CN1401773A

CN1401773A - Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria

Info

Publication number: CN1401773A
Application number: CN 01124164
Authority: CN
Inventors: 张�杰; 黄大昉; 宋福平; 陈中义; 李国勋
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2001-08-20
Filing date: 2001-08-20
Publication date: 2003-03-12
Anticipated expiration: 2021-08-20
Also published as: CN1181203C

Abstract

The present invention relates to a Bt crylBa3 sequence with resistance to the pests in lepidoptera and coleotera, the gene combination mode of crylbalcrl3Aa, the broad-host expression vector pGM1105, and using said dpGM1105 to introduce the cry3Aa gene to the Bt17 strain containing crylBa gene for efficient expression of said two genes.

Description

Bt gene, expression vector and engineering bacteria to Coleoptera and the high virulence of lepidopterous insects

Technical field of the present invention:

The invention belongs to the biological control technical field.Further, the present invention relates to lepidopteran, coleopteran pest are all had Bt gene, expression vector and the engineering bacteria of high virulence.Further, the present invention relates to lepidopteran, coleopteran pest are all had Bt cry1Ba3 gene order, the cry1Ba/cry3Aa assortment of genes mode of resistance, to realize efficiently expressing of two kinds of genes; Also relate to wide host expresses carrier pGM1105.

Research background of the present invention:

Tribactur (Bacillus thuringiensis is called for short Bt) forms the crystallin that the phase can produce a kind of insecticidal activity at gemma, has been widely used in the control of agricultural, forestry, sanitary insect pest.

Nineteen eighty-three, and Krieg etc. (Krieg, A. etc., J.Appli.Entomology, 1983,96:500-508) be separated to the Bt bacterial strain (Bt.tenebrionis) of first strain to the high virulence of coleopteran pest.1987, Herrnstadt (Herrnstadt, C. etc., Gene, 1987,57 (1): 37-46) cloned first, be used to prevent and treat colorado potato bug (Leptinotarsa decernlineata (Say)) to the activated Bt insecticidal protein gene of coleopteran pest cry3Aa.U.S. Ecogen company (Sick, A. etc., Nucl.Acids Res., 1990.18,1305; Entwistle, P.F. etc., An Environmental Biopesticide:Theory andPractice[M], Wiley Chichester Press, UK, 1993,125-146) utilize plasmid to make up lepidopteran and the activated Bt engineering bacteria of Coleoptera in conjunction with cell engineering means such as transfers.

A kind of albumen because term single gene is encoded, insect is very fast to produce resistance (Whalon, M.E. etc., J.ofEconomic Entomology, 1993,86 (2): 226-233 to Cry3A albumen; Nicholas, D. etc., Transgenic Plant and Insect PestsBiocontrol[M], John Wiley ﹠amp; Sons Press, USA, 1997,1-18).Because the rapid generation of pest resistance to insecticide will greatly be shortened the work-ing life of transgenic product, cause huge waste like this.Chang in 1993, (Appl.Environ.Micro.1993 59:815-821.) has reported that there is synergistic function in different crystal albumen to larvae in the Bt Israel subclass to people such as C.Y.M. for Chang, C.Y.M. etc.

Therefore, screen the protein combination of high virulence, and then carry out the assortment of genes, can improve the insecticidal activity of Bt product, enlarge its insecticidal spectrum.The more important thing is that the resistance that the insecticidal proteins of different performance can effectively overcome or delay insect produces, and has very strong practicality.

Content of the present invention:

Purpose of the present invention:

At deficiency such as prevent and treat the employed Bt preparation of coleopteran pest at present in the world and transgenic product gene kind is single, insect is easy to produce resistance, insecticidal spectrum is narrower, the present invention adopts two kinds of gene cry3Aa7 and cry1Ba3 to elytrum height of eye virulence to make up, improve virulence, and utilize the also activated characteristics of cry1Ba3 gene pairs lepidoptera pest, enlarge the insecticidal spectrum of product, to be applied to transform microorganism and plant, make it to show toxicity, and overcome, delay insect engineering bacteria and the drug-fast generation of transgenic plant to relevant insect.

