CN112279902A - Bt protein Cry1A-like and coding gene and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种Bt蛋白Cry1A‑like及其编码基因和应用。该蛋白的氨基酸序列如SEQ ID NO.2所示，或如SEQ ID NO.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的氨基酸序列。本发明提供Cry1A‑like蛋白具有较好的杀虫活性，将其用于制备转基因植物，能够特异性杀灭害虫，并降低农药的使用量，降低成本，减少环境污染。同时，未发现害虫对该蛋白产生抗性的情况。因此，其具有重要的经济价值和应用前景，适合大规模应用于提高植物的抗虫性。The invention discloses a Bt protein Cry1A-like and its encoding gene and application. The amino acid sequence of the protein is shown in SEQ ID NO.2, or the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted and/or added by one or more amino acids and has the same activity. The invention provides a Cry1A-like protein with better insecticidal activity, and when it is used to prepare a transgenic plant, it can specifically kill pests, reduce the usage of pesticides, reduce costs, and reduce environmental pollution. At the same time, no cases of pests developing resistance to this protein were found. Therefore, it has important economic value and application prospect, and is suitable for large-scale application to improve the insect resistance of plants.

Description

Bt protein Cry1A-like and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Bt protein Cry1A-like and a coding gene and application thereof.

Background

In the process of human production, insect pests are important factors causing agricultural production loss and influencing human health. According to FAO statistics, the economic loss caused by insect pests in agricultural production all over the world is up to 14%, the disease loss is up to 12%, and the weed loss is up to 11%. The loss amount is up to 1260 billion dollars, which is equal to half of the total agricultural value in China and more than 4 times of the total agricultural value in the United kingdom. In order to reduce the loss, chemical prevention and control means are generally adopted for preventing and controlling crop pests and mosquitoes for many years, but the long-term and large-scale use of chemical pesticides causes pollution to the environment, pesticide residues in agricultural and sideline products are increased, and harm is brought to the survival and health of human beings. In addition, the chemical pesticide can kill natural enemies and other beneficial substances while killing pests, thereby destroying ecological balance. Compared with chemical control, biological control has the characteristics of safety, effectiveness and durability. And a series of problems caused by chemical control are avoided. Therefore, biological control technology has become a hot spot of research. Among the biological insecticides, bacillus thuringiensis is the one with the widest use and the largest yield in the world at present.

Bacillus thuringiensis (Bt) is a gram-positive bacterium, which is widely distributed, can form parasporal crystals consisting of proteins with insecticidal activity while sporulating, and is also called Insecticidal Crystal Proteins (ICPs) which are coded by cry genes and have strong toxicity to sensitive insects and no toxicity to higher animals and people. Bt has become a powerful substitute for chemically synthesized pesticides in the prevention and treatment of farmland pests, forest pests and sanitary pests at present, and is also an important gene source of transgenic insect-resistant engineering plants.

Since the first gene capable of expressing insecticidal activity was cloned by Schnepf from strain HD-1 in 1981, more than 500 genes encoding insecticidal crystal proteins have been isolated and cloned, and they have been identified as different groups, subgroups, classes and subclasses, respectively, based on the encoded amino acid sequence homology. Generally speaking, toxic proteins such as Cry1, Cry2 and Cry9 are effective against lepidopteran pests; the most studied are Cry1 and Cry9 proteins, the molecular weight of the encoded insecticidal crystal protein is 130-140kD, and a plurality of genes are widely applied to the control of lepidoptera pests of plants at present. Toxin protein produced by bacillus thuringiensis subsp. Meanwhile, Cyt protein has cytolytic property, has synergistic effect on certain Cry proteins and delays insect resistance.

