CN106749566A - The method that insecticidal proteins are combined and its manage insect-resistant - Google Patents
The method that insecticidal proteins are combined and its manage insect-resistant Download PDFInfo
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- CN106749566A CN106749566A CN201611039248.4A CN201611039248A CN106749566A CN 106749566 A CN106749566 A CN 106749566A CN 201611039248 A CN201611039248 A CN 201611039248A CN 106749566 A CN106749566 A CN 106749566A
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- China
- Prior art keywords
- albumen
- cry1ac
- cry2ab
- ostrinia furnacalis
- resistance
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
Abstract
The present invention relates to a kind of combination of insecticidal proteins and its method for management insect-resistant, the method for the management insect-resistant includes at least contacting Ostrinia furnacalis with Cry2Ab albumen and Cry1Ac albumen.The present invention effectively delays by using two kinds of Cry1Ac protein albumen and Cry2Ab albumen or prevents Ostrinia furnacalis from producing resistance, so as to realize the control or preventing and treating to Ostrinia furnacalis, plant is obtained protection and constant rate of production to a greater extent.
Description
Technical field
The present invention relates to a kind of combination of insecticidal proteins and its method for management insect-resistant, more particularly to it is applied in combination
The method of Cry1Ac albumen and Cry2Ab albumen to manage Ostrinia furnacalis resistance.
Background technology
Insect pest makes global production estimation suffer from huge economic loss, and peasant can all face every year due to
Insect pest causes the threat of production loss.Ostrinia furnacalis (Ostrinia furnacalis) popular name borer, belongs to Lepidoptera snout moth
Section, is the primary pest in China's Maize Production, and such insect's food-taking maize leaf is eaten into corn stem or fruit ear, is reduced
Photosynthesis, influences nutrient transport, also results in the generation of various secondary diseases, causes the corn underproduction to degrade.In recent years, with
The change of weather conditions, the change of cropping system, the increasing of corn planting density, the raising of fertilizer and water condition, Ostrinia furnacalis
Harm is increasingly aggravated.
By converting the ability that Bt (Bacillus thuringiensis) insecticidal protein gene produces insect-resistant plants
Revolution is brought to modern agriculture, and improves the importances and value of insecticidal proteins and its gene.Existing several Bt albumen are used
In produce insect-resistant genetically modified plants in, including Cry1Ab albumen, Cry1Ac albumen, Cry1F albumen, Cry2Ab albumen,
Cry3Bb albumen and Vip3A albumen etc..However as the popularization and application of genetically modified crops, insect will under lasting selection pressure
The resistance for the Bt albumen expressed in genetically modified plants, such resistance are evolved once producing and can not be effectively controlled
If, will undoubtedly limit the commercial value of the genetically modified crops kind containing Bt albumen.Only implement rational resistance management plan
Slightly, the lasting utilization of this state-of-the-art technology achievement can just be enable.
Generation in production in order to reduce insect-resistant mainly uses following several resistance management strategies：
1) deployment sanctuary.The purpose for setting sanctuary is the allele for keeping certain proportion non-resistance, with
Delay the generation of the insect of tolerance high dose insect resistance protein, but this kind of strategy can cause that peasant is relatively complicated in practical operation,
And total output is reduced to a certain extent.
2) dosage of single insect resistance protein is improved, this kind of tactful sustainability is poor, and insect is in lasting selection pressure
Under power resistance higher will be produced to single albumen.
3) different insect resistance proteins are used alternatingly or use altogether.In view of Bt albumen specifically combines the acceptor of sensitive insect, because
This superposition need to assess insect to different insect resistance proteins with the presence or absence of cross resistance using insect resistance protein, i.e., whether shared or competition is tied
Site is closed, with uncertainty higher, therefore be there is no so far on Cry2Ab albumen for Ostrinia furnacalis and Cry1Ac albumen
Whether there is the relevant report of cross resistance.
The content of the invention
It is an object of the invention to provide a kind of combination of insecticidal proteins and its method for management insect-resistant, not only propose first
The method of resistance Ostrinia furnacalis is controlled by Cry2Ab albumen and Cry1Ac protein combinations, and has effectively delayed Asia beautiful
Rice snout moth's larva produces resistance to single albumen.
To achieve the above object, the invention provides a kind of method for managing insect-resistant, methods described is included Asia
Corn borer at least contacts with Cry2Ab albumen and Cry1Ac albumen.
Further, the Cry2Ab albumen and Cry1Ac albumen be present at least produce the Cry2Ab albumen and
In the bacterium or genetically modified plants of Cry1Ac albumen, the Ostrinia furnacalis is by the bacterium or the genetically modified plants of ingesting
Tissue at least contacted with the Cry2Ab albumen and Cry1Ac albumen, Ostrinia furnacalis growth is suppressed after contact
And/or cause death, to realize managing the resistance of Ostrinia furnacalis.
Further, the Ostrinia furnacalis of resistance will be produced at least to be connect with the Cry2Ab albumen to Cry1Ac albumen
Touch, the Cry2Ab albumen is present in the bacterium or genetically modified plants at least producing the Cry2Ab albumen, described right
Cry1Ac albumen produce the Ostrinia furnacalis of resistance by the tissue of the ingest bacterium or the genetically modified plants at least with institute
State Cry2Ab albumen contact, after contact it is described to Cry1Ac albumen produce resistance Ostrinia furnacalis growth be suppressed and/or
Cause death, to realize the resistance of the management Ostrinia furnacalis to Cry1Ac albumen generation resistance.
Yet further, the Ostrinia furnacalis of resistance will be produced at least to be connect with the Cry1Ac albumen to Cry2Ab albumen
Touch, the Cry1Ac albumen is present in the bacterium or genetically modified plants at least producing the Cry1Ac albumen, described right
Cry2Ab albumen produce the Ostrinia furnacalis of resistance by the tissue of the ingest bacterium or the genetically modified plants at least with institute
State Cry1Ac albumen contact, after contact it is described to Cry2Ab albumen produce resistance Ostrinia furnacalis growth be suppressed and/or
Cause death, to realize the resistance of the management Ostrinia furnacalis to Cry2Ab albumen generation resistance.
In the above-mentioned technical solutions, the genetically modified plants are in any breeding time.
Further, the genetically modified plants are organized as root, blade, stalk, fruit, tassel, female fringe, flower pesticide or flower
Silk.
Further, the control to Ostrinia furnacalis harm plant is not because of plantation place and/or implantation time
Change and change.
Selectively, the plant is corn, wheat, sorghum, millet, paddy rice or soybean.
In the above-mentioned technical solutions, the amino acid sequence of the Cry2Ab albumen has SEQ ID NO:Amino shown in 1
Acid sequence.
Further, the amino acid sequence of the Cry2Ab albumen has SEQ ID NO:Amino acid sequence shown in 1.
Preferably, the amino acid sequence of the Cry1Ac albumen has SEQ ID NO:Amino acid sequence shown in 2.
Further, the nucleotide sequence of the Cry1Ac albumen has SEQ ID NO:Nucleotide sequence shown in 4.
To achieve the above object, present invention also offers a kind of method for controlling Ostrinia furnacalis, methods described includes will
Ostrinia furnacalis at least contacts with Cry2Ab albumen and Cry1Ac albumen, so as to realize the control to Ostrinia furnacalis.
Further, the Cry2Ab albumen and Cry1Ac albumen be present at least produce the Cry2Ab albumen and
In the bacterium or genetically modified plants of Cry1Ac albumen, the Ostrinia furnacalis is by the bacterium or the genetically modified plants of ingesting
Tissue at least contacted with the Cry2Ab albumen and Cry1Ac albumen, Ostrinia furnacalis growth is suppressed after contact
And/or cause death, to realize endangering Ostrinia furnacalis the control of plant.
Further, the Ostrinia furnacalis of resistance will be produced at least to be connect with the Cry2Ab albumen to Cry1Ac albumen
Touch, the Cry2Ab albumen is present in the bacterium or genetically modified plants at least producing the Cry2Ab albumen, described right
Cry1Ac albumen produce the Ostrinia furnacalis of resistance by the tissue of the ingest bacterium or the genetically modified plants at least with institute
State Cry2Ab albumen contact, after contact it is described to Cry1Ac albumen produce resistance Ostrinia furnacalis growth be suppressed and/or
Cause death, to realize endangering the Ostrinia furnacalis to Cry1Ac albumen generation resistance the control of plant.
Yet further, the Ostrinia furnacalis of resistance will be produced at least to be connect with the Cry1Ac albumen to Cry2Ab albumen
Touch, the Cry1Ac albumen is present in the bacterium or genetically modified plants at least producing the Cry1Ac albumen, described right
Cry2Ab albumen produce the Ostrinia furnacalis of resistance by the tissue of the ingest bacterium or the genetically modified plants at least with institute
State Cry1Ac albumen contact, after contact it is described to Cry2Ab albumen produce resistance Ostrinia furnacalis growth be suppressed and/or
Cause death, to realize endangering the Ostrinia furnacalis to Cry2Ab albumen generation resistance the control of plant.
In the above-mentioned technical solutions, the genetically modified plants are in any breeding time.
Further, the genetically modified plants are organized as root, blade, stalk, fruit, tassel, female fringe, flower pesticide or flower
Silk.
Preferably, the control to Ostrinia furnacalis harm plant is not because of the change of plantation place and/or implantation time
And change.
Selectively, the plant is corn, wheat, sorghum, millet, paddy rice or soybean.
In the above-mentioned technical solutions, the amino acid sequence of the Cry2Ab albumen has SEQ ID NO:Amino shown in 1
Acid sequence.
Further, the nucleotide sequence of the Cry2Ab albumen has SEQ ID NO:Nucleotide sequence shown in 3.
Preferably, the amino acid sequence of the Cry1Ac albumen has SEQ ID NO:Amino acid sequence shown in 2.
Further, the nucleotide sequence of the Cry1Ac albumen has SEQ ID NO:Nucleotide sequence shown in 4.
To achieve the above object, the invention provides a kind of Cry2Ab albumen and Cry1Ac protein combinations using preventing or
Delay Ostrinia furnacalis colony that the purposes of resistance is produced to Cry1Ac albumen or Cry2Ab albumen.
To achieve the above object, present invention also offers a kind of Cry2Ab albumen and Cry1Ac protein combinations using controlling
The purposes of the Ostrinia furnacalis colony of resistance is produced to Cry1Ac albumen or Cry2Ab albumen.
Heretofore described " contact ", refers to insect and/or insect touching, stops and/or feeding plant, plant device
Official, plant tissue or plant cell, the plant, plant organ, plant tissue or plant cell both can be its internal expression
Insecticidal proteins, can also be the plant, plant organ, plant tissue or plant cell surface have insecticidal proteins and/or
With the microorganism for producing insecticidal proteins.