Further, for ease of the expression of research Bt insecticidal protein gene in the different microorganisms recipient bacterium, the present invention make up a kind of can be in three kinds of parent microorganisms the carrier of free shuttling, overcome the narrow deficiency of existing carrier host range, and, finish a series of testing by the Btcry3Aa7 gene is inserted wherein.

Technical scheme of the present invention:

1.Bt22 the clone of cry3Aa gene in the bacterial strain

According to Narva (Narva, K.E. etc., EP0462721 A2,1991,8) etc. method is extracted plasmid DNA from the Bt22 bacterial strain, with HindIII enzyme complete digestion plasmid DNA, carry out Southern hybridization, probe is obtained by cry3Aa gene specific primer amplified plasmid dna, is 1.38kb.Primer is:

5’CGAACAATCGAAGTGAACATGATAC

3’CATCTGTTGTTTCTGGAGGCAAT

Carry out mark with 32p; Hybridizing method is undertaken by " molecular cloning experiment guide ", obtains the results of hybridization (see figure 1).Discovery has hybridization signal at 3kb, from gel, reclaim the plasmid DNA HindIII endonuclease bamhi of 3.0kb, be connected with pBluescript SK (+) behind the purifying, transformed into escherichia coli JM107, carry out PCR with the cry3A gene-specific primer and detect, screen positive recombinant plasmid pBY33 (Fig. 2, Fig. 3).Recombinant plasmid contains the big fragment of cry3A gene of 3.0kb.It is carried out subclone obtain pBY33-5, pBY33-6, three kinds of recombinant plasmids of pBY-16, contain 0.72,1.6 respectively, the exogenous genetic fragment of 0.675kb.Determined dna sequence is finished by the logical bio-engineering corporation of Beijing six directions.This gene is by the international called after cry3Aa7 of NK of Bt δNei Dusu gene.

2. from the Bt17 bacterial strain, clone the cry1Ba gene

Extract Bt17 bacterial strain plasmid DNA, carry out partially digested with Sau3A I enzyme, from gel, reclaim the 2-7kb dna fragmentation, be connected with pBluescript SK (+) carrier behind the purifying through BamHI digestion, alkaline phosphatase treatment, behind the transformed into escherichia coli JM107, to carrying out pcr amplification, screen positive transformant with cry1Ba gene-specific primer Sun1Ba5/3.5 ' and 3 ' end primer is respectively:

Forward primer (Forward primer) TCCTGCAGTTGACTTCAAATAGG;

Reverse primer (Reverse primer) CAGTCGACTCATCCGATAAACACGCCAC '.

Screening obtains recombinant plasmid pHT3-66, and this plasmid contains cry1Ba full length gene dna fragmentation.Carry out dna sequence analysis, the result shows that this gene contains 3687bp, is made up of 1229 amino acid, and molecular weight is 139.5kDa.Because of different with known cry1Ba1 gene, its amino acids coding has also become arginine at the 1055th bit base, and promptly aminoacid sequence and known there are differences in the Bt17 bacterial strain.This gene is by the international called after cry1Ba3 of NK of Bt δNei Dusu gene.

3. the structure of shuttle vectors

The plasmid pHT315 (6.5kb) that will contain the Bt replicon cuts with AatII, mends into flush end with the Klenow enzyme; With StuI and StiI double digestion plasmid pUCP19, what obtain 1.2kb contains pseudomonas replicon plasmid DNA fragment, mend flat with the big fragment of Klenow, these two kinds of DNA are connected, transformed into escherichia coli JM107, the recombinant plasmid of screening 7.7kb, cut evaluation through enzyme and obtain pGM1105 (7.7kb), it is transformed Pseudomonas fluorescens P303 bacterial strain (RifR respectively, Nad) and Bt wild strain Bt17, Bt22, Btk does not have crystal mutant strain BE20 (method for transformation such as Lereclus, Lereclus, D. etc., FEMSMicrobiology Letters, 1989,60:211-217), all can grow positive transformant, and extract all kinds of transformant plasmids and carry out restriction analysis, this carrier of proof pGM1105 can shuttle back and forth in three kinds of bacteriums and genetic stability, and stability is greater than 90%.

