; (b) making the double-stranded random fragment of generation and the mixture sex change of oligonucleotide is single-chain fragment; (c) cultivate the single-chain fragment group who produces with polysaccharase, it carries out under the single-chain fragment annealing formation annealing fragment paired condition that causes in the identity zone, it is fully that this identity district starts duplicating of another for a member of a centering, therefore forms the double-stranded polynucleotide of a mutagenesis; And (d) the second and the 3rd step of repetition at least 2 further circulations, wherein the mixture of gained comprises the double-stranded polynucleotide of the mutagenesis in the 3rd step in the circulation in further round-robin second goes on foot, and wherein forms the double-stranded polynucleotide of further mutagenesis in further circulating.
Others of the present invention and advantage will become clearer by the specification sheets and the unrestricted embodiment of invention for a person skilled in the art.
Definition
" activity " of toxin of the present invention is meant that toxin as the Orally active insect control agent, has toxic effect, maybe can interrupt or prevent the feed of insect, and it may or may not can cause the death of insect.When toxin of the present invention delivered medicine to insect, the result typically was insect death, or insect is not taken food and makes toxin to the available source of insect.
" in conjunction with/effectively connect " be meant that two nucleotide sequences are relevant on structure or the function.For example, if two sequences are effectively to connect or be positioned at a certain position, make this regulating DNA sequence will influence the expression level of coding or structural DNA sequence, then this promotor or regulating DNA sequence are called as " combination " in coding RNA or proteic dna sequence dna.
" mosaic gene " is a recombinant nucleic acid sequence, wherein promotor or regulation and control nucleotide sequence are effectively to be connected in or to be incorporated into a coding mRNA or be expressed as nucleic acid sequences to proteins, thereby this regulation and control nucleotide sequence can be regulated and control transcribing or expressing of combined nucleotide sequence.The regulation and control nucleotide sequence of mosaic gene usually effectively is not connected with combined nucleotide sequence under field conditions (factors).
" encoding sequence " is one and is transcribed into for example mRNA of RNA, rRNA, and tRNA, snRNA has the nucleotide sequence of adopted RNA or sense-rna.Preferably, RNA is translated generation protein then in organism.
" control " insect is meant by the toxin influence and suppresses insect existence, growth, and feed, and/or the ability of breeding, or reduce the loss relevant of farm crop with insect.Kill insects can be meaned or may do not meaned to " control " insect, although preferably refer to kill insects.
" administration " a kind of toxin is meant with toxin and contacts insect, produces toxic effect and control insect.Toxin can be by many generally acknowledged mode administrations, for example express the protein composition of preparation, sprayable protein composition by the insect orally ingestible or by transgenic plant, bait matrix, or other art-recognized toxin drug delivery system contacts insect arbitrarily.
" expression cassette " used herein is meant that one can be instructed the nucleotide sequence of expressing specific nucleotide sequence in appropriate host cell, comprise a promotor that effectively is connected in the purpose nucleotide sequence that effectively links to each other with termination signal.It also typically comprises the needed sequence of nucleotide sequence suitable translation.The expression cassette that comprises the purpose nucleotide sequence may be chimeric, means that one of its component is allogenic with at least a of other component at least.Expression cassette also may be for spontaneous but with a kind of obtained to the useful recombination form of heterogenous expression.Yet typically, expression cassette and host are allogenic, that is, and the not natural generation and must be imported into the ancestors of host cell or host cell in host cell of the specific nucleotide sequence of expression cassette by conversion.The expression of nucleotide sequence can be carried out under the regulation and control of composing type or inducible promoter in the expression cassette, and inducible promoter has only just to start when host cell is exposed to some specific external stimuluss transcribes.In cellulous biology, for example in the plant, promotor can be to specific tissue, or organ, or the etap has specificity.
" gene " is a zone that is defined, it is arranged in genome and except above-mentioned nucleic acid sequence encoding, comprises other basic adjusting, is responsible for the nucleotide sequence of the expression regulation of transcribing and translating of encoding part.A gene also can comprise other 5 ' and 3 ' non-translated sequence and terminator sequence.Other element that may exist is an intron for example.
" goal gene " refers to any gene, it is administered in the plant, give for example antibiotics resistance of the desired feature of plant, virus resistance, insect-resistant, disease resistance, or the resistance of other insect, herbicide tolerance, the nutritive value of improvement, the reproductive performance of improved characteristics or change in the commercial run." goal gene " also is administered into the production of the enzyme that is used for the plant commercially valuable in the plant or meta-bolites.
" allos " nucleotide sequence is that a host cell that is imported into it is not natural bonded nucleotide sequence, comprises the multiple copied that the non-natural of the nucleotide sequence of natural generation produces.
" homology " nucleotide sequence is a natural bonded nucleotide sequence of the host cell that is imported into it.
" homologous recombination " is the mutual exchange of the intermolecular nucleic acid fragment of homologous nucleic acid.
" parasiticidal " is defined as preventing and treating the toxic biological activity that insect preferably passes through kill insects.
When the polypeptide of nucleic acid sequence encoding has identical aminoacid sequence with the polypeptide of the nucleic acid sequence encoding of being quoted, nucleotide sequence and the nucleotide sequence of being quoted " isocoding ".
" isolating " nucleic acid molecule or isolating enzyme are a nucleic acid molecule or enzyme, and it exists and so be not to be natural product by manually breaking away from from its natural surroundings.Isolating nucleic acid or enzyme can exist or be present in non-natural environment with sublimed form, for example, and in the recombinant host cell.
" nucleic acid molecule " or " nucleotide sequence " is can be from the linear fragment of the isolating strand in any source or double-stranded DNA or RNA.In the context of the present invention, nucleic acid molecule is preferably the fragment of DNA.
" ORF " is open reading frame.
" plant " is meant any plant of any etap, is meant spermatophyte especially.
" vegetable cell " is a structure and the physiological unit of plant, comprises a protoplastis and a cell walls.Vegetable cell can be the form of an isolating unicellular or cultured cells, or as more high organized unit for example, plant tissue, plant organ, or the part of whole plants.
" culture plant cell " is meant for example protoplastis of plant unit, cell culture cell, the cell in the plant tissue, pollen, pollen tube, ovulum, blastular, zygote, and the cultivation of the embryo of different developmental phases.
" vegetable material " is meant petal, stem, and root, the parts of flower or flower, fruit, pollen, ovum, zygote, seed is transplanted a cutting, cell or tissue culture, or the other parts of any plant product.
" plant organ " is the significant and tangible structure of plant and the part of differentiation, root for example, stem, leaf, bud, or embryo.
" plant tissue " used herein is meant one group of vegetable cell forming 26S Proteasome Structure and Function unit.In any tissue of plant in plant or the cultivation is included in.This term includes, but not limited to whole plants, plant organ, plant seed, tissue culture and any vegetable cell group of forming structure and/or functional unit.The associating of this term, or lack application above-mentioned or that otherwise be included in the plant tissue of any particular type in this definition and do not get rid of other plant tissue type arbitrarily.
" promotor " is the non-translation DNA sequence of upstream of coding region, and it contains the connection site of RNA polymerase II and starts transcribing of DNA.Promoter region can comprise that also other serves as the element of genetic expression regulon.
" protoplastis " is the isolating vegetable cell that does not have cell walls or the parts of fine cell wall is only arranged.
" controlling element " is meant and relates to the sequence that regulatory nucleotide sequence is expressed.Controlling element contains the promotor and the termination signal that effectively link to each other with the purpose nucleotide sequence.It also typically comprises the needed sequence of suitable translation of nucleotide sequence.
Broadest, term " similar basically " is when being used to refer to nucleotide sequence, be meant a nucleotide sequence corresponding to the nucleotide sequence of quoting, wherein the polypeptide of Dui Ying sequence encoding has identical 26S Proteasome Structure and Function with the nucleotide sequence coded polypeptide of being quoted basically, for example, only amino acid whose variation does not influence the function that produces polypeptide.Similar basically nucleotide sequence coded of ideal by the nucleotide sequence coded polypeptide of being quoted.The per-cent of the identity between similar basically nucleotide sequence and the nucleotide sequence of being quoted is desirably and is at least 80%, more desirably is at least 85%, is preferably at least 90%, more preferably is at least 95%, is more preferably at least 99%." similar basically " is listed in 50 ℃ of 7% sodium lauryl sulphate (SDS) in the nucleotides sequence of the nucleotide sequence of being quoted, 0.5 M NaPO 4, among the 1 mM EDTA, 50 ℃ with 2 * SSC, the 0.1%SDS washing is hybridized in the nucleotide sequence of being quoted, and is comparatively ideal in 50 ℃ of 7% sodium lauryl sulphate (SDS), 0.5 M NaPO 4, among the 1 mM EDTA, 50 ℃ with 1 * SSC, the 0.1%SDS washing is hybridized in the nucleotide sequence of being quoted, and is better in 50 ℃ of 7% sodium lauryl sulphate (SDS), 0.5 M NaPO 4, among the 1 mM EDTA, 50 ℃ with 0.5 * SSC, the 0.1%SDS washing is hybridized in the nucleotide sequence of being quoted, preferably in 50 ℃ of 7% sodium lauryl sulphate (SDS), 0.5 M NaPO 4, among the 1 mM EDTA, 50 ℃ with 0.1 * SSC, the 0.1%SDS washing is hybridized in the nucleotide sequence of being quoted, more preferably in 50 ℃ of 7% sodium lauryl sulphate (SDS), 0.5 M NaPO 4, among 1 mMEDTA, 65 ℃ with 0.1 * SSC, the 0.1%SDS washing is hybridized in the nucleotide sequence of being quoted.
" synthetic " is meant that nucleotide sequence contains the constitutional features that does not exist in native sequences.For example, artificial has closer the sequence that the similar normal codon of G+C content and dicotyledonous and/or unifacial leaf gene distributes and is called as synthetic.
" conversion " is that heterologous nucleic acids is imported host cell or biological process.Specifically, " conversion " refer to that the dna molecular stable integration is in the genome of purpose biology.
" conversion/transgenosis/reorganization " is meant host living beings, and for example bacterium or plant have been imported into the heterologous nucleic acids molecule.Nucleic acid molecule can be incorporated in host's the genome with being stabilized or nucleic acid molecule also can be used as extrachromosomal molecule and exists.This extrachromosomal molecule can self-replicating.Cell transformed, tissue, or plant is appreciated that the end product that not only comprises conversion process, and comprise its genetically modified filial generation." non-conversion ", " not genetically modified ", or " nonrecombinant " host is meant the biology of the wild-type that does not contain the heterologous nucleic acids molecule, for example, and bacterium or plant.
Nucleotide is shown by its base of the abbreviation of following standard:
VITAMIN B4 (A), cytosine(Cyt) (C), thymus pyrimidine [T], and guanine (G).Amino acid is shown by the abbreviation of following standard equally: L-Ala (Ala; A), arginine (Arg; R), l-asparagine (Asn; N), aspartic acid (Asp; D), halfcystine (Cys; C), glutamine (GIn; Q), L-glutamic acid (Glu; E), glycine (Gly; G), Histidine (His; H), Isoleucine (IIe; I), 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; And Xie Ansuan (Val Y); V).In addition, (Xaa; X) represent amino acid arbitrarily.
The concise and to the point description of the sequence in the sequence table
SEQ ID NO:1 is included in the sequence of the about 3.0 kb dna fragmentations among the Xenorhabdus nematophilus clone pCIB9369, and it contains following ORF in the specific nucleotide position:
Title plays connection terminals
orfi????569?????979
orf2????1045????2334
SEQ ID NO:2 encodes～the proteic sequence of 15 kDa for the orf1 that clones pCIB9369.
SEQ ID NO:3 encodes～the proteic sequence of 47.7 kDa juvenile hormone esterase samples for the orf2 that clones pCIB9369.
SEQ ID NO:4 is the sequence of the orf1 of Xenorhabdus nematophilus clone pCIB9381.
SEQ ID NO:5 is the sequence of the orf1 encoded protein of clone pCIB9381.
The dna sequence dna of the orf2 of SEQ ID NO:6 Xenorhabdus nematophilus clone pCIB9381.
SEQ ID NO:7 is the proteic sequence of juvenile hormone esterase sample of the orf2 coding of clone pCIB9381.
SEQ ID NO:8 is the dna sequence dna of the orf1 of Podbielniak Xenorhabdus clone pCIB9354.
SEQ ID NO:9 is the sequence of the orf1 encoded protein of clone pCIB9354.
SEQ ID NO:10 is the dna sequence dna of the orf2 of Podbielniak Xenorhabdus clone pCIB9354.
SEQ ID NO:11 is the proteic sequence of juvenile hormone esterase sample of the orf2 coding of pCIB9354.
The dna sequence dna of the orf1 of the luminous smooth rod bacterium clone pC1B9383-21 of SEQ ID NO:12.
SEQ ID NO:13 is the sequence of the orf1 encoded protein of clone pCIB9383-21.
SEQ ID NO:14 is the dna sequence dna of the orf2 of luminous smooth rod bacterium clone pCIB9383-2.
SEQ ID NO:15 is the sequence of the neotonin sample esterase protein of the orf2 coding of clone pCIB9383-21.
Preservation
Following material is according to the microbial preservation of the international endorsement that is used for the patented procedure purpose of budapest treaty regulation, be deposited in Agricultural Research Service, patent culture collection center (NRRL), 1815 northern university streets, Peoria, Illinois61604.All are awarded on the Patent right basis and will be removed by inalterable being limited in of preserved material availability.
Clone's registration number preservation date
PCIB9369 NRRL B-21883 1997, November 12
PCIB9354 NRRL B-30109 1999, February 25
PCIB9381 NRRL B-30110 1999, February 25
PCIB9383-21 NRRL B-30111 1999, February 25
It expresses the new nucleotide sequence that produces Pesticidal toxins
The present invention relates to express the nucleotide sequence that produces new toxin, and relate to the production and the use of the toxin of pest control.Nucleotide sequence is from Xenorhabdus nematophilus, and Podbielniak Xenorhabdus and luminous smooth rod bacterium are separated among the member of enterobacteria family.Xenorhabdus is the fungal component that Steinemema belongs to nematode.The polished rod Pseudomonas is the fungal component that Heterorhabditis belongs to nematode.Nematode is settled in the larva of insect, it is killed, and their offspring is a food with dead larva.In fact the insecticide activity is produced by symbiotic Xenorhabdus and polished rod Pseudomonas bacterium.The contriver is first people who isolates nucleotide sequence of the present invention.The expression of nucleotide sequence of the present invention has produced and can be used to prevent and treat for example toxin of small cabbage moth (diamondback moth) of lepidopterous insects.
Nucleic acid sequence identity of the present invention among the clone pCIB9369 is the dna fragmentation of about 3.0 kb, and its budapest treaty according to the patent preservation is NRRL B-21883 preservation with the registration number.The sequence of this dna fragmentation is present among the SEQ ID NO:1.Two open reading frames (ORF) are present among the SEQ ID NO:1 and (are respectively Nucleotide 569-979 and Nucleotide 1045-2334), and the coded prediction size is the albumen (being respectively SEQ ID NO:2 and 3) of 15 kDa and 47.7 kDa.These two ORF are arranged in the operon spline structure.Effectively mate and demonstrate between ORF#2 and the bacillus thuringiensis cry3A albumen 21% identity by using the UWGCG Blast and the Gap program pair of research with each ORF demonstration homologous known array not to demonstrate any and ORF#1, it is considered to inapparent in this area.The Gap analysis revealed and the juvenile hormone esterase associated protein to the ORF#2 encoded protein of pCIB9369 of being undertaken by Blast program have 30.6%AA identity and 44.1%AA similarity (GenBank registration number 2921553; Henikoff etc., PNAS USA 89:10915-10919 (1992)).Nucleotide sequence of the present invention also compares with the Xenorhabdus nematophilus sequence (WO 95/00647) of known coded insect-killing toxin toxb4, but does not find significant homology.3.0 the kb dna fragmentation also compares with the nucleotide sequence that is disclosed in WO98/08388.Use UWGCG Gap program, 22 sequences from each 60 Nucleotide (60-mers) of the 38.2 kb dna fragmentations that are described in WO 98/08388 are compared to the present invention's 3.0 kb dna fragmentations.A 60-mer nucleotide sequence that begins at base 1 place of 38.2 kb dna fragmentations and the nucleotide sequence of other 60-mers is positioned at the interval of nearly 2.0 kb.In 22 sequences each and complementary sequence thereof are detected.The highest per-cent of the identity between one of the present invention's 3.0 kb dna fragmentations and these 60-mers is 53%, and it is not significant homology.In addition, 5 of 38.2 kb sequences different dna fragmentations are detected by Southern engram analysis and 3.0 kb fragments of the present invention hybridization.There is not the male hybridization signal to occur.
PCIB9381, pCIB9354, and the nucleotides sequence of pCIB9383-21 be listed in each clone in also demonstrate two open reading frames.Among the nucleotide sequence of two ORF in pCIB9381 and pCIB9383-21 and the pCIB9369 higher homology arranged.Therefore, the ORF#2 albumen of the ORF#2 albumen of pCIB9381 and pCIB9383-21 and pCIB9369 is the same has substantially the same homology with the juvenile hormone esterase associated protein.The nucleotides sequence of the nucleotide sequence of the ORF#1 of pCIB9354 and the ORF#1 of pCIB9369 is shown 77% identity, and the nucleotides sequence of the ORF#2 of the nucleotide sequence of the ORF#2 of pCIB9354 and pCIB9369 is shown 79% identity.The ORF#2 albumen of pCIB9354 also has and juvenile hormone esterase-relevant proteic homology (29.2%AA identity and 42.2%AA similarity).
In a preferred embodiment, invention comprises one basically similar in appearance to the Nucleotide 569-979 of SEQ IDNO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and the nucleotide sequence of SEQ ID NO:14, it is expressed and produces a kind of Pesticidal toxins.The present invention also comprises a recombinant vectors that contains nucleotide sequence of the present invention.In this carrier, nucleotide sequence preferably is contained in the expression cassette that contains controlling element, and controlling element wherein is used for the expression at the nucleotide sequence of the host cell that can express nucleotide sequence.This controlling element contains promotor and termination signal usually and preferably contains permission by the effective element of translation of the polypeptide of nucleic acid sequence encoding of the present invention.The carrier that contains nucleotide sequence can duplicate in particular host cell usually, extrachromosomal molecule preferably, and therefore be used in amplification nucleotide sequence of the present invention in the host cell.In one embodiment, the host cell that is used for this carrier is a microorganism, and for example bacterium is E.coli especially.In another embodiment, the host cell of this recombinant vectors is endophyte or epiphyte.The preferred host cell of this carrier is an eukaryotic cell, yeast for example, vegetable cell, or insect cell.Vegetable cell for example maize cell is most preferred cell, and in another preferred embodiment, this carrier is virus vector and is used to the duplicating of nucleotide sequence in the specific host cell, for example in insect cell or the vegetable cell.Recombinant vectors also is used for transforming nucleotide sequence of the present invention to host cell, and nucleotide sequence is incorporated among the DNA of host cell with being stabilized thus.In one embodiment, host cell is a prokaryotic cell prokaryocyte.In a preferred embodiment, host cell is an eukaryotic cell, yeast cell for example, insect cell, or vegetable cell.In a most preferred embodiment, host cell is vegetable cell, for example maize cell.
Nucleotide of the present invention can be separated by the technology in the embodiment that describes below, or carry out PCR as fundamental construction PCR primer and separate with being described in sequence in the sequence table.For example, have SEQ ID NO:1 orf1 about first and last 20-25 continuous nucleotide sequence oligonucleotide (for example., the Nucleotide 569-588 of SEQ ID NO:1 and Nucleotide 957-976) can be used as the PCR primer and increase directly from the orf1 encoding sequence (the Nucleotide 569-976 of SEQ ID NO:1) of the bacterial strain (Xenorhabdus nematophilus strains A TCC 19061) of originating.Other gene order of the present invention similarly also can by with the end of the encoding sequence that is described in sequence table as the basis of PCR primer from separately source bacterial strain by pcr amplification.
In another preferred embodiment, Pesticidal toxins contains at least a by nucleotide sequence coded polypeptide of the present invention.Molecular weight according to Pesticidal toxins of the present invention records greater than 6000 by the size fractionation experiment.After handling with Proteinase K, in the insect biological assay, only observe the bioactive minimizing of atomic little desinsection, this shows that processing has resistance to Pesticidal toxins basically to Proteinase K.Pesticidal toxins is 22 ℃ or 4 ℃ of its insecticidal activities of 2 week of storage back maintenance.Still keeping its insecticidal activity by freeze-drying and after 22 ℃ of 2 weeks of storage.Pesticidal toxins still has activity at 60 ℃ of incubations after 5 minutes, but it has lost insecticidal activity at 100 ℃ or 80 ℃ of incubations after 5 minutes.
In further embodiment, nucleotide sequence of the present invention can be modified by introduce random mutation in known vitro recombination or DNA shuffling technology.This technology is described in people such as Stemmer., on Nature 370:389-391 (1994) and the US patent 5,605,793, it introduces the present invention as a reference.The sudden change copy of countless nucleotide sequences produces on the basis of nuclei originis nucleotide sequence of the present invention, and having improved characteristics for example increases insecticidal activity, enhanced stability, or the variant of the specificity of different target pests or scope is replied.This method comprises the double-stranded polynucleotide that form a mutagenesis from the double-stranded polynucleotide of the template that contains nucleotide sequence of the present invention, the double-stranded polynucleotide of template wherein are cut into the double-stranded random fragment of desired size, and comprise adding one or more strand or the double chain oligonucleotide step in the colony of the gained of double-stranded random fragment that wherein said oligonucleotide contains a zone and the allogenic zone with double-stranded template polynucleotide homogeny; The double-stranded random fragment that sex change produces becomes single-chain fragment with the mixture of oligonucleotide; Causing described single-chain fragment generated group body with polysaccharase incubation single-chain fragment under the condition of described homogeny regional annealing to form the segmental pairing of annealing, it is enough that described homogeny zone starts duplicating of another for member of a paired, has therefore formed the double-stranded polynucleotide of a mutagenesis; And repeat second and the further circulation of at least 2 of third steps, the mixture that wherein further second step of round-robin generates comprises that a round-robin the 3rd goes on foot the double-stranded polynucleotide of the mutagenesis that generates, and further circulation generates the double-stranded polynucleotide of the further mutagenesis of formation.In a preferred embodiment, single plant the concentration of double-stranded random fragment in the colony of double-stranded random fragment be less than total DNA heavy 1%.In a further embodiment, the double-stranded polynucleotide of template contain at least 100 kinds of polynucleotide, and in another preferred embodiment, the size of double-stranded random fragment is to 5 kb from 5 bp.In a further preferred embodiment, the 4th step of this method comprises repetition second and the 3rd step at least 10 circulations.
Nucleotides sequence is listed in the expression in the allos microorganism host
As biological insect control agent, the expression that Pesticidal toxins is listed in the heterologous host cell that can express Nucleotide by nucleotides sequence is produced.In first embodiment, in its karyomit(e), contain the Xenorhabdus nematophilus of the modification of at least one nucleotide sequence of the present invention, Podbielniak Xenorhabdus or luminous polished rod shape mycetocyte are described.These modifications comprise the sudden change or the disappearance of already present controlling element, and it causes the expression of the change of nucleotide sequence, or the insertion of the new controlling element of regulatory nucleotide sequence expression.In another embodiment, by being inserted in the karyomit(e) or containing the extrachromosomal replication molecule of nucleotide sequence by importing, the extra copy of one or more nucleotide sequences is added to Xenorhabdus nematophilus, the Podbielniak Xenorhabdus, or in the luminous smooth rod bacterium.
