CN106701791B - Cry1Ab13-1 killing gene and application - Google Patents
Cry1Ab13-1 killing gene and application Download PDFInfo
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- CN106701791B CN106701791B CN201710125280.2A CN201710125280A CN106701791B CN 106701791 B CN106701791 B CN 106701791B CN 201710125280 A CN201710125280 A CN 201710125280A CN 106701791 B CN106701791 B CN 106701791B
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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 peptides, i.e. delta-endotoxins
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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
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- 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
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Abstract
The invention disclosesCry1Ab13‑1Killing gene, it is by fallibility PCR method pairCry1Ab13Gene base sequence carries out random mutagenesis acquisition, reach 38.61% with primary sequence homology, Ser 10.1%, Thr8.0%, it is in the majority with Leu7.9%, the content of sequence GC improves 3% after being mutated compared with original sequence, the sequence and known anaphylactogen homology it is lower be not present sensitization, pest-resistant (diamondback moth is carried out with the albumen of inducing expression, corn borer) test, the result shows that the expression albumen has very strong insecticidal activity, make the death rate of diamondback moth larvae up to 87.81%, make the death rate of corn borer larvae up to 80.00%, and the pest growth of survival is also seriously suppressed.The gene has very strong insecticidal activity, can provide candidate gene for the cultivation of transgenic pest-resistant breeding and the building of engineered strain.
Description
Technical field
The invention belongs to field of biotechnology, and in particular toCry1Ab13-1Killing gene and its application.
Background technique
Insect pest is one of an important factor for influencing China's crop yield, and 2016 due to insect pest according to the statistics of State Statistics Bureau
Influence China summer grain crops total output compared with 162.1 ten thousand tons of the last year underproduction, bring serious economic loss.Corn borer (Ostrinia nubilalis), eating-core bean worm (Leguminivora glycinivorella Matsumura)Equal lepidopterous insects are main
The pest to be caused harm, crops not only will receive the influence of these insect pests in the stage of growth and development, the later period transport and
Also there is their invasion in storage, be at present these pests of effectively preventing, used a large amount of chemical pesticide Chinese according to statistics
The unit area usage amount of pesticide is 2.5 times of world average level, however a large amount of extensive sprinkling serious shadows of chemical pesticide
The safety for having rung people and animals and ecological environment, also causes pest to develop drug resistance, while also killing natural enemy and beneficial insect, pollution
It is further aggravated.Therefore being cultivated using technique for gene engineering, there is the kind of pest-resistant character, which to be only, fundamentally solves pest problem
One of effective way.
Bacillus thuringiensis(Bacillus thuringiensis,Abbreviation Bt) it is to be crossed by Japanese scholars stone in 1901
It is isolated for the first time from the silkworm to catch an illness, 1915 by formally named forBacillus thuringiensis , it is that kind of leather is blue
Family name positive soil bacillus can generate the toxicity crystalline protein for killing insect larvae while forming gemma.Nineteen ninety-five
Crickmor etc. is boundary with 45%, 75% and 95% according to the homology of amino acid sequence, carries out system to existing Bt albumen
Classification and name.Since Bt insecticidal crystal protein has the specificity of height, pest natural enemy and beneficial insect nonhazardous are made
With.Only there is insecticidal effect to target pests such as Lepidoptera, coleopteras, not can cause environmental pollution again, thus it is extensive
Prevention and treatment applied to pest.Early in Veack in 1987, just report will for the first timeCry1AbGenetic transformation obtains anti-cigarette into tobacco
The tobacco plant of the trans Bt gene of careless hawkmoth has highlighted the potentiality that Bt gene is applied in genetic engineering.Three heap of Guo etc. is by dividing
Sub- designing technique, it is artificial synthesizedCry1AGene and the gene is transferred in cotton and be widely applied make China become second
The country of a successful incubation Insect Resistant Cotton.But withBtGene and its preparation are largely applied, and pest is to which create different journeys
The resistance of degree[11,12], and wild typeBtThe unstable expression quantity for also resulting in the gene of gene mRNA is extremely low.Then, people
Start to be transformed with Bt gene of the enzyme engineering technology to wild type.Wang Yang etc. is using Domain swapping method to insecticidal crystal
AlbumenCry1AbTransformation is optimized, novel killing gene is obtainedCryFLIa,Prove that the gene pairs is sub- by indoor raw survey
Continent corn borer has apparent resistance.So farcryClassBtGene mining and register share 318.Related virulent property now
It is active with new type disinsectionCry class, cyt class, vip class BtThe excavation of gene and clone are still right both at home and abroadBtGene studies and
The hot spot utilized.