4. the structure of expression vector

Cry3Aa7 gene (3.0kb) is cloned on the HindIII site of pGM1105, transformed into escherichia coli JM107, filter out the recombinant plasmid pLF31105 (10.7kb) that contains the cry3Aa gene, behind its transformed into escherichia coli SCS110 bacterial strain, extract plasmid, these plasmids are transformed Pseudomonas fluorescens P303 bacterial strain and Bt wild strain Bt17 (containing the crylBa3 gene) respectively, carry out Protein Detection respectively, SDS-PAGE analysis revealed cry3A gene is equal energy normal expression 67kDa albumen in P303 and Bt17, and its importing does not influence the albumen of crylBa3 genetic expression 140kDa among the Bt17.The cry3A gene imports Bt17 and obtains transformant, through various Molecular Detection, proves to transform successfully, with this transformant called after engineering bacteria BiotIII-I.

5. the cultivation of engineering bacteria and observation

Adopt GT substratum, 1/2 LB substratum and ZShi substratum (this laboratory self-control), cultivate the BiotIII-I engineering bacteria respectively, scanning electron microscope and observation by light microscope result, in BiotIII-I, exist square and two kinds of crystal of the bipyramid bodily form (seeing Figure 18), prove the coexpression of two kinds of genes.

6. desinsection biological activity determination

The test insect is:

Lepidoptera pest-small cabbage moth (Plutella xylostella);

Coleopteran pest-elm fleautiauxia armata (Pyrrhalta aenescens),

Colorado potato bug (Leplinotarsa decernlineata).

Beneficial effect of the present invention:

The present invention's use all has the Bt cry1Ba gene of resistance to lepidopteran, coleopteran pest and the activated cry3Aa gene of coleopteran pest is made up; The wide host's shuttle expression carrier pGM1105 that utilizes the present invention to make up can import the cry3Aa gene transformation and contain in the Bt17 bacterial strain of cry1Ba gene, realizes efficiently expressing of two kinds of genes.The virulence to coleopteran pest that combination produced of these two kinds of gene expression products is better than similar natural bacterial strain and the engineering strain of finding in the world at present, illustrates that there is remarkable role in synergy in two kinds of gene expression products.

Further, cry1Ba3 gene order, cry1Ba/cry3Aa assortment of genes mode, wide host expresses carrier pGM1105 involved in the present invention are also peculiar by the present invention.

Description of drawings:

Fig. 1 is Bt22 plasmid Southern Blotting result.

Wherein,

road

1,2,3,4,5 is represented cry3Aa1.38kb, λ DNA/HindIII, Bt22 plasmid/PstI, Bt22 plasmid/HindIII, Bt22 plasmid respectively.

Fig. 2 is Bt22, Bt17cry gene identification collection of illustrative plates.

Wherein, on behalf of cry3Aa 1.38kb PCR product, 1.38kb/EcoRI, pUC DNA,

road

1,2,3,4,5,6 mix (1116,883 respectively, 692,501,489,404,331,242,190,147,111,110bps), λ DNA/EcoO130I, cry1Ba1.6kb PCR product, 1.6kb/Bgl II.

Fig. 3 is the restricted enzyme cutting analysis of recombinant plasmid pBY33 (6.0kb).

Wherein,

road

1,2,3,4,5,6 and M represent pBY33/BamHI, pBY33/EcoRI, pBY33/HindIII, pBY33/PstI, pBY33/SalI, pBY33/XbaI, λ/EcoO130I respectively.

Fig. 4 is the subclone schema of cry3Aa gene.

Fig. 5 is three kinds of subclone recombinant plasmids of cry3Aa7 gene restriction analysis result.