In another embodiment, at least one nucleotide sequence of the present invention is inserted in the suitable expression cassette that contains a promotor and termination signal.The expression of nucleotide sequence is a successive, or one starts the evoked promoter of transcribing to dissimilar stimulation responses and is used.In a preferred embodiment, the toxin cell of being expressed is a kind of microorganism therein, for example is a kind of virus, a kind of bacterium, or a kind of fungi.In a preferred embodiment, virus is baculovirus for example, contains a nucleotide sequence of the present invention and express a large amount of corresponding Pesticidal toxins in its genome after infecting the suitable eukaryotic cell that is suitable for virus replication and nucleotide sequence expression.Therefore Pesticidal toxins is produced as sterilant.Perhaps, the baculovirus that is contained nucleotide sequence by genetic modification is used in the body infected insect and by the expression of Pesticidal toxins or by uniting of virus infection and Pesticidal toxins it being killed.
Bacterial cell also can be the host that nucleotide sequence of the present invention is expressed.In a preferred embodiment, use non-pathogenic fungal component, it can be survived in plant tissue and duplicate, so-called endophyte, or non-pathogenic fungal component, and it can be settled in phyllosphere or rhizosphere, so-called epiphyte.This bacterium comprises Agrobacterium, Alcaligenes, Azospirillum, Azobacter, Bacillus, rod shape Bacillaceae, enterobacter, erwinia, Flavobacterium, Klebsiella, Rhodopseudomonas, rhizobium, serratia, streptomyces and Xanthomonas.The symbiosis fungi, for example Trichoderma and Gliocladium also are the possible hosts of the nucleotide sequence of the present invention expression that is used for identical purpose.
These Genetic Manipulative Technology are specific for different hosts and are known by those skilled in the art.For example expression vector pKK223-3 and pKK223-2 can be used to transcribing or translating in E.coli and merge the heterologous gene be expressed in tac or trc promotor back.For the expression of the operon of many ORF that encode, the simplest method is to insert operon for example among the pKK223-3, to allow the related ribosome bind site of heterologous gene to be used to carrier in transcribing fusion.Gram-positive microorganism for example the overexpression technology in the Bacillus also be well known in the art and can be used in the context of the present invention (Quax etc..: industrial microorganism: basis and use molecular genetics, Eds.Baltz etc., American Society forMicrobiology, Washington (1993)).The selectable system of overexpression for example depends on yeast vector and comprises and use Pichia, yeast belong and kluyveromyces spp (Sreekrishna,.: industrial microorganism: basis and application molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society for Microbiology, Washington (1993); Dequin ﹠amp; Barre, biotechnology 12:173-177 (1994); Vanden Berg etc., biotechnology 8:135-139 (1990)).
In another preferred embodiment, at least one nucleotide sequence that is described is administered into and is expressed among Pseudomonas fluorescens (Pseudomonasfluorescens) the bacterial strain CGA267356 (being described on EU 0 472 494 and the WO94/01561) with biological control characteristic, in another preferred embodiment, a nucleotide sequence of the present invention is administered among Pseudomonas aureofaciens (Pseudomonasaureofaciens) the bacterial strain 30-84 that also has the biological control characteristic.Expression in the allos biocontrol strains requires screening to be suitable for the carrier that duplicates and to select suitable promotor in selected host.The technology of expressing in gram-positive microorganism and Gram-negative bacteria and fungi is well known in the art.
Nucleotides sequence is listed in the expression in the plant tissue
In a specific embodiment preferred, a kind of Pesticidal toxins of the present invention at least is at a kind of comparatively high biological expression in vivo, for example in plant.In this case, the transgenic plant protection of having expressed the toxin of significant quantity itself is not subjected to the influence of insect.When insect begins with these transgenic plant is when food, and it has also absorbed the toxin of expressing.This will stop insect further nip plant tissue or may in addition injury or kill this insect.Nucleotide of the present invention is inserted in the expression cassette, in the genome that is incorporated into described plant that it is preferably stable.In another preferred embodiment, nucleotide sequence is included in the non-virulent self-replacation virus.Can be unifacial leaf or dicotyledons and include, but are not limited to corn by plant transformed of the present invention, wheat, barley, rye, sweet potato, beans, pea, witloof, lettuce, Caulis et Folium Brassicae capitatae, cauliflower, Cauliflower, radish, Radix Raphani, spinach, asparagus, onion, garlic, capsicum, celery, cucurbita plants, pumpkin, hemp, summer squash, apple, pears, quince, muskmelon, plum, cherry, peach, honey peach, apricot, strawberry, grape, the red certain kind of berries, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, Chinese sorghum, sugarcane, beet, Sunflower Receptacle, Semen Brassicae campestris, Semen astragali sinici, tobacco, Radix Dauci Sativae, cotton, clover, paddy rice, potato, eggplant, cucumber, Arabidopsis, and xylophyta for example Coniferales with fallen leaves purpose tree.
In a single day desired nucleotide sequence is transformed in the specific plant species, and it can be bred in this kind or move in other mutation mutually of the same race, particularly including the mutation of the commercialization of passing through conventional breeding technique.
Nucleotide sequence of the present invention is preferably expressed in transgenic plant, therefore causes the biosynthesizing of relevant toxin in transgenic plant.Like this, the transgenic plant that have the enhanced insect-resistant have been produced.In order to express in transgenic plant, nucleotide sequence of the present invention may need to modify and optimize.Although in many cases, from the not adorned gene of microorganism in plant with high level expression, the low expression in the transgenic plant may be because the nucleotide sequence with preferred codon of right and wrong in plant of microorganism.All biologies known in this field all have the certain preference that codon uses, and the codon of nucleotide sequence of the present invention can be changed meeting the plant preference, and keep the amino acid that is encoded.In addition, the high expression level in plant is about 35% available from GC content at least preferably, is preferably greater than 45%, and is preferred greater than 50%, and most preferred greater than 60% encoding sequence.It is owing to remove to stablize courier's ATTTA motif that microorganism nucleotides sequence with low GC content is listed in the plant bad expression, and the existence that may cause the AATAAA motif of unsuitable polyadenylation.Although preferred gene order may give full expression in monocotyledons and dicotyledons kind, sequence can be modified satisfies monocotyledons and dicotyledons specificity codon preference and GC content preference, and it is different (Murray etc. that these parameters are selected to be shown.Nucleic acids research.17:477-498(1989))。In addition, Nucleotide can be caused the existence of the undesired splice site that the courier is truncate by examination.Change Example in all nucleotide sequences that need such as above-mentioned change are by utilizing known site-directed mutagenesis, PCR, and utilization is described in disclosed patent application EP 0 385 962 (Monsanto), (Lubrizol produces with the technology of the synthetic gene construction of method on the WO 93/07278 (Ciba-Geigy) EP 0 359 472.
In order effectively to start translation, the sequence that is adjacent to initial methionine(Met) need be modified.For example, can be modified by containing in plant effectively known array.Joshi has proposed one suitable consensus of plant (NAR 15:6643-6653 (1987)) and Clontech has been proposed further consensus translation initiation codon (1993/1994 catalog, 210 pages).These consensuses are fit to use with nucleotide sequence of the present invention.This sequence is integrated in the construction that contains this nucleotide sequence, up to and comprise ATG (allowing second amino acid do not modified simultaneously), or selectively, up to and comprise ATG after GTC (have and modify the genetically modified second amino acid whose possibility).
Nucleotides sequence is listed in the promoters driven that the expression in the transgenic plant is worked in plant.The selection of promotor will be along with the requirement in expression time and place and target species and is changed.Therefore, nucleotide sequence of the present invention preferably in leaf, in fringe, inflorescence (for example. spike, panicle, cob, or the like.) in, in root, and/or express in the seedling.In many cases, yet, need the protection of anti-more than one type insect, and therefore to express be desired in multiple tissue.Although it is effectively and vice versa that many promotors from dicotyledons show in monocotyledons, ideal, the promotor of dicotyledons screened being used for expresses in dicotyledons, and monocotyledonous promotor screened being used for expresses in monocotyledons.Yet, without limits to the origin source of the promotor of being screened; As long as it is just enough effectively to drive the expression of nucleotide sequence in required cell.
Preferred group moulding expression promoter comprises coming promotor and the CaMV 35S and the 19S promotor of own coding Actin muscle or ubiquitin.Nucleotide sequence of the present invention can be expressed under the regulation and control of the promotor of chemically regulating and control.This makes Pesticidal toxins only can be synthesized when inducing chemical agent to handle when farm crop.The technology of preferred genetic expression chemical induction specifically describes on application EP 0 332 104 (Ciba-Geigy) and US patent 5,614,395.The promotor of preferred chemical induction is a tobacco PR-1a promotor.
A preferred class promotor is the wound induction type.The countless promotors that are described are expressed at wound site and are also being caused the sites of infection local expression of phytopathogen.Ideally, such promotor will be only effectively local in the site of infecting, and only be accumulated in by this mode Pesticidal toxins and to need the synthetic insecticide toxin to kill in the cell of invasion insect.The preferred promotor of this type comprises that those are by people such as Stanford.Mol.Gen.Genet.215:200-208 (1989), people such as Xu, molecular biology of plants (Plant Molec.Biol.) 22:573-588 (1993), people's vegetable cell 1:151-158 (1989) such as Logemann, Rohrmeier ﹠amp; Lehle, molecular biology of plants 22:783-792 (1993), people such as Firek, molecular biology of plants, 22:129-142 (1993), and people such as Warner, Plant be (1993) promotor of describing J.3:191-201.
Preferred tissue specific expression pattern comprises the chlorenchyma specificity, root-specific, stem specificity, and flower specific.Be suitable for comprising promotor and many promotors of from monocotyledons and dicotyledons, cloning of the gene that many adjustings are relevant with photosynthesis in the chlorenchyma expression promoter.Preferred promotor is corn PEPC promotor (the Hudspeth ﹠amp from phosphoric acid enol carboxylase gene; Grula, molecular biology of plants 12:579-589 (1989)).Preferred root-specific expression promoter is by de Framond (FEBS 290:103-106 (1991); EP 0 452 269 (Ciba-Geigy) describes.The US patent 5,625,136 (Ciba-Geigy) that is described in preferred stem specificity promoter goes up and startup corn trpA expression of gene.
Particularly preferred embodiment of the present invention is expressed the transgenic plant of at least one nucleotide sequence of the present invention for or root-specific mode preferred with root.Further preferred embodiment is for wound-induced or express the transgenic plant of nucleotide sequence in the mode of infection induced property.
Except screening suitable promotor, the construction of expressing Pesticidal toxins in plant needs a suitable transcription terminator, and its downstream in heterologous nucleotide sequence connects.Several this terminators can be obtained and be technician known (for example from the tml of CaMV, from the E9 of rbcS).Any known its has the got terminator of function can be used in the context of the present invention in plant.
Other numerous sequences can be integrated in the expression cassette described in the invention.These comprise and are shown sequence that strengthen to express for example intron sequences (for example: from Adh 1 and bronze 1) and virus leader sequence (for example from TMV, MCMV and AMV).
The targeted expression of nucleotide sequence of the present invention will be preferred to the different cells site in the plant.In some cases, the location in cytosol will be expected, yet in other cases, the location will be preferred in some subcellular organelles.Carry out the genetically modified Subcellular Localization of codase by technology well known in the art.Typically, coding is handled from the DNA of the purpose peptide of a known organoid target gene product and is merged upstream at nucleotide sequence.The target sequence of known many such chloroplast(id)s and be shown in the function of allos construction.The expression of nucleotide sequence of the present invention also target in the endoplasmic reticulum or the vacuole of host cell.The technology that reaches this purpose is known in the field.
The suitable carrier that is used for Plant Transformation is described in other place of this specification sheets.For the conversion of Agrobacterium mediation, binary vector or the carrier that carries at least one T-DNA edge sequence are suitable, yet for the direct gene administration, carrier is suitable and the linear DNA that only contains the purpose construction will be preferred arbitrarily.Under the situation of direct gene drug delivery, single DNA kind transforms or cotransformation can be used people such as (, biotechnology 4:1093-1096 (1986)) Schocher.For direct gene transforms and the conversion of Agrobacterium mediation, transform common (but optionally) with the mark that screens that can provide to microbiotic (kantlex, Totomycin or methotrexate) resistance or weedicide (basta) resistance.The embodiment of this mark is the Xin Meisu transphosphorylase, hygromix phosphotransferase, dihydrofolate reductase; phosphinothricin acetyl transferase; 2,2 dichloropropionic acid dehalogenase N-acetylhydroxylamine synthetic enzyme, the 5-enolpyruvyl-shikimate-phosphate synthase; aromatic base nitrile salt hydrolysis enzyme; proporphyrinogen oxidase, ethanoyl coenzyme A carboxylic acid, dihydropteroate synthase; chloramphenicol acetyltransferase, and GRD beta-glucuronidase.Yet the selection that is used for the selectable mark that maybe can screen of plant is not a key of the present invention.
Above-mentioned recombinant DNA can be imported in the vegetable cell by mode known in the field.It will be appreciated by those skilled in the art that the selection of method depends on the type of the purpose plant that is transformed.The method of suitable transformed plant cells comprise microinjection (people such as Crossway., biotechnology 4:320-334 (1986)), electroporation (people such as Riggs., Proc.Nat I .Acad.Sci.USA 83:5602-5606 (1986), the conversion of Agrobacterium mediation (people such as Hinchee., biotechnology 6:915-921 (1988); Also referring to, people such as Ish ida., Nature Biotechnol 14:745-750 (June 1996) is used for corn and transforms), directly transgenosis (people such as Paszkowski., EMBO J 3.2717-2722 (1984); People such as Hayashimoto, plant physiology .93:857-863 (1990) (paddy rice)), and utilize from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, the trajectory particle acceleration of the device that Delaware obtains (referring to, for example, people such as Sanford., U.S. patent 4,945, and 050; And people such as McCabe., biotechnology 6.923-926 (1988)). also referring to, people such as Weissinger, Annual Rev.Genet.22:421-477 (1988); People such as Sanford., particulate science and technology 5:27-37 91987) (onion); People such as Svab., Proc.Nat I .Acad.Sci.USA 87:8526-8530 (1990) (tobacco chloroplast); People such as Christou., plant physiology .87:671-674 (1988) (soya bean); People such as McCabe., biotechnology 6:923-926 (1988) (soya bean); People such as Klein., Proc.Nat I .Acad.Sci.USA, 85:4305-4309 (1988) (corn); People such as Klein, biotechnology 6:559-563 (1988) (corn); People such as Klein., plant physiology .91:440-444 (1988) (corn); People such as Fromm., biology/technology 8:833-839 (1990); And people such as Gordon-Kamm., vegetable cell 2:603-618 (1990) (corn); People such as Koziel., biotechnology 11:194-200 (1993) (corn); People such as Shimamoto, natural 338:274-277 (1989) (paddy rice); People such as Christou, biotechnology 9:957-962 (1991) (paddy rice); People such as Datta., biology/technology 8:736-740 (1990) (paddy rice); European patent application EP 0 332 581 (orchard grass and other Pooideae); People such as Vasil, biotechnology 11:1553-1558 (1993) (wheat); People such as Weeks., plant physiology 102:1077-1084 (1993) (wheat); People such as Wan, plant physiology .104:37-48 (1994) (barley); People such as Jahne., Theor.App I .GeneL 89:525-533 (1994) (barley); People such as Umbeck, biology/technology 5:263-266 (1987) (cotton); People such as Casas., Proc.NatL Acad.Sci.USA90:11212-11216 (Dec.1993) is (sorghum); People such as Somers, biotechnology 10:1589-1594 (Dec.1992) (oat); Torbert etc., Plant Cell Reports14:635-640 (1995) (oat); Week etc., Plant Physiol.102:1077-1084 (1993) (wheat); Chang etc., WO 94/13822 (wheat) and Nehra etc., ThePlant Journal 5.285-297 (1994) (wheat).A series of especially preferred embodiments that recombinant DNA molecules imported in the corn by microparticle bombardment can be at Koziel etc., Biotechnology 11:194-200 (1993), Hill etc., Euphytica85:119-123 (1995) and Koziel etc. find among the Annals of the New YorkAcademy of Sciences 792:164-171 (1996).Other preferred embodiment is the method as the protoplast transformation that is used for corn of EP 0 292 435.The available single DNA kind of the conversion of plant or many DNA kind carry out (for example. cotransformation) and these two kinds of technology be applicable to peroxidase coding sequence.
In another preferred embodiment, nucleotide sequence of the present invention is directly imported in the plastid genome.The major advantage that plastid transforms is that plastid can be expressed the regulation and control that do not have substantive bacterial gene of modifying and plastid can be expressed in single promotor usually and expressed multiple open reading frame down.The plastid transformation technology is described in U.S. patent No.5 widely, and 451,513,5,545,817 and 5,545,818, PCT application WO 95/16783 and at people such as McBride (1994) Proc.Nat I .Acad.Sci.USA 91, on the 7301-7305.The basic fundamental that the green grain of leaf transforms comprises that importing side joint clones in zone to the suitable target tissue of plastid DNA in a selection markers that links with goal gene, for example utilize biolistics or protoplast transformation (for example, the conversion of calcium chloride or PEG mediation).Therefore 1 to 1.5 kb flanking region is called target sequence, has promoted with the genomic homologous recombination of plastid and allows the replacement or the modification of the specific regions of plastom(e).At first, given the alternative mark (Svab that the point mutation of the resistance of spectinomycin and/or Streptomycin sulphate is used as conversion in chloroplast(id) 16S rRNA and rpsl 2 genes, Z., Hajdukiewicz, P., with Maliga, P. (1990) Proc.Nat I .Acad.Sci.USA 87,8526-8530; Staub, J.M., and Maliga, P. (1992) Plant Cell 4,39-45).It produces stable homogeneity transformant with per 100 frequencies that produced 1 in the target leaf that bombards approximately.The generation of the plastid targeting vector that the existence of cloning site permission foreign gene imports between these marks (Staub, J.M., with Maliga, P. (1993) EMBO J 12,601-606).By separate toxenzyme aminoglycoside-3 '-VITAMIN B4 transferring enzyme (Svab with the coding spectinomycin, Z., and Maliga, P. (1993) Proc.NatL Acad.Sci.USA 90, bacterium aadA gene dominant selectable marker 913-917) is replaced recessive rRNA or r-albumen antibiotics resistance gene, and transformation frequency has obtained significantly to improve.In the past, this mark was transformed by the genomic high frequency of the plastid that is used for green alga Chiamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) NucL Acids Res.19:4083-4089) of success.Other the selective marker that plastid transforms of being used for is well known in the art and within the scope of the present invention.Typically, need to transform about 15-20 the cell division cycle in back to reach a kind of homoplasmon state.Its gene is inserted into all by homologous recombination and is present in plastid in genomic several thousand copies of ring-type plastid in each vegetable cell and expresses the advantage of utilizing the huge copy number that surpasses the nuclear expression gene and allow expression level can surpass 10% of total soluble vegetable-protein easily.In a preferred embodiment, nucleotide sequence of the present invention is inserted in the plastid targeting vector and is transformed in the plastom of purpose plant host.The plant phenogen (homoplastic) of plastom that contains nucleotide sequence of the present invention is obtained, and deflection can efficiently express nucleotide sequence.
The preparation of insecticides
The present invention also comprises the composition that contains at least a Pesticidal toxins of the present invention.For the effectively preventing insect, this composition preferably contains the toxin of capacity.This tittle is along with protected farm crop, specificity purpose insect, envrionment conditions, humidity for example, temperature or soil type and change.In a preferred embodiment, the composition that contains Pesticidal toxins comprises the host cell of expressing the toxin that does not have additional purification.In another preferred embodiment, the cell of expressing Pesticidal toxins before it is used as sterilant by freeze-drying.In another embodiment, Pesticidal toxins is transformed from cell host and is secreted.Being purified at some toxin of expressing from host cell is under the desirable situation, and the purifying in various degree of Pesticidal toxins is reached.
The present invention further comprises the preparation of the composition that contains at least a Pesticidal toxins of the present invention, and it is evenly mixed with one or more compounds or compound group described herein.The present invention also relates to methods for the treatment of plants, it comprises Pesticidal toxins or the composition that contains Pesticidal toxins is applied to plant.Pesticidal toxins can be applied to crop plant area or be applied to by compound while or processed continuously plant with other by the form with composition.These compounds can be fertilizer or micro-nutrients donor or other the preparation that influences plant-growth.They also can be selective herbicides, sterilant, mycocide, sterilant, nematocides, molluscacide, or the mixture of several these preparations, if desired, with other the carrier of this area routine, tensio-active agent or use and promote adjuvant to unite use together.Suitable carriers and adjuvant can be the material of solid or liquid and the routine relevant with blending process, for example natural or regeneration mineral substance, solvent, dispersion agent, wetting agent, tackifier, tackiness agent or fertilizer.
The method that preferred Pesticidal toxins of the present invention is used is by for example soil to parasitic insect, water, or spray in the environment of the leaf of plant etc.Type and intensity that number of using and ratio depend on killed insect infection.Pesticidal toxins beats into the plant place with liquid composition and is penetrated in the plant by root via soil (systemic effect), or by with the solid form for example the compound of particulate form be applied to (soil application) in the soil.Pesticidal toxins can be by beating into seed (bag by) with the liquid preparation that contains Pesticidal toxins, or be applied to seed with the mode that solid preparation is applied to seed.Under special situation, the application of other type also is possible, and for example selectivity is handled stem or the bud of plant.Pesticidal toxins also can be provided as the soil that bait places upper strata or lower floor.
Pesticidal toxins use with the form of unmodified or, preferred, unite use with the adjuvant of the routine of this formulation art, and be made into emulsifiable concentrate in known manner, can wrap the paste of quilt, directly the solution that sprays or dilute dilutes emulsion, wettable powder, soluble powder, pulvis, granule, and also can wrap quilt, for example in polymkeric substance.As composition, the method for application is for example sprayed, efflorescence, and spreading, diffusion or cast are selected according to target of being planned and main environment.
The preparation that contains the adjuvant of Pesticidal toxins and solid in case of necessity or liquid, composition or preparation are prepared by known methods, for example by uniform mixing and/or grinding Pesticidal toxins and extender, for example solvent, solid carrier and suitable surfactant (tensio-active agent).
Suitable solvent comprises aromatic hydrocarbon, be preferably the fraction that contains 8 to 12 carbon atoms, for example, the naphthalene of xylene mixture or replacement, phthalic ester such as dibutyl phthalate or dioctyl phthalic ester, aliphatic hydrocarbon is hexanaphthene or paraffinic hydrocarbon for example, pure and mild ethylene glycol and ether thereof and ester, ethanol for example, ethene monomethyl ethylene glycol or monoethyl ether, ketone is pimelinketone for example, and intensive polar solvent is the N-N-methyl-2-2-pyrrolidone N-for example, dimethyl sulfoxide (DMSO) or dimethyl formamide, and epoxidized vegetable oil class for example epoxidation Oleum Cocois or soybean oil or water.
Used solid carrier for example is used for pulvis and dispersible pulvis, and normally natural mineral filler is calcite for example, talcum, kaolin, montmorillonite or attapulgite.In order to increase the performance of material, also may add the silicic acid of high dispersive or the absorbing polymeric of high dispersive.Suitable granular adsorption carrier is a multi-hole type, float stone for example, brickbat, sepiolite or soap clay; And suitable non-absorption carrier is for example calcite or sand.In addition, the particulate material of a large amount of inorganic or organic character can be used, for example, and particularly rhombspar or powdery plant residue.Suitable surface active cpd is to have good emulsification, non-ionic type, positively charged ion and/or the anion surfactant of dispersion and wetting property.Term " tensio-active agent " also can be regarded as and comprises surfactant mixtures.Suitable anion surfactant can be water miscible soap and water miscible synthetic surface active cpd.