" control " of the present invention and/or " preventing and treating " refer to Ostrinia furnacalis insect at least with Cry2Ab albumen and
Cry1Ac albumen is contacted, and the growth of Ostrinia furnacalis insect is suppressed and/or causes death after contact.Further, Asia is beautiful
Borer pest worm is at least contacted rice by feeding plant tissue with Cry2Ab albumen and Cry1Ac albumen, all or part of Asia after contact
The growth of corn borer insect is suppressed and/or causes death.Suppression refer to it is sub- lethal, i.e., it is not yet lethal but growth can be caused to send out
Educate, behavior, physiology, certain effect of the aspect such as biochemistry and tissue, slow and/or stopping of such as growing.Meanwhile, plant is in shape
Should be normal in state, and can under conventional approaches cultivate the consumption and/or generation for product.Additionally, containing coding
The plant of the control Ostrinia furnacalis insect of the polynucleotide sequence of Cry2Ab albumen and Cry1Ac albumen and/or vegetable seeds,
It is and non-transgenic under conditions of artificial infection Ostrinia furnacalis insect and/or Ostrinia furnacalis insect naturally-occurring harm
Compared to the plant injury with decrease, specific manifestation includes but is not limited to stalk resistance, and/or the raising for improving to WT lines
Kernel weight, and/or volume increase etc.." control " and/or " preventing and treating " of Cry2Ab albumen and Cry1Ac albumen to Ostrinia furnacalis
Effect can be it is self-existent, not because other " can be controlled " and/or the presence of the material of " preventing and treating " Ostrinia furnacalis insect and
Weaken and/or disappear.Specifically, genetically modified plants (polynucleotide sequence containing coding Cry2Ab albumen and Cry1Ac albumen)
Any tissue simultaneously and/or asynchronously, exist and/or produce, Cry2Ab albumen and Cry1Ac albumen and/or controllable Asia
Another material of continent corn borer insect, the then presence of another material neither influences Cry2Ab albumen and Cry1Ac albumen
" control " and/or " preventing and treating " effect to Ostrinia furnacalis, can not cause " control " and/or " preventing and treating " effect complete
And/or part is realized by another material, and it is unrelated with Cry2Ab albumen and Cry1Ac albumen.Under normal circumstances, big
Field, the process of Ostrinia furnacalis insect feeding plant tissue is of short duration and is difficult to observe with the naked eye, therefore, in artificial infection Asia
Under conditions of corn borer insect and/or Ostrinia furnacalis insect naturally-occurring harm, such as genetically modified plants (contain coding Cry2Ab
The polynucleotide sequence of albumen and Cry1Ac albumen) any tissue there is dead Ostrinia furnacalis insect, and/or thereon
Stop the growth Ostrinia furnacalis insect being suppressed, and/or the plant compared with non-transgenic WT lines with decrease
Thing damage, as realize the method for the present invention and/or purposes, i.e., by Ostrinia furnacalis insect at least with Cry2Ab albumen and
Cry1Ac albumen contacts to realize controlling the method and/or purposes of Ostrinia furnacalis insect.
Term " plant " includes whole plant, plant cell, plant organ, plant protoplast, plant can therefrom again
It is complete in raw plant cell tissue cultures, plant callus, vegetation bed (plant clumps) and plant or plant part
Whole plant cell, the plant part such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stalk, root, the tip of a root, flower
Medicine etc..
The genome of heretofore described plant, plant tissue or plant cell, refers to plant, plant tissue or plant
Intracellular any inhereditary material, and including nucleus and plastid and mitochondrial genomes.
Heretofore described " restructuring " refers to that generally can not be found in nature and therefore by manual intervention product
Raw DNA and/or the form of albumen and/or organism.This manual intervention can produce recombinant DNA molecules and/or recombinant plant.
" recombinant DNA molecules " are to be in other cases separate sequence section and obtain by two kinds of artificial combination, for example
Separate nucleic acid segment is operated by chemical synthesis or by genetic engineering technology.The technology for carrying out nucleic-acid manipulation is well-known
's.
Term " parent toxin " of the present invention or " toxin " or " Pesticidal toxins " refer to and encoded before there is any fracture in middle intestines
The initial translation product of the full-length gene of insecticidal proteins.Term " toxin " of the present invention or " minimum toxic fragment " are interpreted as desinsection
The part of albumen such as Cry2Ab or Cry1Ac albumen, its can by Trypsin Induced or by (targeted insect, for example
Ostrinia furnacalis) in midgut fluid hydrolysis and obtains and still with insecticidal activity.Generally on PAGE gel, Cry1 poison
Molecular weight of the element with about 60-65kD, Cry2A toxin has the molecular weight of about 50-58kD.
In the present invention, Cry2Ab albumen and Cry1Ac albumen are expressed in a kind of genetically modified plants.It is this more than one kind
Pesticidal toxins in same strain genetically modified plants co expression plant can be made to include and needed for expressing by genetic engineering
Gene is realized.In addition, a kind of plant (the 1st parent) can express Cry2Ab protein, second by genetic engineering procedure
Plant (the 2nd parent) can express Cry1Ac protein by genetic engineering procedure.Obtained by the 1st parent and the 2nd parents
The progeny plants of all genes for introducing the 1st parent and the 2nd parent must be expressed.
In the present invention, the genetically modified plants of the Cry1Ac albumen are produced to include but is not limited to TT51 transgenic paddy rice things
Part and/or the vegetable material comprising TT51 transgenic paddy rice events are (as described by CN100582223C and CN101302520B
), 223F-S21 transgenic rice lines and/or the vegetable material comprising 223F-S21 transgenic rice lines (such as exist
Described by CN103773759A), Mon15985 transgenic cotton events and/or comprising Mon15985 transgenic cotton events
Vegetable material (as described by CN101413028B), MON531 transgenic cotton events and/or comprising MON531 transgenosis
The vegetable material (as described by the non-control state application 00-342-01p of USDA APHIS) of cotton event, COT67B turn base
Vegetable material because of cotton event and/or comprising COT67B transgenic cotton events is (such as in the non-control state Shens of USDA APHIS
Please be described by 07-108-01p) or 3006-210-23 transgenic cotton events and/or comprising 3006-210-23 transgenosis
The vegetable material (as described by the non-control state application 03-036-02p of USDA APHIS) of cotton event.
In the present invention, the genetically modified plants of the Cry2Ab albumen are produced to include but is not limited to Mon89034 transgenosis jade
Rice event and/or the vegetable material (as described by CN101495635A) comprising Mon89034 transgenic corn events,
MON87751 transgenic soybean events and/or the vegetable material comprising MON87751 transgenic soybean events are (such as in USDA
Described by the non-control state application 13-337-01p of APHIS) or Mon15985 transgenic cotton events and/or comprising
The vegetable material (as described by CN101413028B) of Mon15985 transgenic cotton events.
Well-known to those skilled in the art, DNA typically exists with double chain form.In this arrangement, chain with
Another chain complementation, vice versa.Other complementary strands for generating DNA are replicated in plant due to DNA.So, present invention bag
Include the use to the polynucleotides and its complementary strand of example in sequence table." coding strand " that this area often uses refers to and antisense link
The chain of conjunction.In order to a chain of DNA is transcribed into a complementary strand of mRNA by marking protein in vivo, typical case, it is used as mould
Plate translates protein.MRNA is actually to be transcribed from " antisense " chain of DNA." having justice " or " coding " chain has a series of passwords
Son (codon is three nucleotides, and once reading three can produce specific amino acids), it can read as ORFs (ORF)
Read to form target protein or peptide.Present invention additionally comprises the RNA that the DNA with example has suitable function.
Any conventional nucleic acid hybridization or amplification method may be used to identify the presence of killing gene of the present invention.Nucleic acid point
Son or its fragment can carry out specific hybrid with other nucleic acid molecules in any case.In the present invention, if two nucleic acid
Molecule can form antiparallel double-strandednucleic acid structure, it is possible to say that the two nucleic acid molecules can carry out specificity to each other miscellaneous
Hand over.If two nucleic acid molecules show complete complementarity, one of nucleic acid molecules are claimed to be another nucleic acid molecules
" complement ".In the present invention, when each nucleotides of nucleic acid molecules and the corresponding nucleotides of another nucleic acid molecules
The mutual added time, then the two nucleic acid molecules are claimed to show " complete complementary ".If two nucleic acid molecules can be with enough stabilizations
Property phase mutual cross so that they anneal and are bonded to each other under the conditions of at least conventional " low strict ", then claim the two nucleic acid
Molecule is " minimum level is complementary ".Similarly, if two nucleic acid molecules can with enough stability phase mutual crosses so that
They anneal and are bonded to each other under the conditions of conventional " height is strict ", then claim the two nucleic acid molecules to have " complementarity ".From
It can be permission to deviate in complete complementary, as long as this not exclusively two molecules of prevention that deviate form duplex structures.In order to
Enable a nucleic acid molecules as primer or probe, it is only necessary to ensure that it has in sequence sufficiently complementary, to cause
The duplex structure of stabilization can be formed under the specific solvent and salinity that are used.
In the present invention, substantially homologous sequence is one section of nucleic acid molecules, and the nucleic acid molecules can under high stringency
There is specific hybrid with the complementary strand of another section of nucleic acid molecules for matching.Promote the suitable stringent condition of DNA hybridization, example
Such as, processed with 6.0 × sodium chloride/sodium citrate (SSC) about under the conditions of 45 DEG C, then with 2.0 × SSC under the conditions of 50 DEG C
Washing, these conditions are known to those skilled in the art.For example, the salinity in washing step can be selected from low tight
About 2.0 × the SSC of glazing bar part, 50 DEG C to high stringency about 0.2 × SSC, 50 DEG C.Additionally, the temperature in washing step
Condition can be increased to about 65 DEG C of high stringency from about 22 DEG C of the room temperature of Low stringency conditions.Temperature conditionss and salt are dense
Degree can all change, it is also possible to which one of holding is constant and another variable changes.Preferably, it is of the present invention
Stringent condition can be in 6 × SSC, 0.5%SDS solution, with SEQ ID NO at 65 DEG C:3 and SEQ ID NO:4 there is spy
Specific hybridization, then respectively washes film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.
Therefore, with anti-insect activity and under strict conditions with SEQ ID NO of the present invention:3 or SEQ ID NO:4 hybridization
Sequence is included in the invention.These sequences are homologous with sequence of the present invention at least about 40%-50%, about 60%, 65% or
70% is homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%th, 99% or bigger sequence homology.
Heretofore described gene and protein not only include specific exemplary sequence, also described specific including saving
The part of the insecticidal activity feature of the protein of example and/fragment (lack including including compared with full length protein and/or end
Lose), variant, mutant, the substituent protein of amino acid (have substitute), chimera and fusion protein." variant " or " become
It is different " refer to the nucleotide sequence for encoding same albumen or encoding the equivalent protein for having insecticidal activity." equivalent protein " refers to
With the albumen of the bioactivity that the albumen of claim has identical or essentially identical anti-lepidopteran insect pests.