The use of Bt insecticidal crystal protein-based insecticides has been known for over 50 years and initially no resistance to Bt has been detected in insects, but, beginning in the middle of the last 80 years, the problem of resistance has been continuously demonstrated in laboratory and field trials (McGaughey, W.H.1985.science.229: 193-. In 1985, McGaughey reported that the resistance increased 97-fold after the storehouse grain pest Indian meal moth (Plodia interpunctella) bred for 15 generations under selective pressure of Dipel (commercial preparation of Bt subsp. kurstaki HD-1); at high dose selection pressure, resistance can increase 250-fold. In 1990, in Hawaii, it was first verified that plutella xylostella in the field developed significant resistance to Bt insecticides (Tabashnik, B.E., et al, 1994.Proc. Natl. Acad. Sci. USA.91: 4120) and since the last 90 th century, in Shenzhen, Guangzhou, Shanghai, etc., where Bt insecticides were applied for a long time in China, it was found that the Bt insecticides had a significantly reduced control effect on plutella xylostella, meaning that resistance had developed (Von summer 1996. insect journal, 39(3): 238-) 244; Hofte, H.,1988.appl. environ. Microbiol.54: 2010-2017). At present, at least ten insects in laboratories and fields are found to generate resistance to Bt and the insecticidal crystal protein thereof, and the selection pressure mathematical model predicts that the insects will generate resistance under the condition of Bt transgenic insect-resistant plant selection pressure (Schnepf, E., et al 1998.mol. biol. Rev.65(3): 775-806). In addition, studies have shown that Bti has not found resistance problems in field use, but that mosquito resistance is constantly being demonstrated in the laboratory, which may also occur in fields (Georghiou G P,1997.Applied and Environmental Microbiology,63: 1095-1101.).

In order to avoid the loss caused by resistant insects, the search for new high-toxicity Bt gene resources is an effective way for solving the problem, which has very important significance for biological control in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Bt protein Cry1A-like, a coding gene and application thereof, and can provide a new Bt virulence protein resource.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a Bt protein Cry1A-like has an amino acid sequence shown as SEQ ID NO.2, or has an amino acid sequence with equivalent activity, wherein one or more amino acids are substituted, deleted and/or added in the amino acid sequence shown as SEQ ID NO. 2.

The gene for coding the protein has a nucleotide sequence shown in SEQ ID No.1, or a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides and has the same activity as the nucleotide sequence shown in SEQ ID No. 1.

The invention relates to a novel Bacillus thuringiensis strain BN23-5 separated from soil in urban areas of Sichuan province, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, address: No. 3 of West Luo 1 of Beijing area facing Yang, institute of microbiology of China academy of sciences, postal code 100101) in 7-14 days of 2014, and is classified and named as Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No. 9448.

The toxicity test of BN23-5 shows that BN23-5 has extremely high toxicity to lepidoptera pests and the like. A pair of specific primers is designed according to a Cry1 gene conserved sequence, genomic DNA of the primers is amplified, results show that cryI genes exist in the strain, full-length gene primers of the primers are further designed, a Cry1A-like gene is obtained by cloning, the nucleotide sequence of the Cry1A-like gene is shown in a sequence table SEQ ID No.1, the full length is 3543bp, analysis shows that the GC content is 39.20%, protein consisting of 1181 amino acids is encoded, and the amino acid composition of the Cry1A-like protein is shown in table 1.

TABLE 1 amino acid composition of Cry1A-like proteins

Amino acids	Percent by weight%	Amino acids	Percent by weight%	Amino acids	Percent by weight%
						Ala(A)	6.17	Gly(G)	6.51	Pro(P)	4.37
Arg(R)	6.86	His(H)	2.14	Ser(S)	7.03
						Asn(N)	6.00	Ile(I)	6.68	Ter(.)	0.09
Asp(D)	5.57	Leu(L)	9.43	Thr(T	5.66
						Cys(C)	1.54	Lys(K)	3.00	Trp(W)	1.37
Gln(Q)	3.26	Met(M)	0.51	Tyr(Y)	4.88
						Glu(E)	8.48	Phe(F)	3.86	Val(V)	6.60

The gene and the protein of the invention can be obtained by cloning or separating Bt strain BN23-5 or by a method of DNA or peptide synthesis.