Wherein,

road

1,2,3,4 and M represent pBY33-16/EcoRI, pBY33-6/EcoRI, pBY33-5/EcoRI+HindIII, pBY33/EcoRI, 1kb gradient (Ladder) (1.0～9.5kb) respectively.

Fig. 6 is a Bt17 plasmid DNA Southern results of hybridization.

Wherein,

road

1,2,3,4,5,6 is represented UV17 plasmid DNA/BamHI, UV17 plasmid DNA/SstI, UV17 plasmid DNA/SalI, UV17 plasmid DNA, λ DNA/EcoO130I, cry1Ba1.6kb respectively.

Fig. 7 is a Bt17 plasmid DNA library construction schema.

Fig. 8 is the partially digested result of Bt17 plasmid DNA.

Wherein, on behalf of the enzyme of different enzyme amounts, road 1-7 cut the result respectively, and road M represents λ DNA/Eco130 I.

Fig. 9 is a cry1Ba gene recombination plasmid restriction analysis.

Wherein,

road

1,2,3,4 and M represent pHT3-67/SalI, pHT3-66/SalI, pHT3-66/HindIII, pHT3-66/EcoRI, λ/Eco130I respectively.

Figure 10 is that cry1Ba gene recombination plasmid PCR detects.

Wherein,

road

1,2 and M represent pUC Mix, 1.6kb PCR product, product/PstI respectively.

Figure 11 is a pGM1105 vector construction schema.

Figure 12 cuts the result for pUCP19 and pHT315 enzyme.

Wherein,

road

1,2,3, M ₁And M ₂Represent pUCP19/HindIII, pUCP19/SfiI+StuI, pHT315/AatII, λ/EcoO130I, 1kb gradient respectively.

Figure 13 is that pGM1105 (7.7kb) enzyme is cut detected result.

Wherein,

road

1,2,3,4,5 and M represent pGM1105/BamHI, pGM1105/EcoRI, pGM1105/HindIII, pGM1105/SalI, pHT315/AatII, λ/EcoO130I respectively.

Figure 14 is a recombinant plasmid pLF31105 restriction analysis.

Wherein,

road

1,2,3,4,5 and M represent pLF31105/SalI, pLF31105/HindIII, pLF31105/EcoRI, pBY33/HindIII, pGM1105/HindIII, 1kb gradient respectively.

Figure 15 transforms Pf, Bt, E.coil plasmid and restriction analysis thereof for pLF31105.

Wherein,

road

1,2,3,4,5,6 and M represent pLF31105 (BE20), pLF31105 (Pf), pLF31105 (E.coli), pLF31105/EcoRI, pLF31105/SalI, pLF31105/HindIII, 1kb gradient respectively.

Figure 16 is a cry3Aa gene PCR analytical results in each transformant.

Wherein,

road

1,2,3 is the PCR product, is respectively pLF31105 (BE20), pLF31105 (Pf), pLF31105 (E.coli);

Road

4,5,6 is PCR product/EcoRI, is respectively pLF31105 (BE20), pLF31105 (Pf), pLF31105 (E.coli); Road M ₁And M ₂Represent pUC Mix and λ/EcoO130I respectively.

Figure 17 is a Bt engineering bacteria cry3Aa gene expression results (1/2LB substratum).

Wherein, road M, 1,2,3,4,5 and 6 represents protein label (marker), Btt, Bt22, BiotIII205, BiotIII-I, Bt17, HD-1 respectively.

Figure 18 is bivalent gene engineering mycoprotein crystal scanning electron microscope result.

Wherein, 18a, 18b, 18c represent BiotIII205, BiotIII-I, Bt22 respectively.

Specific embodiments of the present invention:

Below narrate embodiments of the invention.Should be noted that embodiments of the invention have only illustration for the present invention, and effect without limits.

What need particularly point out is, although detailedly in an embodiment described two kinds of gene combination and expression in Bt, yet this does not mean that the assortment of genes of the present invention is only limited to transform and produce with the Bt bacterial strain and has the Bt engineering bacteria that above-mentioned insect is had resistance.

