CN111088265A - Insect-resistant fusion gene, encoding protein thereof, expression vector thereof and application thereof - Google Patents
Insect-resistant fusion gene, encoding protein thereof, expression vector thereof and application thereof Download PDFInfo
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- 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)
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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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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Abstract
The invention discloses an insect-resistant fusion gene, an encoding protein thereof, an expression vector thereof and application thereof, aiming at solving the problem of insect killingThe resistance trend of insect preparations is an irresistible technical problem. The invention designs an insect-resistant gene and an insect-resistant fusion geneCryAb‑VIP3AAlso provides protein coded by the insect-resistant fusion gene, and designs an expression vector constructed by the insect-resistant fusion gene and a recombinant bacterium constructed by the expression vector. The insect-resistant fusion gene is applied to preparing insect-resistant plant cells. The protein coded by the insect-resistant fusion gene is applied to preparing insect-resistant preparations. The insect-resistant fusion gene can be stably expressed in corn, and the original situation is avoidedVIP3A(a)The biotoxicity generated by the gene alone transferred into the plant overcomes the gene silencing phenomenon existing in the expression of exogenous genes, provides more choices for the prevention and the treatment of plant pests, and provides a new method for restraining the drug resistance trend of insects to the pesticide preparation.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an insect-resistant fusion gene, an encoding protein thereof, an expression vector thereof and application thereof.
Background
The pests eating the crops can reach millions, which causes great economic loss and seriously affects the yield and quality of the crops. At present, the prevention and control of crop pests mainly depend on chemical pesticides, and the chemical pesticides are used in large quantities for a long time to cause great harm to the environment and health. In order to reduce environmental pollution and potential harm to human health, development of novel medicaments by using biotechnology becomes a research hotspot.
With the development of molecular biology technology and the emergence of technologies such as gene cloning and DNA operation, humans begin to clone insect-resistant genes into engineering bacteria, and further resistant transgenic plants are obtained, many of which have already entered field experiments, and even some of which have already begun to be planted in large areas.
The selection and cloning of excellent target genes are the basis of genetic engineering research, and the cloning of the existing insect-resistant genes mainly comprises the following steps:
bacillus thuringiensis (B.thuringiensis) (B.thuringiensis)Bacillus thuringiensisBt) insect-resistant gene: after being ingested by sensitive insects, the insecticidal protein crystal generated in the process of forming spores of Bt is hydrolyzed into polypeptide under the alkaline condition of midgut, so that pore canals are formed on cell membranes, the ion balance of cells is further destroyed, and finally the cells are cracked to cause the death of the insects; protease inhibitor gene: plays an important role in maintaining the normal metabolism of organisms and preventing the damage of foreign proteolytic enzymes to a matrix. The compound feed can inhibit the activity of insect intestinal protease to cause insect indigestion, and finally death is caused by limited growth and development due to lack of amino acid; phytohemagglutinin gene: sugar eggs containing divalent metal ionsWhite, which can lead the red blood cells of insects, especially insects with piercing-sucking mouthparts to generate agglutination, thereby further triggering death; chitinase gene: chitin is an important component of fungal cell walls and insect shells, and chitinase is an endogenous protein existing in plants and has important functions of resisting pathogenic fungi and herbivores; scorpion insect toxin gene: can be specifically combined with sodium ion channels on insect nerve cell membranes to cause the permeability change of sodium ions, thereby causing the paralysis and death of insects.
At least 90 genetically encoded insecticidal crystalline proteins are currently isolated from a large number of Bt strains. The insect pathogen Bt gene produces 3 proteins of Cry, Cyt, Viss and the like. Cry and Cyt proteins produce insecticidal proteins that aggregate in inclusion or byhole crystals, and Cry proteins classify these genes into 4 major classes of CryI, CryII, CryIII, and CryIV based on protein structural homology and host range. Wherein CryI is highly resistant to lepidopteran insects, CryII is highly resistant to lepidopteran and dipteran insects, Cry III is highly resistant to coleopteran insects, and CryIV is resistant to dipteran insects. In recent years, Bt toxic protein (CryV) resisting lepidoptera and coleoptera and Bt toxic protein (CryVI) resisting nematode are discovered in sequence. Most strains of Bacillus thuringiensis produce several insecticidal crystal proteins, but the host range for each insecticidal crystal protein is rather narrow.
In addition to the above-mentioned several classes, there are pea lipoxygenase genes, insect female sterility protein factor genes, insect juvenile hormone lipase genes, insect neurohormone genes, and the like.
However, the potential problems of the insect-resistant plants with the transferred insect-resistant genes are increasingly revealed, and the continuous utilization of the insect-resistant plants is influenced. Such as: the insect resistance of the transgenic plants obtained by directly applying the original insect-resistant gene to the transgenic plants is very low, the insect-resistant effect is poor, the expression quantity of toxic protein is also very low, the expression product is unstable, the insect pest control requirements on agricultural production cannot be met, and most of the transgenic plants are difficult to apply; with the wide range of application of insect-resistant genes, insects can also generate resistance to insecticidal proteins; the insect-resistant spectrum of the insect-resistant gene is narrow; the expression of exogenous genes in plants has the phenomenon of gene silencing, and the like.
Therefore, it is urgent to develop new insect-resistant genes or to take full advantage of the existing insect-resistant gene resources to cope with the trend of resistance of insects to insecticidal agents.
Disclosure of Invention
The invention aims to solve the technical problem of how to select and transform excellent insect-resistant genes, further design insect-resistant fusion genes, and apply the insect-resistant fusion genes, the coding proteins and the expression vectors thereof to insect-resistant plant cells or insect-resistant preparations so as to solve the technical problem that the drug resistance trend of insects to the insect-resistant preparations is not suppressible and provide more diversified technical means for controlling plant pests.
In order to solve the technical problems, the invention adopts the following technical scheme:
designs an optimized and modified insect-resistant geneVIP3A(a)The nucleotide sequence is shown in SEQ ID NO. 3.
Based on a large number of long-term experiments and practical experiences, an insect-resistant fusion gene is designedCryAb-VIP3AThe nucleotide sequence is as follows:
(1) a nucleic acid sequence shown as SEQ ID NO. 5; or
(2) A nucleic acid sequence with the same function derived from the nucleic acid sequence shown in SEQ ID NO. 5.
Provides a protein CryAb-VIP3A coded by an insect-resistant fusion gene, the amino acid sequence of which is as follows:
(1) an amino acid sequence shown as SEQ ID NO. 6; or
(2) The sequence of the active fragment or conservative variant is obtained by adding, deleting or replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 6.
Further designs a gene formed by the insect-resistant fusion gene by combining practical characteristicsCryAb-VIP3AThe constructed expression vector and the recombinant bacterium constructed by the expression vector.
Fusing the insect-resistant geneCryAb-VIP3AThe application in preparing insect-resistant plant cells.
Preferably, the plant is a monocotyledonous plant.
Preferably, the monocot is maize.
The protein CryAb-VIP3A coded by the insect-resistant fusion gene is applied to preparing insect-resistant preparations.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the invention selects the existing insect-resistant gene with modification potential, and obtains the brand-new insect-resistant gene by redesigning and modifyingCryAbAnd insect-resistant geneVIP3A(a)The expression quantity of insecticidal protein can be obviously improved, and then a new variety of insect-resistant transgenic corn is cultivated and transformed.