The gene of the invention can be operably connected with an expression vector to obtain a recombinant expression vector capable of expressing the protein of the invention, and the expression vector can be further introduced into a host by a transgenic method such as an agrobacterium-mediated method, a gene gun method, a pollen tube channel method and the like to obtain a transformant of the transgenic Cry1A-like gene, for example, plants such as crops or fruit trees and the like, so that the transgenic plant has insect-resistant activity.

It is understood that one skilled in the art can substitute, delete and/or add one or several amino acids to obtain a mutant sequence of the protein Cry1A-like according to the amino acid sequence (SEQ ID No.2) of the protein disclosed herein, without affecting its activity. For example, in the inactive segment, Lys at position 48 is replaced by Arg or Leu at position 717 is replaced by Ile, without affecting its activity. Therefore, the Bt protein Cry1A-like also comprises a protein which is obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2, has the same activity with the Bt protein Cry1 and is derived from Cry 1A-like.

In one embodiment of the invention, the Bt protein Cry1A-like recombinant expression vector is obtained by inserting Cry1A-like gene into expression vector pET-28a (+) to construct recombinant expression vector pET-1I.

In addition, fermentation liquor containing Cry1A-like protein can be obtained by fermenting the strain BN23-5, and the fermentation liquor is prepared into an insecticide for controlling crop pests. The skilled person can also transform the above genes into bacteria or fungi to produce the Bt protein of the invention by large-scale fermentation.

A recombinant expression vector containing the gene.

A host cell containing the recombinant expression vector.

Further, the host cell is a plant cell.

An insecticide contains the protein as active component and its pharmacologically acceptable auxiliary components.

The application of the protein in improving the insect resistance of plants.

The gene is applied to culturing transgenic plants, preparing pesticides or improving the insect resistance of plants.

The technical personnel in the field can also transform crops such as cotton, corn, rice, vegetables and the like according to the Cry1A-like gene disclosed by the invention so as to enable the crops to have corresponding insect-resistant activity. For example: by utilizing the degeneracy of codons, a Cry1A-like gene is designed into a gene sequence with rice preference codons, then the synthesized Cry1A-like gene sequence is connected with a vector pCAMBIA1300, and is transferred into a corn genome through agrobacterium mediation, so that a transgenic corn variety with insect-resistant activity is obtained.

The invention has the beneficial effects that:

the Cry1A-like protein provided by the invention is a Bt protein, has good insecticidal activity, can specifically kill pests when being used for preparing transgenic plants, and can reduce the usage amount of pesticides, reduce the cost and reduce the environmental pollution. Meanwhile, the situation that pests have resistance to the protein is not found. Therefore, the Bt protein Cry1A-like has important economic value and application prospect, and is suitable for large-scale application in improving the insect resistance of plants.

Drawings

FIG. 1 is a gel electrophoresis diagram of a cloned full-length Cry1A-like gene;

FIG. 2 is a restriction enzyme identification map of recombinant plasmid pET-1I;

FIG. 3 is an SDS-PAGE image of expression of Cry1A-like gene in E.coli BL21(DE 3).

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 cloning of Cry1A-like Gene

1. A novel Bacillus thuringiensis strain BN23-5 separated from soil in the urban area of Sichuan province is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, address: No. 3 of West Luo No.1 of Beijing area facing Yang, institute of microbiology of Chinese academy of sciences, postal code 100101) in 7-14 days in 2014, and is classified and named as Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No. 9448. The toxicity test of BN23-5 shows that BN23-5 has extremely high toxicity to lepidoptera pests and the like.

2. Then, a genome DNA purification kit (purchased from Saibance) is adopted to extract the total DNA of the strain BN23-5 as a template for amplifying the Cry1A-like gene, the designed primer sequences are shown as follows, and the amplification system is shown in Table 2:

P1：5’-ATGGAAGTGAATAATCAAAATC-3；(SEQ ID No.3)

P2：5’-CTATTCCTCCATAAGGAG-3’；(SEQ ID No.4)

TABLE 2PCR reaction System

The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 50s, denaturation at 54 ℃ for 50s, and elongation at 72 ℃ for 2min for 30 cycles; extending for 10min at 72 ℃; the reaction was stopped at 4 ℃.