Original DNA or egg that heretofore described DNA molecular or " fragment " of protein sequence or " truncation " are referred to
A part of Bai Xulie (nucleotides or amino acid) or its artificial reconstructed form (being for example adapted to the sequence of plant expression), foregoing sequence
The length of row there may be change, but length is enough to ensure that (coding) protein is insect toxins.
Gene variant can be built with modifier and readily using standard technique.For example, it is well known that manufacture point
The technology of mutation.Such as U.S. Patent number 5605793 is described to be reassemblied using DNA after random fracture and produces other molecules again
Multifarious method.The fragment of full-length gene can be manufactured using commercialization endonuclease, and can be according to standardization program
Use exonuclease.It is, for example possible to use enzyme such as Bal31 or direct mutagenesis cut off core from the end system of these genes
Thuja acid.The gene of encoding active fragment can also be obtained using various restriction enzymes.Can be directly obtained using protease
The active fragment of these toxin.
The present invention from B.t. separators and/or DNA library can derive equivalent protein and/or encode these equivalent proteins
Gene.There are various methods to obtain insecticidal proteins of the invention.It is, for example possible to use the desinsection that the present invention is disclosed and claimed
The antibody of albumen is identified and isolated from other albumen from protein mixture.Especially, antibody be probably by albumen it is most constant and with
The most different protein part of other Bt albumen causes.May then pass through immunoprecipitation, enzyme linked immunosorbent assay (ELISA) (ELISA)
Or western immunoblot methods exclusively identify the equivalent protein of activity characteristic using these antibody.This area standard can be used
Program readily prepares the antibody of the fragment of the albumen or equivalent protein or this albuminoid disclosed in the present invention.Then can be from micro-
The gene for encoding these albumen is obtained in biology.
Due to the Feng Yuxing of genetic codon, various different DNA sequence dnas can encode identical amino acid sequence.Produce
The alternative DNA sequence dna of the identical or essentially identical albumen of these codings is just in the technical merit of those skilled in the art.This
A little different DNA sequence dnas are included within the scope of the invention." substantially the same " sequence refers to have 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, lack
Lose, addition or insertion but do not influence the sequence of insecticidal activity substantially, also including retaining the fragment of insecticidal activity.
The substitution of amino acid sequence, missing or addition are the ordinary skill in the art in the present invention, preferably this amino acid
Change is turned to：Small characteristic changing, i.e., do not significantly affect the folding of albumen and/or the conserved amino acid substitution of activity；Small missing,
The normally about 1-30 missing of amino acid；Small amino or c-terminus extend, and for example aminoterminal extends a methionine residues；
Small connection peptide, e.g., from about 20-25 residue is long.
The example of conservative replacement is the substitution occurred in following amino acid group：Basic amino acid (such as arginine, lysine
And histidine), it is acidic amino acid (such as glutamic acid and aspartic acid), polar amino acid (such as glutamine, asparagine), hydrophobic
Acidic amino acid (such as leucine, isoleucine and valine), ArAA (such as phenylalanine, tryptophan and tyrosine), with
And small molecule amino acid (such as glycine, alanine, serine, threonine and methionine).Generally do not change given activity
Those 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors are in the art it is well known that and by for example, N.Neurath and R.L.Hill are 1979
What year new york academic publishing house (Academic Press) published《Protein》In be described.Most common exchange has
Ala/Ser, Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/
Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, and their opposite exchanges.
For a person skilled in the art it should be evident that this substitution can play an important role to molecular function
Region outside occur, and still produce active peptides.For by polypeptide of the invention, its activity is required and therefore selects not
Substituted amino acid residue, can be reflected according to methods known in the art, such as direct mutagenesis or alanine scanning mutagenesis
Determine (such as referring to Cunningham and Wells, 1989, Science 244：1081-1085).Latter technique is every in the molecule
Mutation, the anti-insect activity of detection gained mutating molecule, so that it is determined that to the molecular activity are introduced at one positively charged residue
For important amino acid residue.Substrate-enzyme interacting site can also be determined by the analysis of its three-dimensional structure, this
Three-dimensional structure can be determined by technologies such as nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling (referring to, such as de Vos, 1992,
Science 255：306-312；Smith etc., 1992, J.Mol.Biol 224:899-904；Wlodaver etc., 1992, FEBS
Letters 309：59-64).
Therefore, with SEQ ID NO:1 and/or SEQ ID NO:Amino acid sequence shown in 2 has the ammonia of certain homology
Base acid sequence is also included in the present invention.These sequences are typically larger than 78% with sequence similarities/homogeny of the present invention, preferably
More than 85%, more preferably greater than 90%, even more preferably more than 95%, and can be more than 99%.Can also basis
Homogeny and/or similarity scope particularly defines preferred polynucleotides of the invention and protein.For example with the present invention
The sequence of example has 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeny and/or similarity.
Term " cross resistance " of the present invention refers to a strain of insect due to identical resistance mechanism or similar action mechanism
Or similar chemical constitution, also produce anti-medicine from a kind of original medicament or a class medicament for other beyond selection medicament
The phenomenon of property.Particularly, " cross resistance " refers to other beyond the insecticidal proteins that select from original one in the present invention
Plant insecticidal proteins or a class insecticidal proteins also produce the phenomenon of resistance.The protein for being selected for insect-resistant management needs solely
Its insecticidal effect is on the spot played, so as to be assigned to second resistance of protein (i.e. to the resistance that a kind of protein is produced
For second protein is without cross tolerance or has relatively low cross tolerance).Such as resistant to " albumin A " insect populations pair
" protein B " is sensitive, then people are it may be concluded that albumin A and protein B can effectively postpone without cross tolerance and combinations thereof
To the resistance of single albumin A.
Term " resistance ratio " of the present invention refers to Bt resistance populations relative Bt sensitivity group of the same insecticidal proteins in insect
The multiple that the LC50 values of body are improved, i.e. resistance ratio=Bt resistant strains LC50 values/Bt sensitive strain LC50 values.
Used in the present invention, insect species colony is to insecticidal protein and/or expression insecticidal protein plant (plant
The insect populations are controlled or killed before thing) " generation resistance ", " having produced resistance " or " as resistance " refer to, with
It is firstly introduced the control effect that is caused to identical insect species when the insecticidal proteins and/or the plant and/or by identical insect
The production loss level of the plant kind caused is compared, detected in the plant weakened by the insect populations control effect and/
Or repetition the and significant production loss for causing.
Ostrinia furnacalis (Ostriniafurnacalis) and bollworm (HelicoverpaarmigeraHubner) although
Belong to Lepidoptera together, but Ostrinia furnacalis and bollworm are being biologically two species clearly, completely different, are at least deposited
In the following main distinction：1) Ostrinia furnacalis belongs to Pyralidae, genus heliothis Noctuidae.2) feeding habits are different.Ostrinia furnacalis is most normal
Harm corn, can endanger each position of milpa ground, including blade, tassel, stalk, fringe handle, cob, to various regions
Spring, summer, autumn sowing corn have harm in various degree, especially most heavy with Summer Maize；Bollworm is the important of harm cotton cotton buds and bolls phase
Borer pest, main moth food flower bud, flower, bell, also takes food tender leaf.3) distributed areas are different.Ostrinia furnacalis is distributed in Chinese east
Portion and southwestern Major Maize, sorghum producing region；And bollworm is widely distributed in China and all over the world, Chinese cotton region and vegetable cultivation
Qu Junyou occurs, and Huanghe valley cotton region, Yangtze river basin cotton region are aggrieved heavier；In recent years, Xinjiang cotton also happens occasionally.4) form
Feature is different.The ovum of Ostrinia furnacalis and bollworm, larva, pupa, the morphological feature of adult have larger difference.5) habit
It is different with pests occurrence rule.The generation of corn borer has significant difference with latitude：In 1 generation to the north of China, 45 ° of north latitude,
45 ° of -40 ° of 2 generations, 40 ° of -30 ° of 3 generations, 30 ° of -25 ° of 4 generations, 25 ° of -20 ° of 5-6 generations.Height above sea level is higher, and generation is fewer；In Sichuan Province
There is 2-4 generations within 1 year, temperature is high, height above sea level is low, and generation is more, it is in corn stem, cob or high generally with mature larva
Survived the winter in fine strain of millet, the stalk of sunflower, month next year 4-5 pupates, pupa was sprouted wings by 10 days or so.Adult nocturnalism, power of circling in the air is strong,
Have phototaxis, 5~10 days life-spans, like liftoff more than 50 centimetres, grow more luxuriant corn leaf back middle arteries both sides and lay eggs,
One female moth can lay eggs 350-700,3-5 days ovum phase；Corn borer be adapted under high temperature, super-humid conditions develop, winter temperature compared with
Height, natural enemy parasitic amount is few, is conducive to the breeding of corn borer, endangers heavier；Ovum phase arid, maize leaf curling, pieces of an egg are easily from leaf
The back side comes off and dead, endangers lighter.It is annual in Laizhou City of Shandong Province and the algebraically that bollworm occurs is different because of area because of the time
There are for 4 generations, the larva that grows up late September descends tree to bury successively, pupated more in 5-10cm deep soil near nursery stock or under weeds
Winter；The beginning of spring temperature go up more than 15 DEG C when start to sprout wings, the first tenday period of a month in late April to May are emergence peak period, and adult the first generation occurs and exist
Mid or late June, the second generation still has bollworm to go out in mid or late July, the third generation in mid or late August to early September to early October
Existing, adult has phototaxis, is dodged with spawning at night after emergence, and ovum dissipates and produces, and relatively disperses, and a female moth all one's life can lay eggs 500-
1000, reach as high as 2700, ovum fecund in leaf back, also have produce in front, plunger tip, petiole, tender stem or farming, it is miscellaneous
On other plants such as grass；Chorion habit is taken food after larvae hatch, newly hatched larvae has cluster to limit food habit, two or three, three or five
At face of blade or the back side, head is arranged to leaf margin, inwardly taken food from leaf margin, and as a result blade is eaten up, only surplus master pulse and petiole, or
Reticulate withered, cause cured leaf；1-2 instar larvaes along handle come downwards at ginkgo seedling terminal bud from side eat into food or along eat under at terminal bud into
Spray, causes top tip or top fascicled leaves dead, endangers extremely serious；Larva before 3 ages is less, relatively concentrates, with children
Worm grows and gradually disperses, and is increased into 4 age appetite, edible smooth blade, only surplus petiole；Larva 7-8 months cause harm and most contain；Cotton boll
Worm has the habit that transfer endangers, and a larva can endanger many seedlings；Old skin is let the hair grow the habit of shell under each instar larvae has food to fall to slough off
Property, certain difficulty is caused to discriminating worm age, worm age is irregular；The optimal temperature that bollworm occurs is 25-28 DEG C, relative humidity
70-90%；The second generation, the third generation are caused harm the most serious, and seriously piece insect density is up to 98/blinds, worm strain rate 60-70%,
Individually up to 100%, aggrieved blade influences leaf-making quantity 20%, Quality Down at least one grade, seedling growth to piece up to more than 1/3
Amount influence is very big.