2. The invention obtains the brand new insect-resistant fusion gene by further connecting and fusing the modified insect-resistant geneCryAb-VIP3AGreatly enhances the insect-resistant and insecticidal effects of the transformed crops, enlarges the insecticidal spectrum, and obtains new crop varieties which can resist lepidoptera pests or coleoptera pests (such as spodoptera frugiperda, armyworm, cutworms and the like) besides the borer.
3. The invention relates to an insect-resistant fusion geneCryAb-VIP3ACan be stably expressed in corn, and avoids the original expressionVIP3A (a)The biotoxicity generated by the gene alone transferred into the plant overcomes the gene silencing phenomenon existing in the expression of exogenous genes in the plant body, and can obviously improve the insect resistance of the corn leaf.
4. The invention relates to an insect-resistant fusion geneCryAb-VIP3AHas excellent insecticidal activity, provides more choices for the prevention and the treatment of plant pests, and provides a new method for restraining the drug resistance trend of insects to insecticidal preparations.
5. The invention relates to an insect-resistant fusion geneCryAb-VIP3AThe recombinant expression vector and the recombinant bacteria have wide application space and market prospect in providing insect resistance of plants.
6. The invention fuses insect-resistant genesCryAb-VIP3AAfter introduction into maize, stably inherited transformants can be obtained. In addition, the gene can also transform crops such as cotton, rice, vegetables and the like to ensure that the crops have corresponding insect-resistant activity, thereby reducing the using amount of pesticides and reducing environmental pollution, and having important economic value and wide application rangeAnd (5) landscape.
7. The protein CryAb-VIP3A coded by the insect-resistant fusion gene has excellent insecticidal effect, can be applied to preparation of insect-resistant preparations, and is environment-friendly and pollution-free.
Drawings
FIG. 1 is a plasmid map of pCAMBIA3300-CryAb-VIP 3A.
FIG. 2 shows a transformant obtained by Agrobacterium-mediated transformationCryAb-VIP3AScreening the callus of the gene corn.
FIG. 3 shows a transformant obtained by Agrobacterium-mediated transformationCryAb-VIP3ACallus differentiation of gene maize.
FIG. 4 shows a transformant obtained by Agrobacterium-mediated transformationCryAb-VIP3AThe transgenic plant regeneration seedling of the gene corn.
FIG. 5 is T0Generation of transformant target geneCryAb-VIP3A(ii) PCR detection map of (a);
wherein, M: DL2000 Marker; 1: blank control (double distilled water); 2: negative control: (non-transgenic maize); 3-5: a transgenic line; 6: positive control (plasmid pCAMBIA3300-CryAb-VIP 3A).
FIG. 6 is T0PCR detection of the surrogate transformant selection marker gene, Bar;
wherein, M: DL2000 Marker; 1: blank control (double distilled water); 2: negative control: (non-transgenic maize); 3: positive control (plasmid pCAMBIA3300-CryAb-VIP 3A); 4-6, transgenic strains.
FIG. 7 is T0Transfer deviceCryAb-VIP3AImmunological detection of gene transformant target protein CryAb picture (a), Vip3A (a) picture (b) and screening marker protein bar picture (c);
FIG. 8 is a view of a rotary tableCryAb-VIP3AIndoor bioassay contrast chart of resistance of leaf Asiatic corn borers in seedling stage of insect-resistant genes.
FIG. 9 is a view of a rotary tableCryAb-VIP3AIndoor bioassay contrast chart of insect-resistant gene corn seedling stage Spodoptera frugiperda resistance.
In FIGS. 7 to 9, CK is a negative control of a non-transgenic seedling; 1-3 are transgenic strains.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents or products are all conventional reagents or products on the market if not specifically indicated; the test methods involved are conventional methods unless otherwise specified.
The first embodiment is as follows: insect-resistant geneCry1AbAndVIP3A(a)is improved
The inventor bases on extensive experimental study and practical experience for a long time, in the originalCry1Ab(GenBank sequence number is AY 847289.1) insect-resistant gene, mainly including removing part of redundant base sequence, only leaving nucleotide sequence containing functional structure domain, modifying codon, confirming and removing several AT enrichment regions of ATTTA, AATGAA, etc. in original insect-resistant gene DNA sequence and inverted repeat sequence existed in gene sequence, removing part of commonly-used restriction endonuclease recognition site sequence (A) and (B) (A) are addedXbaI、SacI) The sequence of an intron of an undefined eukaryotic DNA sequence and a sequence which can cause the premature termination of the transcription of the gene or cause the instability of mRNA are reduced, the termination codon at the 3' end is modified into GGG, and finally, the optimized insect-resistant gene Cry1Ab suitable for monocotyledons is obtained, and the insect-resistant gene with enhanced resistance function is obtained. The nucleotide sequence is shown in SEQ ID NO. 1.
The insecticidal protein produced by the microorganism bacillus thuringiensis in the vegetative growth phase is VIP protein, and is the insecticidal protein produced by the thallus in the vegetative growth phase and secreted into a culture medium. VIP proteins are divided into four families according to their amino acid properties, and their functions are different. VIP1 and VIP2 proteins are toxic to some members of the orders coleoptera and hemiptera as binary toxins. The VIP1 component is thought to bind to receptors on insect midgut membranes, and the VIP2 component enters cells, where it exhibits ADP ribotransferase activity against actin, preventing microfilament formation. VIP3 has no sequence similarity to VIP1 or VIP 2. For the recently reported VIP4 family, no target insects have been found. The inventors have found that although the VIP3a protein does not share a binding site with the cry protein, the latter property makes them bind well to cry proteins in transgenic plants to prevent or delay pest resistance and broaden the insecticidal spectrum.
In the original placeVIP3A(a)(the nucleotide sequence is shown as SEQ ID NO. 2) is greatly improved and designed on the basis of the insect-resistant gene, and mainly comprises: only a part of the deleted 2373bp base sequence is left; except that 15bp base sequence at the 5' end is changed, the amino acid composition of the base sequence is changed, the rest 2358bp sequence carries out base substitution by using codons preferred by plants under the condition of keeping the total unchanged amino acid composition of the sequence protein, and a modified DNA sequence is obtained preliminarily; exclusion of AT-rich sequences present in DNA sequences which cause instability of plant transcription (e.g.ATTTA, AATGAA, etc.) and commonly used restriction sites: (HindIII、SacI) Then, correcting and eliminating by a method of replacing codons; adding a termination codon TAG at the 3' end; finally, the insect-resistant gene with codon optimization designed according to the coding characteristics of the monocotyledon is obtainedVIP3A(a)The nucleotide sequence is shown in SEQ ID NO. 3.
Modified synthetic bacillus thuringiensis nutritive insecticidal proteinVIP3A(a)Genes, and originalsVIP3A(a)Compared with the gene sequences, the gene has the following characteristics:
(1) the 5' end 15bp base sequence is changed from the original ATGAACATGAACAAG base sequence into a modified GTCCCCGGTAAAGGA base sequence;
(2) the 5' end protein amino acid sequence is transformed from original MNMNK amino acid into VPGKGAN sequence;
(3) the homology between the two sequences is 66%. The statistics of base composition are: the G + C% of the original gene was 30.89%, and the G + C% of the newly synthesized gene was 56.48%.