And (3) carrying out electrophoresis on the amplification reaction products on 1% agarose gel, and observing the PCR amplification result in a gel imaging system. The results are shown in FIG. 1, in which the band M is DNA marker;strip 1 is the Cry1A-like gene.

As is clear from the results of detection in FIG. 1, a sequence of about 3543bp was obtained by amplification, and the sequence was sequenced, and its nucleotide sequence was shown in SEQ ID No.1 and was identical to the target sequence.

Example 2 acquisition of Cry1A-like protein

Designing and synthesizing a pair of specific primers according to two end sequences of Cry1A-like gene open reading frames

1ITF：5'-GCCGGATCCATGGAGATAGTGAATAATCAGAAT-3'；(SEQ ID No.5)

1ITR:5'-GGGGTCGACCTAAAATTCTGCCTCAAAGGTTA-3'；(SEQ ID No.6)

The underlined parts of the 5' primer are BamH I and Sal I restriction sites respectively. Amplifying by taking BN23-5 total DNA as a template, connecting the enzyme digestion product with a vector pET-28a (+) subjected to double enzyme digestion, transforming E.coli DH5 alpha competent cells, extracting a plasmid of the E.coli DH5 alpha competent cells, and carrying out enzyme digestion electrophoresis, wherein the result is shown in figure 2, and astrip 1 in the figure 2 is a BamH I/Sal I double enzyme digestion product of a recombinant plasmid pET-1I; thestrip 2 is a vector pET-28 a; thestrip 3 is a recombinant plasmid pET-1I;band 4 is the inserted DNA; and the band M is DNA marker. As can be seen from the results of the electrophoresis in FIG. 2, the size of the inserted fragment matches the size of the desired target fragment.

Then transferred into recipient bacterium E.coli.BL21(DE3) (purchased from Beijing Quanjin Biotechnology Ltd.). The recombinant plasmid was designated as pET-1I, and the recombinant plasmid-containing recombinant was designated as E.coli.BL21 (2L). Culturing positive transformant in LB culture medium at 200r/min and 37 deg.C overnight, transferring the culture medium to 1L triangular flask containing 400mL LB culture medium at a ratio of 1:100, culturing at 200r/min and 37 deg.C, adding 0.6mmol/L IPTG to induce expression for 12h when OD 600 value of the culture medium reaches 0.6-0.8, centrifuging the culture medium to collect thallus, discarding supernatant, adding 30mL 10mmol/L Tris-HCl (pH 8.0), ultrasonically crushing, and detecting the expressed protein by SDS-PAGE, wherein the band M is protein marker (molecular weight from top to bottom: 120, 100, 80, 65, 50, 40, 25 KDa);

theband 1 is E.coli BL21(DE3) containing recombinant plasmid pET-1I, and the supernatant of the bacterial lysate is not induced by IPTG; theband 2 is E.coli BL21(DE3) containing recombinant plasmid pET-1I, and is precipitated from a thallus lysate after IPTG induction;lane 3 is e.coli BL21(DE3) expression protein containing vector pET-28a (negative control).

As can be seen from the detection map of FIG. 3, SDS-PAGE analysis shows that the molecular weight of Cry1A-like in the precipitate and supernatant after the ultrasonication of the thallus is about 134.3kDa, which is consistent with the predicted molecular weight of the protein.

Example 3Cry1A-like protein insecticidal Activity assay

The Cry1A-like protein obtained in example 2 is used for measuring the insecticidal activity of diamondback moth, corn borer and cotton bollworm, and the specific process is as follows.