Therefore, use cry1B described in the invention and cry3 gene to make up, to improve virulence and resistance to insect, and expansion desinsection scope, use wide host expresses carrier described in the invention, import in any microorganism, plant or its tissue or the cell with any method that those of ordinary skills were had, and the microorganism with any anti-insect activity, the plant that obtain therefrom, and such plant offspring's seed, hybridization and transformation offspring, include within interest field of the presently claimed invention.

The clone of cry3Aa gene in embodiment 1, the Bt22 bacterial strain

According to Narva (Narva, K.E. etc., EP0462721 A2,1991,8) etc. method is extracted plasmid DNA from the Bt22 bacterial strain, with HindIII enzyme complete digestion plasmid DNA, carries out Southern hybridization, probe is obtained by cry3Aa gene specific primer amplified plasmid dna, is 1.38kb (qualification result is seen Fig. 2).Primer is:

5’CGAACAATCGAAGTGAACATGATAC

3’CATCTGTTGTTTCTGGAGGCAAT

Carry out mark with 32P; Hybridizing method is undertaken by " molecular cloning experiment guide ", obtains the results of hybridization (see figure 1).Discovery has hybridization signal at 3kb, from gel, reclaim the plasmid DNA HindIII endonuclease bamhi of 3.0kb, be connected with pBluescript SK (+) behind the purifying, transformed into escherichia coli JM107, carry out PCR with the cry3A gene-specific primer and detect, screen positive recombinant plasmid pBY33 (Fig. 3, Fig. 5).Recombinant plasmid contains the big fragment of cry3A gene of 3.0kb.It is carried out subclone obtain pBY33-5, pBY33-6, three kinds of recombinant plasmids of pBY-16, contain 0.72,1.6 respectively, the exogenous genetic fragment (the subclone schema is seen Fig. 4) of 0.675kb.Determined dna sequence is finished by the logical bio-engineering corporation of Beijing six directions.This gene is by the international called after cry3Aa7 of NK of Bt δNei Dusu gene.

Clone cry1Ba gene in embodiment 2, the Bt17 bacterial strain

Cry1Ba gene in the Bt17 bacterial strain is identified nuclear and location, and the result shows that this gene is positioned on the big plasmid, and qualification result is seen Fig. 2, and the Southern results of hybridization is seen Fig. 6.Extract Bt17 bacterial strain plasmid DNA, carry out partially digested with Sau3A I enzyme, from gel, reclaim the 2-7kb dna fragmentation, be connected with pBluescript SK (+) carrier behind the purifying through BamHI digestion, alkaline phosphatase treatment, behind the transformed into escherichia coli JM107, to carrying out pcr amplification, screen positive transformant (Bt17 plasmid DNA library construction is seen Fig. 7,8) with cry1Ba gene-specific primer Sun1Ba5/3.5 ' and 3 ' end primer is respectively:

Forward primer (Forward primer) TCCTGCAGTTGACTTCAAATAGG;

Reverse primer (Reverse primer) CAGTCGACTCATCCGATAAACACGCCAC '.

Screening obtains recombinant plasmid pHT3-66, and this plasmid contains cry1Ba full length gene dna fragmentation (the results are shown in Figure 9,10).Carry out dna sequence analysis, the result shows that this gene contains 3687bp, is made up of 1229 amino acid, and molecular weight is 139.5kDa.Because of different with known cry1Ba1 gene, its amino acids coding has also become arginine at the 1055th bit base, and promptly aminoacid sequence and known there are differences in the Bt17 bacterial strain.This gene is by the international called after cry1Ba3 of NK of Bt δNei Dusu gene.This gene order is referring to SEQ ID NO 2.