Example two: insect-resistant fusion geneCryAb-VIP3AConstruction of
Optimized modified insect-resistant fusion geneCryAb-VIP3A（CryAb-VIP3A-MR) Modified by the embodimentCryAbGene sequences, linker sequences L andmodified exampleVIP3A(a)Gene sequence composition.Cry1AbAfter the 3-end stop codon of the gene is removed from the code, the reading frame of the gene can be continuously expressed until the gene is optimized and modifiedvip3A(a)Gene, after forming optimized modificationCry1AbGenes andvip3Athe fusion gene of (4). The nucleotide sequence of the connecting sequence L is shown as SEQ ID NO. 4; the nucleotide sequence of the insect-resistant fusion gene is shown as SEQ ID NO.5, and the coded amino acid sequence thereof is shown as SEQ ID NO. 6.
For detecting modificationsCryAb-VIP3AThe prokaryotic expression vector of the fusion gene is constructed by the in vitro expression of the gene and the toxicity to pests such as corn borer and the like. Adding primer sequence at 5' end according to Bt gene cloning requirementNdeI endonuclease recognition site sequence CATATG, 3' end additionHindIII endonuclease recognition site sequence AAGCTT.
The primer sequences were designed as follows:
the upstream primer F1: 5'-CATATGGACAACAACCCAAACATC-3', respectively;
the downstream primer R1: 5'-AAGCTTCTAGTACTCCGCCTCG-3' are provided.
Synthesized by Biotechnology engineering (Shanghai) LtdCryAb-VIP3ApUC-of-genesCryAb- VIP3APlasmid, using the plasmid as a template and F1 and R1 as primers, and amplifyingCryAb-VIP3AGene using restriction enzymeNdeI andHindIII, carrying out enzyme digestion, and recovering and purifying by a gel recovery kitCryAb-VIP3AA gene fragment.
Using restriction endonucleasesNdeI andHindIII digestion of pET28b +, recovery and purification of 5.3kb fragment by gel recovery kit. pET28b + plasmid fragment and purificationCryAb-VIP3AThe gene fragment is subjected to ligation reaction, and the constructed prokaryotic expression plasmid is named as pET-CryAb-VIP 3A. Using restriction endonucleasesNdeI andHindIII, enzyme digestion identification is carried out, which shows that the vector construction is correct. pET-CryAb-VIP3A was transformed into BL21(DE3) competent cells for use.
Example three: insect-resistant fusion geneCryAb-VIP3AExpression in E.coli
1. Construction of recombinant Escherichia coli
The recombinant plasmid pET-CryAb-VIP3A is transformed into Escherichia coliE.coliBL21(DE3) and positive transformants were selected. After the restriction enzyme of the extracted plasmid is verified, selecting a positive transformant, inoculating the positive transformant into a resistant culture medium, carrying out overnight culture at 37 ℃, inoculating the positive transformant with 2 percent of inoculum size, and culturing the positive transformant until OD is reached600The value is about 0.5-0.6, and the product is stored at 4 ℃ for later use.
2. Indoor insecticidal effect test for detecting recombinant bacterium expression product
(1) The experimental treatments were divided into 4 groups:
inducing escherichia coli containing recombinant pET-CryAb-VIP3A by IPTG, carrying out ultrasonic disruption, and collecting supernatant as a test group; cultivation under the same conditions with example onemCryAbRecombinant pET28b vector of Gene sequenceE.coliBL21(DE3) and modified with examplesVIP3A(a)Recombinant pET28b vector of Gene sequenceE.coliBL21(DE3), sonicated and the supernatant collected as a control; clear water was used as a blank group.
(2) And adding the same amount of supernatant liquid into the prepared feed for the ostrinia nubilalis to serve as a test feed for feeding the ostrinia nubilalis for insect test.
The method comprises the following specific steps:
putting a feed into each test tube, and respectively inoculating 10 newly born first-instar larvae (ostrinia nubilalis, spodoptera frugiperda, spodoptera exigua, Bt-resistant cotton bollworms); 10 test tubes were used for each treatment; and (3) placing the mixture into an environment with the temperature of 26-28 ℃ and the relative humidity of about 70% for culturing for 8 days, and detecting the average death rate and the weight of each live insect. Specific results are shown in table 1.
TABLE 1 results of insecticidal test
Table 1 the statistical results show that:
transformation ofmCryAbThe protein expressed by the prokaryotic expression vector has a good insecticidal effect on the ostrinia nubilalis, the death rate of the ostrinia nubilalis reaches 95.2%, the protein also has a certain insecticidal effect on spodoptera frugiperda and spodoptera exigua, but has almost no insecticidal property on Bt resistant cotton bollworm programs;
transformation ofVIP3A(a)The protein expressed by the prokaryotic expression vector has good insecticidal effect on spodoptera frugiperda and spodoptera exigua, the mortality rate respectively reaches 90.5 percent and 89.9 percent, and the protein also has certain insecticidal effect on corn borer and Bt resistant cotton bollworm, the mortality rate of the corn borer is 82.7 percent, and the mortality rate of the Bt resistant cotton bollworm is 70.2 percent;
the anti-insect-fusion protein CryAb-VIP3A is formed by effectively connecting two independent proteins, namely mCryAb and VIP3A (a), into a fusion protein by using a connecting peptide, so that the fusion protein has the functions of the two proteins. The experimental result of the insecticidal test shows that: the CryAb-VIP3A insect-resistant fusion protein has strong insecticidal property, the killing rate of the Cryptocarya paliurus, Spodoptera frugiperda, Spodoptera exigua and Bt resistant cotton bollworm reaches more than 90 percent, and the insecticidal effect is very obvious.
The experiments of the prokaryotic expression insect test show that:CryAb-VIP3Athe Bt insecticidal protein coded by the gene has strong insecticidal effect, which shows thatCryAb-VIP3AThe gene modification can express the insecticidal toxic protein with strong biological activity.
Example four: construction ofCryAb-VIP3ARecombinant expression vector and recombinant expression bacterium of gene
The method comprises the following specific steps:
(1) using T carrier as frame, and synthesizing the protein by biological engineering (Shanghai) GmbHCryAb-VIP3AThe recombinant vector of the gene is named as T-CryAb-VIP 3A;
(2) cloning by using an amplification primer and taking T-CryAb-VIP3A as a templateCryAb-VIP3AA gene; the upstream primer containsXbaI cleavage site, downstream primer containsSacI enzyme cutting site.
(3) By usingXbaI andSaci double restriction enzyme clonedCryAb-VIP3ARecovering gene segment;
(4) is connected with and usedXbaI andSaci, obtaining the product on a genetic transformation high-efficiency plant expression vector pCAMBIA3300 subjected to double enzyme digestion treatmentCryAb-VIP3AThe recombinant expression vector of the gene is named as pCAMBIA3300-CryAb-VIP3A, and the plasmid map is shown in figure 1.
(5) Recombinant plasmid pCAMBIA3300-CryAb-VIP3A is transformed into agrobacterium EHA105, and positive strains are screened to obtainCryAb-VIP3AThe recombinant expression strain of the gene is preserved at low temperature and is used for subsequent tests.