Suitable soap is higher fatty (chains of 10 to 22 carbon atoms) an alkali metal salt, ammonium salt alkaline earth salt or non-replacement or that replace, for example sodium or the sylvite of oleic acid or the stearic acid natural acid mixture that maybe can from for example Oleum Cocois or animal oil, obtain.The fat methyl tauride also can be used.
Yet, more frequent, use so-called synthetic tensio-active agent, special fatty sulfonate, fat sulphate, sulphonated benzimidazole derivative or alkylaryl sulfomates.
Fat sulfonate or vitriol are generally an alkali metal salt, ammonium salt alkaline earth salt or non-replacement or that replace and the form of alkyl with moieties that comprises alkyl of 8 to 22 carbon, for example, lignosulfonic acid, the sodium salt of fatty alcohol sulphuric acid salt mixture dodecyl sulphate or that from natural acid, obtain or calcium salt.These compounds also comprise the salt of the sulfonic acid of the salt of sulfuric ester and Fatty Alcohol(C12-C14 and C12-C18)/ethylene oxide adduct.Sulphonated benzimidazole derivative preferably contains 2 sulfonic acid groups and one and contains the fatty acid-based of 8 to 22 carbon atoms.The embodiment of alkylaryl sulphonate is a Witco 1298 Soft Acid, dibutyl naphthene sulfonic acid, or the sodium of naphthene sulfonic acid/formaldehyde condensation products, calcium or triethanolamine salt.Also suitable is corresponding phosphoric acid salt for example has the salt of phosphoric acid ester of the p-nonylphenol affixture of 4 to 14 oxyethane.
Nonionogenic tenside is preferably aliphatics or ring-shaped fat alcohol, the polyglycol ether derivative of saturated or unsaturated lipid acid and alkylphenol, described derivative contain 3 to 30 ethylene glycol ether groups and 8 to 20 carbon atoms at (aliphatic) hydrocarbon part and 6 to the 18 carbon atoms moieties at alkylphenol.
Further suitable nonionogenic tenside is polyethylene oxide and polypropylene glycol, the water-soluble affixture of quadrol base propylene glycol and alkyl polypropylene glycol, contain 1 to 10 carbon atom in the alkyl chain of alkyl polypropylene glycol wherein, its addition compound contains 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol groups.These compounds contain 1 to 5 ethylene glycol unit in each propylene glycol unit usually.
The exemplary of nonionogenic tenside is the nonylphenol polyethoxyethanols, Viscotrol C polyglycol ether, polypropylene/poly-epoxy cycloalkanes affixture, tributyl phenoxy group polyethoxyethanols, polyoxyethylene glycol and Octylphenoxy ethoxy ethanol.The fatty acid ester of polyethenoxy sorbitan and poly-oxyethylene sorbitan trioleate also are suitable nonionogenic tensides.
Cats product is preferably quaternary ammonium salt, and it has, as the N-substituting group, at least one C8-C22 alkyl group with, as further substituting group, rudimentary non-replacement or halogenated alkyl, phenmethyl or hydroxyalkyl group such as low.Salt is preferably halogenide, the form of Methylsulfate or sulfovinate, for example, stearyl front three ammonium chloride or phenmethyl two (2-chloroethyl) ethyl brometo de amonio.
The normally used tensio-active agent of formulation art is described in, for example, " McCutcheon ' s Detergents and Emulsifiers Annual ", MCPublishing Corp.Ringwood, New Jersey 1979, and Sisely and Wood, " tensio-active agent encyclopedia " Chemical Publishing Co., Inc. New York, 1980.
Embodiment
The following by reference specific embodiment of the present invention further is described.These embodiment only are provided for the purpose explained, rather than are used for restriction, except as otherwise noted.The recombinant DNA of used here standard and molecule clone technology are well known in the art and by Ausubel (ed.), modern molecular biology method, John Wiley and Sons, Inc. (1994); T.Maniatis, E.F.Fritsch and J.Sambrook, molecular cloning: a kind of laboratory manual, cold spring harbor laboratory, cold spring port, NY (1989); And by T.J.Silhavy, M.L.Berman, and L.W.Enquist, Experiments with Gene Fusions, cold spring harbor laboratory, the cold spring port, New York (1984) are described.
A. express the separation that produces the active nucleotide sequence of anti-lepidopterous insects toxin
Embodiment 1: the growth of Xenorhabdus genus and Photobacterium bacterial strain
For the biological assay of insect, according to the growth medium that ATCC introduces, following bacterial strain in the nutrition broth culture 25 ℃ cultivated 3 days.For DNA separates, culture is cultivated 24 hr under identical condition.
Xenorhabdus nematophilus strains A TCC 19061
Xenorhabdus nematophilus bacterial strain Psi, a USDA isolate
Podbielniak Xenorhabdus strains A TCC 49122
Luminous polished rod shape bacteria strain Ps5, a USDA isolate
Embodiment 2: the biological assay of insect
Carry out small cabbage moth (Px) biological assay (Biever and Boldt, Annals of Entomological Society of America, 1971 by five equilibrium 50 each culture of Escherichia coli of μ l on artificial (P.xylostella) food of solid; Shelton waits people J.Ent.Sci 26:17).The food of 4ml is injected in the clean plastic cup of 1 ounce (oz.) (Bioserve product #9051).5 new lives' of the laboratory population that changes from food small cabbage moth is respectively placed in the cup that contains every kind of food and covers (Bioserve product #9049) with blank sheet of paper then.Every kind of concentration is tested with 10 larvas.The pallet of dress cup be placed in the thermostat container with 72 14:10 (hour) light: the dark cycle.Write down the larva number alive in each cup.
Embodiment 3: the result of biological assay
In the insect biological assay of embodiment 2, the substratum of Xenorhabdus nematophilus strains A TCC 19061 has produced 100% mortality ratio of anti-small cabbage moth (Px).Same, in the insect biological assay of embodiment 2, Xenorhabdus nematophilus bacterial strain Ps1, Podbielniak Xenorhabdus strains A TCC49122, and the bacteria culture medium of luminous polished rod shape bacteria strain Ps5 has produced 100% mortality ratio of anti-small cabbage moth (Px).
Embodiment 4: the structure of cosmid library
Be resuspended among 100 mM Tris pH, 8, the 10 mM EDTA cell of the new growth of 37 ℃ of Xenorhabdus nematophilus strains A TCC 19061 of 30 minutes and therefrom separate and obtain DNA by handling with the 2mg/ml N,O-Diacetylmuramidase.Proteinase K be added among the 0.5%SDS to final concentration be 100 μ g/ml and 45 ℃ of cultivations.The solution clarification and the very viscosity that becomes.SDS concentration is added to 1% and 300 mM NaCl and isopyknic phenol-chloroform-primary isoamyl alcohol is added into.Sample is mixed under 5 minutes and the 3K centrifugal lightly.Repeat twice.Water mixes with the Virahol of 0.7 volume then and is centrifugal.The DNA precipitation also leniently is resuspended among the 0.5X TE for 3 times with 70% washing with alcohol.The DNA of 6 μ g by with the Sau3A of every μ g DNA 0.3 unit in the volume of 100 μ l 37 ℃ handled 3.5 minutes.Sample was made enzyme deactivation in 30 minutes by 65 ℃ of heating then, used 37 ℃ of incubations of calf intestinal alkaline phosphatase 30 minutes of 2 units then.Sample mixes with isopyknic phenol-chloroform-primary isoamyl alcohol and is centrifugal.Water is removed and mixes also centrifugal with the Virahol of 0.7 volume.Throw out is resuspended among the 0.5X TE with 100ng/ml concentration.
By utilize BamH I cloning site as method as described in the supplier prepare the SuperCos cosmid vector (Stratagene, La Jolla, CA).The Supercos of 100 μ g/ml of preparation is by with connecting with Sau3A Xenorhabdus nematophilus DNA with 6 ℃ of digested overnight of ratio of 2: 1 in the volume of 5 μ l in advance.Connecting mixture utilizes Gigapack XL III (Stratagene) packaged as described in supplier.Packaged phage is as infected in XL-1 MR Bacillus coli cells (Stratagene) as described in the supplier.Cosmid library be added on the L-agar that contains 50 μ g/ml kantlex and 37 ℃ cultivated 16 hours.500 bacterium colonies are transferred on the fresh L-kan flat board, and density is 50/ flat board.LB substratum washed cell and mix with 20% glycerine and-80 ℃ freezing.
Under the big genomic situation of 4.2 Mb of Xenorhabdus nematophilus, mean size is that 450 clones of 40kb are equivalent to genomic 4 times.Therefore, 450 clones' screening will produce the possibility of 99% any gene of discovery.
From Xenorhabdus nematophilus bacterial strain Ps1, Podbielniak Xenorhabdus strains A TCC 49122, and the cosmid library of luminous polished rod shape bacteria strain Ps5 is established in the same way.
Embodiment 5: the result of clay biological assay and the evaluation with insecticidal activity clone
400 escherichia coli clonings from each cosmid library are screened by the having the active clone of anti-small cabbage moth of insect biological assay generation.Desinsection clay clone from Xenorhabdus nematophilus strains A TCC 19061 is accredited as pCIB9362.Desinsection clay clone from 42 kb of Xenorhabdus nematophilus bacterial strain Ps1 is accredited as pCIB9379.Desinsection clay clone from 42 kb of Podbielniak Xenorhabdus strains A TCC 49122 is accredited as pCIB9354.One is come the desinsection clay clone of 7 kb of luminous polished rod shape bacteria strain Ps5 to be accredited as pCIB9383-21.
Embodiment 6: the separation with subclone of insecticidal activity
Clone pCIB9362 is digested with the Sac II and is separated to one 9 kb dna fragmentation.This fragment is connected on the Bluescript that cuts with the Sac II.Connect mixture such as molecular cloning, second edition (Sambrook etc.) is described to be transformed in the DH5 α Bacillus coli cells.Transformation mixture is assisted on the L agar that has added 100 μ g/ml penbritins and 37 ℃ of incubated overnight.The bacterium colony that is separated to is grown in the L nutrient solution that has added 100 μ g/ml penbritins and by molecular cloning, the described alkaline micropreparation of second edition (Sambrook etc.) separates cosmid DNA.9 kb Sacl I clone is accredited as pCIB9362-3 and produces 100% mortality ratio when the anti-small cabbage moth of biological assay.The pCIB9362-3 of 3 μ g is separated, and every μ g DNA is with 37 ℃ of digestion of Sau3A 4,6 and 8 minutes of 0.3 unit and 75 ℃ of heating 15 minutes.Sample is merged, and is connected to earlier with handling among the pUC19 of BamH I digestion and with calf intestinal alkaline phosphatase.Linker is transformed into DH5 α Bacillus coli cells, is containing on the L agar of Xgal/Amp and 37 ℃ of incubated overnight as described the assisting of molecular cloning.Select white colony and cultivation on the L of the penbritin that contains 100 μ g/ml substratum, cosmid DNA is separated as mentioned above.DNA is checked order with EcoR I/Hind III digestion and new restriction spectrum.Sequencing primer fixed from Genosys Biotechnologies (Woodlands, Tx).Order-checking be undertaken by dideoxy chain termination and use Applied Biosystems Inc.377 type automated DNA sequenator (Foster City CA) finish.Sequence is by Gene Codes Corporation (Ann Arbor, Sequencher 3.0 assemblings Michgan).
After the evaluation of restriction site and potential ORF ' s, pCIB9362-3 is isolated and cloned among the Bluescript by the fragment with digestion of CIa I and one 3.0 kb and is transformed in the DH5 α Bacillus coli cells.Separated bacterium colony is grown as previously described and is passed through the basic process isolated plasmid dna.Subclone is accredited as pCIB9369 and has produced 100% anti-small cabbage moth mortality ratio in biological assay.PCIB9369 is by November 12 in 1997, preservation and be NRRL B-21883 at USDA ARS patent culture center preserving number.
A 20kb is accredited as the fragment of pCIB9381 by Not I digestion subclone from clay pCIB9379.
Embodiment 7: bioassay results
The insect biological assay of the pCIB9369 of different insects
Insect ???pCIB9369
Small cabbage moth Heliothis Virescens Helioverpa zea Spodoptera exguia C.elegans ????+++ ????na ????na ????na ????na
Na=does not have activity
+=significant growth-inhibiting
++=＞40% mortality ratio, but be less than 100%
+++100% mortality ratio
These results show that the Pesticidal toxins that the nucleotide sequence expression of the 3.0kb of pCIB9369 produces has the small cabbage moth resistance of height.
PCIB9381, pCIB9354, and the biological assay of pCIB9383-21 also shows the small cabbage moth resistance of height.
Embodiment 8: the size fractionation of insecticidal activity
Stratagene ' s Blue Script carrier among Xenorhabdus nematophilus clay clone pCIB9369 and the escherichia coli host DH5 is grown on the substratum of being made up of 50%Terrific meat soup and 50%Luria meat soup of the penbritin that has added 50 μ g/ml.Shake-flask culture thing (adorning 300ml in the 1000ml flask with indentation) 250 RPM cultivate, and 37 ℃ are spent the night.The culture of each bacterial strain is centrifugal at 4 ℃ of Sorvall GS-3 rotor 7,000 RPM.Sedimentation cell is resuspended in the 50mM NaCl of 30ml, and 25mM Tris alkali is among the pH 7.0.Spissated cell by use Branson450 type about eight circulations of 10 seconds of ultrasonoscope ultrasonication and between circulation in the cooled on ice ultrasonication.The product of ultrasonication is centrifugal 10 minutes with 4 ℃ of Sorvall SS34 rotor 6,000 RPM.The supernatant liquor that produces filters by one 0.2 μ strainer.Be resuspended in the 50mM NaCl of 30ml from the throw out of centrifugal ultrasonic wave product, 25mM Tris alkali is among the pH 7.0.
The filtrate of 3ml is applied to before using the 50mM NaCl of 10ml, and 25mM Tris alkali is in the pH 7.0 equilibrated Bio-Rad Econo-Pac 10 DG posts.The effluent liquid of collecting in the sample loading procedure is discarded.Add two kinds subsequently and respectively be the NaCl-Tris level pad fractionation sample of 4ml.Three kinds of fractions at first are saved for experiment.First fraction should contain all materials greater than about 6,000 mol.wt.Fraction afterwards should contain the material less than 6,000 mol.wt.
The throw out of resuspension after the sample of the filtrate of ultrasonication and the ultrasonication is with the activity that is used to together from three kinds of fractions of 10DG post be determined in the surface contamination test newborn small cabbage moth.The filtration supernatant liquor of ultrasonication thing and be high activity to small cabbage moth from the first post fraction of 9369 samples.Dividing from second and the third stage of 9369 samples is non-activity.The sample that carries the DH5a of Blue Script plasmid culture does not have activity.These results show the insecticidal activity of Xenorhabdus nematophilus (X.nematophilus) clone pCIB9369 coding with from pCIB9381, pCIB9354, and the homologue of pCIB9383-21 is greater than 6,000 molecular weight.
Embodiment 9: the stability of insecticidal activity
300ml has added the Luria substratum of penbritin and has been inoculated pCIB9369 and 37 ℃ of incubated overnight.Sample is placed in the spiral cover test tube of aseptic 15ml and 22 ℃ and 4 ℃ of preservations.Sample is by centrifugal and remove supernatant liquor, lyophilize and 22 ℃ of preservations.These samples are preserved the biological assay of carrying out anti-Px. 2 week then under these conditions.Cryodesiccated material is resuspended in the equal volume with previous freeze drying example.All samples by vortex by resuspension.Another kind of sample was handled 5 minutes for 100 ℃.
Handle The result
22 ℃ (2 week) ++
4 ℃ (2 week) ++
Lyophilize (2 week) ++
100 ℃ 5 minutes na
Na=does not have activity
Embodiment 10: the heat inactivation of insecticidal activity
The thermostability of toxin is detected.The overnight culture of coli strain pCIB9369 (escherichia coli host DH5a carries 3.0 kbDNA of Xenorhabdus nematophilus) is cultured on 50: 50 mixtures of Luria substratum and terrific substratum.37 ℃ of cultivations of culture in the culture tube are in the test tube cylinder.The sample of the 1ml of every kind of culture is placed in the eppendorf pipe of 1.5ml and places 60 ℃, 80 ℃.After 5 minutes, remove sample and cool to room temperature.This sample and the culture one of processed part not are used from the detection to small cabbage moth.The sample of 50 μ l is coated on the food, and dry and newborn diamondback moth larvae is applied to this surface.Test was at room temperature cultivated 5 days.
Untreated sample and cause 100% mortality ratio at the sample of 60 ℃ of processing.The sample of 80 ℃ of processing and the contrast of independent food do not cause observable mortality ratio.
Embodiment 11: the protease treatment of insecticidal activity
Stratagene ' s Blue Script carrier among Xenorhabdus nematophilus clay clone pCIB9369 and the escherichia coli host DH5 is cultured in and contains 50% Terrific substratum and 50%Luria substratum, has wherein added on the substratum of 50 μ g/ml penbritins.250 RPM shake bottle (adorning 300ml in the 1000ml flask with indentation) and cultivate, and 37 ℃ are spent the night.The culture of each bacterial strain in Sorvall GS-3 rotor 4 ℃ down 7,000RPM is centrifugal.The throw out cell is resuspended in the 50mM NaCl of 30ml, and 25mM Tris alkali is in the solution of pH 7.0..Spissated cell is by using Branson450 type about eight circulations of 10 seconds of ultrasonoscope ultrasonication and being broken in cooled on ice between circulation.The product of ultrasonication is centrifugal 10 minutes with 4 ℃ of Sorvall SS34 rotor 6,000 RPM.The supernatant liquor that produces filters by one 0.2 μ strainer.The supernatant samples of 1ml adds CaCl 2Adjust to 5mM Ca++.Add Proteinase K (Gibco BRL; Gaithersburg is MD) to 500 μ g/ml.Sample was cultivated 2 to 24 hours at 37 ℃.Prepare control sample and add Ca++ but do not add proteolytic enzyme and cultivate.
The pCIB9369 that cultivates under the situation that 5mM Ca++ exists and the ultrasonic wave filtrate of pCIB9369 sample have produced 100% lethality rate to the small cabbage moth newborn larvae.The pCIB9369 sample is cultivated with Proteinase K and Ca++ and is demonstrated less to Plutella, about 90% lethality rate.
Embodiment 12: with the sequence of the protein sequence of the protein sequence of cry3A and juvenile hormone esterase relatively
The nucleotides sequence of pCIB9369 (SEQ ID NO:1) is listed in Nucleotide 569-976 and 1045-2334 shows two open reading frames (ORF).ORF#1 does not find any homologous sequence in Genbank after with UWGCG Blast and Gap program search.Determined that by the Gap analysis that the Blast program is carried out the ORF#2 encoded protein of pCIB9369 itself and bacillus thuringiensis cry3A albumen have certain inapparent homology (21% identity), yet the Gap analysis of pCIB9369 ORF#2 (SEQ ID NO:3) encoded protein being carried out by the Blast program shows that really (the GenBank number of registration is 2921553 to its albumen relevant with juvenile hormone esterase; Henikoff etc., PNAS USA 89:10915-10919 (1992)) have a homology (30.6% AA identity and 44.1%AA similarity).
PCIB9381, pCIB9354 also demonstrates two open reading frames at each among these clones with the nucleotide sequence of pCIB9383-21.The nucleotide sequence of two ORF in each of pCIB9381 and pCIB9383-21 all surpasses 90% with the identity of those sequences in pCIB9369.Therefore, the same albumen relevant with juvenile hormone esterase with ORF#2 among the pCIB9369 of the ORF#2 albumen of pCIB9381 and pCIB9383-21 has identical homology basically.The nucleotides sequence of the nucleotide sequence of pCIB9354 ORF#1 and the ORF#1 of pCIB9369 is shown 77% identity, and the nucleotides sequence of the nucleotide sequence of pCIB9354 ORF#2 and pCIB9369 ORF#2 is shown 79% identity.The albumen that the ORF#2 albumen of pCIB9354 is also relevant with juvenile hormone esterase has homology (29.2%AA identity and 42.2%AA similarity).
Embodiment 13:pCIB9369 with from the sequence of the sequence of WO 98/08388 relatively
From its nucleotide sequence is described in 38.2 kb dna fragmentations on the WO 98/08388, obtain 22 wherein each sequence be the sequence of 60 Nucleotide (60-mers), and be compared to the nucleotide sequence of the pCIB9362-3 that contains pCIB9369.First 60-mer is from the base 1 of 38.2kb dna fragmentation, and other 60-mers being located at interval on the dna fragmentation with about 2kb.They are listed below the position on 38.2 kb dna fragmentations:
1-60；2,041-2,100；4,021-4,080；6,001-6,060；8,041-8,100；10,021-10,080；12,001-12,060；14,041-14,100；16,021-16,080；18,001-18,060；20,041-20,100；22,021-22,080；24,001-24,060；26,041-26,100；28,021-28,080；30,001-30,060；32,041-32,100；34,021-34,080；36,001-36,060；38,041-38,100；38,161-38,220.
It is detected to utilize UWGCG Gap program to carry out each and the complementary sequence thereof of sequence relatively and in 22 60-mer sequences.The correlated result's demonstration of these sequences is up to 53% identity, and it is considered to inapparent homology in this area.
Embodiment 14: use the probe that derives from WO 98/08388 sequence to carry out the Southern engram analysis
Oligonucleotide is disclosed in the dna fragmentation of 38.2 kb dna fragmentations on the WO 98/08388 to being designed to increase.Oligonucleotide order from Genosys Biotechnologies (TheWoodlands, Texas) and their position display in 38.2 kb dna fragmentations as follows.Their the segmental size of amplification PCR also is listed below:
VK1046: position 20-40
VK1047: position 2,078-2,100
Use VK1046 and the segmental size of VK1047 amplification PCR: 2,080 bp
VK1048: position 11,221-11,241
VK1049: position 13,360-13,380
Use VK1 048 and the segmental size of VK1049 amplification PCR: 2,120 bp
VK1050: position 26,581-26,601
VK1051: position 28,537-28,560
Use VK1050 and the segmental size of VK1051 amplification PCR: 1,979 bp
VK1052: position 18,901-18,921
VK1053: position 20,321-20,340
Use VK1052 and the segmental size of VK1053 amplification PCR: 1,439 bp
VK1054: position 34,261-34,281
VK1055: position 35,320-35,340 BP
Use VK1054 and the segmental size of VK1055 amplification PCR: 1,079bp
By using Perkin-Elmer 9600 Thermo-Cycler to finish the PCR reaction under the following conditions: 94 ℃, 2 minutes; 30 circulations then: 94 ℃ 30 seconds, 54 ℃, 30 seconds; 72 ℃, 4 minutes.Sample contains the Xenorhabdus nematophilus DNA of 800ng in 100 μ l final volume, each oligonucleotide of 0.1-0.5 μ M is right, 250 μ M dNTP, 5UTaq polysaccharase and 1x damping fluid (Perkin-Elmer).The reactant ethanol sedimentation of finishing is resuspended among the TE and places 1%SeaPlaque (FMC, Rockland is Maine) on the TBE gel.Behind electrophoresis, ethidium bromide staining and with the UV-light cutting fragment from the gel that develops then.Gel slice dissolves and mixes with μ l distilled water with 10 μ l aliquot sample at 65 ℃, boils 5 minutes.And place on ice.Then, and the random primer labelling damping fluid of 15 μ l (GIBCO-BRL, Gaithersburg, MD), and 6 μ l dNTP mixtures (no dCTP), 80 μ Ci α-dCT32P and 1 μ l Klenow are mixed.The reaction of mark was at room temperature carried out 60 minutes.Sample is cleared up (Pharmacia Biotech) on the Nick post by foundation supplier's specification sheets.Probe is boiled 5 minutes, and places on ice.