Main method, which is transformed, in Bt albumen at present structural domain conversion, codon optimization, DNA Shuffling, fallibility PCR
Deng wherein fallibility round pcr simple and effective can introduce mutation into known dna sequence, and system built by Leung earliest
It is vertical, Cadwell[19]Fallibility PCR, the gene of mutagenesis coding tetrahymena ribozyme, hence it is evident that improve the enzyme are carried out with the system
Activity.Shelly Goomber etc.[20]By fallibility PCR method Evolution in vitro bacillus lipase there is it still at 5 DEG C
Activity enhances the suitable to cold of the enzyme.
Summary of the invention
The problem of polluting environment the purpose of the present invention is to solve chemical pesticide, and one kind is providedCry1Ab13-1Desinsection
Gene and application.
Mutated geneCry1Ab13-1, base sequence is as shown in sequence table SEQ ID NO.1；
The gene is artificial synthesized；
Cry1Ab13-1 albumen, it is the albumen of gene expression shown in sequence table SEQ ID NO.1.
Cry1Ab13-1 albumen is as the application for preparing insecticide；
The worm is diamondback moth or corn borer.
Mutated geneCry1Ab13-1Turn the application of base plant variety in the anti-diamondback moth worm of cultivation or maize borer.
The present invention providesCry1Ab13-1Killing gene, it is by fallibility PCR method pairCry1Ab13Gene base sequence
Column carry out random mutagenesis acquisition, by showing the sequencing results after mutagenesis: reaching with primary sequence homology
38.61%, Ser 10.1%, Thr8.0% and Leu7.9% are in the majority, and the content of sequence GC improves after being mutated compared with original sequence
3%, which is shown by using online allergy original database on-line analysis and known anaphylactogen homology is lower is not present
Sensitization shows an apparent specific band, explanation occur in 79.5KDa or so by SDS-PAGE electrophoretic analysis result
Under the induction of IPTGCry1Ab13-1 Gene has obtained correct expression.With the albumen of inducing expression carry out it is pest-resistant (diamondback moth,
Corn borer) test, the results showed that the expression albumen has very strong insecticidal activity, reaches the death rate of diamondback moth larvae
87.81%, make the death rate of corn borer larvae up to 80.00%, and the pest growth survived also seriously is suppressed, it is poor with compareing
It is different obvious.A kind of this research of preliminary proof success modified form of mutagenesisCry1Ab13Gene, the gene are living with very strong desinsection
Property, candidate gene can be provided for the cultivation of transgenic pest-resistant breeding and the building of engineered strain.
Detailed description of the invention
Fig. 1 difference Mg2+ concentration electrophoretogram；M: DL2000 DNA marker;H: sterile water；1-10: in reaction system
Mg2+Concentration is once 2,2.5,3,3.5,4,4.5,5,5.5,6,6.5mmol/L；
The electrophoretogram of Fig. 2 fallibility PCR；M:DL2000 DNA marker;The fallibility pcr of 1-7:Cry1Ab13 is expanded；
Fig. 3 Cry1Ab13-1Conserved domain；
Fig. 4 recombinant plasmid PCR verifying；M:DL2000 DNA marker；1-7: recombinant plasmid verifying；
The identification of Fig. 5 .CryAb13-1 gene plasmid double digestion, M:DL2000 DNA marker；1-2: digestion verification；
Fig. 6pET28a-Cry1Ab13-1The SDS-PAGE of albumen is analyzed, M: protein standard molecular weight；1:Cry1Ab13-1
It does not induce；2: cry1Ab13Induction；3:cry1Ab13-1Induction；
Fig. 7 prokaryotic expression protein insect resistance identification, A, B, C are respectively 24,48,72h.