Example five: recombinant agrobacterium-mediated transformation of maize immature embryos and calluses
1. Stripping young maize embryo
(1) Removing corn bracts;
(2) cutting off the top end of the fruit cluster by about 1cm, inserting the fruit cluster from the top end by using a pair of tweezers as a handle, then placing the fruit cluster into a beaker containing disinfectant, and placing 4-6 fruit clusters into the same beaker according to actual needs;
(3) adding about 700mL of disinfectant (50% of bleaching agent or 5.25% of sodium hypochlorite, and adding one drop of Tween 20) into the beaker to soak the cluster, and disinfecting for 20 min; during the disinfection period, the ears are rotated at intervals and the beaker is lightly tapped to remove air bubbles on the surface of the seeds, so that the optimal disinfection effect is achieved;
(4) after the disinfection is finished, taking out the clusters, putting the clusters into a beaker filled with sterilized water, washing the clusters in the water for 3 times, and preparing for embryo peeling;
(5) put the one end of the ear of fruit of disinfecting on a big culture dish, cut off the top (1.5 ~ 1.8 mm) of seed grain with big scalpel, in the middle of this process, the used instrument of disinfection of will going on duty, if: surgical blades, culture dishes, embryo peeling knives, and the like; inserting the tip of an embryo peeling knife between an embryo and an endosperm, prying the young embryo upwards slightly, slightly supporting the young embryo by using a small surgical tip to ensure that the young embryo is not damaged, tightly attaching the embryo axial surface of the young embryo to an N6E culture medium containing filter paper, wherein the density of the embryo is about 2 multiplied by 2cm (30 embryos per dish);
(6) sealing the culture dish with a sealing film, and culturing at 28 ℃ in the dark for 2-3 days.
2. Recombinant Agrobacterium infection
(1) Taking the recombinant agrobacterium constructed in the fourth embodiment to perform activated culture on a YEP (containing Kan 50 mg/L and Str100 mg/L antibiotics) culture medium;
(2) streaking was performed in YEP medium (containing Kan 50 mg/L and Str 50 mg/L antibiotics) at 19 ℃ for 3 days;
(3) picking recombinant agrobacterium tumefaciens, putting the recombinant agrobacterium tumefaciens into a 50 mL centrifuge tube containing 5mL of a staining culture medium, adding 100 uM AS (inf + AS) at the same time, and shaking the strain at the rotation speed of 75 rpm at room temperature (25 ℃) for 2-4 h;
(4) the peeled embryos are placed in a centrifuge tube containing inf + AS liquid medium (2 mL) and about 20 to 100 embryos per tube, washed 2 times with such medium, and then 1 to 1.5mL of OD is added550And (3) slightly inverting the centrifuge tube for 20 times by using the recombinant agrobacterium of which the concentration is 0.3-0.4, and then vertically placing the recombinant agrobacterium in a dark box for 5 min to ensure that all the immature embryos are soaked in the agrobacterium liquid, so that vortex oscillation is avoided in the whole process.
3. Co-cultivation
After infection, transferring the impregnated immature embryos to a co-culture medium, enabling the embryonic axis of the immature embryos to contact the surface of the culture medium, and simultaneously, sucking dry by using sterile filter paper to expel redundant agrobacterium on the surface of the culture medium; the petri dish was sealed with a sealing film and incubated at 20 ℃ for 3 days in the dark.
4. At rest
After 3 days of co-cultivation, the embryos were transferred to a resting medium while the dishes were sealed with a sealing film and incubated at 28 ℃ for 7 days in the dark as shown in FIG. 2.
5. Selecting
After 7 days, all the immature embryos were transferred to a selection medium containing 1.5 mg/L bialaphos for two weeks, which was subcultured two weeks later, the concentration of bialaphos was increased to 3 mg/L, which was about 5 weeks after the infection, and the cells containing the transformants grew into visible type II calli, as shown in FIG. 3.
6. Regeneration of transgenic plants
In a light culture room, taking the callus to grow for 3 weeks on a regeneration culture medium I, and then germinating on a regeneration culture medium II; when 3-4 leaves grow out from the transgenic regeneration seedlings, the transgenic regeneration seedlings are transferred to a greenhouse and checked, and positive plants are reserved, as shown in fig. 4. When the plant grows to the stage of spinning and pollen scattering, the plant is pollinated.
Example six: detection ofCryAb-VIP3AThe genes being in maize plantsExpression of
1. PCR detection
When the transgenic plant cultured in the fifth embodiment grows to 5-6 leaf stages, extracting leaf genome DNA of the plant by a CTAB method, and designing a primer for PCR amplificationCryAb-VIP3AGene and glufosinate-resistant gene on plasmid pCAMBIA3300barA gene.
According toCryAb-VIP3AGenes andbarthe PCR detection primer sequence designed by the internal sequence of the gene is as follows:
CryAb-VIP3A-F’： AACCTGGGCAGCGGCACCAGCG；
CryAb-VIP3A-R’： CACGTTGACGTTGATGATGTC；
size of the target fragment: 957 bp.
bar-F：ATGAGCCCAGAACGACGCCCG；
bar-R：TCGGTGACGGGCAGGACCGG；
Size of the target fragment: 545 bp.
The reaction system of PCR is: 2uL of DNA template, 2uL10 XPCR Buffer, 2uL of dNTP (10mM each), 1uL of forward primer (10mM), 1uL of reverse primer (10mM), 0.3uL of Tap enzyme, sterile water make up to 20 uL.
The reaction procedure for PCR is shown in Table 2.
TABLE 2 PCR reaction procedure
The detection results are shown in fig. 5 and 6:
FIG. 5 shows T0PCR detection results of a transformant target gene CryAb-VIP3A are generated; FIG. 6 shows T0PCR detection results of the generation transformant selection marker gene Bar. Demonstration of foreign GeneCryAb-VIP3AGenes andbarthe genes have been integrated into the maize genome.
2. Test strip detection
Detection of target protein CryAb-VIP3A (Bt-Cry 1Ab/1Ac immunology and VIP3A detection):
(1) taking about 1cm2Left and right fresh young leaves are placed in 1.5ml EppendorfIn the tube. The tubes containing leaves of the transgenic plants grown in example five were then inserted in an ice box to maintain freshness.
(2) Liquid nitrogen was taken, the material was snap frozen, ground to powder with a drill bit, and 500 μ L of the lmlSEB4 sample extraction buffer was quickly added to the tube.
(3) The test strip (Beijing Hande Weixin Tech Co., Ltd.) was taken out of the bucket, and the top end of the test strip was held by hand to make a test mark. The protective film is not removed. The test strip is held upright and the labeled end is inserted into a centrifuge tube or extraction bag. The insertion portion does not exceed 0.5 cm. The inserted state is maintained throughout the detection process.
(4) And a quality control line appears within 3-5 minutes, the longest reaction time is 30 minutes, and the detection strip can be taken out at the moment. The quality control line is used to ensure the accuracy of the test results. If the quality control line does not appear, the detection is invalid. The time at which the signal is generated varies due to the difference in fluidity of the sample. If the sample is positive, a test line will appear. If the sample is negative, the test line will not appear. If the test results are to be stored for a long period of time, the sample pad can be cut off and blotted dry with a paper towel, which will prevent the remaining liquid from interfering with the results. The depth of the detection line reflects the content of the detected protein.
The results are shown in FIG. 7:
indicating insect resistance genesCryAb-VIP3AExpressed target protein CryAb-L, VIP3A (a) and screening marker genebarHigh expression in transgenic corn.
Example seven: identification of insect resistance in transgenic maize plants
1. Rotating shaftCryAb-VIP3AIndoor bioassay identification of resistance of insect-resistant gene corn leaf Asiatic corn borer at seedling stage
Transfer in greenhouseCryAb-VIP3AThe method comprises the following steps of growing seedlings of the gene corn plants to be corn leaf planting leaves in a 5-8 leaf stage (taking the unextended tender heart leaves), shearing the corn leaf planting leaves into 2-3 cm in size by using disinfection scissors, placing the corn leaf planting leaves in a 24-hole cell culture plate, and connecting 3 newly hatched larvae in each hole. Using leaf of common corn plant as control group, transferringCryAb-VIP3AThe leaf of the gene plant is used as a test group, and the insect-resistant effect of the transgenic corn is observed after 1 week of culture.