Although summary, Ostrinia furnacalis belongs to Lepidoptera together with bollworm, but only in morphological feature and Damage habits
On there is all many differences, and the two affiliation is farther out, it is impossible to which mating produces offspring.Therefore, the two midgut epithelium
The acceptor combined from Bt toxin on cell membrane upper surface is also different.
Insect is not single to the resistance mechanism of Bt albumen, and Heckel (1994) etc. analyzes insect and Bt albumen is produced
The potential mechanism of resistance, it is believed that the generation of resistance is main relevant with following factor：1) dissolubility of Bt insecticidal proteins：Parent toxin is not
Can dissolving or dissolubility reduction；2) proteolytic of Bt parent toxins：Hydrolyze insufficient or excessive hydrolysis；3) Bt albumen and cell
The combination of acceptor on film：The combination of Bt albumen and acceptor is obstructed due to Reverse transcriptase, the primary structure of acceptor or two grades knot
The modification of structure changes, and causes Bt albumen to be reduced with the binding site of acceptor；4) formation empty on cell membrane：Cavity
Formation is obstructed or is blocked；5) repair of midgut epithelium；6) behavioral mechanism etc..Wherein toxin and acceptor combination energy on cell membrane
The change of power is key resistance mechanism.
When two kinds or more different Bt albumen share binding site in insect, they can not be provided for insect
The good combination of resistance management purpose.Under actual conditions, which kind of resistance management strategy is taken to have for different insects higher
Uncertainty.Two larger Bt albumen of amino acid sequence differences are likely in a certain insect species be combined with high-affinity
Common binding site, such as Cry1Ab and Cry1F albumen are in diamond-back moth (Plutella xylostella).And, it has been found that
Two kinds of albumen without shared binding site in an insect species can share combination in another insect species
Site, such as Fiuza et al. (1996) find Cry1Ac and Cry1Ba albumen in striped rice borer (Chilo suppressalis)
Shared binding site, and Ballester et al. (1999) has found that both the above albumen combines different binding sites in diamond-back moth.
In the present invention, the Cry2Ab albumen can have SEQ ID NO in sequence table:Amino acid sequence shown in 1；Institute
Stating Cry1Ac albumen can have SEQ ID NO in sequence table:Amino acid sequence shown in 2.Except comprising Cry2Ab albumen and
Outside the code area of Cry1Ac albumen, other elements, the code area of such as encoding transit peptides or encoding selection markers can be also included
Protein, the Cry2Ab albumen and the nucleotide sequence of Cry1Ac albumen that the present invention is provided can form structure by conventional meanses
Build body.
Additionally, comprising encode the construct of Cry2Ab albumen of the present invention and Cry1Ac albumen in plant can also with least
A kind of protein of encoding herbicide resistance gene is expressed together, and the herbicide resistance gene is included but is not limited to, phosphine oxamate
Resistant gene (such as bar genes, pat genes), phenmedipham resistant gene (such as pmph genes), Glyphosate resistance gene (such as EPSPS
Gene), Brominal (bromoxynil) resistant gene, sulfonylurea resistance gene, the resistant gene to herbicide Dalapon, to ammonia
The resistant gene of the resistant gene or glutamine synthetase inhibitor (such as PPT) of nitrile, thus obtain both have insecticidal activity high,
There are the genetically modified plants of Herbicid resistant again.
Heretofore described regulating and controlling sequence include but is not limited to promoter, transit peptides, terminator, enhancer, targeting sequencing,
Introne and other be operably connected to the regulatory sequence of the Cry2Ab albumen or the Cry1Ac albumen.
The promoter is effable promoter in plant, and described " effable promoter in plant " refers to ensure
The promoter that connected coded sequence is expressed in plant cell.Effable promoter can be composing type in plant
Promoter.The example of the promoter of constitutive expression in plant is instructed to include but is not limited to, from cauliflower mosaic virus
35S promoter, corn Ubi promoters, promoter of paddy rice GOS2 genes etc..Alternatively, effable promoter can in plant
It is the promoter of organizing specific, i.e., the promoter such as instructs the table of coded sequence in some tissues of plant in chlorenchyma
Its hetero-organization (can be tested by conventional RNA and be measured), such as PEP carboxylase promoters up to level higher than plant.Alternatively,
Effable promoter can be wound-induced promoter in plant.Wound-induced promoter instructs the expression pattern of wound-induced
Promoter refer to undergo machinery or when gnawing the wound for causing by insect when plant, the table of the coded sequence under promoter regulation
It is significantly increased up under the conditions of compared with normal growth.The example of wound-induced promoter is included but is not limited to, potato and tomato
Protease suppressor (pin I and pin II) and zein enzyme level gene (MPI) promoter.
The transit peptides (also known as secretory signal sequence or targeting sequencing) are to instruct transgene product to specific organelle
Or cellular compartment, for receptor protein, the transit peptides can be heterologous, for example, using encoding chloroplast transit peptide
Sequence targets chloroplaset, or utilizes ' KDEL ' to retain sequence targeting endoplasmic reticulum, or using barley plants agglutinin gene
CTPP targets vacuole.
The targeting sequencing is including but not limited to picornavirus targeting sequencing, such as EMCV targeting sequencings (encephalomyo-carditis disease
Malicious 5 ' noncoding regions)；Potyvirus leaders, such as MDMV (Maize Dwarf Mosaic Virus) targeting sequencing；Human immunity
Globular protein heavy-chain binding protein matter (BiP)；The coat protein mRNA's of alfalfa mosaic virus does not translate targeting sequencing (AMV
RNA4)；Tobacco mosaic virus (TMV) (TMV) targeting sequencing.
The enhancer is including but not limited to cauliflower mosaic virus (CaMV) enhancer, figwort mosaic virus (FMV) increase
Hadron, carnation weathering circovirus virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, Mirabilis jalapa mosaic virus
(MMV) enhancer, dama de noche tomato yellow leaf curl China virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV), duck plantar
Straw colour mottle virus (CoYMV) and peanut chlorisis streak mosaic virus (PCLSV) enhancer.
For monocotyledon is applied, the introne is including but not limited to corn hsp70 intrones, corn are general
Plain introne, Adh introne 1s, crose synthase intron or paddy rice Act1 intrones.For dicotyledon is applied, institute
Introne is stated including but not limited to CAT-1 intrones, pKANNIBAL intrones, PIV2 intrones and " super ubiquitin " are included
Son.
The terminator can be the suitable polyadenylation signal sequence worked in plant, including but not limit
In from the Polyadenylation of Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) gene
Signal sequence, the polyadenylation signal sequence from protease-inhibitor Ⅱ (pin II) gene, from pea
The polyadenylation signal sequence of ssRUBISCO E9 genes and the poly from alpha-tubulin (α-tubulin) gene
Polyadenylation signal sequence.
Heretofore described " effectively connection " represents the connection of nucleotide sequence, and the connection causes that a sequence can provide right
The function of being needed for linked sequence.Described in the present invention " effectively connection " can be by promoter and sequence phase interested
Even so that the transcription of the sequence interested is subject to the promoter to control and regulate and control.When sequential coding albumen interested and
Go for " effectively connection " expression during expression of the albumen：Promoter is connected with the sequence, and connected mode causes to obtain
Transcript efficient translation.If promoter is that transcript merges and wants to realize the albumen of coding with the connection of coded sequence
Expression when, the such connection of manufacture so that the first translation initiation codon is the starting of coded sequence in the transcript for obtaining
Codon.Alternatively, if promoter and the table that the connection of coded sequence is that the albumen for realizing coding is merged and wanted in translation
Up to when, the such connection of manufacture so that the first translation initiation codon and the promoter contained in 5 ' non-translated sequences are connected,
And connected mode causes that the relation of the translation opening code-reading frame of the translation product for the obtaining albumen desired with coding is to meet reading
Code frame.Can be included but is not limited to the nucleotide sequence of " effectively connection "：Sequence (the i.e. gene expression of gene expression function is provided
Element, such as promoter, 5 ' untranslated regions, introne, protein encoding regions, 3 ' untranslated regions, poly- putative adenylylation site and/
Or transcription terminator), sequence (i.e. T-DNA border sequences, the locus specificity recombinase of DNA transfers and/or integration function are provided
Recognition site, integrate enzyme recognition site), sequence (i.e. antibiotic resistance markers, the biosynthesis base of selectivity function are provided
Cause), the sequence of the label function that can score, sequence that is external or assisting series of operations in vivo (i.e. polylinker sequence, site are provided
Specific recombination sites) and sequence (the i.e. replication orgin of bacterium, autonomously replicating sequence, the centromere sequence of copy function are provided
Row).
It to crop pests is poisonous that heretofore described " desinsection " or " pest-resistant " refers to, so as to realize " control "
And/or " preventing and treating " crop pests.Preferably, described " desinsection " or " pest-resistant " refer to kill crop pests.More specifically, mesh
Mark insect is Ostrinia furnacalis insect.
Plant in the present invention, particularly corn, contain exogenous DNA in its genome, and the exogenous DNA includes coding
The nucleotide sequence of Cry1Ac albumen and Cry2Ab albumen, Ostrinia furnacalis insect is by feeding plant tissue and Cry1Ac albumen
With the contact of Cry2Ab albumen, the growth of Ostrinia furnacalis insect is suppressed and/or causes death after contact, while Ostrinia furnacalis
Can delay to produce the resistance for Cry1Ac albumen.Suppression refers to lethal or sub- lethal.Meanwhile, plant should be morphologically normal
, and can under conventional approaches cultivate the consumption and/or generation for product.Additionally, the plant can be substantially eliminated to chemistry
Or the need for biological insecticides.
In the present invention, by Exogenous DNA transfered plant, Cry2Ab albumen and/or the Cry1Ac albumen as described in will encode
Gene or expression cassette or recombinant vector import plant cell, conventional method for transformation is included but is not limited to, agriculture bacillus mediated
Convert, micro transmitting is bombarded, DNA is directly taken in the DNA importings of protoplast, electroporation or silicon whisker mediation.
The invention provides a kind of combination of insecticidal proteins and its method for management insect-resistant, with advantages below：
1st, delaying drug resistance.Insect can produce resistance under lasting selection pressure to single Bt albumen, and the present invention is by profit
Effectively delayed with two kinds of Cry1Ac protein albumen and Cry2Ab albumen or prevent Ostrinia furnacalis from producing resistance, so as to realize
Control or preventing and treating to Ostrinia furnacalis.