The structure of embodiment 3, shuttle vectors

The plasmid pHT315 (6.5kb) that will contain the Bt replicon cuts with AatII, mends into flush end with the Klenow enzyme; With StuI and StiI double digestion plasmid pUCP19, what obtain 1.2kb contains pseudomonas replicon plasmid DNA fragment, mend flat with the big fragment of Klenow, these two kinds of DNA are connected, transformed into escherichia coli JM107, the recombinant plasmid of screening 7.7kb is cut evaluation through enzyme and is obtained pGM1105 (7.7kb) (analytical results is seen Figure 11,12,13).It is transformed Pseudomonas fluorescens P303 bacterial strain (having Rifampin Rif and nalidixic acid Nad resistance) respectively and Bt wild strain Bt17, Bt22, Btk do not have crystal mutant strain BE20 (method for transformation such as Lereclus, Lereclus, D. etc., FEMS Microbiology Letters, 1989,60:211-217).

The pseudomonas method for transformation is: 28 ℃, 220r/pm, and LB culture medium culturing bacterial strain spends the night, and carries out 1% inoculation next day, and culture condition is constant.Treat O.D ₆₀₀Be, get cultured pseudomonas bacterium liquid and carry out ice bath that 4 ℃ are descended centrifugal collection thalline, add the 0.1M MgCl of 1/2 volume at 0.5 o'clock ₂Ice bath 5min, centrifugal collection thalline adds the 0.05MCaCl of 1/2 volume ₂Ice bath 30min, centrifugal collection thalline adds the above-mentioned CaCl of 1/10 volume ₂Standby.Remaining part is the same with intestinal bacteria.

Above-mentioned two kinds of bacterium all can grow positive transformant, extract all kinds of transformant plasmids and carry out restriction analysis, prove that this carrier of pGM1105 can shuttle back and forth in three kinds of bacteriums and genetic stability, and stability is greater than 90%.

The structure of embodiment 4, expression vector

Cry3Aa7 gene (3.0kb) is cloned on the HindIII site of pGM1105, transformed into escherichia coli JM107 filters out the recombinant plasmid pLF31105 (10.7kb) (seeing Figure 14) that contains the cry3Aa gene; Behind its transformed into escherichia coli SCS110 bacterial strain, extract plasmid, these plasmids are transformed Pseudomonas fluorescens P303 bacterial strain and Bt wild strain Bt17 (containing the cry1Ba3 gene) (Analysis and Identification the results are shown in Figure 15,16) respectively; Carry out Protein Detection respectively, SDS-PAGE analysis revealed cry3A gene is equal energy normal expression 67kDa albumen in P303 and Bt17, and its importing does not influence the albumen of cry1Ba3 genetic expression 140kDa among the Bt17.The cry3A gene imports Bt17 and obtains transformant, through various Molecular Detection, proves to transform successfully, with this transformant called after engineering bacteria BiotIII-I (analysis of protein the results are shown in Figure 17).

The cultivation of embodiment 5, engineering bacteria, observation and detection

Adopt GT substratum, 1/2LB substratum and ZShi substratum (develop voluntarily in this laboratory), cultivate the BiotIII-I engineering bacteria respectively, scanning electron microscope and observation by light microscope result, in BiotIII-I, exist square and two kinds of crystal of the bipyramid bodily form (seeing Figure 18), prove the coexpression of two kinds of genes.

Embodiment 6, engineering bacteria are to several insect toxicity tests

The test insect:

Lepidoptera pest-small cabbage moth (Plutella xylostella) second instar larvae;

Coleopteran pest-elm fleautiauxia armata (Pyrrhalta aenescens) two～four-age larva;

Coleopteran pest-colorado potato bug (Leplinotarsa decernlineata) two～four-age larva.

The desinsection bioassay method:

Small cabbage moth: leaf dipping method behind the employing cabbage leaves weighing, 96 hours investigation results;

Elm fleautiauxia armata: adopt fresh elm blade leaf dipping method, 96 hours investigation results;

Colorado potato bug: adopt the foliage-spray method, 96 hours investigation results.