The total DNA of Xenorhabdus nematophilus by digestion derives from clay pCIB9362 and pCIB9363 (the overlapping dna fragmentation that surpasses 25 kb and contain pCIB9369 of these clays; PCIB9362 is used to subclone) DNA, the DNA that derives from subclone pCIB9362-3 (9 kb Sac II fragment) and pCIB9369 (2.96 kb Cla I fragment) carries out Southern hybridization, use the Cla I, Sac II or Hind III digest.The digestion reaction thing is added on the 0.75% agarose TBE gel and electrophoresis spends the night.Take pictures and gel is used for trace to Zeta-probe hybridization film as handling as described in the Bio-Rad.Behind the trace, film was by 80 ℃ of oven dry 30 minutes.Film is placed to 7%SDS then, 250 mM sodium phosphates, among the pH 7.2 and 67 ℃ cultivated 30 minutes.Add fresh solution and after equilibrating to 67 ℃, aforesaid radioactive probe is added into and allows hybridization to spend the night, film 2X SSC, and 67 ℃ of washings of 0.5%SDS 30 minutes then, at 0.5 * SSC, were washed 30 minutes down in 67 ℃ among the 0.5%SDS.Film is exposed on film 1 and 3 hour.Colour developing film and result show and do not hybridize in the DNA of clay of the present invention or the DNA of subclone from the PCR probe of WO 98/08388 sequence.Yet observe with Xenorhabdus nematophilus DNA the intensive hybridization signal is arranged.
These results have confirmed the result of sequence comparison and have shown that clone pCIB9369 is different with the nucleotide sequence described in the WO98/08388.
B. the expression of nucleotide sequence of the present invention in the allos microorganism host
The microorganism that is suitable for the heterogenous expression of nucleotide sequence of the present invention is all microorganisms that can be settled in plant or rhizosphere.Itself will be used to contact insect.These comprise for example Rhodopseudomonas of gram-negative micro-organism, and enterobacter and serratia, gram-positive microorganism be Bacillus and fungi trichoderma for example, Gliocladium, and cereuisiae fermentum.Especially preferred heterologous host is a Pseudomonas fluorescens, pseudomonas putida, pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantica, enterobacter cloacae, serratia marcescens, Bacillus subtillis, cured shape bacillus, Trichoderma viride, Trichodermaharizianum, Gilocladium virens, and yeast saccharomyces cerevisiae.
Embodiment 19: nucleotides sequence is listed in the expression in intestinal bacteria and other the gram negative bacterium
Many genes are expressed in allogenic mode in gram negative bacterium.Expression vector pKK223-3 (Pharmacia catalogue#27-4935-01) can be at expression in escherichia coli.This carrier have one by lac regulation and control and IPTG inductive intensive tac promotor (Brosius, people such as J.., Proc.Nat ' .Acad.Sd.USA 81).A large amount of expression systems is modified and is used for intestinal bacteria.Thermal induction expression vector pPL (Pharmacia#27-4946-01) uses stringent controlled phage promotor, and it allows proteic high level expression.But whether the lac promotor provide the mode promotor of another kind of expression or not high level as the tac promotor.Along with wide scope host's replicon is joined in the carrier of these expression systems, nucleotides sequence is listed in for example Rhodopseudomonas of tight relevant gram negative bacterium, enterobacteria, and the expression in serratia and the Erwinia is possible.For example, pLRKD211 (Kaiser ﹠amp; Kroos, Proc.Nat I .Acad.Sci.USA 81:5816-5820 (1984)) contains it and allow wide scope host's replicon ori T of in many gram negative bacteriums, duplicating.
In intestinal bacteria, the expression of IPTG derived need tac (being trp-lac) promotor.When this identical promotor (as broad host range plasmid PLRKD211) when being imported into Rhodopseudomonas, it is not for there being PTG inductive constitutive activity.This trp-lac promotor can be placed in arbitrarily the goal gene of expressing or operon in the tight relevant bacterium of Rhodopseudomonas or other front is used for the purpose of the constitutive expression of this gene.Therefore, the nucleotide sequence of expressing the generation Pesticidal toxins can be placed in the back of constitutive promoter, is administered into to have in the bacterium of settling down plant or rhizosphere characteristic, and transforming this microorganism is sterilant.Other possible promotor can be used to the constitutive expression of the nucleotide sequence of Gram-negative bacteria.These promotors comprise, for example from Rhodopseudomonas regulatory gene gafA and lemA (WO 94/01561) and Sa Shi pseudomonas IAA operon promotor (people such as Gaffney., J.Bactedol.172:5593-5601 (1990).
Embodiment 20: nucleotides sequence is listed in the expression in the gram positive bacterium
The heterogenous expression that nucleotides sequence is listed in the gram positive bacterium is the another kind of mode that produces Pesticidal toxins.The expression system that is used for Bacillus and streptomyces is characterized by fullest ground.Be displayed in Gram-positive aerophil and anerobe and the intestinal bacteria (people such as Trieu-Cuot, Nud AcidsRes 18:3660 (1990)) from the promotor of the erythromycin resistance gene (ermR) of streptococcus pneumoniae and have activity.Another is used in (Bibb, Mol Gen Ge net 199:26-36 (1985)) in the streptomyces cloning vector from thiostrepton gene antibiotics resistance promotor.Shuttle vectors pHT3101 also is suitable for expressing (Lereclus, FEMS Microbiol Lett 60:211-218 (1989)) in Bacillus.A significant advantage of this method is that many gram positive bacteriums produce the spore that can be used to prepare persistent pesticide.Bacillus and streptomyces kind are the aggressiveness settlers of soil.
Embodiment 21: nucleotides sequence is listed in the expression in the fungi
Trichodema harzianum and Gliocladium virens are shown biology regulation and control that different levels in the field is provided (US 5,165,928 and US 4,996,157, authorize Cornell Research Foundation).Expressing the nucleotide sequence that produces Pesticidal toxins for one can express in this fungi.This can finish by a large amount of modes well known in the art.The conversion that one of them protoplastis that is fungi is undertaken by the technology of PEG or electroporation mediation mediates.Selectively, microparticle bombardment can be used to transform and have the regenerate protoplastis of ripe structural capacity or other fungal cell.Originally by development be used for Aspergillus transform be widely used in now fungi conversion carrier pAN7-1 (Curragh etc., Mycol.Res.97 (3): 313-317 (1992); Tooley etc., Curr.Genet.21:55-60 (1992); Punt etc., Gene 56:117-124 (1987)) by the engineered nucleotide sequence that contains.This plasmid contains the hygromycin B resistant gene (Punt etc., Gene 56:117-124 (1987)) of intestinal bacteria side joint in Aspergillus nidulans gpd promotor and trpC terminator.
In a preferred embodiment, nucleotides sequence of the present invention is listed in the yeast saccharomyces cerevisiae and expresses.For example, from pCIB9369, pCIB9381, pCIB9354, or among two ORF ' s of pCIB9383 each is cloned in each carrier that has GALl inducible promoter and CYCl terminator.Each carrier has amicillin resistance and 2 microns replicons.Carrier preferably has the zymic of being different from growth mark.Construction independently and together is transformed in the yeast saccharomyces cerevisiae.ORF is expressed and is used for detecting protein expression and insecticidal activity together.
C. the preparation of Pesticidal toxins
The activeconstituents that use contains separative toxin or described toxigenic cell suspending liquid of the foregoing description or concentrated solution makes pesticide preparation.The intestinal bacteria of for example expressing Pesticidal toxins can be used to pest control.Preparation is made and is described as follows with liquid or solid-state form.
Embodiment 18: the liquid formulation of insecticide ingredient
In the following embodiments, the percentage of composition represents 1. emulsible concentrates with weight: a b c active component 20% 40% 50% calcium dodecyl benzene sulfonates 5% 8% 6% castor oil polyglycol ethers 5%--(36 moles oxirane) tributyl phenol polyglycol ether-12% 4% (30 moles the oxirane) cyclohexanone-15% 20% xylene mixture 70% 25% 20% arbitrarily emulsion of desired concn can produce by this concentrate of dilute with water. 2. solution: a b c d active component 80% 10% 5% 95% ethylene glycol monomethyl ether 20%---PEG400-70%--METHYLPYRROLIDONE-20%--epoxidation coconut oil--1% 5% oil distillate--94%-(160～190 ° of boiling ranges)
These solution are fit to use with the form of droplet.3. the silicic acid 1%-Attapulgit-90% of granule a b active ingredient 5% 10% kaolin 94%-high dispersing
Active composition is dissolved in the methylene dichloride, and solution is injected on the carrier, and solvent is evaporated subsequently in a vacuum.4. pulvis: the silicic acid of a b active ingredient 2% 5% high dispersing 1% 5% talcum 97%-kaolin-90%
By mixed carrier and active ingredient have obtained the i.e. pulvis of usefulness up hill and dale.
Embodiment 19: the solid formulation of insect-killing composition
In the following embodiments, the percentage of composition represents 1. wettable powders with weight: the silicic acid of a b c active component 20% 60% 75% sodium lignosulfonate 5% 5%-lauryl sodium sulfate 3%-5% 2 isobutyl sodium naphthalene sulfonates-6% 10% octylphenol polyethylene glycol ether (epoxy of 7-8 mole-2%-ethane) high degree of dispersion 5% 27% 10% kaolin 67%--
Activeconstituents mixes up hill and dale with auxiliary agent and mixture is ground in suitable runner milling completely, makes wettable powder, can be produced the suspension of desired concn by dilute with water.2. emulsifiable concentrate: activeconstituents 10% octylphenol polyethylene glycol ether, (oxyethane of 4-5 mole) 3% calcium dodecylbenzene sulphonate 3% Viscotrol C polyglycol ether, (36 moles oxyethane) 4% pimelinketone 30% xylene mixture 50%
The emulsion of desired concn can produce by this concentrated solution of dilute with water arbitrarily.3. pulvis: a b active ingredient 5% 8% talcum 95%-kaolin-92%
By mixed active composition and carrier and in a suitable runner milling, grind, obtained the i.e. pulvis of usefulness.4. extruding granule: activeconstituents 10% sodium lignosulfonate 2% carboxymethyl cellulose 1% kaolin 87%
Active ingredient is mixed with auxiliary agent and is ground, and it is moistening that mixture is followed water.Mixture is extruded dry in air-flow then.5. wrap by particle: activeconstituents 3% Macrogol 200 3% kaolin 94%
The activeconstituents of fine grinding is applied in stirrer uniformly with in the moistening kaolin of polyoxyethylene glycol.Obtain non-powdered bag by particle by this mode.6. suspending concentrate: silicone oil 0.8% water 32% of active component 40% ethylene glycol 10% nonylphenol polyethylene glycol (15 moles oxirane) 6% sodium lignosulfonate 10% carboxymethyl cellulose 10%37% aqueous formaldehyde solution 0.2%75% aqueous emulsion
The activeconstituents of fine grinding is mixed with auxiliary agent completely, produces a suspending concentrate, from then on can obtain required arbitrarily concentration suspension by dilute with water.
Above-mentioned pesticide preparation is applied to plant by well known to a person skilled in the art technology with the amount that insect is prevented and treated by Pesticidal toxins.
D. nucleotides sequence is listed in the expression in the transgenic plant
The described nucleotide sequence of the application can be introduced in the vegetable cell by the recombinant DNA technology of routine.Normally, this relates to that to insert encoding sequence to of the present invention its encoding sequence by standard cloning process well known in the art be in the allos expression system of (that is, not existing usually).Carrier contain insertion albumen coded sequence transcribe and translate essential element.Well known to a person skilled in the art that a large amount of expression systems can be used, for example, plasmid, phage virus and other adorned virus.Suitable carriers includes, but not limited to for example λ carrier system λ gtl1 of virus vector, λ gtl0 and Charon carrier 4; Plasmid vector is pB1121 for example, pBR322, and pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pR1 (290, pKC37, pKC101, pCDNA II; And other similar system.The element of expression system can be modified increases expression.For example truncate sequence, Nucleotide is replaced or other modification can be used.Expression system described herein can be used for transforming in fact any crop plants cell under appropriate condition.Cell transformed regeneration whole plants, nucleotide sequence of the present invention has so just been given the transgenic plant insect-resistant.
Embodiment 22: the modification of encoding sequence and flanking sequence
The described nucleotide sequence of the application can be used for expressing transgenic plant by modification.Expressing host plant nucleotide sequence and produce Pesticidal toxins in its cell has strengthened the resistance of attack of insect and has therefore reduced the losses of the farm crop that cause because of attack of insect preferably.
The transgene expression of microbe-derived gene in plant may need the modification of these genes to obtain and the expression of optimization in plant.Especially, coding independently but the bacterium ORF of the enzyme of in natural microbial, being encoded by identical transcription be preferably in the plant and independently express on the transcription.In order to reach above-mentioned target, the ORF of each microorganism is separated respectively and clone in box, and box wherein provides a plant promoter sequence and provides a plant transcription terminator at the 3 ' end of ORF at the 5 ' end of ORF.Isolating ORF sequence preference comprises initial ATG codon and stops the STOP codon, but may comprise other sequence outside initial ATG codon and STOP codon.In addition, ORF can be by brachymemma, but still keeps required activity; For special long ORF, still the clipped form of retentive activity can preferentially be expressed in transgenic organism." plant promoter " and " plant transcription terminator " refers to effective promotor and transcription terminator in vegetable cell.It comprises the promotor and the transcription terminator that can obtain from for example virus (example is a cauliflower mosaic virus) in non-plant source.
In some cases, do not need ORF encoding sequence and flanking sequence thereof are modified.It is just enough with the downstream that is inserted into plant promoter to separate the fragment that contains purpose ORF.For example, people such as Gaffney (Science 261:754-756 (1993)) in transgenic plant under the regulation and control of CaMV 35S promoter and CaMV tml terminator successful expression Rhodopseudomonas nahG gene, and do not have the modification of encoding sequence and the Rhodopseudomonas gene xbp of ATG upstream and the downstream ybp of STOP codon still to link to each other with nahG ORF.The sequence of preferred as little adjacent microorganism will be left on the upstream of ATG and the downstream of STOP codon and link to each other.In fact, this construction depends on the availability of restriction site.
In other cases, in expression, can produce some problems from microbe-derived genetic expression.These problems have fully been described in this area and particularly for example the gene of Bacillus is common from certain source.These problems can be applied to the modification of nucleotide sequence of the present invention and these genes and can be undertaken by well known to a person skilled in the art technology.Following problem will be run into.
1. codon is selected
The codon that preferred codon in the plant is selected to be different from specific microorganism is selected.The codon of cloned microorganism ORF is selected relatively can identify codon among the preferred reformed ORF with the codon of plant gene (and from target plant specific gene) is selected.Typically, plant evolution is tending towards using Nucleotide C and G strongly in monocotyledonous the 3rd base position, yet dicotyledons is used Nucleotide A and T in this position usually.Introduce for the preferred codon selection of specific purpose transformed variety by modifying a gene, the many following GC/AT content and the difficult problem of undesired montage will be overcome.
2.GC/AT content
Plant gene typically contains the GC content greater than 35%.The ORF sequence that is rich in A and T Nucleotide can cause some difficult problems in plant.At first, it is believed that the ATTTA primitive causes courier's destabilization and is found at the 3 ' end of many short-lived mRNA.The second, it is believed that polyadenylation signal for example the generation in the suitable site of AATAA in the courier cause blocking too early of transcribing.In addition, monocotyledons can to discern the sequence that is rich in AT be splice site (face as follows).
3. the sequence adjacent with initial methionine
Plant is different from the microorganism part and is that its courier does not have definite ribosome binding site.On the contrary, it is believed that rrna is connected in courier's 5 ' end and scans the ATG that first can get, and begins translation herein.Yet, it is believed that some Nucleotide preferentially can be enhanced by the introducing of consensus translation initiation codon at the ATG place of an eucaryon adjacent to the expression of ATG and microbial gene.Clontech (1993/1994 catalog, is hereby incorporated by by the 210th page) has proposed a sequence and has been used for the expression of intestinal bacteria uidA gene plant as a consensus translation initiation codon.In addition, Joshi (NAR L5:6643-6653 (1987) is hereby incorporated by) compared many adjacent to ATG the plant sequence and other consensus sequence has been proposed.Run under the situation of the difficult problem in the expression of microorganism ORF in plant, in these sequences one can improve translation in comprising of initial ATG place.In the case, last three Nucleotide of consensus may be unsuitable for being included in the sequence of modification, and this is because it modifies second AA residue.Preferably the sequence adjacent to initial methionine may be different in different floristics.Research to 14 corn genes in the GenBank database has obtained following result:
Position before the initial ATG of 14 corn genes:
-10???-9???-8???-7???-6???-5???-4???-3????-2????-1
C????3????8????4????6????2????5????6????0????10????7
T????3????0????3????4????3????2????1????1????1?????0
A????2????3????1????4????3????2????3????7????2?????3
G????6????3????6????0????6????5????4????6????1?????5
Being used for the floristic this analysis that this desired nucleotide sequence is introduced into can be carried out, and the sequence that is adjacent to ATG is modified and introduced preferred Nucleotide.
4. the removal of undesired splice site.
It is also cut that clone from the non-plant source and not optimised gene of expressing plant also may contain the primitive that is identified as 5 ' or 3 ' splice site in plant, therefore produces courier brachymemma or disappearance.These sites can be removed by technology well known in the art.
The modification technique of encoding sequence and flanking sequence is well known in the art.The initial expression of microorganism ORF very low and its be considered to be fit to produce under the change situation of sequence as mentioned above, then the structure of synthetic gene can be done by means commonly known in the art.These for example, are described in disclosed patent EP 0 385 962 (Monsanto), and among EP 0 359 472 (Lubrizol) and the WO 93/07278 (Ciba-Geigy), all these are hereby incorporated by.Under most situation, be preferred by using transient analysis method (it is being known in the art) expression of analyzing gene construction before it changes transgenic plant over to.
Embodiment 23: the structure of expression of plants box
The encoding sequence that is intended for use in expressing in transgenic plant at first is assembled in the back of the suitable promotor of expressing in the expression cassette in plant.Expression cassette also can contain any other needs or select to be used for the sequence of transgene expression.This sequence includes, but not limited to transcription terminator, and the additional sequence that enhancing is expressed is intron for example, viable sequence, and be used for the sequence of gene product to the targetting of specific organoid and cellular compartment.These expression cassettes can be administered into the plant conversion carrier that is described below then easily.Following is the description of the difference composition of typical expression cassette.
1. promotor
The selection of used promotor will determine the expression pattern of genetically modified room and time in the transgenic plant in the expression cassette.The promotor of selecting will be at the specific cell type (cell of leaf epidermis for example, mesophyll cell, the root tegumental cell) or at specific tissue or organ (for example, root, leaf, or flower) in express transgenic and select the place will reflect that desired gene product accumulates.Selectively, selected promotor drives expression of gene under different inductive conditions.It is different that promotor for example starts in its intensity on the ability of transcribing.Depend on the host cell systems of being utilized, any one suitable promotor can be used, and comprises the natural promoter of gene.Be the non-limiting instance that can be used on the promotor in the expression cassette below.
A. constitutive expression, the ubiquitin promotor:
Ubiqutin be a kind of gene product known in many cell types accumulation and its promotor be used for transgenic plant (as Plant Science 79:87-94 (1991) such as Sunflower Receptacle-Binet by clone from some are planted; People .PlantMolec.Biol.12:619-632 (1989) such as corn-Christensen; And people such as Arabidopsis-Norris., Plant MoL BIoL 21:895-906 (1993)).Corn ubiqutin promotor is modified in transgenosis monocotyledons system and it is used for the monocotyledons conversion sequence and carrier are disclosed in patent application EP 0 342 926 (to Lubrizol), and it is closed by reference at this.People such as Taylor (Plant Cell Rep.12:491-495 (1993)) described a carrier (pAHC25) its contain corn ubiqutin promotor and first intron with and when bombardment imports through micropartical the high reactivity in a lot of monocotyledonous cell suspending liquids.Arabidopsis ubiqutin promotor is used for nucleotide sequence of the present invention ideally.The ubiqutin promotor is suitable for transgenic plant, the genetic expression in monocotyledons and the dicotyledons.Suitable carriers be the derivative of pAHC25 or passing through of describing in this application arbitrarily import that suitable ubiqutin promotor and/or intron sequences modify conversion carrier.
B. constitutive expression, the CaMV 35S promoter
The structure of plasmid pCGN1761 is described in disclosed patent application EP 0 392 225 (embodiment 23), and it is hereby incorporated by.PCGN1761 contains " two " CaMV 35S promoter and tml transcription terminator, has the EcoR I site of a uniqueness and have a pUC type main chain between promotor and terminator.The derivative of pCGN1761 be fabricated have a modification comprise the Notl site except that the EcoR I site that exists and the polylinker in Xhol site.This derivative is designed to pCGN1761ENX.The clone who is used for the expression purpose under the regulation and control of transgenic plant 35S promoter in its polylinker is useful to pCGN1761ENX for cDNA sequence or encoding sequence (comprising microorganism ORF sequence).Whole 35S promoter-the encoding sequence of this construction-tml terminator can be by the Hind III, the Sph I, Sall is in the excision from 5 ' site to promotor, and Xba I, BamH I and Bg II are administered into conversion carrier as described below excising from 3 ' site to terminator.In addition, two 35S promoter fragments can be removed by using the Hind III, the Sph I, Sall, the Xba I, or the 5 ' excision of Pst I and arbitrarily polylinker restriction site (EcoR I, Not I or Xho I) with other promotor replacement.If desired, the modification around the cloning site can produce by importing the sequence that can strengthen translation.Served as scale and reached when being supposed to, this is particularly useful.For example, pCGN1761ENX can be modified by the optimization of translation initiation site, and as the US patent No.5 among the embodiment 37,639,949 is described, and it is incorporated herein by reference.
C. constitutive expression, actin promoter
So some isomeric form of Actin muscle are known in most cell type, express and therefore Actin muscle be the good selection of constitutive promoter.Specific, cloned and identified (people Plant Cell 2:163-171 (1990) such as McElroy) from the promotor of paddy rice Act I gene.The fragment of a 1.3kb of promotor is found and contains all controlling elements of expressing needs at rice protoplast.In addition, countless based on the AcTl promotor specific for the expression vector on basis is fabricated be used for monocotyledons people Mol.Gen.Genet232:150-160 (1991) such as () McElroy.These comprise the Acti-intron, Adhl 5 ' flanking sequence and Adhl-intron (from the maize alcohol dehydrogenase gene) and from the sequence of CaMV 35S promoter.The carrier that demonstrates high expression level is the fusions of 35S and Acti-intron or Adhl 5 ' flanking sequence and Adhl-intron.The optimization of initiator codon ATG (GUS reporter gene) sequence has on every side also strengthened expression.The promoter expression cassettes that people such as McElroy (Mol.Gen.Genet.231:150-160 (1991)) describe can be used for genetic expression and be particularly suitable for the monocotyledons host by modification easily.For example, the fragment that contains promotor is removed and is used for replacing two 35S promoters of pCGNl761 ENX from the McElroy construction, and it is effective to the insertion of specific gene sequence.Fusion gene so is fabricated and is administered into then in the suitable conversion carrier.At one independently in the report, the paddy rice Act I promotor of carrying its first intron also has been found in and instructs in the barley cell of plantation and efficiently express people .Plant Cell Rep.12:506-509 (1993) such as () Chibbar.