2nd, resistant insect is effectively controlled.Cry2Ab is applied for the Ostrinia furnacalis for producing resistance to Cry1Ac albumen
Albumen can effectively control resistance Ostrinia furnacalis to cause harm plant, so that plant obtains protection and constant rate of production to a greater extent.
3rd, the present invention is Cry1Ac albumen and Cry2Ab albumen is expressed in plant, and need to only plant and can be somebody's turn to do
Genetically modified plants, without using other measures, so as to save a large amount of human and material resources and financial resources, while effect is steady
It is fixed, thorough.
Below by drawings and Examples, technical scheme is described in further detail.
Brief description of the drawings
Fig. 1 is that the method for insecticidal proteins of the present invention combination and its management insect-resistant contains Cry2Ab nucleotide sequences
The structural representation of recombinant expression carrier Cry2Ab-pET30；
Fig. 2 is the people of the feeding Cry1Ac albumen treatment of the method for insecticidal proteins combination of the present invention and its management insect-resistant
The larva corrected mortality schematic diagram of the ACB-BtS and ACB-AcR of work feed；
Fig. 3 is the people of the feeding Cry2Ab albumen treatment of the method for insecticidal proteins combination of the present invention and its management insect-resistant
The larva corrected mortality schematic diagram of the ACB-BtS and ACB-AcR of work feed；
Fig. 4 is the people of the feeding Cry1Ac albumen treatment of the method for insecticidal proteins combination of the present invention and its management insect-resistant
The larval growth inhibiting rate schematic diagram of the ACB-BtS and ACB-AcR of work feed；
Fig. 5 is the people of the feeding Cry2Ab albumen treatment of the method for insecticidal proteins combination of the present invention and its management insect-resistant
The larval growth inhibiting rate schematic diagram of the ACB-BtS and ACB-AcR of work feed.
Specific embodiment
The method that insecticidal proteins combination of the present invention and its management insect-resistant are further illustrated below by specific embodiment
Technical scheme.
First embodiment, the expression of Cry2Ab albumen, purifying and activation
The structure of the recombinant expression carrier the 1st, containing Cry2Ab nucleotide sequences
SEQ ID NO in the amino acid sequence (634 amino acid) of Cry2Ab insect-killing proteins, such as sequence table:Shown in 1；
Preference according to e. coli codon is optimized to the nucleotide sequence of Cry2Ab insect-killing proteins, obtains coding corresponding
In the Cry2Ab nucleotide sequences (1905 nucleotides) of the amino acid sequence of the Cry2Ab insect-killing proteins, such as in sequence table
SEQ ID NO:Shown in 3.
The Cry2Ab nucleotide sequences (SEQ ID NO such as in sequence table:Shown in 3) had by Nanjing Jin Sirui biotechnologies
Limit company synthesizes, and by structural representation (wherein, the Kan as shown in Figure 1 of its recombinant expression carrier Cry2Ab-pET30 for building
Represent kalamycin resistance gene；F1 represents the replication orgin of bacteriophage f1；Cry2Ab is Cry2Ab nucleotide sequences (SEQ ID
NO:3)；Lac I represent operator；Ori represents replication orgin).
Then recombinant expression carrier Cry2Ab-pET30 is converted into Escherichia coli T1 competent cells with heat shock method
(Transgen, Beijing, China, CAT：CD501), its hot shock condition is：50 μ l Escherichia coli T1 competent cells, 10 μ l
DNA (recombinant expression carrier Cry2Ab-pET30), 42 DEG C of water-baths 30 seconds；37 DEG C of shaken cultivations 1 hour are (under 100rpm rotating speeds
Shaking table shakes), surface scribble IPTG (isopropylthio-β-D-galactoside) and X-gal (the chloro- 3- indoles-β of the bromo- 4- of 5--
D- galactosides) ampicillin (100mg/L) LB flat boards (tryptone 10g/L, yeast extract 5g/L, NaCl
10g/L, agar 15g/L, pH is adjusted to growing overnight on 7.5) with NaOH.Picking white colony, in LB fluid nutrient medium (tryptoses
Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, with NaOH adjust pH in 7.5) in temperature
Overnight incubation under the conditions of 37 DEG C.Alkalinity extraction its plasmid：Bacterium solution is centrifuged 1min under 12000rpm rotating speeds, supernatant is removed, sunk
Shallow lake thalline with the solution I of 100 μ l ice precoolings (25mM Tris-HCl, 10mM EDTA (ethylenediamine tetra-acetic acid), 50mM glucose,
PH8.0) suspend；The solution II (0.2M NaOH, 1%SDS (lauryl sodium sulfate)) for adding 150 μ l newly to prepare, pipe is run
4 times, 3-5min on ice is put in mixing；The ice-cold solution IIIs of 150 μ l (4M potassium acetates, 2M acetic acid) are added, are fully mixed immediately,
5-10min is placed on ice；5min is centrifuged under the conditions of 4 DEG C of temperature, rotating speed 12000rpm, in supernatant add 2 times of volumes without
Water-ethanol, room temperature places 5min after mixing；5min is centrifuged under the conditions of 4 DEG C of temperature, rotating speed 12000rpm, supernatant is abandoned, precipitates
Dried after being washed with the ethanol that concentration (V/V) is 70%；Add 30 μ l containing RNase (20 μ g/ml) TE (10mM Tris-HCl,
1mM EDTA, pH8.0) dissolution precipitation；In water-bath 30min at 37 DEG C of temperature, RNA is digested；In temperature, -20 DEG C save backup.
After the plasmid of extraction is identified through KpnI and HindIII digestions, sequence verification is carried out to positive colony, as a result show weight
The Cry2Ab nucleotides sequences inserted in group expression vector Cry2Ab-pET30 are classified as SEQ ID NO in sequence table:Shown in 3
Nucleotide sequence, i.e. Cry2Ab nucleotide sequences are correctly inserted into.
2nd, the expression of Cry2Ab albumen
Correct recombinant expression carrier Cry2Ab-pET30 will be sequenced and be transformed into Bacillus coli expression host BL21 (DE3)
In (being purchased from Beijing Tiangeng biochemical technology Co., Ltd), specific method for transformation is：Take BL21 (DE3) impressions of 50 μ L thawed on ice
State cell, adds DNA (recombinant expression carrier Cry2Ab-pET30) simultaneously gently to mix, in standing 25min on ice；In temperature
Water-bath 90s at 42 DEG C, then puts back to rapidly on ice and stands 2min；To the LB liquid for adding 800 μ L to be free of antibiotic in centrifuge tube
(tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L adjust pH to 7.5), in temperature after mixing to body culture medium with NaOH
Recovery 60min under the conditions of 37 DEG C of degree, rotating speed 150rpm；Then 1min is centrifuged under the conditions of rotating speed 4000rpm, collects thalline is stayed
Take resuspended after 100 μ L or so supernatant is gently blown and beaten and be coated on LB flat boards (the tryptone 10g/ containing 50 μ g/mL kanamycins
L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH is adjusted to growing overnight on 7.5) with NaOH.Obtain expression bacterial strain
Afterwards, induced expression is carried out as steps described below：
Step 121, one positive monoclonal containing recombinant expression carrier Cry2Ab-pET30 of picking, in the LB liquid of 5mL
(tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, the μ g/mL of kanamycins 50 adjust pH extremely to body culture medium with NaOH
7.5) under the conditions of 37 DEG C of temperature concussion and cultivate, make its OD600Value reaches 0.5-0.6；
Step 122, take the above-mentioned bacterium solutions of 0.5mL 10min is centrifuged under the conditions of rotating speed 12000rpm, supernatant precipitation is taken respectively
As the negative control of non-induced expression；
Step 123, in the above-mentioned bacterium solutions of remaining 4.5mL isopropylthiogalactoside (IPTG) is added to final concentration of
1mM, continues Fiber differentiation 20h under the conditions of rotating speed 150rpm, 16 DEG C of temperature；
Step 124, the collects thalline after the bacterium solution after inducing is centrifuged 10min under the conditions of rotating speed 12000rpm that will continue, and
10mM Tris (pH 8.0) is added to suspend；
Thalline after step 125, ultrasonic disruption suspension, is centrifuged 10min under the conditions of rotating speed 12000rpm, collects respectively
Supernatant is precipitated and carries out SDS-PAGE protein electrophoresises detection (reference《Protein electrophorese experimental technique (second edition)》)Cry2Ab
The expression of albumen, testing result shows：There are Cry2Ab protein expressions (72KDa) in supernatant precipitation.
3rd, the purifying of Cry2Ab albumen
According to a large amount of induced expression Cry2Ab albumen of above-mentioned steps 121-125, the crude protein that will be collected into crosses ni-sepharose purification
(His-Trap, FFGE Healthcare), comprises the following steps that：
Step 131, by above-mentioned steps 124 collect thalline be dissolved in combination buffer (50mM NaH2PO4、500mM
NaCl, 20mM imidazoles, pH 8.0) in, then ultrasonication on ice is placed in, received after 30min is centrifuged under the conditions of rotating speed 12000rpm
Collection supernatant, 0.45uM filter membrane removal of impurities is crossed by supernatant；
Step 132, with the combination buffer balance nickel post, wash 5 column volumes, flow velocity is 2mL/min；
Step 133, by the supernatant loading after filtering, flow velocity is 0.5mL/min；
Step 134, wash 5 column volumes again with the combination buffer, flow velocity is 2mL/min, makes Cry2Ab albumen abundant
Hanging column；
Step 135, with the combination buffer continue rinse, wash away foreign protein, flow velocity is 2mL/min；
Step 136, with elution buffer (50mM NaH2PO4, 500mM NaCl, 500mM imidazoles, pH 8.0) carry out gradient
Wash-out (is followed successively by：Percentage is 10% elution buffer 3 column volumes of wash-out, and percentage is 20%
The elution buffer elutes 3 column volumes, and percentage is 40% elution buffer 3 column volumes of wash-out, dense
Degree percentage be 100% the elution buffer elute 5 column volumes), flow velocity is 2mL/min, respectively collect eluting peak and
Eluent, by obtaining Cry2Ab albumen after purification after the detection of SDS-PAGE protein electrophoresises.
4th, the activation of Cry2Ab albumen
Above-mentioned Cry2Ab albumen after purification is dissolved in 50mM sodium carbonate buffers (pH 10.0)；Trypsase is matched somebody with somebody
The aqueous solution that concentration is 1mg/mL is made, and by 100:1 (Cry2Ab albumen:Trypsase) mass ratio in Cry2Ab albumen
Add the trypsase aqueous solution；Digested 1-3 hours at 37 DEG C of temperature after mixing.SDS- is carried out to the Cry2Ab albumen after activation
PAGE protein electrophoresises detect, as a result show to obtain the Cry2Ab albumen after digestion activation.
Cry1Ac albumen for following experiments is bought from Beijing Le Shining Science and Technology Ltd.s.