Specific embodiment
1 fallibility PCR amplification of embodiment
Use software Primer5.0 design primer
s:TGGACAACAATCCTAACATCAAC,
As: TTATCAAAGTTCATCCTTCTCGG (drawing horizontal line part is random permutation point),
To containpUC57-Cry1Ab13Plasmid is template, carries out fallibility PCR, and fallibility PCR 50ul reaction system includes
DDH2O 17.8ul、Buffer 5ul、Mgcl220ul, dNTP 1ul, Primer1 2ul, primer2 2ul, 1 ul of template,
Taq archaeal dna polymerase 1.2ul.Response procedures: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 40s；40 DEG C of annealing 40s, 72 DEG C are prolonged
Stretch 3min；70 × circulation;Extend 10min after 72 DEG C;4 DEG C of preservations.Without thermal starting.1% agarose gel electrophoresis is pure
Change recycling target fragment, using this segment as template, the second wheel fallibility PCR is carried out with same condition[22,23], through 1% agarose
After gel electrophoresis, purifies and be sequenced.Sequencing result is compared using software DNAman6.0, passes through SWISS-MODEL
(www.expasy.org) homologous to sequencing result to model and carry out physical property analysis, it is analyzed according to exogenous proteins anaphylaxis
National standard, its sensitization is carried out using the online allergy original database website (www.allergenonline.org) online
Analysis.
With different magnesium ion concentrations, Lo-Fi is usedTaq Archaeal dna polymerase extends the time of each circulation, reduces starting
The final concentration of template angularly sets out without thermal starting using replacement site primer is had and carries out fallibility PCR amplification, passes through
Preliminary gropes, final to determine the Mg for adding 5mmol/L2+(such as Fig. 1), 70 circulations are suitable conditions, and carrying out fallibility PCR can be with
Amplification obtains clearly specific band (such as Fig. 2).
The building of 2 cloning vector of embodiment
Two-wheeled fallibility PCR will be passed through, the target fragment and carrier PMD-18T of purification and recovery carry out 16 DEG C, connect overnight.It will
Connection product is transformed into Escherichia coliE.coli In DH5 α competent cell, next day picking single colonie is incubated overnight, and extracts plasmid
PCR verifying is carried out, purification and recovery PCR product send Jilin provincial treasury U.S. Bioisystech Co., Ltd sequence verification.It is named asCry1Ab13-1Gene order.
Cry1Ab13-1Gene order and conserved structure domain analysis and function prediction
It is derived by DNA sequence dna, the protein of coding includes that wherein hydrophilic amino acid accounts for 41.3% to 706 amino acid, hydrophobic
Amino acid accounts for 39.1%, and acidic amino acid accounts for 10.6%, and basic amino acid accounts for 9%, and theoretical molecular weight size is 79.5KDa, isoelectric point
5.19.In website, NCBI carries out CDD (Convsed Domain Database) to the amino acid sequence of the DNA encoding the protein
Analysis shows thatCry1Ab13-1The Domain I range for encoding albumen is Ser58~Asp265Be primarily involved in film penetrate and hole
Formation, the corresponding range of Domain II is Thr273~Glu485The combination of receptor is primarily involved in, corresponding to Domain III
Range is Asn487~Pro632,And the combination of carbohydrate is related, is a part of δ endotoxin region of activation, can produce and kill
Worm poison element.Such as figure (3).It is compared with former sequence preservative structural domain, and the 130th Ser is transformed into Pro in structural domain I, is tying
The 383rd Thr is transformed into Ser in the II of structure domain.Pass through online blast(NCBI) compare homology and show, the gene withCry1A
The most similar, homology is up to 78%.Online sensitization analysis the results show thatCry1Ab1380 ammonia of protein of -1 gene coding
The sequence homology of base acid sequence and known anaphylactogen is respectively less than 35%, i.e.,CryAb13Sensitization is not present in the albumen of -1 gene coding
Property.