D. inducible expression, the PR-1 promotor:
Two 35S promoters among the pCGN1761ENX can be replaced by the promotor that any other meeting produces suitable high expression level.For example, one is described in US patent No.5, replaceable pair of 35S promoter of promotor of the chemical regulation on 614,395.Selected promotor preferably being limited property enzyme is cut from its source, carries the primer of suitable termination restriction site by pcr amplification but selectively utilize.If PCR-amplification will be carried out, then promotor should be after the clone of promotor in targeting vector of amplification the quilt preface of being resurveyed check the amplification mistake.Chemistry/pathogenic agent regulation and control tobacco PR-1a promotor separated from plasmid pCIB1004 (be used for making up, referring to the embodiment 21 of EP 0,332 104, it is hereby incorporated by) and be administered into plasmid pCGN1761ENX (people such as Uknes., 1992) in.PCIB1004 is passed through to handle rust with the T4 archaeal dna polymerase with 3 ' overhang of the linear fragment of cutting of Nco I and gained.Then fragment with Hind III cutting and the fragment that contains the PR-1a promotor that produces by gel-purified and be cloned among the removed pCGN1761 ENX of its 35S promoter.This finishes by following step: with Xho1 cutting and with the passivation of T4 polysaccharase, then cut with the Hind III and separation pCIB1004 promotor is cloned into contains more greatly-fragment of carrier terminator.This has produced and has had PR-1a promotor and tml terminator and one and have the unique EcoR I and the pCGN1761ENX derivative that interleaves polylinker in Not I site.Selected encoding sequence can be inserted in this carrier, and the product (being promotor-gene-terminator) that merges can be acted as in the selected conversion carrier of meaning by administration subsequently, comprises that those are described below.Different chemical regulators can be used to induce selected encoding sequence by the expression in the plant transformed of the present invention, comprises to be disclosed in US patent No.5 the diazosulfide on 523,311 and 5,614,395, Yi Yansuan, and salicylic acid compound.
E. inducible expression, ethanol-inducible promoter:
By specific alcohol or ketone, for example alcohol induced promotor can be used to give encoding sequence of the present invention derivable expression.This kind promotor is for example from the alcA gene promoter (people (1998) Nat.Biotechnol16:177-180 such as Caddick) of Aspergillus nidulans.In A.nidulans, alcA genes encoding ethanol dehydrogenase, it is expressed under the existence of chemical inducer to be regulated and control by the AIcR transcription factor.In order to reach purpose of the present invention, the CAT encoding sequence in containing the plasmid palcA:CAT that is fused to the alcA gene promoter sequence on minimum 35S promoter people (1998) Nat.Biotechnol 16:177-180 such as () Caddick is replaced by an encoding sequence of the present invention and is formed an expression cassette with the encoding sequence under the regulation and control of alcA gene promoter.This is to carry out by means commonly known in the art.
F. inducible expression, the glucocorticoid inducible promoter:
Utilization of the present invention is considered based on the inducing also of expression of the nucleotide sequence of the system of steroid hormone.For example, the application inducible gene expression that the inducible system of glucocorticoid mediation is used (Aoyama and Chua (1997) The Plant Journal 11:605-612) and passes through glucocorticoid, for example, a synthetic glucocorticoid, dexamethasone preferably, preferred concentration be 0.1mM to 1mM, more preferably arrive 100mM for 10mM.In order to reach purpose of the present invention, luciferase gene sequence is replaced by one section nucleotide sequence of the present invention and is produced an expression cassette with nucleotide sequence of the present invention, and it is under the regulation and control of the activation sequence upstream GAL4 that is fused to the 35S minimal promoter of 6 copies.This carries out by means commonly known in the art.Trans-acting factor contains the GAL4 DNA-land (people (1986) Science 231:699-704 such as Keegan) that is fused to scar exanthema virus protein VP16 (people (1988) Genes Devel.2:718-729 such as Triezenberg) trans-activation district, and protein herpesvirus matter VP16 wherein is fused to the hormone binding domain of mouse glucocorticoid acceptor (people (1988) Cell54:1073-1080 such as Picard).Any promotor that Expression of Fusion Protein is applied to express in the plant known in the art or described herein is regulated and control.This expression cassette is also contained in the plant of the nucleotide sequence that contains the 6xGAL4/ of being fused to minimal promoter of the present invention.Therefore, obtained derivable tissue or the organ specificity that produces Pesticidal toxins of the tissue of fusion rotein or organ specificity.
G. root-specific is expressed:
The pattern of another kind of genetic expression is that root is expressed.Suitable root promotor is described (FEBS 290:103-106 (1991)) by deFramond and is described on the disclosed patent application EP 0 452 269, and it is hereby incorporated by.This promotor is transferred in the suitable for example pCGN1761 ENX carrier, is used for inserting a selecteed gene and shifts complete promotor-gene-terminator box subsequently to the purpose conversion carrier.
H. callus-evoked promoter:
Callus-evoked promoter also is suitable for genetic expression.A large amount of this promotors be described (as people Plant Molec.Biol.22:573-588 (1993) such as Xu, people Plant Cell 1:151-158 (1989) such as Logemann, Rohrmeier ﹠amp; Lehle, Plant Molec.Biol.22:783-792 (1993), people .Plant Molec.Biol.22:129-142 (1993) such as Firek, people .Plant such as Warner are (1993) J.3:191-201) and all be applicable to the present invention.People such as Logemann have described 5 ' upstream sequence of dicotyledonous potato wunL gene.People such as Xu show that the callus-evoked promoter that derives from dicotyledonous potato (pin2) also has activity in monocotyledon rice.In addition, Rohrmeier ﹠amp; Lehle describes the clone of corn Wipl cDNA of callus of induce and it can be used for separating related promotor by routine techniques.Similar, people such as people such as Firek and Warner has described a callus-induced gene that comes from monocotyledons Asparagus officinalls, and it is expressed in local callus district and pathogenic agent infects the site.Utilize clone technology well known in the art, these promotors can be transferred in the suitable carriers, are fused in the gene that the present invention relates to, and are used for expressing these genes at plant callus point.
I. pith-preferred expression:
Patent WO 93/07278, and it is incorporated herein by reference, and described the separation of the corn trpA gene of preferably expressing in the pith cell.The gene order and the promotor that extend to-1726 bp from transcription initiation are described.Utilize conventional Protocols in Molecular Biology, this promotor or its part can be transferred to one as in the pCGN1761 carrier, and its replaceable 35S promoter also is used to drive foreign gene so that pith-preferably mode is expressed.In fact, the fragment that contains pith-preferred promotor or its part can be transferred to any carrier and be modified and be used for transgenic plant.
J. leaf-specific expressed:
The corn gene of coding phosphoric acid enol carboxylase (PEPC) is by Hudspeth; Grula describes (Plant Molec Biol 12:579-589 (1989)).Utilize conventional Protocols in Molecular Biology, the promotor of this gene can be used to drive any gene and express in transgenic plant in leaf-specific mode.
K. pollen-specific expressed:
WO 93/07278 has described the separation of protein kinase (CDPK) gene of the corn calcium-dependence that is expressed in the pollen cell.This gene order and promotor extend to 1400bp from transcription initiation.Utilize conventional Protocols in Molecular Biology, this promotor or its part can be transferred to as replacing 35S promoter in the pCGN1761 carrier and being used to drive nucleotide sequence of the present invention and express in pollen-specificity mode.
2. transcription terminator
The transcription terminator that can obtain many types is used for expression cassette.These terminators be responsible for to remove transgenosis and the outside the pale of civilization Transcription Termination of polyadenylic acid accurately.Suitable transcription terminator has the terminator of function and comprises CaMV 35S terminator, tml terminator, nopaline synthase terminator and pea rbcS E9 terminator for those are known in plant.These can be used to unifacial leaf and dicotyledons.In addition, the natural transcription terminator of a gene also can be utilized.
3. be used to strengthen or the sequence of regulating and expressing
A large amount of sequences has been found and can be used to unite gene of the present invention from the inner reinforcing gene expression of transcriptional units and these sequences and increases its expression transgenic plant.
Different intron sequences has been shown particularly to strengthen in monocotyledonous cell and has expressed.For example, the intron of corn Adhl gene has been found the expression of significantly strengthening the wild type gene under its related promotor control after being directed to maize cell.Introne 1 be found especially effectively and strengthen have chloramphenicol acetyl transferasegene (people such as Callis., Genes Develop.1:1183-1200 (1987)) fusion construct in expression.In identical experimental system, has similar enhancing expression activity from the intron of corn bronzel gene.Intron sequences has been incorporated into plant conversion carrier routinely, typically in the untranslated leader.
The also known a large amount of untranslated leaders that derive from virus strengthen expresses, and these are effective especially in the dicotyledons cell.Particularly, from tobacco mosaic virus (TMV) (TMV, " W-sequence "), maize pinta poison (MCMV), and the leader sequence of alfalfa mosaic virus (AMV) has been shown to strengthen expressing effectively (people Nud.Acids Res.15:8693-8711 (1987) such as e.g.Gallie; People Plant Molec.Biol.15:65-79 (1990) such as Skuzeski).
4. the targetting of gene product in the cell
The different mechanisms of target gene product is known to be present in the plant and the sequence of regulating and control these machine-processed functions is identified on some details.For example, gene product is regulated and control at the aminoterminal signal sequence of different proteins by a discovery the targetting of chloroplast(id), and it is cut in the process of chloroplast(id) input and produces sophisticated protein (as people J.Biol.Chem.263:15104-15109 (1988) such as Comai).These signal sequences can be fused to and influence the allos product in the heterologous gene products and be transported to (van den Broeck waits people .Nature313:358-363 (1985)) in the chloroplast(id).The DNA of coding proper signal sequence can be by from coding RUBISCO albumen, CAB albumen, and the EPSP synthase, G52 albumen and many other known locations are 5 ' the terminal separation of the proteic cDNA of chloroplast(id).Also referring to, U.S. patent No.5, the life of 639,949 embodiment 37 is called " expression of chloroplast(id) targetting ".
The organoid that other gene product is positioned in other is (as people .Plant Molec.Biol.13:411-418 (1989) such as Unger) in plastosome and the peroxisome for example.The cDNA of these products of encoding is influenced the targetting of heterologous gene products to these organoids by manual handling.The example of this sequence is that nuclear-encoded adenosine triphosphatase and mitochondrial specificity aspartate transaminase are with the I type.The targeted cells proteoplast is described (Proc.Nat I .Acad.Sci.USA 82:6512-6516 (1985)) by people such as Rogers.
In addition, identified and caused the sequence of gene product the targetting of other Cytology Lab.The N-terminal sequence is responsible for ER, apoplast, and from targetting (the Koehler ﹠amp of aleurone cell's cell exocrine thing; Ho, Plant Cell 2:769-783 (1990)).In addition, N-terminal sequence and C-terminal sequence association are responsible for Plant Molec.Biol.14:357-368 (1990) such as () Shinshi to the targetting of the vacuole of gene product.
By above-mentioned suitable targetting sequence is fused in the purpose transgenic sequence, it may guide transgene product in any organoid or cell cell.For the targetting of chloroplast(id), for example, derive from the RUBISCO gene, the CAB gene, epsp synthase gene, or the chloroplast(id) signal sequence of GS2 gene is fused in the genetically modified N-terminal ATG frame.The signal sequence of selecting should comprise known cleavage site, and the fusions that makes up should consider to cut the arbitrary amino acid behind the required cleavage site.In some cases, this needs can pass through to add the amino acid of a little between cleavage site and transgenosis ATG, or selectively, satisfy at inner some amino acid of replacing of transgenosis.The structure fusions that is used for the chloroplast(id) input can be followed the efficient that external chloroplast(id) absorption is used to detect the chloroplast(id) absorption by the external translation of in-vitro transcription construction, the technology of utilizing exists for people such as Bartlett: Edelmann etc. (Eds.) Methodsin Chloroplast Molecular Biology, Mol.Gen.Genet.205:446-453 (1986) such as Elsevier pp 1081-1091 (1982) and Wasmann describe.These construction technology are well known in the art and are equally applicable to plastosome and peroxisome.
Above-mentioned be used for the cell targeting mechanism of action can by not only with its related promotor associating, and can unite with allogeneic promoter and use so that reach the purpose of the specific cell-targetting under the transcriptional control of the promotor of an expression pattern with the promotor that is different from target signal source.
Embodiment 24: make up plant conversion carrier
A large amount of conversion carriers that is applicable to Plant Transformation is known for the those of ordinary skill in Plant Transformation field, and gene related to the present invention can be used to be connected in so arbitrarily carrier.The target kind that the selection of carrier will depend on preferred transformation technology and be used to transform.For the kind of certain target, different microbiotic or weedicide selective marker will be preferred.The conventional selective marker of using comprises the nptll gene in the conversion, and it gives kantlex and relevant antibiotic resistance (Messing ﹠amp; Vierra. gene 19:259-268 (1982); People such as Bevan, Nature 304:184-187 (1983)), the bar gene, its conferring herbicide phosphine silk mycin resistance (people such as White., Nud.Acids Res 18:1062 (1990), people .Theor.Appl.Genet 79:625-631 (1990) such as Spencer), the hph gene, it gives antibiotic hygromycin resistance (Blochinger ﹠amp; Diggelmann, MolCell Biol 4:2929-2931), and the dhfr gene, its give the methatrexate resistance (people such as Bourouis., EMBO is (7) 1099-1104 (1983) J.2), and EPSPS gene, its conferring glyphosate resistance (U.S. patent No.4,940,935 and 5,188,642).
1. be suitable for the carrier that edaphic bacillus transforms
Many carriers are suitable for utilizing the conversion of Agrobacterium tumefaciems.These typically carry at least one T-DNA border sequence and comprise for example carrier of pBINl 9 (Bevan, Nud.Acids Res. (1984)) and pXYZ.Below, two kinds of structures that typically are applicable to the carrier that edaphic bacillus transforms are described.
A.pCIB200 and pCIB2001:
Binary vector pcIB200 and pCIB2001 are used to make up the recombinant vectors that is used for edaphic bacillus and make up in following mode.Nar I digestion (Schmidhauser﹠amp by pTJS75; Helinski, J.Bacteriol.164:446-455 (1985)) allows the excision of tsiklomitsin-resistant gene, then insert an Acc I fragment that derives from the pUC4K that carries the NPT II, produced pTJS75kan (Messing ﹠amp; Vierra, gene 19:259-268 (1982): people such as Bevan., Nature 304:184-187 (1983): people such as McBride. and, Plant Molecular Biology 14:266-276 (1990)).Xho I joint is connected on the EcoRV fragment of PCIB7, EcoRV fragment wherein contains a left side or right T-DNA border, plant selectivity nos/nptll mosaic gene and pUC polylinker (people such as Rothstein., gene 53:153-161 (1987)), and the fragment of Xhol-digestion is cloned on the pTJS75kan fragment of Sall-digestion and is produced pCIB200 (also referring to EP 0,332 104, embodiment 19).PCIB200 contains the polylinker restriction site of following uniqueness: EcoR I, Sst I, Kpn I, Bg III, Xba I and Sa II.PCIB2001 is by inserting the derivative of the pCIB200 that other restriction site produces in the polylinker.Restriction site unique in the pCIB2001 polylinker is the EcoR I, Sst I, Kpn I, Bg III, Xba I, Sa II, MIu I, Bc II, Avr II, Apal, Hpa I, and Stu I.PCIB2001 also contains plant and bacterium kantlex selectivity except containing unique restriction site, the left side of agrobacterium-mediated conversion and right T-DNA border, the trfA function that is used for the RK2-source of the migration between E.coil and other host, and the OriT and the OriV function that also come from RK2.The pCIB2001 polylinker is applicable to the clone of the expression of plants box that contains they self conditioning signal.
B.pCIB10 with and Totomycin selective derivatization thing:
Binary vector pCIB10 contains that coding is used for the kalamycin resistance gene that plant selects and T-DNA is right and left margin sequence and introducing come from the sequence of its plasmid pRK252 that duplicates of permission of host's wide spectrum in E.coli and edaphic bacillus.Its construction is described (gene 53:153-161 (1987)) by people such as Rothstein.The different derivatives of pCIB10 are fabricated, and it has introduced the hygromycin B phosphotransferase gene, such as Gritz etc. description (Gene 25:179-188 (1983)).These derivatives can only contain Totomycin (pCIB743), or Totomycin and kantlex (pCIB715, pCIB717) last screening transgenic plant cells.
2. be applicable to the carrier that non--edaphic bacillus transforms
Do not utilize the conversions of Agrobacterium tumefaciems to avoid, and therefore except such as the above-mentioned carrier that contains the T-DNA sequence, the carrier that lacks these sequences can be utilized to requirement in the T-DNA sequence of selecting conversion carrier.The transformation technology that does not rely on edaphic bacillus comprises by microparticle bombardment, the conversion of protoplastis picked-up (for example .PEG and electroporation) and microinjection.The selection of carrier depends on the preferred selection that is transformed kind morely.Below, be applicable to that the structure of the typical carriers that non-agrobacterium transforms is described.
a．pCIB3064:
PC1B3064 is the carrier of a pUC-origin, is applicable to the direct gene transfer techniques of uniting with the selection of weedicide basta (or phosphine silk mycin).Plasmid pCIB246 contains CaMV 35S promoter (operationally being fused to the E.coil gus gene) and CaMV 35S transcription terminator and is described in disclosed PCT application WO 93/07278.The 35S promoter of this carrier contains 5 ' ATG sequence of two initiation sites.These sites utilize the round pcr of standard to be made by mutagenesis and remove ATG and produce Sspl and Pvu II restriction site.New restriction site is apart from Sa II site 96 and 37 bp and the actual initiation site 101 and 42 bp of distance of uniqueness.The derivative of the pCIB246 that produces is named as pCIB3025.Then, gus gene is excised from pCIB3025 by the digestion of Sa II and Sac I, and terminal also being reconnected by rust produces plasmid pCIB3060.Plasmid pJIT82 is from John InnesCentre, Norwich obtain and one 400 bp contain cut and be inserted into Hpal site EMBO J 6:2519-2523 (1987) such as () Thompson of pCIB3060 from the Smal fragment of the bar gene of Streptomycesviridochrormogenes.Produce pCIB3064, it contains at the CaMV 35S promoter and is used for bar gene under the regulation and control of the terminator that weedicide selects, and an ampicillin resistance gene (being used for selecting at E.coil) and one have specific site Sph I, Pst I, the polylinker of Hind III and BamH I.This carrier is suitable for containing the clone of the expression of plants box of autogenous control signal.
B.pSOG19 and pSOG35:
PSOG35 is a conversion carrier, and it utilizes E.coli dihydrofolate reductase gene (DFR) to give the methotrexate resistance as selected marker.The PCR 35S promoter (800 bp) that is used to increase is from the intron 6 (550 bp) of corn Adhl gene and from the GUS untranslated leader of the 18bp of pSOG10.The 250-bp fragment of a coding E.coli Tetrahydrofolate dehydrogenase II type gene also is amplified by PCR and these two PCR fragments use the Sac I-Pst I fragment from pBl221 (Clontech) to assemble, and fragment wherein contains pUC19 carrier main chain and nopaline synthase terminator.These segmental assemblings produce pSOG19, and it contains fragment and intron 6 sequences, GUS leader sequence, the 35S promoter that DHFR gene and nopaline synthase terminator merge.GUS leader sequence among the pSOG19 is replaced from maize pinta poison (Maize Chlorotic Mottle Virus) leader sequence (MCMV) has produced carrier pSOG35.PSOG19 and pSOG35 carry the pUC gene of amicillin resistance and have Hind III, Sph I, Pst I and the EcoR I site that is suitable for the allogenic material clone.
3. be applicable to the carrier that chloroplast(id) transforms
For nucleotides sequence of the present invention is listed in expression in the plant plastid, plastid conversion carrier pPH143 (WO 97/32011, and embodiment 36) is utilized.Nucleotide sequence is inserted into pPH143, therefore replaces the PROTOX encoding sequence.This carrier is used to the selection of the transformant of plastid conversion and spectinomycin resistance then.Selectively, thus nucleotide sequence is inserted into pPH143 has replaced the aadH gene.In this case, select the inhibitor resistance to PROTOX of transformant.
Embodiment 25: transform
In case nucleotide sequence of the present invention is cloned in the expression system, it is transformed into vegetable cell.Conversion of plant and regenerated method are well known in the art.For example, the Ti-plasmids carrier has been beneficial to the transhipment foreign DNA, and directly DNA picked-up, liposome, electroporation, microinjection, and particulate.In addition, the bacterium that derives from Agrobacterium can be used to transformed plant cells.Representational technology and the representative plasmid transformation technology of transforming monocots and dicotyledons are described below.
1. the conversion of dicotyledons
The transformation technology of dicotyledons is well known in the art and comprises based on the technology of edaphic bacillus and do not need the technology of edaphic bacillus.The non-agrobacterium technology comprises by protoplastis or cell directly absorbs exogenous genetic material.This can be by the picked-up of PEG or electroporation mediation, the transhipment of microparticle bombardment mediation, or microinjection is done.The example of these technology is by Paszkowski etc., EMBO J 3:2717-2722 (1984), Potrykus etc., Mol.Gen.Genet.199:169-177 (1985), Reich etc., Biotechnology 4:1001-1004 (1986), and people such as Klein, Nature 327:70-73 (1987) describes.Under each situation, be regenerated as whole plant by routine techniques well known in the art by plant transformed.
Agrobacterium-mediated conversion is the optimization technique that dicotyledons transforms, because its efficient conversion and the widespread use in a lot of different sortses.Edaphic bacillus transforms and to comprise that typically the binary vector (for example pCIB200 or pCIB2001) that carries the external source target DNA is transformed in the suitable edaphic bacillus bacterial strain, and it may depend on by edaphic bacillus bacterial strain (complementary sequence (complement) of the entrained vir gene of the bacterial strain CIB542 of pCIB200 and pCIB2001 Plant Cell 5:159-169 (1993) such as () Uknes for example on the Ti-plasmids or karyomit(e) of living altogether.The edaphic bacillus that forwards to of reorganization binary vector carries the E.coil of reorganization binary vector by utilization, carries the plasmid of pRK2013 for example and can transport the binary vector of recombinating to be done to three parent's mating methods of the auxiliary E.coil bacterial strain of target edaphic bacillus bacterial strain.Selectively, the reorganization binary vector can transform by DNA and be transported to edaphic bacillus (HOfgen ﹠amp; Willmitzer, Nud.Acids Res.16:9877 (1988)).
The conversion of target plant species by the reorganization edaphic bacillus generally includes from the common cultivation of explant of plant and edaphic bacillus and according to method well known in the art.What transformed is organized in to contain on the selective medium of microbiotic between the binary plasmid T-DNA border or Herbicid resistant mark and regenerates.
Another kind of method with gene-transformed plant cell relates to propelling inertia or bioactive particles in plant tissue and cell.This technology is disclosed in people's such as Sanford U.S. patent No.4, on 945,050,5,036,006 and 5,100,792.Normally, this method relates at the outside surface of effective penetration cell and with mixing and advances inertia or bioactive particles under its inner condition.When inert particle was utilized, carrier can be directed in the cell by the particle that is coated with the carrier that contains goal gene.Optionally, carrier is directed in the cell by exciting of particle but the target cell suppressed by vector surrounds.Bioactive particles (for example, dry yeast cell, dried bacterium or phage respectively contain the DNA that will be imported into) also can be advanced in the plant cell tissue.
2. monocotyledonous conversion
The conversion of most monocotyledons kind has become conventional technology now.Preferred technology comprises utilizes PEG or electroporation technology and particulate to attack the direct transforming gene of callus in protoplastis.Transform available single DNA kind or many DNA kind carry out (for example. cotransformation) and these two kinds of technology all be applicable to the present invention.Cotransformation can have the advantage of the transgenic plant of the disconnected locus that the structure of avoiding complete carrier and generation have goal gene and alternative mark, can remove selected marker in the offspring, and these are desired.Yet a shortcoming utilizing cotransformation is that the frequency that the DNA isolation kind is integrated in the genome is lower than 100% (Biotechnology 4:1093-1096 (1986) such as Schocher).