The life of second embodiment, Bt responsive type Ostrinia furnacalis strains and Ostrinia furnacalis resistance strain (Cry1Ac albumen)
Thing is determined
1st, for examination insect source
For trying Bt sensitivity strain of the insect for Ostrinia furnacalis (Ostrinia furnacalis) (hereafter with ACB-BtS generations
Table Ostrinia furnacalis Bt sensitivity strain) and Ostrinia furnacalis (Ostrinia furnacalis) Bt resistances strain (hereafter with
ACB-AcR represents Ostrinia furnacalis Bt resistances strain), the insect of above two strain is both from Chinese Academy of Agricultural Sciences plant
Protective strategy institute.
2nd, Ostrinia furnacalis bioassay method
Biologicall test is carried out using various concentrations for every kind of Bt albumen, the concentration range for determining Cry1Ac albumen is 0 μ g/
The μ g/g of g to 50, the concentration range for determining Cry2Ab albumen is 0 μ of μ g/g to 100 g/g.The preparation method of Bt protein solutions is：Will
Cry1Ac albumen is mixed and made into concentration and is respectively 0 μ g/g, 0.01 μ g/g, 0.05 according to a certain percentage with distilled water (or buffer solution)
The Cry1Ac protein solutions of μ g/g, 0.1 μ g/g, 0.5 μ g/g, 1 μ g/g, 5 μ g/g, 10 μ g/g and 50 μ g/g.By Cry2Ab albumen
Concentration is mixed and made into according to a certain percentage with distilled water (or buffer solution) is respectively 0 μ g/g, 0.01 μ g/g, 0.05 μ g/g, 0.1 μ
The Cry2Ab protein solutions of g/g, 0.5 μ g/g, 1 μ g/g, 5 μ g/g, 10 μ g/g, 50 μ g/g and 100 μ g/g.By the Bt of various concentrations
(man-made feeds come from Scientia Agricultura Sinica to protein solution (Cry1Ac protein solutions or Cry2Ab protein solutions) with man-made feeds
Institute's Plant Protection Institute) press 1.5：1 is mixed into mixed fodder respectively, and mixed fodder dispersion is positioned over the biologicall test in 48 holes
In plate, about 0.5g mixed fodders are put into each hole, will (Cry1Ac protein solutions or Cry2Ab albumen be molten with Bt protein solutions
Liquid) equivalent distilled water (or buffer solution) treatment man-made feeds as blank.In the mixing in each hole of biometric fixed board
(the survival of 1 newborn larvae of Ostrinia furnacalis is inoculated with feed surface<24h), hole is covered with sealed membrane.Biometric fixed board is existed
28 DEG C of temperature, relative humidity 80%, photoperiod (light dark) 16:Placed 6 days under conditions of 8, the 7th day start recording children after inoculation
The worm death rate and larval growth inhibiting rate.Insect strains and Bt protein solutions (Cry1Ac protein solutions or Cry2Ab protein solutions)
Every group of the combination of concentration is repeated 3 times, and each repeats to include 48 larvas.
Larval mortality is embodied with " actual " (the hereinafter referred to as death rate), and calculating the death rate need to consider actual dead
The larva for dying and non-display body dramatically increase again (<0.1mg/ larvas) survival larva.The death rate of Ostrinia furnacalis is used as follows
Formula calculate：The death rate (%)=100 × [display body of dead larvae number+not dramatically increase again (<0.1mg/ larvas) deposit
Larva number living]/test insect sum.The death rate of each larva carries out school according to the larval mortality of feeding blank
Just.Then probit analysis is carried out to corrected dosage and Mortality data, determines to cause 50% using POLO statistical softwares
(LC50) the Bt protein concentrations (Cry1Ac protein solutions or Cry2Ab protein solutions) and corresponding 95% confidential interval of the death rate.
Resistance ratio is calculated by the LC50 values by the LC50 values of ACB-AcR divided by ACB-BtS.
The larval growth inhibiting rate for feeding the Ostrinia furnacalis of mixed fodder is calculated with below equation：Larval growth suppresses
(%)=100 × (with blank feed larval weight-with mixed fodder feed larval weight)/(raised with blank
The larval weight fed).If no body weight dramatically increase (<0.1mg/ larvas) larva, then specify this be repeated as 100% children
Worm growth inhibition.Growth inhibition data are analyzed with two-way ANOVA, using insect strains and Bt protein solutions concentration as two
Main Factors.The processing difference for determining α=0.05 level is checked using LSMEANS.
3rd, the larval mortality and growth inhibition ratio measurement result of the insect strains of feeding mixed fodder
The larval mortality of the ACB-BtS and ACB-AcR of the mixed fodder of feeding Cry1Ac albumen treatment with
The increase of Cry1Ac protein concentrations and improve, and the larval mortality of ACB-BtS is significantly higher than ACB-AcR under same concentration.
As shown in table 1 and Fig. 2, the LC50 values that the larval mortality based on ACB-BtS is calculated are 0.21 μ g/g；Children based on ACB-AcR
The LC50 values that the worm death rate is calculated are more than 1000 μ g/g, therefore the difference of the LC50 values of ACB-BtS and ACB-AcR is extremely notable
, both are also extremely significant for the Resistant Difference of Cry1Ac albumen, i.e., Cry1Ac albumen has extremely weak for ACB-AcR
Insecticidal activity.
The larval mortality of the ACB-BtS and ACB-AcR of the mixed fodder of feeding Cry2Ab albumen treatment with
The increase of Cry2Ab protein concentrations and improve, and under same concentration ACB-BtS larval mortality be higher than ACB-AcR.Such as table 1
With shown in Fig. 3, the LC50 values that the larval mortality based on ACB-BtS is calculated are 1.23 μ g/g；Dead larvae based on ACB-AcR
The μ g/g of LC50 values 6.71 that rate is calculated, therefore the difference of the LC50 values of ACB-BtS and ACB-AcR is significant, but Cry2Ab eggs
In vain insecticidal activity is respectively provided with for ACB-BtS and ACB-AcR.
As shown in figure 4, abscissa is the logarithm value of Bt protein concentrations (μ g/g) used, ordinate is correspondence Ostrinia furnacalis
Growth inhibition ratio, the larval growth inhibiting rate of the ACB-BtS and ACB-AcR of the mixed fodder of feeding Cry1Ac albumen treatment is equal
Improved with the increase of Cry1Ac protein concentrations, and the larval growth inhibiting rate of ACB-BtS is significantly higher than under same concentration
ACB-AcR；When reaching 70% growth inhibition ratio, the concentration of Cry1Ac albumen needed for ACB-BtS is for about 0.1 μ g/g, ACB-AcR institutes
The concentration for needing Cry1Ac albumen is for about 500 μ g/g, shows that Cry1Ac albumen has weaker inhibitory activity for ACB-AcR.
As shown in figure 5, the larval growth suppression of the ACB-BtS and ACB-AcR of the mixed fodder of feeding Cry2Ab albumen treatment
Rate processed is improved with the increase of Cry2Ab protein concentrations, and the larval growth inhibiting rate of ACB-BtS is high under same concentration
In ACB-AcR；When reaching 100% growth inhibition ratio, the concentration of Cry2Ab albumen needed for ACB-BtS is for about 1 μ g/g, ACB-AcR
The concentration of required Cry2Ab albumen is for about 5 μ g/g, shows that Cry2Ab albumen has suitable suppression for ACB-BtS and ACB-AcR
System activity.
Table 1：Toxicity data of the Bt albumen to ACB-BtS and ACB-AcR newborn larvaes
The above results show that ACB-AcR is to Cry1Ac albumen with (i.e. resistance ratio is more than more than 5000 times of resistances
5000), and Cry2Ab albumen is respectively provided with insecticidal activity for ACB-BtS and ACB-AcR, thus ACB-AcR is shown to Cry2Ab
The relatively low cross resistance of albumen, it is 5.4 that resistance ratio is significantly reduced.As can be seen here, the friendship of Cry2Ab albumen and Cry1Ac albumen
Mutual resistance is relatively low, and Cry2Ab albumen can delay or postpone Ostrinia furnacalis to Cry1Ac albumen with the application of Cry1Ac protein combinations
Resistance is produced, while Cry2Ab albumen can effectively manage the resistance that Ostrinia furnacalis species are produced to Cry1Ac albumen.
The purposes of 3rd embodiment, Cry2Ab albumen and Cry1Ac albumen in insect-resistant transgenic plants are produced
By the experimental result of second embodiment, Cry2Ab albumen is expected to can be used for turning base with Cry1Ac albumen
Because in plant such as corn plant combinational expression with delay or prevent Ostrinia furnacalis to the plant produce resistance.
First method is subsequent transformation：Wherein the plant for having converted the first gene (such as Cry1Ac genes) is carried out
Convert again, to introduce the second gene (such as Cry2Ab genes).The subsequent transformation is preferably used two different selected markers
The phosphinothricin acetyl transferase gene of gene, such as kalamycin resistance gene and imparting to the resistance of glufosinate-ammonium herbicide
(such as pat or bar genes).
Second method is co-transformation method of particle：The nucleotides sequence for encoding Cry1Ac albumen is listed in plant and coding Cry2Ab
The nucleotide sequence of albumen is expressed together, can be by using the selected marker being connected with respective gene, and overall screening includes two
The plant of individual Select gene.
The third method is independent transformation event, and two insecticidal protein genes are each individually transferred into different plants
Genome in, can be then combined in single plant by hybridization, and can be selected using DNA marker technology
Select the plant comprising these different genes.
In sum, the present invention effectively delays or prevents by using two kinds of Cry1Ac protein albumen and Cry2Ab albumen
Only Ostrinia furnacalis produces resistance, so as to realize the control or preventing and treating to Ostrinia furnacalis, plant is protected to a greater extent
Protect and constant rate of production；The present invention makes Cry1Ac albumen and Cry2Ab albumen be expressed in plant simultaneously, and only needs plantation
Can the genetically modified plants, without use other measures, so as to save a large amount of human and material resources and financial resources, while
Effect stability, thoroughly.