The results are shown in FIG. 8, where the non-transgenic maize leaves were completely eaten by Asian corn borers, and appeared to be hypersensitive insects, and in turn, were transformedCryAb-VIP3AThe gene corn leaf has no Asiatic corn borer pest, high resistance and high transgenic effectCryAb-VIP3AThe gene insect-resistant corn is expressed as high resistance to Asiatic corn borer.
2. Rotating shaftCryAb-VIP3AInsect-resistant gene indoor bioassay identification of spodoptera frugiperda resistance of maize seedling stage leaf
The Spodoptera frugiperda population to be tested was collected from fresh corn fields in Delhong State of Yunnan province in 2019, 1 month and 19 days. Transfer in greenhouseCryAb-VIP3AThe method comprises the following steps of growing seedlings of the gene corn plants to the leaf planting stage of 5-8, cutting the corn plants into 2-3 cm-sized leaves by using disinfection scissors, and placing the leaves in the leaves, wherein each hole is connected with 5 adults of Spodoptera frugiperda. Using leaf of common corn plant as control group, transferringCryAb-VIP3AThe leaf of the gene plant is a test group, and the insect-resistant effect of the transgenic corn is observed after 24 hours.
As shown in FIG. 9, after only 24 hours, the leaves of non-transgenic maize were almost completely consumed by Spodoptera frugiperda, which appeared to be spodoptera frugiperda-sensitive, and in turn, were exposed to Spodoptera frugiperdaCryAb-VIP3AThe gene corn leaf is nearly not complete, has no spodoptera frugiperda harm and extremely strong resistance, and shows thatCryAb-VIP3AThe gene insect-resistant corn is expressed as high-resistance Spodoptera frugiperda.
To sum up, turn toCryAb-VIP3AThe indoor bioassay identification result of the insect-resistant genes of the Asiatic corn borer and the Spodoptera frugiperda in the seedling stage of the corn shows that: through mCryAbGenes andVIP3A(a)the gene transformation and fusion increase the insect resistance of the single gene; in addition, the single gene is transformed and fused, so that the protein expressed by the fusion gene is highly resistant to Asiatic corn borers and Spodoptera frugiperda, the insecticidal effect of crops can be enhanced, the insecticidal spectrum is expanded, and new varieties of crops which are resistant to other Lepidoptera pests or Coleoptera pests (Spodoptera frugiperda) besides the Ostrinia frugiperda are obtained.
While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.
SEQUENCE LISTING
<110> Guangzhou grass agricultural science and technology Co., Ltd
<120> insect-resistant fusion gene, encoding protein thereof, expression vector thereof and application thereof
<130>2019
<160>5
<170>PatentIn version 3.2
<210>1
<211>1848
<212>DNA
<213> Artificial Synthesis
<400>1
atggacaaca acccaaacat caacgagtgc atcccctaca actgcctctc gaacccggag 60
gtggaggtgc tgggcggcga gcgcatcgag acggggtata cgccgatcga tatctcgctc 120
agcctgaccc agttcctgct gagcgagttc gtcccgggcg cgggtttcgt ccttgggctc 180
gtggacatca tctggggcat cttcggcccc tcccagtggg acgcgttcct cgtccagatc 240
gagcagctga tcaaccagcg catcgaggag ttcgcgcgga atcaagcgat cagccggctc 300
gagggcctga gcaacctgta ccaaatctac gcggaatcct tccgggaatg ggaggcggac 360
cccaccaacc ccgccctgcg cgaggagatg aggatccagt tcaacgacat gaactccgcg 420
ctgaccaccg ccatccccct gttcgccgtg cagaactatc aggtcccgct actctcggtc 480
tacgtgcagg ccgccaacct gcacctgagc gtcctccggg acgtgtcggt cttcggtcag 540
cgctggggct tcgacgccgc caccatcaac tcgcggtaca atgacctcac gcgcctcatc 600
ggcaactaca ccgaccacgc cgtgcgctgg tacaacacgg ggctcgagcg ggtgtggggc 660
cccgacagcc gcgactggat caggtacaac caattccgtc gggagcttac gttgacggtc 720
ctggacatcg tgagcctgtt ccccaactac gattcgagga cgtatccgat ccggacggtc 780
agccagctga cccgcgagat ttacaccaac ccggtcctcg agaacttcga tgggtccttc 840
cgcggcagcg cccagggcat cgagggcagc atccgctccc cgcacctcat ggatatcctc 900
aacagcatca ccatctacac cgacgcccac cggggggagt actattggtc cgggcaccag 960
atcatggcca gccccgtcgg cttcagcggc ccggagttca cgttcccgct gtacgggacg 1020
atgggcaacg ctgcacctca gcagcgcatc gtcgcgcagc tcgggcaggg cgtgtaccgc 1080
accctgagca gcaccctgta ccgtcgacct ttcaacatcg ggatcaacaa ccaacagctc 1140
agcgtgctgg acggcaccga gttcgcctac gggacgagca gcaacctccc gtcggcggtc 1200
taccgcaaga gcggcaccgt ggacagcctg gatgagatcc cgccgcagaa caataacgtc 1260
ccacctcgac agggcttcag ccaccgtctg tcgcacgtct cgatgttccg gtcggggttc 1320
agcaacagca gcgtgagcat catccgtgca ccgatgttct cgtggatcca ccggtcggcg 1380
gagttcaaca acatcatccc cagcagccag atcacgcaga tcccgctcac gaagtcgacg 1440
aacctgggca gcggcaccag cgtggtgaag gggccggggt tcacgggtgg ggacatcctc 1500
cgccgcacca gccccggcca gatcagcacc ttgcgggtca acatcacggc gccgctctcc 1560
cagcgctacc gcgtccgcat ccgctacgcc tcgacgacga acctccagtt ccacacgtcg 1620
atcgacggcc gccccatcaa ccagggcaac ttctcggcga cgatgtcctc ggggtcgaac 1680
ctgcagagcg gcagcttccg caccgtgggc ttcacgacgc cgttcaactt ctccaacggc 1740
agcagcgtgt tcaccctgag cgcccacgtg ttcaactccg ggaacgaggt ctacatcgac 1800