The building and identification of 3 prokaryotic expression carrier of embodiment
It extractspMD18T-Cry1Ab13-1Plasmid DNA use with BamHI and SalI restriction enzyme site seamless primer into
The normal PCR amplification of row, amplification condition: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 40s；62 DEG C of annealing 40s, 72 DEG C of extensions
2min；Extend 10min after 72 DEG C；35 × circulation.Product carries out 1% agarose gel electrophoresis, purification and recovery purpose piece after PCR
Section, and using seamless connection kit, it is attached with prokaryotic expression carrier pET-28a, then convert Escherichia coliE.coli In DH5 α competent cell, next day picking whole single colonie is incubated overnight, and extracts its Plasmid DNA, carry out PCR and
Digestion verification send Jilin provincial treasury U.S. Bioisystech Co., Ltd sequence verification for primarily determining for positive PCR product.
Cry1Ab13-1Prokaryotic expression vector building
Picking whole bacterium colony is incubated overnight, and next day extracts plasmid PCR verifying (such as Fig. 4), filters out stable positive colony,
Digestion verification is carried out to recombinant plasmid dna, obtains 2 stripe size difference 5.3kb and 2033bp(such as Fig. 5), sequencing analysis table
Bright sequence is correct, and illustration purpose genetic fragment is successfully connected to prokaryotic expression carrier pET28a(+) in.
Embodiment 4 Cry1Ab13-1Gene is in expression in escherichia coli
It extractspET28a-Cry1Ab13-1Plasmid DNA, be transformed into e. coli bl21 (DE3) competent cell
In, picking single colonie is incubated overnight, and bacterium solution is inoculated into the LB liquid medium equipped with 50ml by next day by the ratio column of 1:100
(containing Kan100mg/ml), 37 DEG C of shaking table 198rpm shaken cultivation 3.5h make OD600When reaching 0.6, it is added the IPTG's of 100mM
Mother liquor continues shaken cultivation 3h to its final concentration 1mM.Triangular flask is placed in 5min on ice, thalline were collected by centrifugation (4 DEG C,
10000r/min, 8min), and thallus is resuspended with the sterile water of pre-cooling and is centrifuged again, it repeats twice.Abandon supernatant PBS Buffer
It is resuspended.By suspension with ultrasonic disruption (1200W, 6s on, 5s off 10min), be centrifuged (4 DEG C, 10000r/min,
It 10min) collects thallus and supernatant is spare.Carry out SDS-PAGE electrophoretic analysis verifying.
1.2.4 Cry1Ab13-1Gene is in expression in escherichia coli
It extractspET28a-Cry1Ab13-1Plasmid DNA, be transformed into e. coli bl21 (DE3) competent cell
In, picking single colonie is incubated overnight, and bacterium solution is inoculated into the LB liquid medium equipped with 50ml by next day by the ratio column of 1:100
(containing Kan100mg/ml), 37 DEG C of shaking table 198rpm shaken cultivation 3.5h make OD600When reaching 0.6, it is added the IPTG's of 100mM
Mother liquor continues shaken cultivation 3h to its final concentration 1mM.Triangular flask is placed in 5min on ice, thalline were collected by centrifugation (4 DEG C,
10000r/min, 8min), and thallus is resuspended with the sterile water of pre-cooling and is centrifuged again, it repeats twice.Abandon supernatant PBS Buffer
It is resuspended.By suspension with ultrasonic disruption (1200W, 6s on, 5s off 10min), be centrifuged (4 DEG C, 10000r/min,
It 10min) collects thallus and supernatant is spare.Carry out SDS-PAGE electrophoretic analysis verifying.