1. And (3) raw testing of the plutella xylostella: preparing Cry1A-like protein into 6 different concentration gradients of 4, 2, 1, 0.5, 0.25, 0.01 mu g/mL and the like; selecting cabbage leaves with moderate age and tenderness, cleaning and airing; irradiating under ultraviolet lamp for 15min, and cutting into 2 × 2cm²The size of the mixture is divided into bacterial liquids with different concentrations, and the mixture is soaked for 5 min; taking out, draining off excessive liquid, air drying in sterilized culture dish, and placing 4 leaves in each culture dish with E.coli.BL21(DE3) as negative control and clear water as blank control; selecting and placing 20 healthy diamondback moths of 2-3 ages; each treatment was repeated 3 times, housed and larval mortality was investigated after 3 days.

2. Bioassay of corn borers: cry1A-like protein is prepared into 6 different concentration gradients of 200, 100, 50, 25, 12.5, 0.1 mu g/mL and the like, the protein is added into feed for raising corn borers and mixed evenly, E.coli.BL21(DE3) is used as negative control, clear water is used as blank control, 20 corn borers with 2-3 ages are added into each treatment, each treatment is repeated for 3 times, and the results are counted after 7 days.

3. The birth test of the cotton bollworm: preparing Cry1A-like protein into 6 different concentration gradients of 200, 100, 50, 25, 12.5, 0.1 μ g/mL, etc., adding protein into feed for feeding cotton bollworm, mixing, using E.coli.BL21(DE3) as negative control and clear water as blank control, and processing each position with the above-mentioned materialsAnd (4) throwing 20 cotton bollworms of 2-3 years into the cotton bollworms, repeating the treatment for 3 times, and counting the result after 3 days. Finally, LC is calculated by SPSS 10.0 software₅₀The results are shown in Table 3.

TABLE 3 insecticidal Activity of Cry1A-like

As can be seen from the results in Table 3, the expression products have high insecticidal activity against diamond back moth, corn borer and cotton bollworm, LC₅₀0.17. mu.g/mL, 1.66. mu.g/mL and 26.31. mu.g/mL, respectively; bioassay results showed that e.coli.bl21(DE3) and blank controls had no insecticidal activity against diamondback moth, corn borer and cotton bollworm. The Cry1A-like protein developed by the invention has excellent insecticidal activity, and can be used for improving insect resistance of plants, preparation of insecticides and the like.

Sequence listing

<110> Sichuan university of agriculture

<120> Bt protein Cry1A-like and coding gene and application thereof

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<170> SIPOSequenceListing 1.0

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atggagatag tgaataatca gaatcaatgc gtgccttata attgtttgaa taatcctgaa 60