It should be noted last that, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although ginseng
The present invention has been described in detail according to preferred embodiment, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
SEQUENCE LISTING
<110>BeiJing DaBei farm Science Group Co., Ltd
Beijing great Bei agricultures Bioisystech Co., Ltd
<120>The method that insecticidal proteins are combined and its manage insect-resistant
<130> DBNBC116
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 634
<212> PRT
<213> Artificial Sequence
<220>
<223>Cry2Ab amino acid sequences
<400> 1
Met Asp Asn Ser Val Leu Asn Ser Gly Arg Thr Thr Ile Cys Asp Ala
1 5 10 15
Tyr Asn Val Ala Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu
20 25 30
Asp Thr Val Gln Lys Glu Trp Thr Glu Trp Lys Lys Asn Asn His Ser
35 40 45
Leu Tyr Leu Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys
50 55 60
Lys Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu Leu Arg Asn
65 70 75 80
Leu Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg
85 90 95
Glu Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala
100 105 110
Arg Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe
115 120 125
Asn Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro
130 135 140
Leu Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn
145 150 155 160
Arg Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro
165 170 175
Leu Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val
180 185 190
Ile Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr
195 200 205
Tyr Arg Asp Tyr Leu Lys Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys
210 215 220
Ile Asn Thr Tyr Gln Ser Ala Phe Lys Gly Leu Asn Thr Arg Leu His
225 230 235 240
Asp Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr
245 250 255
Val Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser
260 265 270
Gly Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser
275 280 285
Phe Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn
290 295 300
Ser Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Ser Asn Thr
305 310 315 320
Phe Pro Asn Ile Val Gly Leu Pro Gly Ser Thr Thr Thr His Ala Leu
325 330 335
Leu Ala Ala Arg Val Asn Tyr Ser Gly Gly Ile Ser Ser Gly Asp Ile
340 345 350
Gly Ala Ser Pro Phe Asn Gln Asn Phe Asn Cys Ser Thr Phe Leu Pro
355 360 365
Pro Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp
370 375 380
Arg Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu
385 390 395 400
Thr Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser
405 410 415
Asn Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu
420 425 430
Val Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile
435 440 445
Arg Asn Ile Ala Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr
450 455 460
Met Val Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu
465 470 475 480
Asn Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr
485 490 495
Ile Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe
500 505 510
Ile Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln
515 520 525
Asn Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr
530 535 540
Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val
545 550 555 560
Thr Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr
565 570 575
Asn Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn
580 585 590
Ile Gly Asn Val Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile
595 600 605
Asn Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Ile Met
610 615 620
Leu Val Pro Thr Asn Ile Ser Pro Leu Tyr
625 630
<210> 2
<211> 1178
<212> PRT
<213> Artificial Sequence
<220>
<223>Cry1Ac amino acid sequences
<400> 2
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn
465 470 475 480
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly
485 490 495
Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly
500 505 510
Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg
515 520 525
Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn
530 535 540
Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr
545 550 555 560
Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala
565 570 575
Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe
580 585 590
Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val
595 600 605
Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala
610 615 620
Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn
625 630 635 640
Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu
645 650 655
Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val
660 665 670
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser
675 680 685
Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser
690 695 700
Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr
705 710 715 720
Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr
725 730 735
Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu
740 745 750
Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg
755 760 765
Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu
770 775 780
Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn
785 790 795 800
Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys
805 810 815
Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp
820 825 830
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val
835 840 845
Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu
850 855 860
Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val
865 870 875 880
Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp
885 890 895
Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu
900 905 910
Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala
915 920 925
Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr
930 935 940
Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu
945 950 955 960
Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg
965 970 975
Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn
980 985 990
Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val
995 1000 1005
Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg
1010 1015 1020
Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys
1025 1030 1035
Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn
1040 1045 1050
Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile
1055 1060 1065
Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln
1070 1075 1080
Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn
1085 1090 1095
Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu
1100 1105 1110
Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn
1115 1120 1125
Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr
1130 1135 1140
Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
1145 1150 1155
Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu
1160 1165 1170
Leu Leu Met Glu Glu
1175
<210> 3
<211> 1905
<212> DNA
<213> Artificial Sequence
<220>
<223>Cry2Ab nucleotide sequences
<400> 3
atggataact cagttctgaa ttcgggccgt accacgattt gcgatgccta taatgtcgcg 60
gcccatgacc cgtttagctt ccaacacaaa tctctggata ccgtgcagaa agaatggacg 120
gaatggaaga aaaacaatca tagcctgtac ctggacccga ttgtgggcac cgttgcgagc 180
tttctgctga agaaagtggg ctctctggtc ggtaaacgta ttctgagtga actgcgcaat 240
ctgatctttc cgtcaggttc gaccaacctg atgcaagata tcctgcgtga aacggaaaaa 300
ttcctgaacc agcgtctgaa taccgacacg ctggcgcgcg ttaacgccga actgaccggt 360
ctgcaagcaa acgtggaaga atttaatcgt caggttgata acttcctgaa cccgaatcgc 420
aacgctgtcc cgctgtctat taccagctct gtgaatacga tgcagcaact gtttctgaac 480
cgcctgccgc agttccaaat gcagggctat caactgctgc tgctgccgct gtttgcgcag 540
gcagctaatc tgcatctgag cttcattcgt gatgtgatcc tgaacgccga cgaatggggt 600
atttctgcgg ccaccctgcg tacgtatcgc gattacctga aaaattatac ccgcgactat 660
tccaactact gcatcaatac gtaccagtca gcatttaaag gcctgaatac ccgtctgcac 720
gatatgctgg aattccgcac ctatatgttt ctgaacgtgt tcgaatacgt tagcatttgg 780
tctctgttta aatatcagag cctgctggtt agctccggtg caaacctgta cgctagcggc 840
tctggtccgc agcaaaccca aagtttcacg tcccaggatt ggccgtttct gtattcactg 900
ttccaggtca attcgaacta cgtgctgaat ggctttagtg gtgcccgtct gtccaacacc 960
ttcccgaata tcgtgggtct gccgggttca accacgaccc acgcactgct ggcagctcgt 1020
gttaactatt cgggcggtat ttcatcgggc gatatcggtg cctcgccgtt taatcagaac 1080
ttcaattgta gcacctttct gccgccgctg ctgacgccgt tcgttcgtag ctggctggat 1140
agtggttccg accgtgaagg tgtcgcaacc gtgacgaatt ggcagaccga atcttttgaa 1200
acgaccctgg gcctgcgtag tggtgcattc accgctcgcg gcaactccaa ctacttcccg 1260
gattacttca tccgtaacat cagcggcgtg ccgctggttg tgcgcaacga agacctgcgt 1320
cgcccgctgc attataacga aattcgcaat atcgcctcac cgtcgggcac cccgggcggt 1380
gcacgtgcat acatggtttc tgtccacaac cgcaaaaaca atattcatgc ggtccacgaa 1440
aatggcagta tgatccatct ggccccgaac gattataccg gttttacgat ttccccgatc 1500
cacgcaaccc aagtgaacaa tcagacccgt acgtttattt cagaaaaatt cggcaatcag 1560
ggtgattcgc tgcgctttga acagaacaat acgaccgctc gttataccct gcgcggcaac 1620
ggtaatagtt ataacctgta cctgcgtgtt agctctatcg gcaattccac cattcgtgtt 1680
acgatcaacg gtcgcgtcta caccgcgacg aacgtgaata cgaccacgaa caatgatggc 1740
gtgaacgaca atggtgcacg ctttagcgat attaacatcg gcaatgtcgt ggctagttcc 1800
aatagcgatg ttccgctgga cattaacgtc accctgaatt ctggtacgca gtttgacctg 1860
atgaatatta tgctggtgcc gaccaacatc agcccgctgt attaa 1905
<210> 4
<211> 3537
<212> DNA
<213> Artificial Sequence
<220>
<223>Cry1Ac nucleotide sequences
<400> 4
atggacaaca acccaaacat caacgaatgc attccataca actgcttgag taacccagaa 60
gttgaagtac ttggtggaga acgcattgaa accggttaca ctcccatcga catctccttg 120
tccttgacac agtttctgct cagcgagttc gtgccaggtg ctgggttcgt tctcggacta 180
gttgacatca tctggggtat ctttggtcca tctcaatggg atgcattcct ggtgcaaatt 240
gagcagttga tcaaccagag gatcgaagag ttcgccagga accaggccat ctctaggttg 300
gaaggattga gcaatctcta ccaaatctat gcagagagct tcagagagtg ggaagccgat 360
cctactaacc cagctctccg cgaggaaatg cgtattcaat tcaacgacat gaacagcgcc 420
ttgaccacag ctatcccatt gttcgcagtc cagaactacc aagttcctct cttgtccgtg 480
tacgttcaag cagctaatct tcacctcagc gtgcttcgag acgttagcgt gtttgggcaa 540
aggtggggat tcgatgctgc aaccatcaat agccgttaca acgaccttac taggctgatt 600
ggaaactaca ccgaccacgc tgttcgttgg tacaacactg gcttggagcg tgtctggggt 660
cctgattcta gagattggat tagatacaac cagttcagga gagaattgac cctcacagtt 720
ttggacattg tgtctctctt cccgaactat gactccagaa cctaccctat ccgtacagtg 780
tcccaactta ccagagaaat ctatactaac ccagttcttg agaacttcga cggtagcttc 840
cgtggttctg cccaaggtat cgaaggctcc atcaggagcc cacacttgat ggacatcttg 900
aacagcataa ctatctacac cgatgctcac agaggagagt attactggtc tggacaccag 960
atcatggcct ctccagttgg attcagcggg cccgagttta cctttcctct ctatggaact 1020
atgggaaacg ccgctccaca acaacgtatc gttgctcaac taggtcaggg tgtctacaga 1080
accttgtctt ccaccttgta cagaagaccc ttcaatatcg gtatcaacaa ccagcaactt 1140
tccgttcttg acggaacaga gttcgcctat ggaacctctt ctaacttgcc atccgctgtt 1200
tacagaaaga gcggaaccgt tgattccttg gacgaaatcc caccacagaa caacaatgtg 1260
ccacccaggc aaggattctc ccacaggttg agccacgtgt ccatgttccg ttccggattc 1320
agcaacagtt ccgtgagcat catcagagct cctatgttct cttggataca tcgtagtgct 1380
gagttcaaca acatcatcgc atccgatagt attactcaaa tccctgcagt gaagggaaac 1440
tttctcttca acggttctgt catttcagga ccaggattca ctggtggaga cctcgttaga 1500
ctcaacagca gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560
ccatccacat ctaccagata tagagttcgt gtgaggtatg cttctgtgac ccctattcac 1620
ctcaacgtta attggggtaa ttcatccatc ttctccaata cagttccagc tacagctacc 1680
tccttggata atctccaatc cagcgatttc ggttactttg aaagtgccaa tgcttttaca 1740
tcttcactcg gtaacatcgt gggtgttaga aactttagtg ggactgcagg agtgattatc 1800
gacagattcg agttcattcc agttactgca acactcgagg ctgagtacaa ccttgagaga 1860
gcccagaagg ctgtgaacgc cctctttacc tccaccaatc agcttggctt gaaaactaac 1920
gttactgact atcacattga ccaagtgtcc aacttggtca cctaccttag cgatgagttc 1980
tgcctcgacg agaagcgtga actctccgag aaagttaaac acgccaagcg tctcagcgac 2040
gagaggaatc tcttgcaaga ctccaacttc aaagacatca acaggcagcc agaacgtggt 2100
tggggtggaa gcaccgggat caccatccaa ggaggcgacg atgtgttcaa ggagaactac 2160
gtcaccctct ccggaacttt cgacgagtgc taccctacct acttgtacca gaagatcgat 2220
gagtccaaac tcaaagcctt caccaggtat caacttagag gctacatcga agacagccaa 2280
gaccttgaaa tctactcgat caggtacaat gccaagcacg agaccgtgaa tgtcccaggt 2340
actggttccc tctggccact ttctgcccaa tctcccattg ggaagtgtgg agagcctaac 2400
agatgcgctc cacaccttga gtggaatcct gacttggact gctcctgcag ggatggcgag 2460
aagtgtgccc accattctca tcacttctcc ttggacatcg atgtgggatg tactgacctg 2520
aatgaggacc tcggagtctg ggtcatcttc aagatcaaga cccaagacgg acacgcaaga 2580
cttggcaacc ttgagtttct cgaagagaaa ccattggtcg gtgaagctct cgctcgtgtg 2640
aagagagcag agaagaagtg gagggacaaa cgtgagaaac tcgaatggga aactaacatc 2700
gtttacaagg aggccaaaga gtccgtggat gctttgttcg tgaactccca atatgatcag 2760
ttgcaagccg acaccaacat cgccatgatc cacgccgcag acaaacgtgt gcacagcatt 2820
cgtgaggctt acttgcctga gttgtccgtg atccctggtg tgaacgctgc catcttcgag 2880
gaacttgagg gacgtatctt taccgcattc tccttgtacg atgccagaaa cgtcatcaag 2940
aacggtgact tcaacaatgg cctcagctgc tggaatgtga aaggtcatgt ggacgtggag 3000
gaacagaaca atcagcgttc cgtcctggtt gtgcctgagt gggaagctga agtgtcccaa 3060
gaggttagag tctgtccagg tagaggctac attctccgtg tgaccgctta caaggaggga 3120
tacggtgagg gttgcgtgac catccacgag atcgagaaca acaccgacga gcttaagttc 3180
tccaactgcg tcgaggaaga aatctatccc aacaacaccg ttacttgcaa cgactacact 3240
gtgaatcagg aagagtacgg aggtgcctac actagccgta acagaggtta caacgaagct 3300
ccttccgttc ctgctgacta tgcctccgtg tacgaggaga aatcctacac agatggcaga 3360
cgtgagaacc cttgcgagtt caacagaggt tacagggact acacaccact tccagttggc 3420
tatgttacca aggagcttga gtactttcct gagaccgaca aagtgtggat cgagatcggt 3480
gaaaccgagg gaaccttcat cgtggacagc gtggagcttc tcttgatgga ggaataa 3537
Claims (26)
1. it is a kind of manage insect-resistant method, it is characterised in that including by Ostrinia furnacalis at least with Cry2Ab albumen and
Cry1Ac albumen is contacted.