Cry1Ab13-1Gene is analyzed in expression in escherichia coli
SDS-PAGE electrophoretic analysis contains after IPTG is induced as the result is shown (Fig. 6)CryAb13-1Engineering bacteria (non-solubility
Component) in 79.5KDa or so, an apparent specific band is given expression to, matches, shows with expected sizeCry1Ab13-1Base
Because the insecticidal proteins of coding are correctly expressed in e. coli bl21.
Embodiment 5 Cry1Ab13-1The bioassay of albumen insecticidal activity
Ultrasonic disruption is carried out after recombinant bacterium thallus inducing expression and recycles Cry1Ab13-1 albumen, it is living for following desinsection
The identification of property.
The identification of diamondback moth insecticidal activity uses leaf dipping method: the tender consistent blade of wild cabbage children is chosen in the lab, with clear
Water, which is cleaned, to be dried, and is divided into bulk similar in size, these cabbage leaves of the same size are put into sample to be tested and are soaked
10min is steeped, dries, is laid in glass culture dish later, diamondback moth larvae 12 of 2-3 age are met in each culture dish, each
Processing repeats three times, and culture dish is put into growth cabinet and is cultivated[24](culture environment in case, temperature be (25 ±
1.5) DEG C, relative humidity 50%-60%, periodicity of illumination 14h, dark 10h).
The insecticidal activity identification of corn borer uses man-made feeds method: method and item of the preparation of man-made feeds referring to Qiao Li
Part.To containCry1Ab13The broken bacterium solution of carrier is as positive control, using the broken bacterium solution containing empty carrier as negative control, respectively
In for 24 hours, 48h, 72h investigation result with software DPS9.5 statistically analyze, repeat three times.
Cry1Ab13-1The bioassay of albumen insecticidal activity
Diamondback moth insecticidal test the result shows that: three groups of diamondback moths have a large amount of feeding blade within for 24 hours, with processing
The extension experimental group of time and the diamondback moth feeding blade amount of positive controls gradually decrease (hole of blade is reduced), and growth is tight
Be obstructed again, and dead worm occurs in atrophy, and the normal feeding of negative control group (such as Fig. 7), for 24 hours, 48h, 72h experimental group diamondback moth it is dead
The rate of dying is respectively 15.15%, 33.31%, 87.81% to be higher than positive controls (such as table 2).In the different processing time, diamondback moth
Mortality difference reach extremely significant level.The death rate of positive controls and experimental group is reached with negative control group between carrier
To extremely significant level of difference (such as table 1).
Corn borer insecticidal test the result shows that: in corn borer larvae room raising for 24 hours after, processing group occur soften, black and
Dead larva, and the larva normal growth of negative control group, with the extension of processing time, the larva of processing group is tapered off
The number of activity, dead worm increases.For 24 hours, 48h, 72h experimental group larval mortality are respectively 11.11%, 23.89%, 80%(such as table
4).The difference of the death rate of different disposal time each group reaches extremely significant level, and between different carriers, experimental group and the positive are right
The difference of the death rate and negative control group according to group larva reaches extremely significant level (such as table 3).And the corn borer growth survived
Also it is seriously suppressed, it is 7% before processing, negative control group worm living that only the live weight of worm of experimental group list, which is only 0.18g, after 72h
Weight is 98% times (such as table 5) that 0.252g is before processing.
Insecticidal activity bioassay the result shows that: the gene of new mutagenesis can encode the toxalbumin for providing insecticidal activity,
And there is very strong toxic action to diamondback moth and corn borer.