atcgaaatat tagaaggcga aagaatatca gttggtaaca ccccgatcga tatttctctg 120

tcgctggtgg aacttcttat tagtgaattt gttccaggcg gtggaattat aacgggattg 180

ttgaacattg tatggggatt tgtaggtcct tcccaatggg acgcatttct tgctcaagtg 240

gaacagttaa ttaaccaaag aatagctgaa gctgtaagaa atacagcaat tcaggaatta 300

gagggaatgg cacgagttta tagaacctat gctactgctt ttgctgagtg ggaaaaagct 360

cctgatgacc cagagctaag ggaagcacta cgtacacaat ttacagcaac tgagacttat 420

ataagtggaa gaatatccgt tttaaaaatt caaaattttg aagtacagct gttatcggtg 480

tttgcccaag ctgcaaattt acatttatct ttattaagag acgttgtgtt ttttgggcaa 540

agatggggtg tttcaacgac aaccgtaaat aattactaca atgatttaac agaagagatt 600

agtacctata cagattatgc tgtacgctgg tacaatacgg gattagaacg tgtatgggga 660

ccggattcta gagactgggt aagatataat caatttagaa gagaattaac actaactgta 720

ttagatatcg tttctctatt tccgaactat gatagtagaa ggtatccaat tcgaacagtt 780

tcccaattaa caagagaaat ttatacaaac ccagtattag aaaattttga tggtagtttt 840

cgtggaatgg ctcagagaat agaacagaat attaggcaac cacatcttat ggatatcctt 900

aatacaataa ccatttatac tgatgtgcat agaggcttta attattggtc aggacatcaa 960

ataactgctt ctcctgtagg gttttcagga ccagaattta cattcccttt atttggtaat 1020

gcagggaacg cagctccacc cgtacttgtc tcattaacgg gtttggggat ttttagaaca 1080

ttatcttcac ctttttacag aagaattata ctcggttcag gtccaaataa tcaggaactg 1140

tttgtccttg atggaacgga attttctttt gcctccctaa caactaatat accttctact 1200

atatacagac aaagaggaac ggtcgattca ctagatttaa taccaccaca agataatagt 1260

gtgcccgctc gtgcgggatt tagtcatcga ttaagccatg ttacaatgct gagccaagca 1320

gccggagcag tttacacctt gagagctcca acgttttctt ggcagcatcg tagtgctgaa 1380

tttaataata taattccttc atcacaaatt acacaaatac ctttaacaaa atctactaat 1440

cttggctctg gaactactgt cgttaaagga ccaggattta caggaggaga tattctacga 1500

agaacttcac ctggccagat ttcaacctta agagtaaata ttactgcacc attatcacaa 1560

agatatcgcg taagaattcg ttatgcctct actacaaatt tacaattcca tacatcaatt 1620

gatggaagac ctattaatca ggggaatttt tcagcaacta tgagtagtgg gggtaattta 1680

cagtccggaa gctttaggac tttaggtttt actactccgt ttaacttttc aaatggatca 1740

agtgtattta cgttaagtgc tcaggtcttc aattcaggca atgaagttta tatagataga 1800

attgaatttg ttccggcaga agtaaccttt gaggcagaat ttgatttaga aagagcccaa 1860

gaggcggtga atgcgctgtt tacttctccc aatcaaatcg ggttaaaaac agatataacg 1920

gattatcata ttgatcaagt atccaatcta gttgagtgtt tatcagatga attttgtctg 1980

gatgaaaaga gagaattatc cgaaaaagtc aaacatgcga agcgactcag tgatgagcgg 2040

aatttacttc aagatccaaa ctttagaggc atcaatagac aaccagaccg tggttggaga 2100

ggaagtacgg atattaccat ccaaggtgga gatgacgtat tcaaagagaa ttacgtcaca 2160

ctaccgggca cttttgatga gtgctatcca acgtatttgt atcaaaaaat agatgaatca 2220

aaattaaaag cctacactcg ttaccgatta agagggtaca tcgaggatag tcaagactta 2280

gaaatctatt taatccgcta caatgcaaaa cacgaaaccg taaatgtacc aggtaccggg 2340

ggggtatggc cactttctgt agaaaattca attggacctt gtggcgaacc gaatcgatgc 2400

gcgccacacc ttgaatggaa tcctaatcta gagtgttcct gcagagacgg ggaaaaatgt 2460

gcacaccatt cgcatcattt ctccttggac attgatgttg gatgtacaga cttaaatgag 2520

gacttaggtg tatgggtgat attcaagatt aagacgcaag atggccatgc aagactagga 2580

aatctagagt ttctcgaaga gaaaccatta ttaggggaag cactagctcg tgtgaaaaga 2640

gcggagaaaa aatggagaga caaatgtgaa aaattggaat tggaaacaaa tattgtttat 2700

aaagaggcaa aaaaatctgt agatgcttta tttgtgaact ctcaatatga tagattacag 2760

gcggatacga atattgccat gattcatgcg gcagataaac gtgttcatag aatccgagaa 2820

gcgtatcttc cagagttatc tgtgattccg ggtgtaaatg cagacatttt cgaagaatta 2880

gaagggcgta ttttcacagc ctactctcta tatgatgcga gaaatgtcat taaaaacggt 2940