2. the method for managing insect-resistant according to claim 1, it is characterised in that the Cry2Ab albumen and Cry1Ac eggs
It is present in the bacterium or genetically modified plants that at least produce the Cry2Ab albumen and Cry1Ac albumen in vain, the Ostrinia furnacalis
At least contacted with the Cry2Ab albumen and Cry1Ac albumen by the tissue of the ingest bacterium or the genetically modified plants, connect
The Ostrinia furnacalis growth after touch is suppressed and/or causes death, to realize managing the resistance of Ostrinia furnacalis.
3. the method for managing insect-resistant according to claim 1, it is characterised in that resistance will be produced to Cry1Ac albumen
Ostrinia furnacalis at least contacts with the Cry2Ab albumen, and the Cry2Ab albumen is present in and at least produce the Cry2Ab albumen
Bacterium or genetically modified plants in, it is described to Cry1Ac albumen produce resistance Ostrinia furnacalis by the bacterium or the institute of ingesting
The tissue for stating genetically modified plants is at least contacted with the Cry2Ab albumen, the Asia to Cry1Ac albumen generation resistance after contact
Continent corn borer growth is suppressed and/or causes death, beautiful to realize the management Asia that resistance is produced to Cry1Ac albumen
The resistance of rice snout moth's larva.
4. the method for managing insect-resistant according to claim 1, it is characterised in that resistance will be produced to Cry2Ab albumen
Ostrinia furnacalis at least contacts with the Cry1Ac albumen, and the Cry1Ac albumen is present in and at least produce the Cry1Ac albumen
Bacterium or genetically modified plants in, it is described to Cry2Ab albumen produce resistance Ostrinia furnacalis by the bacterium or the institute of ingesting
The tissue for stating genetically modified plants is at least contacted with the Cry1Ac albumen, the Asia to Cry2Ab albumen generation resistance after contact
Continent corn borer growth is suppressed and/or causes death, beautiful to realize the management Asia that resistance is produced to Cry2Ab albumen
The resistance of rice snout moth's larva.
5. the method that insect-resistant is managed according to claim any one of 2-4, it is characterised in that at the genetically modified plants
In any breeding time.
6. the method that insect-resistant is managed according to claim any one of 2-4, it is characterised in that the genetically modified plants
It is organized as root, blade, stalk, fruit, tassel, female fringe, flower pesticide or filigree.
7. the method that insect-resistant is managed according to claim any one of 2-4, it is characterised in that described to Ostrinia furnacalis
The control of harm plant does not change because of the change of plantation place and/or implantation time.
8. according to claim any one of 2-7 manage insect-resistant method, it is characterised in that the plant be corn,
Wheat, sorghum, millet, paddy rice or soybean.
9. the method that insect-resistant is managed according to claim any one of 1-8, it is characterised in that the Cry2Ab albumen
Amino acid sequence has SEQ ID NO:Amino acid sequence shown in 1.
10. the method for managing insect-resistant according to claim 9, it is characterised in that the nucleotides of the Cry2Ab albumen
Sequence has SEQ ID NO:Nucleotide sequence shown in 3.
11. methods that insect-resistant is managed according to claim any one of 1-10, it is characterised in that the Cry1Ac albumen
Amino acid sequence there is SEQ ID NO:Amino acid sequence shown in 2.
12. methods that insect-resistant is managed according to claim 11, it is characterised in that the nucleotides of the Cry1Ac albumen
Sequence has SEQ ID NO:Nucleotide sequence shown in 4.
13. it is a kind of control Ostrinia furnacalis methods, it is characterised in that including by Ostrinia furnacalis at least with Cry2Ab albumen and
Cry1Ac albumen is contacted, so as to realize the control to Ostrinia furnacalis.
14. according to claim 13 control Ostrinia furnacalis methods, it is characterised in that the Cry2Ab albumen and
Cry1Ac albumen is present in the bacterium or genetically modified plants that at least produce the Cry2Ab albumen and Cry1Ac albumen, the Asia
Continent corn borer by the tissue of the ingest bacterium or the genetically modified plants at least with the Cry2Ab albumen and Cry1Ac eggs
White contact, the Ostrinia furnacalis growth is suppressed and/or causes death after contact, is planted with realizing endangering Ostrinia furnacalis
The control of thing.
15. methods that Ostrinia furnacalis is controlled according to claim 13, it is characterised in that Cry1Ac albumen will be produced anti-
The Ostrinia furnacalis of property at least contacts with the Cry2Ab albumen, and the Cry2Ab albumen is present in and at least produce the Cry2Ab
It is described that the Ostrinia furnacalis of resistance is produced to Cry1Ac albumen by the bacterium that ingests in the bacterium or genetically modified plants of albumen
It is described after contact that resistance is produced to Cry1Ac albumen or the tissue of the genetically modified plants is at least contacted with the Cry2Ab albumen
Ostrinia furnacalis growth be suppressed and/or cause death, with realize to it is described to Cry1Ac albumen produce resistance Asia
Corn borer endangers the control of plant.
16. methods that Ostrinia furnacalis is controlled according to claim 13, it is characterised in that Cry2Ab albumen will be produced anti-
The Ostrinia furnacalis of property at least contacts with the Cry1Ac albumen, and the Cry1Ac albumen is present in and at least produce the Cry1Ac
It is described that the Ostrinia furnacalis of resistance is produced to Cry2Ab albumen by the bacterium that ingests in the bacterium or genetically modified plants of albumen
It is described after contact that resistance is produced to Cry2Ab albumen or the tissue of the genetically modified plants is at least contacted with the Cry1Ac albumen
Ostrinia furnacalis growth be suppressed and/or cause death, with realize to it is described to Cry2Ab albumen produce resistance Asia
Corn borer endangers the control of plant.
17. methods that Ostrinia furnacalis is controlled according to claim any one of 14-16, it is characterised in that the transgenosis
Plant is in any breeding time.
18. methods that Ostrinia furnacalis is controlled according to claim any one of 14-16, it is characterised in that the transgenosis
Plant is organized as root, blade, stalk, fruit, tassel, female fringe, flower pesticide or filigree.
19. methods that Ostrinia furnacalis is controlled according to claim any one of 14-16, it is characterised in that described to Asia
The control of corn borer harm plant does not change because of the change of plantation place and/or implantation time.
20. methods that Ostrinia furnacalis is controlled according to claim any one of 14-19, it is characterised in that the plant is
Corn, wheat, sorghum, millet, paddy rice or soybean.
21. methods that Ostrinia furnacalis is controlled according to claim any one of 13-20, it is characterised in that the Cry2Ab
The amino acid sequence of albumen has SEQ ID NO:Amino acid sequence shown in 1.
22. methods that Ostrinia furnacalis is controlled according to claim 21, it is characterised in that the nucleosides of the Cry2Ab albumen
Acid sequence has SEQ ID NO:Nucleotide sequence shown in 3.
23. methods that Ostrinia furnacalis is controlled according to claim any one of 13-22, it is characterised in that the Cry1Ac
The amino acid sequence of albumen has SEQ ID NO:Amino acid sequence shown in 2.
24. methods that Ostrinia furnacalis is controlled according to claim 23, it is characterised in that the nucleosides of the Cry1Ac albumen
Acid sequence has SEQ ID NO:Nucleotide sequence shown in 4.
A kind of 25. Cry2Ab albumen and Cry1Ac protein combinations are using preventing or delay Ostrinia furnacalis colony to Cry1Ac eggs
White or Cry2Ab albumen produces the purposes of resistance.
A kind of 26. Cry2Ab albumen and Cry1Ac protein combinations are anti-to Cry1Ac albumen or the generation of Cry2Ab albumen using controlling
The purposes of the Ostrinia furnacalis colony of property.
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| PCT/CN2017/102442 WO2018090714A1 (en) | 2016-11-21 | 2017-09-20 | Insecticidal protein combination, and insect resistance management method for same |
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| WO2018090714A1 (en) * | 2016-11-21 | 2018-05-24 | 北京大北农科技集团股份有限公司 | Insecticidal protein combination, and insect resistance management method for same |
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| CN117396495A (en) * | 2022-05-11 | 2024-01-12 | 北京大北农生物技术有限公司 | Use of insecticidal proteins |
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