The present invention passes through fallibility PCR method pairCry1Ab13Gene base sequence carries out random mutagenesis research, by mutagenesis
The sequencing results afterwards show: reaching 38.61%, Ser 10.1%, Thr8.0% with primary sequence homology and Leu7.9% is occupied
More, the content of sequence GC improves 3% after being mutated compared with original sequence, is divided online by using online allergy original database
Analysis shows that the sequence and known anaphylactogen homology are lower there is no sensitization, is shown by SDS-PAGE electrophoretic analysis result
There is an apparent specific band in 79.5KDa or so, illustrates under the induction of IPTGCry1Ab13-1 Gene has obtained just
Really expression.Pest-resistant (diamondback moth, corn borer) test is carried out with the albumen of inducing expression, the results showed that the expression albumen has very strong
Insecticidal activity, make the death rate of diamondback moth larvae up to 87.81%, make the death rate of corn borer larvae up to 80.00%, and survive
Pest growth be also seriously suppressed, it is obvious with contrast difference.A kind of this research of preliminary proof success modified form of mutagenesisCry1Ab13Gene, the gene have very strong insecticidal activity, can be cultivation and the engineered strain of transgenic pest-resistant breeding
Building provides candidate gene.
<110>Jilin Agriculture University
<120>Cry1Ab13-1 killing gene and application
<160> 1
<210> 1
<211> 2036
<212> DNA
<213>artificial
<400> 1
cttgcatgcc tgcaggtcga cgattatgga caacaatcct aacatcaacg aatgtatccc 60
atacaactgt ttgtcaaatc ctgaggttga ggtgcttggt ggtgagagga tcgagacagg 120
atacactcca atcgacatat cactttctct tacacagttt cttctttctg agttcgtgcc 180
tggtgctgga ttcgtgcttg gacttgtgga catcatctgg ggtatctttg gaccttcaca 240
gtgggacgca ttccttgtgc agatcgagca gttgatcaat cagagaatcg aagagttcgc 300
taggaaccag gctatctcaa ggcttgaagg actttctaac ttgtaccaga tctacgctga 360
atctttcagg gagtgggagg ctgactcaac taatcctgca cttagggaag aaatgagaat 420
acagttcaac gacatgaact ctgctcttac tactgctatc cctcttttcg ctgtgcaaaa 480
ctaccaagtg ccacttcttt cagtgtacgt gcaggcagct aaccttcacc tttctgtgct 540
tagggacgtg tcagtgttcg gtcagaggtg gggttttgac gctgcaacaa tcaattcaag 600
gtacaacgat cttactaggc ttatcggtaa ctatactgac cacgctgtga ggtggtacaa 660
cactggtttg gagagagtgt ggggtccaga ctcaagggac tggatcaggt acaaccagtt 720
cagaagggag ttgactttga ctgttcttga cattgtgtca ttgttcccta actacgattc 780
taggacttat cctatcagga ctgtttctca attgactagg gaaatctaca ctaacccagt 840
tcttgagaac ttcgacggtt ctttcagggg ttcagctcag ggtatcgagg gatctatcag 900
atcaccacac cttatggaca tcttgaactc aatcacaatc tacacagacg cacacagggg 960
tgagtactac tggtcaggtc accagatcat ggcatctcca gtgggtttct ctggtcctga 1020
gttcacattc cctttgtatg gaactatggg aaacgcagct cctcagcaga gaattgtggc 1080
tcagttggga cagggagtgt ataggactct ttcaacaaca ttgtatagga gaccattcaa 1140
cattggaatc aacaatcagc agctttcagt gcttgatggt acagagttcg catacggtac 1200
atcatcaaac ttgccttctg ctgtgtacag gaagtctgga actgtggatt cattggacga 1260
gattccacct caaaacaaca acgtgcctcc aaggcaggga ttctcacaca ggttgtctca 1320
cgtgtctatg ttcaggtctg gattctcaaa ctcatctgtt tctattatca gggcacctat 1380
gttctcatgg atacacaggt cagcagagtt caacaatatc atcccttctt ctcaaatcac 1440
tcagatccct cttactaagt ctacaaactt gggatctgga acatcagtgg tgaagggtcc 1500
aggattcact ggtggtgaca tcttgaggag gacatctcca ggtcagatct ctacacttag 1560
agttaacatc acagctcctc tttctcagag gtacagggtt aggatcaggt atgcatctac 1620
aactaacttg cagttccaca cttcaattga tggaaggcca atcaaccagg gtaacttctc 1680
tgcaactatg tcatcaggtt caaaccttca gtcaggatca ttcaggacag ttggtttcac 1740
aacaccattc aacttctcta atggatcatc agttttcaca ttgtcagcac acgtgttcaa 1800
ctcaggtaat gaagtgtata tcgacaggat tgagttcgtt ccagcagagg ttacattcga 1860
ggcagagtac gacttggaga gggcacagaa ggcagtgaac gagcttttca catcttcaaa 1920
tcaaatcggt ttgaaaacag atgttacaga ctaccatatc gaccaggtgt ctaactccga 1980
gaaggatgaa ctttgataaa atctctagag gatccccggg taccgagctc gaatcg 2036
Claims (5)
1. mutated geneCry1Ab13-1, base sequence is as shown in sequence table SEQ ID NO.1.