aatttcaata atggcttatc atgttggaac gtgaaagggc atgtagatgt agaagaacaa 3000

aacaaccacc gttcagtcct tgtggtccca gaatgggaag cagaagtgtc ccaagaagtt 3060

cgtgtctgtc caggacgtgg ctatatcctt cgcgttacag cgtacaaaga aggatatgga 3120

gagggctgcg taacgatcca tgagattgaa gacaatacag acgaactgaa attcagcaac 3180

tgtgtagaag aggaagtata tccaaataac acggtaacgt gtaatgatta tactgcgact 3240

caagaagaat acgggggtgt atacacttcc cgtaatcgtg gatatgacga aacttatgga 3300

agcaattctt ccgtatcagc agattatgcg tcagtctatg aagcaaaagc gtatacagat 3360

ggacgaagag agaatccttg tgaatttaac agaggatatg gggattacac accactacca 3420

gctggctatg tgacaaaaga attagagtac ttcccagaaa ccgataaggt atggattgag 3480

atcggagaaa cagaaggaac attcatcgtg gacagcgtgg aattactcct tatggaggaa 3540

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Glu Gln Leu Ile Asn Gln Arg Ile Ala Glu Ala Val Arg Asn Thr Ala

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Ala Phe Ala Glu Trp Glu Lys Ala Pro Asp Asp Pro Glu Leu Arg Glu

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Ala Leu Arg Thr Gln Phe Thr Ala Thr Glu Thr Tyr Ile Ser Gly Arg

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Ile Ser Val Leu Lys Ile Gln Asn Phe Glu Val Gln Leu Leu Ser Val

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Phe Ala Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Val Val

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Phe Phe Gly Gln Arg Trp Gly Val Ser Thr Thr Thr Val Asn Asn Tyr

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Tyr Asn Asp Leu Thr Glu Glu Ile Ser Thr Tyr Thr Asp Tyr Ala Val

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Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

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Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val

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Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro

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Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val

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Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Met Ala Gln Arg Ile Glu

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Gln Asn Ile Arg Gln Pro His Leu Met Asp Ile Leu Asn Thr Ile Thr

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Ile Tyr Thr Asp Val His Arg Gly Phe Asn Tyr Trp Ser Gly His Gln

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Ile Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro

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Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu

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Thr Gly Leu Gly Ile Phe Arg His Leu Ser Ser Pro Phe Tyr Arg Arg

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Ile Ile Leu Gly Ser Gly Pro Asn Asn Gln Glu Leu Phe Val Leu Asp

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Gly Thr Glu Phe Ser Phe Ala Ser Leu Thr Thr Asn Ile Pro Ser Thr

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Ile Tyr Arg Gln Arg Gly Thr Val Asp Ser Leu Asp Leu Ile Pro Pro

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Gln Asp Asn Ser Val Pro Ala Arg Ala Gly Phe Ser His Arg Leu Ser

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His Val Thr Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr Leu Arg

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Ala Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu Phe Asn Asn Ile