cgcatcgagt tcgtgcccgc cgaggttacc ttcgaggcgg agtacggg 1848
<210>2
<211>2376
<212>DNA
<213>Bacillus thuringiensis
<400>2
atgaacatga acaagaataa tactaaatta agcacaagag ccttaccaag ttttattgat 60
tattttaatg gcatttatgg atttgccact ggtatcaaag acattatgaa catgattttt 120
aaaacggata caggtggtga tctaacccta gacgaaattt taaagaatca gcagttacta 180
aatgatattt ctggtaaatt ggatggggtg aatggaagct taaatgatct tatcgcacag 240
ggaaacttaa atacagaatt atctaaggaa atattaaaaa ttgcaaatga acaaaatcaa 300
gttttaaatg atgttaataa caaactcgat gcgataaata cgatgcttcg ggtatatcta 360
cctaaaatta cctctatgtt gagtgatgta atgaaacaaa attatgcgct aagtctgcaa 420
atagaatact taagtaaaca attgcaagag atttctgata agttggatat tattaatgta 480
aatgtactta ttaactctac acttactgaa attacacctg cgtatcaaag gattaaatat 540
gtgaacgaaa aatttgagga attaactttt gctacagaaa ctagttcaaa agtaaaaaag 600
gatggctctc ctgcagatat tcttgatgag ttaactgagt taactgaact agcgaaaagt 660
gtaacaaaaa atgatgtgga tggttttgaa ttttacctta atacattcca cgatgtaatg 720
gtaggaaata atttattcgg gcgttcagct ttaaaaactg catcggaatt aattactaaa 780
gaaaatgtga aaacaagtgg cagtgaggtc ggaaatgttt ataacttctt aattgtatta 840
acagctctgc aagcccaagc ttttcttact ttaacaacat gccgaaaatt attaggctta 900
gcagatattg attatacttc tattatgaat gaacatttaa ataaggaaaa agaggaattt 960
agagtaaaca tcctccctac actttctaat actttttcta atcctaatta tgcaaaagtt 1020
aaaggaagtg atgaagatgc aaagatgatt gtggaagcta aaccaggaca tgcattgatt 1080
gggtttgaaa ttagtaatga ttcaattaca gtattaaaag tatatgaggc taagctaaaa 1140
caaaattatc aagtcgataa ggattcctta tcggaagtta tttatggtga tatggataaa 1200
ttattgtgcc cagatcaatc tgaacaaatc tattatacaa ataacatagt atttccaaat 1260
gaatatgtaa ttactaaaat tgatttcact aaaaaaatga aaactttaag atatgaggta 1320
acagcgaatt tttatgattc ttctacagga gaaattgact taaataagaa aaaagtagaa 1380
tcaagtgaag cggagtatag aacgttaagt gctaatgatg atggggtgta tatgccgtta 1440
ggtgtcatca gtgaaacatt tttgactccg attaatgggt ttggcctcca agctgatgaa 1500
aattcaagat taattacttt aacatgtaaa tcatatttaa gagaactact gctagcaaca 1560
gacttaagca ataaagaaac taaattgatc gtcccgccaa gtggttttat tagcaatatt 1620
gtagagaacg ggtccataga agaggacaat ttagagccgt ggaaagcaaa taataagaat 1680
gcgtatgtag atcatacagg cggagtgaat ggaactaaag ctttatatgt tcataaggac 1740
ggaggaattt cacaatttat tggagataag ttaaaaccga aaactgagta tgtaatccaa 1800
tatactgtta aaggaaaacc ttctattcat ttaaaagatg aaaatactgg atatattcat 1860
tatgaagata caaataataa tttagaagat tatcaaacta ttaataaacg ttttactaca 1920
ggaactgatt taaagggagt gtatttaatt ttaaaaagtc aaaatggaga tgaagcttgg 1980
ggagataact ttattatttt ggaaattagt ccttctgaaa agttattaag tccagaatta 2040
attaatacaa ataattggac gagtacggga tcaactaata ttagcggtaa tacactcact 2100
ctttatcagg gaggacgagg gattctaaaa caaaaccttc aattagatag tttttcaact 2160
tatagagtgt atttttctgt gtccggagat gctaatgtaa ggattagaaa ttctagggaa 2220
gtgttatttg aaaaaagata tatgagcggt gctaaagatg tttctgaaat gttcactaca 2280
aaatttgaga aagataactt ttatatagag ctttctcaag ggaataattt atatggtggt 2340
cctattgtac atttttacga tgtctctatt aagtaa 2376
<210>3
<211>2376
<212>DNA
<213> Artificial Synthesis
<400>3
gtccccggta aaggaaacaa caccaagttg agcaccaggg cgttgccgag cttcatcgac 60
tacttcaacg gcatctacgg attcgcgacc ggcatcaagg acatcatgaa catgatcttc 120
aagacggaca cgggcggcga cctgacgctg gacgagatct tgaagaacca gcagctgctg 180
aacgacatct ccggcaagtt ggacggggtg aacggcagct tgaacgacct gatcgcacag 240
ggcaacttga acacggagtt gtcgaaggag atcttgaaga tcgccaacga gcagaaccag 300
gtcttgaacg acgtcaacaa caagctcgac gcgatcaaca cgatgctgcg ggtctacctg 360
ccgaagatca cctcgatgtt gagcgacgtc atgaagcaga actacgcgct gagcctgcag 420
atcgagtact tgagcaagca gttgcaggag atctccgaca agttggacat catcaacgtc 480
aacgtgctga tcaactcgac gctcaccgag atcacgcctg cgtaccagag gatcaagtac 540
gtgaacgaga agttcgagga gttgaccttc gcgaccgaga ccagctcgaa ggtcaagaag 600
gacggctcgc cggccgacat cctcgacgag ttgaccgagt tgaccgagct agcgaagagc 660
gtcaccaaga acgacgtcga cggcttcgag ttctacctca acacgttcca cgacgtcatg 720
gtcggcaaca acttgttcgg gcgctccgcc ttgaagaccg cctccgagtt gatcacgaag 780
gagaacgtga agaccagcgg cagcgaggtc ggcaacgtct acaacttcct catcgtcttg 840
accgcgctgc aggcgcaggc cttcctcacg ttgacgacgt gccgcaagtt gttgggcttg 900
gccgacatcg actacacgtc catcatgaac gagcacttga acaaggagaa ggaggagttc 960
agggtcaaca tcctgccgac gctgtcgaac acgttctcga acccgaacta cgcgaaggtc 1020
aagggcagcg acgaggacgc gaagatgatc gtggaggcga agccgggcca cgccttgatc 1080
ggcttcgaga tcagcaacga ctcgatcacg gtcttgaagg tctacgaggc gaagctgaag 1140
cagaactacc aggtcgacaa ggactccttg tcggaggtca tctacggcga catggacaag 1200
ttgttgtgcc cggaccagtc cgagcagatc tactacacga acaacatcgt cttcccgaac 1260
gagtacgtca tcacgaagat cgacttcacg aagaagatga agacgttgag gtacgaggtc 1320
acggcgaact tctacgactc gtccacgggc gagatcgact tgaacaagaa gaaggtcgag 1380
tcgagcgagg cggagtacag gacgttgagc gccaacgacg acggcgtgta catgccgttg 1440
ggcgtcatca gtgagacgtt cttgacgccg atcaacgggt tcggcctcca ggccgacgag 1500
aactccaggt tgatcacgtt gacgtgcaag tcctacttga gggagctgct gctcgccacg 1560
gacttgagca acaaggagac gaagttgatc gtcccgccga gcggcttcat cagcaacatc 1620
gtcgagaacg ggtccatcga ggaggacaac ttggagccgt ggaaggcgaa caacaagaac 1680