Patent application EP 0 292435, EP 0 392 225, and WO 93/07278 has described and is used to prepare from the callus of the good inbred lines of corn and the technology of protoplastis, utilize PEG or electroporation to transform the technology of protoplastis, and from the regeneration of the milpa of the protoplastis that is transformed.(Biotechnology 8:833-839 (1990)) such as Gordon-Kamm etc. (Plant Cell 2:603-618 (1990)) and Fromm disclose and have utilized microparticle bombardment to transform the derive technology of corn system of Al88.In addition, (Biotechnology il:194-200 (1993)) such as WO 93/07278 and Koziel have described the technology of utilizing the good inbred lines of microparticle bombardment maize transformation.The long non-ripe maize and the PDS-1000He Biolistics device that is used to bombard of 1.5-2.5mm that this technology utilization is cut from mealie from the back 14-15 that pollinates.
The conversion of paddy rice is also by utilizing the direct gene transfer technique of protoplastis or microparticle bombardment to carry out.The conversion of the protoplastis of Japonica-type and Indica-type mediation is described (Plant Cell Rep Z:379-384 (1988) such as Zhang; Nature338:274-277 such as Shimamoto (1989); Biotechnology 8:736-740 (1990) such as Datta).Two types also can be utilized microparticle bombardment by the conversion of routine (Biotechnology 9:957-962 (1991) such as Christou).In addition, WO 93/21335 has described the technology of utilizing electroporation to come rice transformation.
Patent application EP 0 332 581 describes the generation of Pooideae protoplastis, transforms and regenerating technique.These technology allow the conversion of Dactyils and wheat.In addition, wheat transforms to be described by (Biotechnology 10:667-674 (1992)) such as Vasil and utilizes the cell of microparticle bombardment to the long-term reproducible callus of C type, also be described the callus that utilizes microparticle bombardment immature embryos and immature embryos source, (Plant Physiol.102:1077-1084 (1993)) such as people such as Vasil (Biotechnology11:1553-1558 (1993)) and Weeks.Yet the preferred technology that wheat transforms comprises by the immature paotoblastic wheat conversion of particle bombardment and high-sucrose or high malt sugar step before being included in gene delivery.Before bombardment, any amount of embryo (0.75-1mm is long) has been placed in 3% sucrose (Murashiga ﹠amp; Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3mg/l2, being used for the inductor somatic embryo on the MS substratum of 4-D, it carries out in the dark.In that day of selected bombardment, indusium is removed and is placed on the osmoticum from inducing culture and (has the sucrose or the maltose that are added to desired concn, typically be 15% inducing culture).Indusium allows plasmolysis 2-3h and is bombarded.Typical 20 embryos of each purpose plate, but be not crucial.A suitable plasmid that carries gene (for example pCIB3064 or pSG35) utilizes conventional method to be deposited upon the gold grain of micron size.Each embryo plate DuPontBiolistics Helium equipment is depressed in the burst of 1000 psi and is utilized standard 80 purposes to sieve to shoot.After bombardment, indusium is put back into and recovers 24 hours (still on permeate agent) in the dark.After 24 hours, embryo is removed and is taken back on the inducing culture and cultivated before regeneration about 1 month from permeate agent.After about 1 month, embryo explants with callus that the embryo of growth takes place is transformed into regeneration culture medium, and (MS+1mg/ rises NAA, 5mg/ rises GA), contain suitable selective agent 0 (being 10mg/l basta under the pCIB3064 situation, is the 2mg/I methotrexate) in addition under the pSOG35 situation.After about 1 month, the bud of growth is transferred to bigger sterile chamber, and it contains half amount MS, 2% sucrose, and the selective agent of same concentrations.
Utilize the monocotyledons conversion of edaphic bacillus also to be described.Referring to WO 94/00977 and U.S. patent No.5,591,616, this two be incorporated herein by reference.
3. the conversion of plastid
The seed of Nicotiana tabacurn c.v. ' Xanthi nc ' is on a T nutrient agar 1 " have in the every ware of circular permutation 7 germinate and sowing after 12-14 days be coated with 1 μ m tungsten particle from the DNA of plasmid pPH143 and PPHI45 (M10; Biorad; Hercules; CA) bombardment; as described below basically (Svab; Z. and Maliga, P. (1993) PNAS 90,913-917).Two days later, leaf is cut and incites somebody to action axle side far away up in light (350-500 pmol photon/m to the seed culture of being bombarded on the T substratum 2/ (Sigma, St.Louis is on RMOP substratum MO) in the spectinomycin dihydrochloride that contains 500 μ g/ml in s).After 3 to 8 weeks of bombardment, the resistant buds that occurs below that the decolouring leaf arranged to identical selective medium, allow to be formed callus and the separated and subclone of the bud of secondary by subclone.The plastom of the conversion in independent subclone copy separate fully by the Southern engram technology of standard evaluated (Sambrook etc., (1989) molecular cloning: laboratory manual, Cold Spring Harbor Laboratory, cold spring port).Total cell dna (the Mettler of BamH/EcoRl-digestion, I.J. (1987) Plant Mol BiolReporter 5,346-349) separated on 1%Tris-boric acid (TBE) sepharose, be transferred to (Amersham) on the nylon film and use corresponding to from 0.7 kb BamH I/Hind III dna fragmentation of the pC8 that contains part rps7/12 plastid target sequence 32The random primer dna sequence dna of p mark is surveyed.The homogeneity bud contain sterile rootage on the MS/IBA substratum of spectinomycin (McBride, people such as K.E.. (1994) PNAS 91,7301-7305) and be transferred in the greenhouse.
E. breeding and seed production
Embodiment 26: breeding
By transforming the plant variety that the plant that obtains can be any kind, comprise monocotyledons and dicotyledons with nucleotide sequence of the present invention; Yet the agricultural that is selected from that is used for the plant optimization of the inventive method goes up important purpose crop as indicated above.Expression of gene of the present invention and other important yield and quality characteristic can be integrated into plant lines by breeding.The method of breeding and technology are known in the art.Referring to, for example, Welsh J.R., plant genetic and breeding basis, John Wiley ﹠amp; Sons, NY (1981); Crop breeding, Wood D.R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., plant breeding principle, second edition, Clarendon press, Oxford (1987); Singh, D.P. is used for the breeding of anti-disease and anti-insect, Springer-Verlag, NY (1986); And Wricke and Weber, Quantitative Genetics andSelection Plant Breeding, Walter de Gruyter and Co., Berlin (1986).
By engineered hereditary property in above-mentioned transgenic seed and plant by sexual propagation or asexual growth by administration and therefore in the progeny plants plant, keep and propagate.Usually said maintenance and propagation have utilized the known Agricultural methods that are modified to be suitable for specific purpose, for example cultivate sowing, or harvesting.Special method for example water culture or greenhouse technology also can be employed.Because the crop that growing up is attacked and damage the influence that equally also is subjected to weed competition easily by insect, some are as controlling weeds, plant disease, and insect, the measure of nematode and other unfavourable condition is used and improves output.These comprise mechanical method for example farming or the impurity elimination grass and the infected plant of soil, equally also comprise for example weedicide of agrochemicals medicine, sterilant, gametocide, nematocides, growth regulator, the application of ripener and sterilant.
The purposes of the favourable hereditary property of transgenic plant of the present invention and seed can further be used in the plant breeding, its purpose is to cultivate the plant of improved characteristics, for example insect patience and herbicide tolerance, or stress tolerance, the nutritive value of improvement, the output that increases causes to reduce owing to parasitism or come off to cause the improved structure of loss.Different breeding steps are characterized by clear and definite human intervention, for example screen the strain that is used to hybridize, the pollination of guiding parent system, or screen suitable progeny plant.Depend on desired characteristic, taked different breeding measures.Relevant technology is well known in the art and includes but not limited to hybridization, inbreeding, and back cross breeding, multi-thread breeding, kind is mixed and is handed over species hybridization, aneuploid technology or the like.Hybridization technique also comprises by machinery, chemistry, or biochemical mode sterilization plant produces male or female sterile plants.Crossing pollination between the pollen of male sterile plants and different strains guarantees male sterile but the genome of female child care plant will obtain the characteristic of two parental lines consistently.Therefore, the breeding of the plant lines that transgenic seed of the present invention and plant can be used to improve for example, increases the traditional method for example validity or because their the adorned hereditary property and can save said method of weedicide or pesticide treatments.Selectively, the new crop that has the stress patience of improvement can be obtained, and it depends on their optimised heredity " equipment ", compares the results product that has produced better quality with the product that can not tolerate similar bad developmental condition.
Embodiment 27: the production of seed
In the production of seed, the homogeneity of germination quality and seed is important product performance, yet the germination quality of the seed that the peasant gathered in the crops and sell and the homogeneity of seed are unessential.Owing to keep a kind of crop not to be subjected to other the crop and the influence of weed seed, control kind of biography is sick, and produce that to have good seeds germinated be very difficult, quite extensive and clear and definite seed production operation is improved by the seed manufacturer, wherein the manufacturer is in the growth of purebred son, conditioning, and the sale aspect is exper ienced.Therefore, using the seed of buying that meets the extra fine quality standard to replace using the seed of oneself gathering in the crops is the experience that has to the peasant.The breeding material that is used as seed is usually with containing weedicide, sterilant, mycocide, sterilant, nematocides, molluscacide, or the protective material Cotton seeds of its mixture.Usually, used protective material dressing contains for example Vancide 89 of compound, carboxin, thiram (TMTD ), methalaxyl (Apron ), and methyl pirimiphosmethyl (ActelIic).As needs, these compounds and other the carrier that is generally used for formulation art, the auxiliary agent that tensio-active agent or promotion are used is prepared together and is provided antibacterium, the protection of the infringement that fungi or animal pest cause.The protective material dressing can be by being employed with liquid formulation infiltration breeding material or by the wet or dry preparation bag with associating.It also is suitable that the method for other application is for example directly handled bud or fruit.
Another aspect of the present invention is by new Agricultural methods, and for example the method for the foregoing description is characterized in that transgenic plant of the present invention, transgenic plant material, or the application of transgenic seed.
Seed can be with the sack of being made up of suitable wrapping material, and the form in vessel or the container provides, and this sack and container can sealedly comprise seed.Sack, vessel or container can be designed to short-term or long-term or the short-term and the secular storage of seed.The example of suitable wrapping material comprises paper, for example kraft paper, hard or soft plastics or other polymeric material, glass or metal.The ideal sack, vessel or container are made of identical or dissimilar multiwalled wrapping material.In one embodiment, provide sack, thereby vessel or container are got rid of or restriction water and moisture contact seed.In one embodiment, sack, vessel or container are sealed, and for example heat-sealing stops moisture or moisture to enter.In another embodiment, the water absorbing material is placed in wrapping material interlayer or adjacent with the packing timber bed of material.In another embodiment, sack, vessel or container, or it constitutes its processed restriction of wrapping material, inhibition or prevention disease, pollution or other the influence bad with transportation to seed storage.An embodiment of this processing is sterilization, for example by chemical mode or by being exposed in the ray.It is a commercial sack that the present invention comprises, it contains in described conversion plant the seed with the transgenic plant of the gene of the present invention of expressing than the higher level in the wild-type plant, with a suitable carriers, and the label specification sheets of giving plant spectrum of diseases resistance.
Above-mentioned disclosed embodiment illustrates.Content disclosed by the invention will make those skilled in the art have many versions of the present invention.Therefore, these all tangible and foreseeable variations are comprised in the scope of additional claim.
＜110〉Novartis AG＜120〉＜130〉30472/A/CGC1992＜140〉＜141〉＜160〉15＜170〉PatentIn Ver.2.0＜210〉1＜211〉2978＜212〉DNA＜213〉＜220〉＜221〉CDS＜222〉 ( 569 ) .. ( 976 )＜223〉orf1＜220〉＜221〉CDS＜222〉 ( 1045 ) .. ( 2331 )＜223〉JHE-orf2＜220〉＜223〉pCIB9369＜400〉1atcgatgtga cggcagagta tttttattcc tgtaaactga cgacaatgca tttctaagat 60atcaatataa taatgataaa tttattgatc atatatctgt tatattttga ttgaaaatta 120ttgaatatac ctcttgtact aaattcatta catttttttt actttaaaca acattaaatt 180cacacataat acagcttaaa tataacatgt gatatatatt atgattataa aaaacattaa 240aataaataat acgccacata tattaacaat atctaattac tgatgatact attttctgag 300tatatataaa tcttaaagaa aataattatt ttttatattt cacatcaatt taaaatctgc 360ttagaatgcc ccccggcatc acaagaaaac aaaatcattc aagtaataca atagagttaa 420atttaaaaat aacatgtata acaaaataca tagacaatta tacatgtaaa tgacagacaa 480ctgacaaaac atagcaaaaa aacgccttaa atattaaggt atcaaaacaa tatatcagac 540tatcttaaat ctaataggag aatccctc atg att aca ata cat atc agt ggt 592
Met?Ile?Thr?Ile?His?Ile?Ser?Gly
1???????????????5ggt?agt?gta?aca?att?aat?aac?aat?ata?gta?aca?gaa?act?gat?gtc?caa???640Gly?Ser?Val?Thr?Ile?Asn?Asn?Asn?Ile?Val?Thr?Glu?Thr?Asp?Val?Gln
10??????????????????15??????????????????20aat?aca?ccc?gct?tca?gcg?cct?tta?tca?att?act?aat?ttt?agg?gat?atg???688Asn?Thr?Pro?Ala?Ser?Ala?Pro?Leu?Ser?Ile?Thr?Asn?Phe?Arg?Asp?Met?25??????????????????30??????????????????35??????????????????40aca?ata?gaa?cct?cat?tca?tct?gtt?gag?gcg?ata?aga?acc?gat?aca?ccg????736Thr?Ile?Glu?Pro?His?Ser?Ser?Val?Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro
45??????????????????50??????????????????55att?att?cct?gaa?tca?cga?cca?aat?tac?tat?gtt?gct?aat?tct?ggc?ccg????784Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn?Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro
60??????????????????65??????????????????70gcc?tca?tca?gtc?aga?gct?gtt?ttc?tat?tgg?tcc?cac?tct?ttt?aca?tca????832Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser
75??????????????????80??????????????????85gaa?tgg?ttt?gaa?tct?tcc?tct?att?att?gta?aaa?gca?ggc?gaa?gac?gga????880Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile?Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly
90??????????????????95?????????????????100gtc?tta?cat?tca?ccg?ggt?aat?tct?tta?tat?tac?agc?aag?gtt?gta?att????928Val?Leu?His?Ser?Pro?Gly?Asn?Ser?Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile105?????????????????110?????????????????115?????????????????120tat?aac?gat?aca?gac?aaa?cgt?gct?ttt?gtt?acc?ggc?tac?aat?cta?taa????976Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala?Phe?Val?Thr?Gly?Tyr?Asn?Leu
125?????????????????130?????????????????135taacgcagaa?atacaatcca?tatttccaat?gaatttcaaa?taacatcctt?aaggcaagaa??1036acaaaatc?atg?aat?aat?gaa?ccg?atg?aat?act?aat?gaa?tca?caa?gct?tca???1086
Met?Asn?Asn?Glu?Pro?Met?Asn?Thr?Asn?Glu?Ser?Gln?Ala?Ser
140?????????????????145?????????????????150gag?ata?gta?ccc?tca?atg?aat?gaa?tct?ata?tta?gca?gca?cct?tat?tca????1134Glu?Ile?Val?Pro?Ser?Met?Asn?Glu?Ser?Ile?Leu?Ala?Ala?Pro?Tyr?Ser
155?????????????????160?????????????????165att?tct?aca?cct?aat?tat?gaa?tgg?gat?atg?tca?tca?ata?ata?aaa?gat????1182Ile?Ser?Thr?Pro?Asn?Tyr?Glu?Trp?Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp
170?????????????????175?????????????????180gct?att?att?ggt?ggt?ata?ggc?ttt?att?cct?ggt?ccg?ggc?tca?gca?ata????1230Ala?Ile?Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile
185?????????????????190?????????????????195tca?ttt?ttg?tta?ggg?tta?ttt?tgg?cca?caa?caa?acc?gac?aat?act?tgg????1278Ser?Phe?Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp
200?????????????????205?????????????????210gag?caa?att?ctc?caa?aaa?gta?gaa?caa?atg?atc?gag?caa?gcc?aat?ctc????1326Glu?Gln?Ile?Leu?Gln?Lys?Val?Glu?Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu215?????????????????220?????????????????225?????????????????230aaa?act?att?caa?gga?ata?ttg?aac?ggc?gat?ata?caa?gaa?att?aaa?ggc????1374Lys?Thr?Ile?Gln?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly
235?????????????????240?????????????????245aaa?atg?gaa?cat?gtg?caa?ttc?atg?cta?gaa?tcc?tca?cct?ggc?act?caa????1422Lys?Met?Glu?His?Val?Gln?Phe?Met?Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln
250?????????????????255?????????????????260gaa?agc?cat?gac?gca?tac?atg?ttt?ctg?gcg?aga?tat?ctg?gtc?agt?ata????1470Glu?Ser?His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile
265?????????????????270?????????????????275gac?gaa?aaa?ttc?aag?tct?ttt?gat?aac?aaa?aca?aat?tat?caa?att?ctt????1518Asp?Glu?Lys?Phe?Lys?Ser?Phe?Asp?Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu
280?????????????????285?????????????????290ccc?atg?tat?acc?aat?acg?att?atg?tta?caa?gcc?cct?tat?tgg?aaa?atg????1566Pro?Met?Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met295?????????????????300?????????????????305?????????????????310ggt?ata?gag?aga?aaa?gat?gag?atc?aaa?cta?aca?gat?ata?gaa?gtt?aat????1614Gly?Ile?Glu?Arg?Lys?Asp?Glu?Ile?Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn
315?????????????????320?????????????????325gaa?tta?aaa?gag?ctg?ata?gga?aaa?tta?tct?acc?agc?gcc?gat?aaa?tat????1662Glu?Leu?Lys?Glu?Leu?Ile?Gly?Lys?Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr
330?????????????????335?????????????????340att?cat?gat?gtc?tat?act?cgt?gaa?tat?gat?aat?gcg?atg?aac?act?tca????1710Ile?His?Asp?Val?Tyr?Thr?Arg?Glu?Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser
345?????????????????350?????????????????355aca?gca?gca?aat?atc?acc?aat?aat?tta?tta?tct?gta?aga?ggc?tat?tgt????1758Thr?Ala?Ala?Asn?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys
360?????????????????365?????????????????370tta?tta?cat?ggt?tta?gaa?tgt?ctc?gaa?gtc?att?aac?cat?ata?caa?aat????1806Leu?Leu?His?Gly?Leu?Glu?Cys?Leu?Glu?Val?Ile?Asn?His?Ile?Gln?Asn375?????????????????380?????????????????385?????????????????390aat?agc?ctt?gag?caa?agt?ttt?tat?cct?aaa?act?atc?agc?tac?tcc?acc????1854Asn?Ser?Leu?Glu?Gln?Ser?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr
395?????????????????400?????????????????405gta?ttc?gat?cgc?cag?aca?aat?aaa?aca?agg?gtt?caa?gcc?ctg?aca?gaa????1902Val?Phe?Asp?Arg?Gln?Thr?Asn?Lys?Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu
410?????????????????415?????????????????420gac?gat?caa?atg?caa?gag?cca?ttc?aag?cct?gct?tta?att?aat?ggg?aag????1950Asp?Asp?Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys
425?????????????????430?????????????????435tac?aac?aaa?ata?aaa?tca?ttg?att?ggg?tat?gta?caa?aga?atc?gga?aac????1998Tyr?Asn?Lys?Ile?Lys?Ser?Leu?Ile?Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn
440?????????????????445?????????????????450gca?ccc?aga?gtt?gga?ggc?att?aaa?gtc?aca?ttt?gca?aac?gat?gca?tct????2046Ala?Pro?Arg?Val?Gly?Gly?Ile?Lys?Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser455?????????????????460?????????????????465?????????????????470tat?acc?ctc?ggt?aca?gta?act?tca?gaa?gta?aac?tca?att?gaa?ctg?aat????2094Tyr?Thr?Leu?Gly?Thr?Val?Thr?Ser?Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn
475?????????????????480?????????????????485gac?agc?gtt?ata?acc?agc?ctg?gaa?gta?tgg?gga?aat?ggc?gct?att?gat????2142Asp?Ser?Val?Ile?Thr?Ser?Leu?Glu?Val?Trp?Gly?Asn?Gly?Ala?Ile?Asp
490?????????????????495?????????????????500gag?gca?ttc?ttt?aca?tta?agt?gac?gga?cgt?caa?ttt?agg?ctt?ggc?caa????2190Glu?Ala?Phe?Phe?Thr?Leu?Ser?Asp?Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln
505?????????????????510?????????????????515cgc?tat?gcc?agt?aac?tat?aga?aaa?tat?gct?gtc?gat?aac?cac?tat?att????2238Arg?Tyr?Ala?Ser?Asn?Tyr?Arg?Lys?Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile
520?????????????????525?????????????????530tca?gga?ttg?tac?tta?gcc?agt?gat?gaa?cct?tca?ttg?gca?ggt?caa?gca????2286Ser?Gly?Leu?Tyr?Leu?Ala?Ser?Asp?Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala535?????????????????540?????????????????545?????????????????550gca?ggc?att?gca?gtt?tca?tac?cat?atg?ata?gct?gat?aaa?aaa?tca???????2331Ala?Gly?Ile?Ala?Val?Ser?Tyr?His?Met?Ile?Ala?Asp?Lys?Lys?Ser
555 560 565tagtattaac aggcttctga tttcagacta agcaagtaag cggatcttca gagtcatata 2391gcatgctata tgactgaaga gttatccgct cctcactttt aactaaactc attacatcct 2451ccactaattt attcagcgat aaaaaacaca caatgaaaac caaacatttg tttgttattt 2511tcataaaaaa tgcaactctt tgtttaataa aaatttatcg cagtataaaa tattgccagt 2571tttatagact atattatatt ctcactttat ctatattttt agtttaaaat tcaagactaa 2631aatcacactt ttatgcaaaa tgttcacttt atataactta cgatcgtact ctcataatta 2691gaatcaaata tcaaaataat ctttactgtt tatcagacat gcaatacaac attaatacaa 2751aaaatagcta aggacatgat atgttgaaaa gggaaaatca gatattgcaa ctactgaagg 2811gcgatccttt catgcagcag caagaaatcg ctgatatcct tggaattagc cgctcgtgtg 2871ttgcaggaca tattatgaac ctaagcaaaa aaggatatat taaaggcaaa gggtatatct 2931tatctaatga tgtttatact gttacaattg gtgctgccaa tatcgat 2978＜210〉2＜211〉135＜212〉PRT＜213〉＜400〉2Met Ile Thr Ile His Ile Ser Gly Gly Ser Val Thr Ile Asn Asn Asn 1 5 10 15Ile Val Thr Glu Thr Asp Val Gln Asn Thr Pro Ala Ser Ala Pro Leu
20??????????????????25??????????????????30Ser?Ile?Thr?Asn?Phe?Arg?Asp?Met?Thr?Ile?Glu?Pro?His?Ser?Ser?Val
35??????????????????40??????????????????45Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn
50??????????????????55??????????????????60Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile
85??????????????????90??????????????????95Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly?Val?Leu?His?Ser?Pro?Gly?Asn?Ser
100?????????????????105?????????????????110Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125Phe?Val?Thr?Gly?Tyr?Asn?Leu
130 135＜210〉3＜211〉429＜212〉PRT＜213〉Xenorhabdus nematophilus＜400〉3Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile, 15 10 15Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser
20??????????????????25??????????????????30Thr?Pro?Asn?Tyr?Glu?Trp?Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp?Ala?Ile
35??????????????????40??????????????????45Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile?Ser?Phe
50??????????????????55??????????????????60Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp?Glu?Gln?65??????????????????70??????????????????75??????????????????80Ile?Leu?Gln?Lys?Val?Glu?Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu?Lys?Thr
85??????????????????90??????????????????95Ile?Gln?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly?Lys?Met
100?????????????????105?????????????????110Glu?His?Val?Gln?Phe?Met?Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln?Glu?Ser
115?????????????????120?????????????????125His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile?Asp?Glu
130?????????????????135?????????????????140Lys?Phe?Lys?Ser?Phe?Asp?Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu?Pro?Met145?????????????????150?????????????????155?????????????????160Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met?Gly?Ile
165?????????????????170?????????????????175Glu?Arg?Lys?Asp?Glu?Ile?Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn?Glu?Leu
180?????????????????185?????????????????190Lys?Glu?Leu?Ile?Gly?Lys?Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr?Ile?His
195?????????????????200?????????????????205Asp?Val?Tyr?Thr?Arg?Glu?Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser?Thr?Ala
210?????????????????215?????????????????220Ala?Asn?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys?Leu?Leu225?????????????????230?????????????????235?????????????????240His?Gly?Leu?Glu?Cys?Leu?Glu?Val?Ile?Asn?His?Ile?Gln?Asn?Asn?Ser
245?????????????????250?????????????????255Leu?Glu?Gln?Ser?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr?Val?Phe
260?????????????????265?????????????????270Asp?Arg?Gln?Thr?Asn?Lys?Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu?Asp?Asp
275?????????????????280?????????????????285Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys?Tyr?Asn
290?????????????????295?????????????????300Lys?Ile?Lys?Ser?Leu?Ile?Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn?Ala?Pro305?????????????????310?????????????????315?????????????????320Arg?Val?Gly?Gly?Ile?Lys?Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser?Tyr?Thr
325?????????????????330?????????????????335Leu?Gly?Thr?Val?Thr?Ser?Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn?Asp?Ser
340?????????????????345?????????????????350Val?Ile?Thr?Ser?Leu?Glu?Val?Trp?Gly?Asn?Gly?Ala?Ile?Asp?Glu?Ala
355?????????????????360?????????????????365Phe?Phe?Thr?Leu?Ser?Asp?Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln?Arg?Tyr????370?????????????????375?????????????????380Ala?Ser?Asn?Tyr?Arg?Lys?Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile?Ser?Gly385?????????????????390?????????????????395?????????????????400Leu?Tyr?Leu?Ala?Ser?Asp?Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala?Ala?Gly
405?????????????????410?????????????????415Ile?Ala?Val?Ser?Tyr?His?Met?Ile?Ala?Asp?Lys?Lys?Ser
420 425＜210〉4＜211〉408＜212〉DNA＜213〉Xenorhabdus nematophilus＜220〉＜221〉CDS＜222〉(1) .. (405)＜223〉orf1＜400〉4atg att aca atc aat atc act ggt gat aat gta aga gtt aat aac aat 48Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn, 15 10 15ata gca aca gaa acc gac ctc caa aat aca cct gct tca gca ccc tta 96Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu of pCIB9381
20??????????????????25??????????????????30tca?att?att?aat?ttt?agg?gat?atg?aca?ata?gaa?cct?cat?tca?tct?gtt????144Ser?Ile?Ile?Asn?Phe?Arg?Asp?Met?Thr?Ile?Glu?Pro?His?Ser?Ser?Val
35??????????????????40??????????????????45gag?gcg?ata?aga?acc?gat?aca?ccg?att?att?cct?gaa?tca?cga?cca?aat????192Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn
50??????????????????55??????????????????60tac?tat?gtt?gct?aat?tct?ggc?ccg?gcc?tca?tca?gtc?aga?gct?gtt?ttc????240Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80tat?tgg?tcc?cac?tct?ttt?aca?tca?gaa?tgg?ttt?gaa?tct?tcc?tct?att????288Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile
85??????????????????90??????????????????95att?gta?aaa?gca?ggc?gaa?gac?gga?gtc?tta?cat?tca?ccg?ggt?aat?tct????336Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly?Val?Leu?His?Ser?Pro?Gly?Asn?Ser
100?????????????????105?????????????????110tta?tat?tac?agc?aag?gtt?gLa?att?tat?aac?gat?aca?gac?aaa?cgt?gct????384Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125ttt?gtt?acc?ggc?tac?aat?cta?taa????????????????????????????????????408Phe?Val?Thr?Gly?Tyr?Asn?Leu
130 135＜210〉5＜211〉135＜212〉PRT＜213〉Xenorhabdus nematophilus＜400〉5Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn, 15 10 15Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20??????????????????25??????????????????30Ser?Ile?Ile?Asn?Phe?Arg?Asp?Met?Thr?Ile?Glu?Pro?His?Ser?Ser?Val
35??????????????????40??????????????????45Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn
50??????????????????55??????????????????60Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile
85??????????????????90??????????????????95Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly?Val?Leu?His?Ser?Pro?Gly?Asn?Ser
100?????????????????105?????????????????110Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125Phe?Val?Thr?Gly?Tyr?Asn?Leu
130 135＜210〉6＜211〉1290＜212〉DNA＜213〉Xenorhabdus nematophilus＜220〉＜221〉CDS＜222〉(1) .. (1287)＜223〉the same orf2 of JHE＜400〉6atg aat aat gaa ccg atg aat act aat gaa tca caa gtt tca gag ata 48Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Val Ser Glu Ile, 15 10 15gta ccc tca atg aat gaa tct ata tta gca gca cct tat tca att tct 96Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser of pCIB9381
20??????????????????25??????????????????30aca?cct?aat?tat?gaa?tgg?gat?atg?tca?tca?ata?ata?aaa?gat?gcc?att????144Thr?Pro?Asn?Tyr?Glu?Trp?Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp?Ala?Ile
35??????????????????40??????????????????45att?ggt?ggt?ata?ggc?ttt?att?cct?ggt?ccg?ggc?tca?gca?ata?tca?ttt????192Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile?Ser?Phe
50??????????????????55??????????????????60ttg?tta?ggg?tta?ttt?tgg?cca?caa?caa?acc?gac?aat?act?tgg?gag?caa????240Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp?Glu?Gln?65??????????????????70??????????????????75??????????????????80att?ctc?caa?aaa?gta?gaa?caa?atg?atc?gag?caa?gcc?aat?ctc?aaa?act????288Ile?Leu?Gln?Lys?Val?Glu?Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu?Lys?Thr
85??????????????????90??????????????????95att?caa?gga?ata?ttg?aac?ggc?gat?ata?caa?gaa?att?aaa?ggc?aaa?atg????336Ile?Gln?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly?Lys?Met
100?????????????????105?????????????????110gaa?cat?gtg?caa?ttc?atg?cta?gaa?tcc?tca?cct?ggc?act?caa?gaa?agc????384Glu?His?Val?Gln?Phe?Met?Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln?Glu?Ser
115?????????????????120?????????????????125cat?gac?gca?tac?atg?ttt?ctg?gcg?aga?tat?ctg?gtc?agt?ata?gac?gaa????432His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile?Asp?Glu
130?????????????????135?????????????????140aaa?ttc?aag?tct?ttt?gat?aac?aaa?aca?aat?tat?caa?att?ctt?ccc?atg????480Lys?Phe?Lys?Ser?Phe?Asp?Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu?Pro?Met145?????????????????150?????????????????155?????????????????160tat?acc?aat?acg?att?atg?tta?caa?gcc?cct?tat?tgg?aaa?atg?ggt?ata????528Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met?Gly?Ile
165?????????????????170?????????????????175gag?aga?aaa?gat?gag?ata?aaa?cta?aca?gat?ata?gaa?gtt?aat?gaa?tta????576Glu?Arg?Lys?Asp?Glu?Ile?Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn?Glu?Leu
180?????????????????185?????????????????190aaa?gag?ctg?ata?gga?aaa?tta?tct?acc?agc?gcc?gat?aaa?tat?att?cat????624Lys?Glu?Leu?Ile?Gly?Lys?Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr?Ile?His
195?????????????????200?????????????????205gat?gtc?tat?act?cgt?gaa?tat?gat?aat?gcg?atg?aac?act?tca?aca?gca????672Asp?Val?Tyr?Thr?Arg?Glu?Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser?Thr?Ala
210?????????????????215?????????????????220gca?aat?atc?acc?aat?aat?tta?tta?tct?gta?aga?ggc?tat?tgt?tta?tta????720Ala?Asn?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys?Leu?Leu225?????????????????230?????????????????235?????????????????240cat?ggt?tta?gaa?tgt?ctc?gaa?gtc?att?aac?cat?ata?caa?aat?aat?agc????768His?Gly?Leu?Glu?Cys?Leu?Glu?Val?Ile?Asn?His?Ile?Gln?Asn?Asn?Ser
245?????????????????250?????????????????255ctt?gag?caa?agt?ttt?tat?cct?aaa?act?atc?agc?tac?tcc?acc?gta?ttc????816Leu?Glu?Gln?Ser?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr?Val?Phe
260?????????????????265?????????????????270gat?cgc?cag?aca?aat?aaa?aca?agg?gtt?caa?gcc?ctg?aca?gaa?gac?gat????864Asp?Arg?Gln?Thr?Asn?Lys?Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu?Asp?Asp
275?????????????????280?????????????????285caa?atg?caa?gag?cca?ttc?aag?cct?gct?tta?att?aat?ggg?aag?tac?aac????912Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys?Tyr?Asn
290?????????????????295?????????????????300aaa?ata?aaa?tca?ttg?att?ggg?tat?gta?caa?aga?atc?gga?aac?gca?ccc????960Lys?Ile?Lys?Ser?Leu?Ile?Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn?Ala?Pro305?????????????????310?????????????????315?????????????????320aga?gtt?gga?ggc?att?aaa?gtc?aca?ttt?gca?aac?gat?gca?tct?tat?acc????1008Arg?Val?Gly?Gly?Ile?Lys?Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser?Tyr?Thr
325?????????????????330?????????????????335ctc?ggt?aca?gta?act?tca?gaa?gta?aac?tca?att?gaa?ctg?aat?gac?agc????1056Leu?Gly?Thr?Val?Thr?Ser?Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn?Asp?Ser
340?????????????????345?????????????????350gtt?ata?acc?agc?ctg?gaa?gta?tgg?gga?aat?ggc?gct?gtt?gat?gag?gca????1104Val?Ile?Thr?Set?Leu?Glu?Val?Trp?Gly?Asn?Gly?Ala?Val?Asp?Glu?Ala
355?????????????????360?????????????????365ttc?ttt?aca?tta?agt?gac?gga?cgt?caa?ttt?agg?ctt?ggc?caa?cgc?tat????1152Phe?Phe?Thr?Leu?Ser?Asp?Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln?Arg?Tyr
370?????????????????375?????????????????380gcc?agt?aac?tat?aga?aaa?tat?gct?gtc?gat?aac?cac?tat?att?tca?gga????1200Ala?Ser?Asn?Tyr?Arg?Lys?Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile?Ser?Gly385?????????????????390?????????????????395?????????????????400ttg?tac?tta?gcc?agt?gat?gaa?cct?tca?ttg?gca?ggt?caa?gca?gca?ggc????1248Leu?Tyr?Leu?Ala?Ser?Asp?Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala?Ala?Gly
405?????????????????410?????????????????415att?gca?gtt?tca?tac?cat?atg?ata?gct?gat?aaa?aaa?tca?tag????????????1290Ile?Ala?Val?Ser?Tyr?His?Met?Ile?Ala?Asp?Lys?Lys?Ser
420 425＜210〉7＜211〉429＜212〉PRT＜213〉Xenorhabdus nematophilus＜400〉7Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Val Ser Glu Ile, 15 10 15Val Pro Ser Met Asn Glu Ser Ile Leu Ala Ala Pro Tyr Ser Ile Ser
20??????????????????25??????????????????30Thr?Pro?Asn?Tyr?Glu?Trp?Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp?Ala?Ile
35??????????????????40??????????????????45Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile?Ser?Phe
50??????????????????55??????????????????60Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp?Glu?Gln?65??????????????????70??????????????????75??????????????????80Ile?Leu?Gln?Lys?Val?Glu?Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu?Lys?Thr
85??????????????????90??????????????????95Ile?Gln?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly?Lys?Met
100?????????????????105?????????????????110Glu?His?Val?Gln?Phe?Met?Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln?Glu?Ser
115?????????????????120?????????????????125His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile?Asp?Glu
130?????????????????135?????????????????140Lys?Phe?Lys?Ser?Phe?Asp?Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu?Pro?Met145?????????????????150?????????????????155?????????????????160Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met?Gly?Ile
165?????????????????170?????????????????175Glu?Arg?Lys?Asp?Glu?Ile?Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn?Glu?Leu
180?????????????????185?????????????????190Lys?Glu?Leu?Ile?Gly?Lys?Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr?Ile?His
195?????????????????200?????????????????205Asp?Val?Tyr?Thr?Arg?Glu?Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser?Thr?Ala
210?????????????????215?????????????????220Ala?Asn?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys?Leu?Leu225?????????????????230?????????????????235?????????????????240His?Gly?Leu?Glu?Cys?Leu?Glu?Val?Ile?Asn?His?Ile?Gln?Asn?Asn?Ser
245?????????????????250?????????????????255Leu?Glu?Gln?Ser?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr?Val?Phe
260?????????????????265?????????????????270Asp?Arg?Gln?Thr?Asn?Lys?Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu?Asp?Asp
275?????????????????280?????????????????285Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys?Tyr?Asn
290?????????????????295?????????????????300Lys?Ile?Lys?Ser?Leu?Ile?Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn?Ala?Pro305?????????????????310?????????????????315?????????????????320Arg?Val?Gly?Gly?Ile?Lys?Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser?Tyr?Thr
325?????????????????330?????????????????335Leu?Gly?Thr?Val?Thr?Ser?Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn?Asp?Ser
340?????????????????345?????????????????350Val?Ile?Thr?Ser?Leu?Glu?Val?Trp?Gly?Asn?Gly?Ala?Val?Asp?Glu?Ala
355?????????????????360?????????????????365Phe?Phe?Thr?Leu?Ser?Asp?Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln?Arg?Tyr
370?????????????????375?????????????????380Ala?Ser?Asn?Tyr?Arg?Lys?Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile?Ser?Gly385?????????????????390?????????????????395?????????????????400Leu?Tyr?Leu?Ala?Ser?Asp?Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala?Ala?Gly
405?????????????????410?????????????????415Ile?Ala?Val?Ser?Tyr?His?Met?Ile?Ala?Asp?Lys?Lys?Ser
420 425＜210〉8＜211〉408＜212〉DNA＜213〉Podbielniak Xenorhabdus＜220〉＜221〉CDS＜222〉(1) .. (405)＜223〉orf1＜400〉8atg atc aca atc aat atc agt ggt ggt aat gta aca att aat aac aat 48Met Ile Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn, 15 10 15atc agt tca gta acg gat atc caa aaa ccc ctt gat gca gaa ccc ctc 96Ile Ser Ser Val Thr Asp Ile Gln Lys Pro Leu Asp Ala Glu Pro Leu of pCIB9354
20??????????????????25??????????????????30tca?gtc?acg?aat?tat?aga?gat?ctg?aca?ata?gag?ccg?cac?tca?tct?att????144Ser?Val?Thr?Asn?Tyr?Arg?Asp?Leu?Thr?Ile?Glu?Pro?His?Ser?Ser?Ile
35??????????????????40??????????????????45caa?gca?gac?aga?acg?gac?acc?ccc?att?att?cct?gaa?aca?cgc?cct?gat????192Gln?Ala?Asp?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Thr?Arg?Pro?Asp
50??????????????????55??????????????????60tat?tat?atc?gct?aac?tca?ggc?cct?gct?tca?tca?gtc?aaa?gct?gtg?ttt????240Tyr?Tyr?Ile?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Lys?Ala?Val?Phe??65??????????????????70??????????????????75??????????????????80tat?tgg?tcg?cat?tcg?ttt?aca?tcg?gaa?tgg?ttc?gag?tat?tca?tct?atc????288Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Tyr?Ser?Ser?Ile
85??????????????????90??????????????????95acg?gta?aaa?gca?gga?gaa?gat?gga?ata?tta?aaa?tca?ccg?agt?aat?gct????336Thr?Val?Lys?Ala?Gly?Glu?Asp?Gly?Ile?Leu?Lys?Ser?Pro?Ser?Asn?Ala
100?????????????????105?????????????????110gta?tat?tac?agt?aaa?gta?gtc?att?tat?aat?gat?aca?gat?aag?cgg?gct????384Val?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125ttt?gtg?act?gga?tat?aac?atg?taa????????????????????????????????????408Phe?Val?Thr?Gly?Tyr?Asn?Met
130 135＜210〉9＜211〉135＜212〉PRT＜213〉Podbielniak Xenorhabdus＜400〉9Met Ile Thr Ile Asn Ile Ser Gly Gly Asn Val Thr Ile Asn Asn Asn 15 10 15Ile Ser Ser Val Thr Asp Ile Gln Lys Pro Leu Asp Ala Glu Pro Leu
20??????????????????25??????????????????30Ser?Val?Thr?Asn?Tyr?Arg?Asp?Leu?Thr?Ile?Glu?Pro?His?Ser?Ser?Ile
35??????????????????40??????????????????45Gln?Ala?Asp?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Thr?Arg?Pro?Asp
50??????????????????55??????????????????60Tyr?Tyr?Ile?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Lys?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Tyr?Ser?Ser?Ile
85??????????????????90??????????????????95Thr?Val?Lys?Ala?Gly?Glu?Asp?Gly?Ile?Leu?Lys?Ser?Pro?Ser?Asn?Ala
100?????????????????105?????????????????110Val?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125Phe?Val?Thr?Gly?Tyr?Asn?Met
130 135＜210〉10＜211〉1056＜212〉DNA＜213〉Podbielniak Xenorhabdus＜220〉＜221〉CDS＜222〉(1) .. (1053)＜223〉the JHE-sample orf2＜400〉10atg aat aat agt cca atg aat gat cag tta tca aca gcg cct tat tca 48Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser, 15 10 15att tcg aca ccc aat tat gaa tgg gat atg tca tca atc ata aaa gat 96Ile Ser Thr Pro Asn Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp of pCIB9354
20??????????????????25??????????????????30gcc?att?atc?ggt?ggc?ata?gga?ttt?att?ccc?gga?cca?ggc?cct?gca?atc????144Ala?Ile?Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Pro?Ala?Ile
35??????????????????40??????????????????45tct?ttt?tta?tta?gga?ctg?ttc?tgg?cca?caa?cag?aca?gac?aat?acc?tgg????192Ser?Phe?Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp
50??????????????????55??????????????????60gat?caa?atc?ctc?caa?aaa?atc?gaa?caa?atg?ata?gaa?gaa?gcg?aat?tta????240Asp?Gln?Ile?Leu?Gln?Lys?Ile?Glu?Gln?Met?Ile?Glu?Glu?Ala?Asn?Leu?65??????????????????70??????????????????75??????????????????80aaa?acc?att?aaa?ggt?ata?tta?aat?gga?gat?ata?caa?gaa?att?aaa?gga????288Lys?Thr?Ile?Lys?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly
85??????????????????90??????????????????95aaa?atg?gac?cat?gtg?aaa?tct?atg?cta?gag?aat?tct?cct?ggc?agc?cag????336Lys?Met?Asp?His?Val?Lys?Ser?Met?Leu?Glu?Asn?Ser?Pro?Gly?Ser?Gln
100?????????????????105?????????????????110gaa?agc?cat?gat?gct?tat?atg?ttt?ctg?gca?agg?ttt?ttg?gtc?agt?att????384Glu?Ser?His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Phe?Leu?Val?Ser?Ile
115?????????????????120?????????????????125gat?gaa?aaa?ttc?aaa?tct?ttc?gat?gat?aga?aca?aat?tat?caa?att?ctt????432Asp?Glu?Lys?Phe?Lys?Ser?Phe?Asp?Asp?Arg?Thr?Asn?Tyr?Gln?Ile?Leu
130?????????????????135?????????????????140ccc?atg?tac?acg?aat?aca?att?atg?tta?caa?gcg?cct?tat?tgg?aaa?atg????480Pro?Met?Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met145?????????????????150?????????????????155?????????????????160ggc?atc?gaa?aag?aaa?gag?gat?atc?ggt?tta?acc?gat?att?gaa?gtt?ggt????528Gly?Ile?Glu?Lys?Lys?Glu?Asp?Ile?Gly?Leu?Thr?Asp?Ile?Glu?Val?Gly
165?????????????????????170?????????????????175gaa?tta?aaa?gaa?ctt?atc?gat?aaa?tta?tat?act?aaa?tca?tat?gat?tat????576Glu?Leu?Lys?Glu?Leu?Ile?Asp?Lys?Leu?Tyr?Thr?Lys?Ser?Tyr?Asp?Tyr
180??????????????????????185?????????????????190atc?aat?aat?acg?tat?aat?cgt?gaa?tat?aat?aat?gca?atc?aat?acg?tca????624Ile?Asn?Asn?Thr?Tyr?Asn?Arg?Glu?Tyr?Asn?Asn?Ala?Ile?Asn?Thr?Ser
195?????????????????????200?????????????????205acc?gca?gag?agt?atc?acc?aat?aat?tta?ttg?tct?gtc?aga?gga?tat?tgt????672Thr?Ala?Glu?Ser?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys
210?????????????????215?????????????????220tta?tta?cat?ggt?tgt?gaa?tgc?ctt?gaa?gtt?att?gcg?cat?ata?caa?aac????720Leu?Leu?His?Gly?Cys?Glu?Cys?Leu?Glu?Val?Ile?Ala?His?Ile?Gln?Asn225?????????????????230?????????????????235?????????????????240aat?agt?ctt?gat?aaa?ggc?ttc?tac?cct?aaa?acg?atc?agc?tat?tcg?agt????768Asn?Ser?Leu?Asp?Lys?Gly?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Ser
245?????????????????250?????????????????255gtt?ttc?gat?cgt?cct?aca?aac?aaa?atg?aga?att?cag?gcg?ctt?aca?gaa????816Val?Phe?Asp?Arg?Pro?Thr?Asn?Lys?Met?Arg?Ile?Gln?Ala?Leu?Thr?Glu
260?????????????????265?????????????????270gat?gac?caa?atg?caa?gaa?ccg?ttc?aaa?cct?tct?ttc?gtc?aat?ggt?caa????864Asp?Asp?Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ser?Phe?Val?Asn?Gly?Gln
275?????????????????280?????????????????285tat?aat?aaa?ata?aaa?tca?ttg?gag?ggt?tat?gtc?aca?agg?atc?ggc?aat????912Tyr?Asn?Lys?Ile?Lys?Ser?Leu?Glu?Gly?Tyr?Val?Thr?Arg?Ile?Gly?Asn
290?????????????????295?????????????????300gcc?ccc?cga?gtc?ggc?gga?att?aaa?atc?aca?ttt?gaa?aac?aac?gca?tct????960Ala?Pro?Arg?Val?Gly?Gly?Ile?Lys?Ile?Thr?Phe?Glu?Asn?Asn?Ala?Ser305?????????????????310?????????????????315?????????????????320tat?act?ctt?ggc?act?gta?act?tca?gaa?aca?acc?tct?att?gaa?ctc?aat????1008Tyr?Thr?Leu?Gly?Thr?Val?Thr?Ser?Glu?Thr?Thr?Ser?Ile?Glu?Leu?Asn
325?????????????????330?????????????????335gag?agt?gtt?ata?acc?agc?ata?gaa?gtg?tgg?gga?gag?tgg?tgc?cgt?tga????1056Glu?Ser?Val?Ile?Thr?Ser?Ile?Glu?Val?Trp?Gly?Glu?Trp?Cys?Arg
340 345 350＜210〉11＜211〉351＜212〉PRT＜213〉Podbielniak Xenorhabdus＜400〉11Met Asn Asn Ser Pro Met Asn Asp Gln Leu Ser Thr Ala Pro Tyr Ser 15 10 15Ile Ser Thr Pro Ash Tyr Glu Trp Asp Met Ser Ser Ile Ile Lys Asp
20??????????????????25??????????????????30Ala?Ile?Ile?Gly?Gly?Ile?Gly?Phe?Ile?Pro?Gly?Pro?Gly?Pro?Ala?Ile
35??????????????????40??????????????????45Ser?Phe?Leu?Leu?Gly?Leu?Phe?Trp?Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp
50??????????????????55??????????????????60Asp?Gln?Ile?Leu?Gln?Lys?Ile?Glu?Gln?Met?Ile?Glu?Glu?Ala?Asn?Leu?65??????????????????70??????????????????75??????????????????80Lys?Thr?Ile?Lys?Gly?Ile?Leu?Asn?Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly
85??????????????????90??????????????????95Lys?Met?Asp?His?Val?Lys?Ser?Met?Leu?Glu?Asn?Ser?Pro?Gly?Ser?Gln
100?????????????????105?????????????????110Glu?Ser?His?Asp?Ala?Tyr?Met?Phe?Leu?Ala?Arg?Phe?Leu?Val?Ser?Ile
115?????????????????120?????????????????125Asp?Glu?Lys?Phe?Lys?Ser?Phe?Asp?Asp?Arg?Thr?Asn?Tyr?Gln?Ile?Leu
130?????????????????135?????????????????140Pro?Met?Tyr?Thr?Asn?Thr?Ile?Met?Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met145?????????????????150?????????????????155?????????????????160Gly?Ile?Glu?Lys?Lys?Glu?Asp?Ile?Gly?Leu?Thr?Asp?Ile?Glu?Val?Gly
165?????????????????170?????????????????175Glu?Leu?Lys?Glu?Leu?Ile?Asp?Lys?Leu?Tyr?Thr?Lys?Ser?Tyr?Asp?Tyr
180?????????????????185?????????????????190Ile?Asn?Asn?Thr?Tyr?Asn?Arg?Glu?Tyr?Asn?Asn?Ala?Ile?Asn?Thr?Ser
195??????????????????200?????????????????205Thr?Ala?Glu?Ser?Ile?Thr?Asn?Asn?Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys
210?????????????????215?????????????????220Leu?Leu?His?Gly?Cys?Glu?Cys?Leu?Glu?Val?Ile?Ala?His?Ile?Gln?Asn225?????????????????230?????????????????235?????????????????240Asn?Ser?Leu?Asp?Lys?Gly?Phe?Tyr?Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Ser
245?????????????????250?????????????????255Val?Phe?Asp?Arg?Pro?Thr?Asn?Lys?Met?Arg?Ile?Gln?Ala?Leu?Thr?Glu
260?????????????????265?????????????????270Asp?Asp?Gln?Met?Gln?Glu?Pro?Phe?Lys?Pro?Ser?Phe?Val?Asn?Gly?Gln
275?????????????????280?????????????????285Tyr?Asn?Lys?Ile?Lys?Ser?Leu?Glu?Gly?Tyr?Val?Thr?Arg?Ile?Gly?Asn
290?????????????????295?????????????????300Ala?Pro?Arg?Val?Gly?Gly?Ile?Lys?Ile?Thr?Phe?Glu?Asn?Asn?Ala?Ser305?????????????????310?????????????????315?????????????????320Tyr?Thr?Leu?Gly?Thr?Val?Thr?Ser?Glu?Thr?Thr?Ser?Ile?Glu?Leu?Asn
325?????????????????330?????????????????335Glu?Ser?Val?Ile?Thr?Ser?Ile?Glu?Val?Trp?Gly?Glu?Trp?Cys?Arg
340 345 350＜210〉12＜211〉408＜212〉DNA＜213〉luminous smooth rod bacterium＜220〉＜221〉CDS＜222〉(1) .. (405)＜223〉orf1＜400〉12atg att aca atc aat atc act ggt gat aat gta aga gtt aat aac aat 48Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn, 15 10 15ata gca aca gaa acc gac ctc caa aat aca cct gct tca gca ccc tta 96Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu of pCIB9383-21
20??????????????????25??????????????????30tca?att?att?aat?ttt?agg?gat?atg?aca?ata?gaa?cct?cat?tca?tct?gtt????144Ser?Ile?Ile?Asn?Phe?Arg?Asp?Met?Thr?Ile?Glu?Pro?His?Ser?Ser?Val
35??????????????????40??????????????????45gag?gcg?ata?aga?acc?gat?aca?ccg?att?att?cct?gaa?tca?cga?cca?aat????192Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn
50??????????????????55??????????????????60tac?tat?gtt?gct?aat?tct?ggc?ccg?gcc?tca?tca?gtc?aga?gct?gtt?ttc????240Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80tat?tgg?tcc?cac?tct?ttt?aca?tca?gaa?tgg?ttt?gaa?tct?tcc?tct?att????288Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile
85??????????????????90??????????????95att?gta?aaa?gca?ggc?gaa?gac?gga?gtc?tta?cat?tca?ccg?ggt?aat?tct????336Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly?Val?Leu?His?Ser?Pro?Gly?Asn?Ser
100?????????????????105?????????????????110tta?tat?tac?agc?aag?gtt?gta?att?tat?aac?gat?aca?gac?aaa?cgt?gct????384Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125ttt?gtt?acc?ggc?tac?aat?cta?taa????????????????????????????????????408Phe?Val?Thr?Gly?Tyr?Asn?Leu
130 135＜210〉13＜211〉135＜212〉PRT＜213〉luminous smooth rod bacterium＜400〉13Met Ile Thr Ile Asn Ile Thr Gly Asp Asn Val Arg Val Asn Asn Asn, 15 10 15Ile Ala Thr Glu Thr Asp Leu Gln Asn Thr Pro Ala Ser Ala Pro Leu
20??????????????????25??????????????????30Ser?Ile?Ile?Asn?Phe?Arg?Asp?Met?Thr?Ile?Glu?Pro?His?Ser?Ser?Val
35??????????????????40??????????????????45Glu?Ala?Ile?Arg?Thr?Asp?Thr?Pro?Ile?Ile?Pro?Glu?Ser?Arg?Pro?Asn
50??????????????????55??????????????????60Tyr?Tyr?Val?Ala?Asn?Ser?Gly?Pro?Ala?Ser?Ser?Val?Arg?Ala?Val?Phe?65??????????????????70??????????????????75??????????????????80Tyr?Trp?Ser?His?Ser?Phe?Thr?Ser?Glu?Trp?Phe?Glu?Ser?Ser?Ser?Ile
85??????????????????90??????????????????95Ile?Val?Lys?Ala?Gly?Glu?Asp?Gly?Val?Leu?His?Ser?Pro?Gly?Asn?Ser
100?????????????????105?????????????????110Leu?Tyr?Tyr?Ser?Lys?Val?Val?Ile?Tyr?Asn?Asp?Thr?Asp?Lys?Arg?Ala
115?????????????????120?????????????????125Phe?Val?Thr?Gly?Tyr?Asn?Leu
130 135＜210〉14＜211〉1320＜212〉DNA＜213〉luminous smooth rod bacterium＜220〉＜221〉CDS＜222〉(1) .. (1317)＜223〉the JHE-sample orf2＜400〉14atg aat aat gaa ccg atg aat act aat gaa tca caa gct tca gag ata 48Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile, 15 10 15gta ccc tca atg aat gaa tct ata tta aat gaa tct ata tta aat gaa 96Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu of pCIB9383-21
20??????????????????25??????????????????30tct?ata?tta?gca?gca?cct?tat?tca?att?tct?aca?cct?aat?tat?gaa?tgg????144Ser?Ile?Leu?Ala?Ala?Pro?Tyr?Ser?Ile?Ser?Thr?Pro?Asn?Tyr?Glu?Trp
35??????????????????40??????????????????45gat?atg?tca?tca?ata?ata?aaa?gat?gcc?att?att?ggt?ggt?ata?ggc?ttt????192Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp?Ala?Ile?Ile?Gly?Gly?Ile?Gly?Phe
50??????????????????55??????????????????60att?cct?ggt?ccg?ggc?tca?gca?ata?tca?ttt?ttg?tta?ggg?tta?ttt?tgg????240Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile?Ser?Phe?Leu?Leu?Gly?Leu?Phe?Trp?65??????????????????70??????????????????75??????????????????80cca?caa?caa?acc?gac?aat?act?tgg?gag?caa?att?ctc?caa?aaa?gta?gaa????288Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp?Glu?Gln?Ile?Leu?Gln?Lys?Val?Glu
85??????????????????90??????????????????95caa?atg?atc?gag?caa?gcc?aat?ctc?aaa?act?att?caa?gga?ata?ttg?aac????336Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu?Lys?Thr?Ile?Gln?Gly?Ile?Leu?Asn
100?????????????????105?????????????????110ggc?gat?ata?caa?gaa?att?aaa?ggc?aaa?atg?gaa?cat?gtg?caa?ttc?atg????384Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly?Lys?Met?Glu?His?Val?Gln?Phe?Met
115?????????????????120?????????????????125cta?gaa?tcc?tca?cct?ggc?act?caa?gaa?agc?cat?gac?gca?tac?atg?ttt????432Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln?Glu?Ser?His?Asp?Ala?Tyr?Met?Phe
130?????????????????135?????????????????140ctg?gcg?aga?tat?ctg?gtc?agt?ata?gac?gaa?aaa?ttc?aag?tct?ttt?gat????480Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile?Asp?Glu?Lys?Phe?Lys?Ser?Phe?Asp145?????????????????150?????????????????155?????????????????160aac?aaa?aca?aat?tat?caa?att?ctt?ccc?atg?tat?acc?aat?acg?att?atg????528Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu?Pro?Met?Tyr?Thr?Asn?Thr?Ile?Met
165?????????????????170?????????????????175tta?caa?gcc?cct?tat?tgg?aaa?atg?ggt?ata?gag?aga?aaa?gat?gag?ata????576Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met?Gly?Ile?Glu?Arg?Lys?Asp?Glu?Ile
180?????????????????185?????????????????190aaa?cta?aca?gat?ata?gaa?gtt?aat?gaa?tta?aaa?gag?ctg?ata?gga?aaa????624Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn?Glu?Leu?Lys?Glu?Leu?Ile?Gly?Lys
195?????????????????200?????????????????205tta?tct?acc?agc?gcc?gat?aaa?tat?att?cat?gat?gtc?tat?act?cgt?gaa????672Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr?Ile?His?Asp?Val?Tyr?Thr?Arg?Glu
210?????????????????215?????????????????220tat?gat?aat?gcg?atg?aac?act?tca?aca?gca?gca?aat?atc?acc?aat?aat????720Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser?Thr?Ala?Ala?Asn?Ile?Thr?Asn?Asn225?????????????????230?????????????????235?????????????????240tta?tta?tct?gta?aga?ggc?tat?tgt?tta?tta?cat?ggt?tta?gaa?tgt?ctc????768Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys?Leu?Leu?His?Gly?Leu?Glu?Cys?Leu
245?????????????????250?????????????????255gaa?gtc?att?aac?cat?ata?caa?aat?aat?agc?ctt?gag?caa?agt?ttt?tat????816Glu?Val?Ile?Asn?His?Ile?Gln?Asn?Asn?Ser?Leu?Glu?Gln?Ser?Phe?Tyr
260?????????????????265?????????????????270cct?aaa?act?atc?agc?tac?tcc?acc?gta?ttc?gat?cgc?cag?aca?aat?aaa????864Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr?Val?Phe?Asp?Arg?Gln?Thr?Asn?Lys
275?????????????????280?????????????????285aca?agg?gtt?caa?gcc?ctg?aca?gaa?gac?gat?caa?atg?caa?gag?cca?ttc????912Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu?Asp?Asp?Gln?Met?Gln?Glu?Pro?Phe
290?????????????????295?????????????????300aag?cct?gct?tta?att?aat?ggg?aag?tac?aac?aaa?ata?aaa?tca?ttg?att????960Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys?Tyr?Asn?Lys?Ile?Lys?Ser?Leu?Ile305?????????????????310?????????????????315?????????????????320ggg?tat?gta?caa?aga?atc?gga?aac?gca?ccc?aga?gtt?gga?ggc?att?aaa????1008Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn?Ala?Pro?Arg?Val?Gly?Gly?Ile?Lys
325?????????????????330?????????????????335gtc?aca?ttt?gca?aac?gat?gca?tct?tat?acc?ctc?ggt?aca?gta?act?tca????1056Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser?Tyr?Thr?Leu?Gly?Thr?Val?Thr?Ser
340?????????????????345?????????????????350gaa?gta?aac?tca?att?gaa?ctg?aat?gac?agc?gtt?ata?acc?agc?ctg?gaa????1104Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn?Asp?Ser?Val?Ile?Thr?Ser?Leu?Glu
355?????????????????360?????????????????365gta?tgg?gga?aat?ggc?gct?gtt?gat?gag?gca?ttc?ttt?aca?tta?agt?gac????1152Val?Trp?Gly?Asn?Gly?Ala?Val?Asp?Glu?Ala?Phe?Phe?Thr?Leu?Ser?Asp
370?????????????????375?????????????????380gga?cgt?caa?ttt?agg?ctt?ggc?caa?cgc?tat?gcc?agt?aac?tat?aga?aaa????1200Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln?Arg?Tyr?Ala?Ser?Asn?Tyr?Arg?Lys385?????????????????390?????????????????395?????????????????400tat?gct?gtc?gat?aac?cac?tat?att?tca?gga?ttg?tac?tta?gcc?agt?gat????1248Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile?Ser?Gly?Leu?Tyr?Leu?Ala?Ser?Asp
405?????????????????410?????????????????415gaa?cct?tca?ttg?gca?ggt?caa?gca?gca?ggc?att?gca?gtt?tca?tac?cat????1296Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala?Ala?Gly?Ile?Ala?Val?Ser?Tyr?His
420?????????????425?????????????????430atg?ata?gct?gat?aaa?aaa?tca?tag????????????????????????????????????1320Met?Ile?Ala?Asp?Lys?Lys?Ser
435＜210〉15＜211〉439＜212〉PRT＜213〉luminous smooth rod bacterium＜400〉15Met Asn Asn Glu Pro Met Asn Thr Asn Glu Ser Gln Ala Ser Glu Ile, 15 10 15Val Pro Ser Met Asn Glu Ser Ile Leu Asn Glu Ser Ile Leu Asn Glu
20??????????????????25??????????????????30Ser?Ile?Leu?Ala?Ala?Pro?Tyr?Ser?Ile?Ser?Thr?Pro?Asn?Tyr?Glu?Trp
35??????????????????40??????????????????45Asp?Met?Ser?Ser?Ile?Ile?Lys?Asp?Ala?Ile?Ile?Gly?Gly?Ile?Gly?Phe
50??????????????????55??????????????????60Ile?Pro?Gly?Pro?Gly?Ser?Ala?Ile?Ser?Phe?Leu?Leu?Gly?Leu?Phe?Trp?65??????????????????70??????????????????75??????????????????80Pro?Gln?Gln?Thr?Asp?Asn?Thr?Trp?Glu?Gln?Ile?Leu?Gln?Lys?Val?Glu
85??????????????????90??????????????????95Gln?Met?Ile?Glu?Gln?Ala?Asn?Leu?Lys?Thr?Ile?Gln?Gly?Ile?Leu?Asn
100?????????????????105?????????????????110Gly?Asp?Ile?Gln?Glu?Ile?Lys?Gly?Lys?Met?Glu?His?Val?Gln?Phe?Met
115?????????????????120?????????????????125Leu?Glu?Ser?Ser?Pro?Gly?Thr?Gln?Glu?Ser?His?Asp?Ala?Tyr?Met?Phe
130?????????????????135?????????????????140Leu?Ala?Arg?Tyr?Leu?Val?Ser?Ile?Asp?Glu?Lys?Phe?Lys?Ser?Phe?Asp145?????????????????150?????????????????155?????????????????160Asn?Lys?Thr?Asn?Tyr?Gln?Ile?Leu?Pro?Met?Tyr?Thr?Asn?Thr?Ile?Met
165?????????????????170?????????????????175Leu?Gln?Ala?Pro?Tyr?Trp?Lys?Met?Gly?Ile?Glu?Arg?Lys?Asp?Glu?Ile
180?????????????????185?????????????????190Lys?Leu?Thr?Asp?Ile?Glu?Val?Asn?Glu?Leu?Lys?Glu?Leu?Ile?Gly?Lys
195?????????????????200?????????????????205Leu?Ser?Thr?Ser?Ala?Asp?Lys?Tyr?Ile?His?Asp?Val?Tyr?Thr?Arg?Glu
210?????????????????215?????????????????220Tyr?Asp?Asn?Ala?Met?Asn?Thr?Ser?Thr?Ala?Ala?Asn?Ile?Thr?Asn?Asn225?????????????????230?????????????????235?????????????????240Leu?Leu?Ser?Val?Arg?Gly?Tyr?Cys?Leu?Leu?His?Gly?Leu?Glu?Cys?Leu
245?????????????????250?????????????????255Glu?Val?Ile?Asn?His?Ile?Gln?Asn?Asn?Ser?Leu?Glu?Gln?Ser?Phe?Tyr
260?????????????????265?????????????????270Pro?Lys?Thr?Ile?Ser?Tyr?Ser?Thr?Val?Phe?Asp?Arg?Gln?Thr?Asn?Lys
275?????????????????280?????????????????285Thr?Arg?Val?Gln?Ala?Leu?Thr?Glu?Asp?Asp?Gln?Met?Gln?Glu?Pro?Phe
290?????????????????295?????????????????300Lys?Pro?Ala?Leu?Ile?Asn?Gly?Lys?Tyr?Asn?Lys?Ile?Lys?Ser?Leu?Ile305?????????????????310?????????????????315?????????????????320Gly?Tyr?Val?Gln?Arg?Ile?Gly?Asn?Ala?Pro?Arg?Val?Gly?Gly?Ile?Lys
325?????????????????330?????????????????335Val?Thr?Phe?Ala?Asn?Asp?Ala?Ser?Tyr?Thr?Leu?Gly?Thr?Val?Thr?Ser
340?????????????????345?????????????????350Glu?Val?Asn?Ser?Ile?Glu?Leu?Asn?Asp?Ser?Val?Ile?Thr?Ser?Leu?Glu
355?????????????????360?????????????????365Val?Trp?Gly?Asn?Gly?Ala?Val?Asp?Glu?Ala?Phe?Phe?Thr?Leu?Ser?Asp
370?????????????????375?????????????????380Gly?Arg?Gln?Phe?Arg?Leu?Gly?Gln?Arg?Tyr?Ala?Ser?Asn?Tyr?Arg?Lys385?????????????????390?????????????????395?????????????????400Tyr?Ala?Val?Asp?Asn?His?Tyr?Ile?Ser?Gly?Leu?Tyr?Leu?Ala?Ser?Asp
405?????????????????410?????????????????415Glu?Pro?Ser?Leu?Ala?Gly?Gln?Ala?Ala?Gly?Ile?Ala?Val?Ser?Tyr?His
420?????????????????425?????????????????430Met?Ile?Ala?Asp?Lys?Lys?Ser
435

Claims (34)

1. isolated nucleic acid molecule comprises:
(a) a kind of basically similar in appearance to the nucleotide sequence that is selected from following nucleotide sequence: the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; Or
(b) with the nucleotide sequence of (a) nucleotide sequence isocoding;
The expression of wherein said nucleic acid molecule produces at least a toxin that anti-insect active is arranged.
2. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence with basically similar in appearance to the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, or the nucleotide sequence isocoding of SEQ ID NO:14.
3. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence is basically similar in appearance to the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
4. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence coded SEQ ID NO:2,3,5,7,9,11,13 and 15 the aminoacid sequence of being selected from.
5. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence contains the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
6. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence be basically similar in appearance to the Nucleotide 569-979 of SEQ ID NO:1, SEQ ID NO:4, SEQID NO:8, or SEQ ID NO:12.
7. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence coded SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:9, or the aminoacid sequence shown in the SEQ ID NO:13.
8. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence be basically similar in appearance to the Nucleotide 1045-2334 of SEQ ID NO:1, SEQ ID NO:6, SEQID NO:10, or SEQ ID NO:14.
9. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence coded SEQ ID NO:3, SEQ ID NO:7, the aminoacid sequence shown in SEQ ID NO11 and the SEQ ID NO:15.
10. isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence contain the about 3.0 kb dna fragmentations that are included among the pCIB9369 (NRRL B-21883).
11. isolated nucleic acid molecule according to claim 1, wherein said toxin have anti-small cabbage moth activity.
12. isolated nucleic acid molecule, contain 20 base pair nucleotide segments that on sequence, are equal to 20 the base pair nucleotide segments of successive that are selected from following nucleotide sequence: the Nucleotide 569-979 of SEQ ID NO:1, the Nucleotide 1045-2334 of SEQ ID NO1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14, the expression of wherein said nucleic acid molecule produces at least a toxin that anti-insect active is arranged.
13. a mosaic gene contains the allogeneic promoter sequence on the nucleic acid molecule that is connected to claim 1 or claim 12 effectively.
14. recombinant vectors that contains the mosaic gene of claim 13.
15. host cell that contains the mosaic gene of claim 13.
16. according to the host cell of claim 15, it is a bacterial cell.
17. according to the host cell of claim 15, it is a yeast cell.
18. according to the host cell of claim 15, it is a vegetable cell.
19. plant that contains the vegetable cell of claim 18.
20. according to the plant of claim 18, it is a corn.
21. toxin that produces by the expression of the dna molecular of claim 1 or 12.
22. according to the toxin of claim 21, wherein said toxin has anti-small cabbage moth activity.
23. according to the toxin of claim 21, wherein said toxin produces by the coli strain of called after NRRL registration number B-21883.
24. according to the toxin of claim 21, wherein said toxin contains and is selected from SEQ IDNO:2, and 3,5,7,9,11,13,15 aminoacid sequence.
25. according to the toxin of claim 24, wherein said toxin contains and is selected from SEQ IDNO:2,5,9 and 13 aminoacid sequence.
26. according to the toxin of claim 24, wherein said toxin contains and is selected from SEQ IDNO:3,7,11 and 15 aminoacid sequence.
27. the composition of the toxin of a claim 21 that contains insecticidal effective dose.
28. a production has the method for the toxin of anti-insect active, comprising:
(a) obtain host cell as claimed in claim 15; And
(b) express nucleic acid sequence in described cell, it produces at least a toxin that anti-insect active is arranged.
29. a method that produces insect-resistant plants comprises importing claim 1 or 12 described nucleic acid molecule in described plant, wherein said nucleic acid molecule can be expressed with control insect significant quantity in described plant.
30. according to the method for claim 29, insect wherein is a small cabbage moth.
31. a method of preventing and treating insect comprises the described toxin of the claim 21 of insect effective dosage.
32. according to the method for claim 31, insect wherein is a small cabbage moth.
33. according to the method for claim 32, toxin wherein is administered orally in insect.
34. the method for mutagenesis claim 1 or the described nucleic acid molecule of claim 12, nucleic acid molecule wherein is cut into the colony of the double-stranded random fragment of required size, comprising:
(a) add one or more lists-or double chain oligonucleotide in the colony of double-stranded random fragment, wherein said oligonucleotide respectively contain one with the identity of double-stranded template polynucleotide zone and a heterology zone;
(b) the double-stranded random fragment of sex change generation and the mixture of oligonucleotide become single-chain fragment;
(c) form under the segmental paired condition of annealed in of the annealing of the described single-chain fragment of generation in described same district, the colony of the single-chain fragment that produces with the polysaccharase incubation, it is enough that described identity district starts duplicating of another for a member of a centering, has therefore formed a kind of double-stranded polynucleotide of mutagenesis; And
(d) repeat at least two the further circulations of the second and the 3rd step, wherein the mixture that further round-robin produced in the 2nd step comprise from the double-stranded polynucleotide of mutagenesis in the 3rd step an of round-robin, and further circulation has wherein formed the double-stranded polynucleotide of further mutagenesis.
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