2. mutated gene as described in claim 1Cry1Ab13-1, it is characterised in that: the gene is artificial synthesized.
3. Cry1Ab13-1 albumen, it is the albumen of gene expression shown in sequence table SEQ ID NO.1.
4. Cry1Ab13-1 albumen as claimed in claim 3, as the application for preparing insecticide, the worm is diamondback moth or jade
Rice snout moth's larva.
5. mutated gene described in claim 1Cry1Ab13-1Cultivating anti-diamondback moth worm or maize borer genetically modified plants
The application of kind.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926365A (en) * | 2009-11-26 | 2010-12-29 | 中国农业科学院植物保护研究所 | Use of bacillus thuringiensis cry1Ai in pest control, modified mcry1Ai gene and use of modified mcry1Ai gene |
| CN102010873A (en) * | 2010-11-22 | 2011-04-13 | 河南省农业科学院 | Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof |
| CN102660560A (en) * | 2012-04-26 | 2012-09-12 | 河南省农业科学院 | Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof |
| CN104292314A (en) * | 2013-07-19 | 2015-01-21 | 中国科学院亚热带农业生态研究所 | Codon optimized Cry1Ca<#> gene, recombinant vector and method for improving crop resistance |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7449552B2 (en) * | 2006-04-14 | 2008-11-11 | Pioneer Hi-Bred International, Inc. | Bacillus thuringiensis cry gene and protein |
| US20110239334A1 (en) * | 2008-08-25 | 2011-09-29 | Dow Agrosciences Llc | Nematode-resistant plants, and modified bacillus thuringiensis cry genes and proteins |
-
2017
- 2017-03-04 CN CN201710125280.2A patent/CN106701791B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926365A (en) * | 2009-11-26 | 2010-12-29 | 中国农业科学院植物保护研究所 | Use of bacillus thuringiensis cry1Ai in pest control, modified mcry1Ai gene and use of modified mcry1Ai gene |
| CN102010873A (en) * | 2010-11-22 | 2011-04-13 | 河南省农业科学院 | Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof |
| CN102660560A (en) * | 2012-04-26 | 2012-09-12 | 河南省农业科学院 | Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof |
| CN104292314A (en) * | 2013-07-19 | 2015-01-21 | 中国科学院亚热带农业生态研究所 | Codon optimized Cry1Ca<#> gene, recombinant vector and method for improving crop resistance |
Non-Patent Citations (4)
| Title |
|---|
| Cry1Ab13杀虫基因的PCR诱变及功能验证;闫鸽等;《西北农林科技大学学报（自然科学版）》;20180531;第46卷(第5期);第141-148页 |
| Oryza sativa Japonica Group transgenic CryIA(a)(cryIA(a))gene,partial cds;wang,X. et al.;《GenBank》;20110123;全文 |
| 杀虫基因Cry1Ab的合成、表达及其抗虫性鉴定;岳润清等;《华北农学报》;20141231;第29卷(第6期);第131-135页 |
| 苏云金芽孢杆菌cry1Ab13基因的克隆及表达研究;檀建新等;《微生物学报》;20020228;第42卷(第1期);第40-44页 |
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