gcgtacgtcg accacaccgg cggcgtgaac ggcacgaagg cgttgtacgt ccacaaggac 1740
ggcggcatct cgcagttcat cggcgacaag ttgaagccga agacggagta cgtcatccag 1800
tacacggtca agggcaagcc gtcgatccac ttgaaggacg agaacacggg ctacatccac 1860
tacgaggaca cgaacaacaa cttggaggac taccagacga tcaacaagcg cttcacgacc 1920
ggcacggact tgaagggcgt gtacttgatc ttgaagagcc agaacggcga cgaggcgtgg 1980
ggcgacaact tcatcatctt ggagatcagc ccgtcggaga agttgttgag cccggagttg 2040
atcaacacga acaactggac gagcacgggc tcgacgaaca tcagcggcaa cacgctcacg 2100
ctctaccagg gcggacgcgg catcctgaag cagaacctgc agttggacag cttctcgacg 2160
tacagagtgt acttctcggt gtccggagac gcgaacgtca ggatcaggaa ctctagggaa 2220
gtgttgttcg agaagaggta catgagcggt gcgaaggacg tctccgagat gttcacgacg 2280
aagttcgaga aggacaactt ctacatcgag ctgtcgcaag ggaacaactt gtacggtggt 2340
cctatcgtcc acttctacga cgtctcgatc aagtaa 2376
<210>4
<211>63
<212>DNA
<213> Artificial Synthesis
<400>4
tgcaggagcg gtggaggcgg aggtggcagc agcggtggtg gcggagccaa cgtcgccagc 60
gtc 63
<210>5
<211>4287
<212>DNA
<213> Artificial Synthesis
<400>5
atggacaaca acccaaacat caacgagtgc atcccctaca actgcctctc gaacccggag 60
gtggaggtgc tgggcggcga gcgcatcgag acggggtata cgccgatcga tatctcgctc 120
agcctgaccc agttcctgct gagcgagttc gtcccgggcg cgggtttcgt ccttgggctc 180
gtggacatca tctggggcat cttcggcccc tcccagtggg acgcgttcct cgtccagatc 240
gagcagctga tcaaccagcg catcgaggag ttcgcgcgga atcaagcgat cagccggctc 300
gagggcctga gcaacctgta ccaaatctac gcggaatcct tccgggaatg ggaggcggac 360
cccaccaacc ccgccctgcg cgaggagatg aggatccagt tcaacgacat gaactccgcg 420
ctgaccaccg ccatccccct gttcgccgtg cagaactatc aggtcccgct actctcggtc 480
tacgtgcagg ccgccaacct gcacctgagc gtcctccggg acgtgtcggt cttcggtcag 540
cgctggggct tcgacgccgc caccatcaac tcgcggtaca atgacctcac gcgcctcatc 600
ggcaactaca ccgaccacgc cgtgcgctgg tacaacacgg ggctcgagcg ggtgtggggc 660
cccgacagcc gcgactggat caggtacaac caattccgtc gggagcttac gttgacggtc 720
ctggacatcg tgagcctgtt ccccaactac gattcgagga cgtatccgat ccggacggtc 780
agccagctga cccgcgagat ttacaccaac ccggtcctcg agaacttcga tgggtccttc 840
cgcggcagcg cccagggcat cgagggcagc atccgctccc cgcacctcat ggatatcctc 900
aacagcatca ccatctacac cgacgcccac cggggggagt actattggtc cgggcaccag 960
atcatggcca gccccgtcgg cttcagcggc ccggagttca cgttcccgct gtacgggacg 1020
atgggcaacg ctgcacctca gcagcgcatc gtcgcgcagc tcgggcaggg cgtgtaccgc 1080
accctgagca gcaccctgta ccgtcgacct ttcaacatcg ggatcaacaa ccaacagctc 1140
agcgtgctgg acggcaccga gttcgcctac gggacgagca gcaacctccc gtcggcggtc 1200
taccgcaaga gcggcaccgt ggacagcctg gatgagatcc cgccgcagaa caataacgtc 1260
ccacctcgac agggcttcag ccaccgtctg tcgcacgtct cgatgttccg gtcggggttc 1320
agcaacagca gcgtgagcat catccgtgca ccgatgttct cgtggatcca ccggtcggcg 1380
gagttcaaca acatcatccc cagcagccag atcacgcaga tcccgctcac gaagtcgacg 1440
aacctgggca gcggcaccag cgtggtgaag gggccggggt tcacgggtgg ggacatcctc 1500
cgccgcacca gccccggcca gatcagcacc ttgcgggtca acatcacggc gccgctctcc 1560
cagcgctacc gcgtccgcat ccgctacgcc tcgacgacga acctccagtt ccacacgtcg 1620
atcgacggcc gccccatcaa ccagggcaac ttctcggcga cgatgtcctc ggggtcgaac 1680
ctgcagagcg gcagcttccg caccgtgggc ttcacgacgc cgttcaactt ctccaacggc 1740
agcagcgtgt tcaccctgag cgcccacgtg ttcaactccg ggaacgaggt ctacatcgac 1800
cgcatcgagt tcgtgcccgc cgaggttacc ttcgaggcgg agtacgggtg caggagcggt 1860
ggaggcggag gtggcagcag cggtggtggc ggagccaacg tcgccagcgt cgtccccggt 1920
aaaggaaaca acaccaagtt gagcaccagg gcgttgccga gcttcatcga ctacttcaac 1980
ggcatctacg gattcgcgac cggcatcaag gacatcatga acatgatctt caagacggac 2040
acgggcggcg acctgacgct ggacgagatc ttgaagaacc agcagctgct gaacgacatc 2100
tccggcaagt tggacggggt gaacggcagc ttgaacgacc tgatcgcaca gggcaacttg 2160
aacacggagt tgtcgaagga gatcttgaag atcgccaacg agcagaacca ggtcttgaac 2220
gacgtcaaca acaagctcga cgcgatcaac acgatgctgc gggtctacct gccgaagatc 2280
acctcgatgt tgagcgacgt catgaagcag aactacgcgc tgagcctgca gatcgagtac 2340
ttgagcaagc agttgcagga gatctccgac aagttggaca tcatcaacgt caacgtgctg 2400
atcaactcga cgctcaccga gatcacgcct gcgtaccaga ggatcaagta cgtgaacgag 2460
aagttcgagg agttgacctt cgcgaccgag accagctcga aggtcaagaa ggacggctcg 2520
ccggccgaca tcctcgacga gttgaccgag ttgaccgagc tagcgaagag cgtcaccaag 2580
aacgacgtcg acggcttcga gttctacctc aacacgttcc acgacgtcat ggtcggcaac 2640
aacttgttcg ggcgctccgc cttgaagacc gcctccgagt tgatcacgaa ggagaacgtg 2700
aagaccagcg gcagcgaggt cggcaacgtc tacaacttcc tcatcgtctt gaccgcgctg 2760
caggcgcagg ccttcctcac gttgacgacg tgccgcaagt tgttgggctt ggccgacatc 2820
gactacacgt ccatcatgaa cgagcacttg aacaaggaga aggaggagtt cagggtcaac 2880
atcctgccga cgctgtcgaa cacgttctcg aacccgaact acgcgaaggt caagggcagc 2940
gacgaggacg cgaagatgat cgtggaggcg aagccgggcc acgccttgat cggcttcgag 3000
atcagcaacg actcgatcac ggtcttgaag gtctacgagg cgaagctgaa gcagaactac 3060
caggtcgaca aggactcctt gtcggaggtc atctacggcg acatggacaa gttgttgtgc 3120
ccggaccagt ccgagcagat ctactacacg aacaacatcg tcttcccgaa cgagtacgtc 3180
atcacgaaga tcgacttcac gaagaagatg aagacgttga ggtacgaggt cacggcgaac 3240
ttctacgact cgtccacggg cgagatcgac ttgaacaaga agaaggtcga gtcgagcgag 3300
gcggagtaca ggacgttgag cgccaacgac gacggcgtgt acatgccgtt gggcgtcatc 3360