450 455 460

Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr Asn

465 470 475 480

Leu Gly Ser Gly Ala Ala Val Val Lys Gly Pro Gly Phe Thr Gly Gly

485 490 495

Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val

500 505 510

Asn Ile Ala Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr

515 520 525

Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro

530 535 540

Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Gly Ile Leu

545 550 555 560

Gln Ser Gly Ser Phe Arg Thr Leu Gly Phe Thr Thr Pro Phe Asn Phe

565 570 575

Ser Ile Gly Ser Ser Val Phe Thr Leu Ser Ala Gln Val Phe Ile Ser

580 585 590

Gly Asn Gly Val Tyr Ile Asp Arg Ile Gly Phe Val Pro Ala Gly Val

595 600 605

Thr Phe Glu Ala Gly Phe Asp Leu Gly Arg Ala Gln Glu Ala Val Ile

610 615 620

Ala Leu Phe Thr Ser Pro Asn Gln Ile Gly Leu Lys Thr Asp Ile Thr

625 630 635 640

Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp

645 650 655

Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His

660 665 670

Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe

675 680 685

Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser Thr Asp

690 695 700

Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr

705 710 715 720

Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys

725 730 735

Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Arg Leu Arg Gly

740 745 750

Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn

755 760 765

Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Gly Val Trp Pro

770 775 780

Leu Ser Val Glu Asn Ser Ile Gly Pro Cys Gly Glu Pro Asn Arg Cys

785 790 795 800

Ala Pro His Leu Glu Trp Asn Pro Asn Leu Glu Cys Ser Cys Arg Asp

805 810 815

Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp

820 825 830

Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe

835 840 845

Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe

850 855 860

Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val Lys Arg

865 870 875 880

Ala Glu Lys Lys Trp Arg Asp Lys Cys Glu Lys Leu Glu Leu Glu Thr

885 890 895

Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val Asp Ala Leu Phe Val

900 905 910

Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile

915 920 925

His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr Leu Pro

930 935 940

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

945 950 955 960

Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr Asp Ala Arg Asn Val

965 970 975

Ile Lys Asn Gly Asn Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys

980 985 990

Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val

995 1000 1005

Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro

1010 1015 1020

Glu Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Glu

1025 1030 1035 1040

Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp Asn Thr Asp Glu Leu

1045 1050 1055

Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val

1060 1065 1070

Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu Tyr Gly Gly Val Tyr

1075 1080 1085

Thr Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser

1090 1095 1100

Val Ser Ala Asp Tyr Ala Ser Val Tyr Glu Ala Lys Ala Tyr Thr Asp

1105 1110 1115 1120

Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly Tyr Gly Asp Tyr

1125 1130 1135

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro

1140 1145 1150

Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Gly Thr Glu Gly Thr Phe

1155 1160 1165

Ile Val Asp Ser Val Glu Leu Ser Leu Met Glu Glu Ter

1170 1175 1180

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaagtga ataatcaaaa tc 22

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctattcctcc ataaggag 18

<210> 5

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gccggatcca tggagatagt gaataatcag aat 33

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggggtcgacc taaaattctg cctcaaaggt ta 32

Claims (8)

Translated fromChinese

1.一种Bt蛋白Cry1A-like，其特征在于，所述蛋白的氨基酸序列如SEQ ID NO.2所示，或如SEQ ID NO.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的氨基酸序列。1. a Bt protein Cry1A-like, is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ ID NO.2, or the aminoacid sequence as shown in SEQ ID NO.2 through substitution, deletion and/or increase one or amino acid sequences of more than one amino acid with the same activity.2.一种编码权利要求1所述蛋白的基因，其特征在于，所述基因的核苷酸序列如SEQ IDNO.1所示，或如SEQ ID NO.1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且具有同等活性的核苷酸序列。2. A gene encoding the protein of claim 1, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO.1, or the nucleotide sequence shown in SEQ ID NO.1 is substituted , deletion and/or addition of one or more nucleotides and nucleotide sequences with equivalent activity.3.一种含有权利要求2所述基因的重组表达载体。3. A recombinant expression vector containing the gene of claim 2.4.一种含有权利要求4所述重组表达载体的宿主细胞。4. A host cell containing the recombinant expression vector of claim 4.5.根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为植物细胞。5. The host cell according to claim 5, wherein the host cell is a plant cell.6.一种杀虫剂，其特征在于，以权利要求1所述蛋白为活性成分，以及其在药学上可接受的辅助成分。6. An insecticide, characterized in that the protein of claim 1 is used as an active ingredient, and a pharmaceutically acceptable auxiliary ingredient thereof.7.权利要求1所述蛋白在提高植物抗虫性能中的应用。7. The application of the protein of claim 1 in improving plant insect resistance.8.权利要求2所述基因在培育转基因植物、制备杀虫剂或提高植物抗虫性能中的应用。8. The application of the gene of claim 2 in cultivating transgenic plants, preparing insecticides or improving plant insect resistance.

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