agtgagacgt tcttgacgcc gatcaacggg ttcggcctcc aggccgacga gaactccagg 3420
ttgatcacgt tgacgtgcaa gtcctacttg agggagctgc tgctcgccac ggacttgagc 3480
aacaaggaga cgaagttgat cgtcccgccg agcggcttca tcagcaacat cgtcgagaac 3540
gggtccatcg aggaggacaa cttggagccg tggaaggcga acaacaagaa cgcgtacgtc 3600
gaccacaccg gcggcgtgaa cggcacgaag gcgttgtacg tccacaagga cggcggcatc 3660
tcgcagttca tcggcgacaa gttgaagccg aagacggagt acgtcatcca gtacacggtc 3720
aagggcaagc cgtcgatcca cttgaaggac gagaacacgg gctacatcca ctacgaggac 3780
acgaacaaca acttggagga ctaccagacg atcaacaagc gcttcacgac cggcacggac 3840
ttgaagggcg tgtacttgat cttgaagagc cagaacggcg acgaggcgtg gggcgacaac 3900
ttcatcatct tggagatcag cccgtcggag aagttgttga gcccggagtt gatcaacacg 3960
aacaactgga cgagcacggg ctcgacgaac atcagcggca acacgctcac gctctaccag 4020
ggcggacgcg gcatcctgaa gcagaacctg cagttggaca gcttctcgac gtacagagtg 4080
tacttctcgg tgtccggaga cgcgaacgtc aggatcagga actctaggga agtgttgttc 4140
gagaagaggt acatgagcgg tgcgaaggac gtctccgaga tgttcacgac gaagttcgag 4200
aaggacaact tctacatcga gctgtcgcaa gggaacaact tgtacggtgg tcctatcgtc 4260
cacttctacg acgtctcgat caagtaa 4287
<210>6
<211>4287
<212>PROTEIN
<213> Artificial Synthesis
<400>6
MDNNPNINEC IPYNCLSNPE VEVLGGERIE TGYTPIDISL SLTQFLLSEF VPGAGFVLGL 60
VDIIWGIFGP SQWDAFLVQI EQLINQRIEE FARNQAISRL EGLSNLYQIY AESFREWEAD 120
PTNPALREEM RIQFNDMNSA LTTAIPLFAV QNYQVPLLSV YVQAANLHLS VLRDVSVFGQ 180
RWGFDAATIN SRYNDLTRLI GNYTDHAVRW YNTGLERVWG PDSRDWIRYN QFRRELTLTV 240
LDIVSLFPNY DSRTYPIRTV SQLTREIYTN PVLENFDGSF RGSAQGIEGS IRSPHLMDIL 300
NSITIYTDAH RGEYYWSGHQ IMASPVGFSG PEFTFPLYGT MGNAAPQQRI VAQLGQGVYR 360
TLSSTLYRRP FNIGINNQQL SVLDGTEFAY GTSSNLPSAV YRKSGTVDSL DEIPPQNNNV 420
PPRQGFSHRL SHVSMFRSGF SNSSVSIIRA PMFSWIHRSA EFNNIIPSSQ ITQIPLTKST 480
NLGSGTSVVK GPGFTGGDIL RRTSPGQIST LRVNITAPLS QRYRVRIRYA STTNLQFHTS 540
IDGRPINQGN FSATMSSGSN LQSGSFRTVG FTTPFNFSNG SSVFTLSAHV FNSGNEVYID 600
RIEFVPAEVT FEAEYGCRSG GGGGGSSGGG GANVASVVPG KGNNTKLSTR ALPSFIDYFN 660
GIYGFATGIK DIMNMIFKTD TGGDLTLDEI LKNQQLLNDI SGKLDGVNGS LNDLIAQGNL 720
NTELSKEILK IANEQNQVLN DVNNKLDAIN TMLRVYLPKI TSMLSDVMKQ NYALSLQIEY 780
LSKQLQEISD KLDIINVNVL INSTLTEITP AYQRIKYVNE KFEELTFATE TSSKVKKDGS 840
PADILDELTE LTELAKSVTK NDVDGFEFYL NTFHDVMVGN NLFGRSALKT ASELITKENV 900
KTSGSEVGNV YNFLIVLTAL QAQAFLTLTT CRKLLGLADI DYTSIMNEHL NKEKEEFRVN 960
ILPTLSNTFS NPNYAKVKGS DEDAKMIVEA KPGHALIGFE ISNDSITVLK VYEAKLKQNY 1020
QVDKDSLSEV IYGDMDKLLC PDQSEQIYYT NNIVFPNEYV ITKIDFTKKM KTLRYEVTAN 1080
FYDSSTGEID LNKKKVESSE AEYRTLSAND DGVYMPLGVI SETFLTPING FGLQADENSR 1140
LITLTCKSYL RELLLATDLS NKETKLIVPP SGFISNIVEN GSIEEDNLEP WKANNKNAYV 1200
DHTGGVNGTK ALYVHKDGGI SQFIGDKLKP KTEYVIQYTV KGKPSIHLKD ENTGYIHYED 1260
TNNNLEDYQT INKRFTTGTD LKGVYLILKS QNGDEAWGDN FIILEISPSE KLLSPELINT 1320
NNWTSTGSTN ISGNTLTLYQ GGRGILKQNL QLDSFSTYRV YFSVSGDANV RIRNSREVLF 1380
EKRYMSGAKD VSEMFTTKFE KDNFYIELSQ GNNLYGGPIV HFYDVSIK 1428
<210>7
<211>24
<212>DNA
<213> Artificial Synthesis
<400>7
catatggaca acaacccaaa catc 24
<210>8
<211>22
<212>DNA
<213> Artificial Synthesis
<400>8
aagcttctag tactccgcct cg 22
<210>9
<211>22
<212>DNA
<213> Artificial Synthesis
<400>9
aacctgggca gcggcaccag cg 22
<210>10
<211>21
<212>DNA
<213> Artificial Synthesis
<400>10
cacgttgacg ttgatgatgt c 21
<210>11
<211>21
<212>DNA
<213> Artificial Synthesis
<400>11
atgagcccag aacgacgccc g 21
<210>12
<211>20
<212>DNA
<213> Artificial Synthesis
<400>12
tcggtgacgg gcaggaccgg 20
Claims (9)
1. Insect-resistant geneVIP3A(a)The nucleotide sequence is shown in SEQ ID NO. 3.
2. An insect-resistant fusion gene, namedCryAb-VIP3AThe nucleotide sequence is as follows:
(1) a nucleic acid sequence shown as SEQ ID NO. 5; or
(2) A nucleic acid sequence with the same function derived from the nucleic acid sequence shown in SEQ ID NO. 5.
3. A protein CryAb-VIP3A coded by an insect-resistant fusion gene has an amino acid sequence as follows:
(1) an amino acid sequence shown as SEQ ID NO. 6; or
(2) The sequence of the active fragment or conservative variant is obtained by adding, deleting or/and replacing one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 6.
4. An insect-resistant fusion gene according to claim 2CryAb-VIP3AAnd (3) constructing an expression vector.
5. A recombinant bacterium constructed from the expression vector of claim 4.
6. The insect-resistant fusion gene of claim 2CryAb-VIP3AApplication in preparing insect-resistant plant cells.
7. Use according to claim 6, wherein the plant is a monocotyledonous plant.
8. Use according to claim 7, wherein the monocotyledonous plant is maize.
9. The use of the protein CryAb-VIP3A encoded by the insect-resistant fusion gene of claim 3 in the preparation of an insect-resistant agent.
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| CN114107344A (en) * | 2021-11-15 | 2022-03-01 | 山东省农业科学院 | Insect-resistant fusion gene M2CryAb-VIP3A, expression vector and product thereof, and application thereof |
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