(1) engineering bacteria is to the small cabbage moth desinsection result (referring to table 1) of different population:

The extension rate that the engineering bacteria BiotIII-I of bivalent gene combination is different is suitable with starting strain Bt17 to the toxic effect of small cabbage moth, and for example, 180 times of dilute samples, BiotIII-I corrected mortality (CM) are that 92.56%, 540 times of CM is 84.62%; And the Bt small cabbage moth population from Hainan still had stronger toxic effect, 180 times of CM are 57.82%, and produce the bacterial strain BtC005 that uses 180 times of CM of this resistant population are had only 36%.

(2) engineering bacteria is to elm fleautiauxia armata desinsection result (referring to table 2, table 3):

Find these in the test agent from table 2, engineering bacteria BiotIII-I, Bt22, Bt17 insecticidal effect CM are respectively 63.33%, 43.33%, 36.67%.The engineering bacteria BiotIII-I virulence of bivalent gene combination is the strongest, is better than two kinds of starting strain Bt22 and Bt17.

According to table 3 result, two kinds of gene expression products of cry1Ba and cry3A (protein concentration is respectively 0.5mg/mL) are during according to the mixed proportioning of 50: 50 and 40: 60, the insecticidal toxicity maximum, and CM is respectively 68.75% and 72.73%; Prove this two kinds of proteic obvious synergy.

Table 1, engineering bacteria are to different population small cabbage moth desinsection measurement result

Beijing examination worm (sensitivity) Hainan examination worm (resistance) sample

48 hours 96 hours 48 hours 96 hours

Proofread and correct the dead dead dead dead total dead borer population of the dead borer population of the total borer population of the dead borer population of the dead borer population of borer population of proofreading and correct of proofreading and correct of proofreading and correct

530535540 <210> 55 <211> 1916 <212> DNA <213> Arabidopsis (Arabidopsis thaliana) <400> 55 gtcgacccac gcgtccgcaa cagaaaccac aacaaaaact ttgagtcctc ttcttctcta 60 tacacaaaca tgaacacttt tacctcaaac tcttcggatc tcactaccac tgcaaccgaa 120 acatcgtcct ttagcacctt gtatctcctc tcaacacttc aagcttttgt ggctataacc 180 ttagtgatgc tactcaagaa attgatgacg gatcccaaca aaaagaaacc gtatctgcca 240 ccgggtccca caggatggcc gatcattgga atgattccga cgatgctaaa gagccggccc 300 gttttccggt ggctccacag catcatgaag cagctcaata ctgagatagc atgcgtgaag 360 ttaggaaaca ctcatgtgat caccgtcacg tgccctaaga tagcacgtga gatactcaag 420 caacaagacg ctctcttcgc gtcgaggcct ttaacttacg ctcagaagat cctctctaac 480 ggctacaaaa cctgcgtgat cactcccttt ggtgaccaat tcaagaaaat gaggaaagtt 540 gtgatgacgg aactcgtatg tccagcgaga cacaggtggc tccaccagaa gagatcagaa 600 gaaaacgatc atttaaccgc ttgggtatac aacatggtta agaactcggg ctctgtcgat 660 ttccggttca tgactaggca ttactgtgga aatgcaatca agaagcttat gttcgggacg 720 agaacgttct ctaagaacac tgcacctgac ggtggaccca ccgtagaaga tgtagagcac 780 atggaagcaa tgtttgaagc attagggttt accttcgctt tttgcatctc tgattatctg 840 ccgatgctca ctggacttga tcttaacggt cacgagaaga ttatgagaga atcaagtgcg 900 attatggaca agtatcatga cccaatcatc gacgagagga tcaagatgtg gagagaagga 960 aagagaactc aaatcgaaga ttttcttgat attttcatct ctatcaaaga cgaacaaggc 1020 aacccattgc ttaccgccga tgaaatcaaa cccaccatta aggagcttgt aatggcggcg 1080 ccagacaatc catcaaacgc cgtggaatgg gccatggcgg agatggtgaa caaaccggag 1140 attctccgta aagcaatgga agagatcgac agagtcgtcg ggaaagagag actcgttcaa 1200 gaatccgaca tcccaaaact aaactacgtc aaagctatcc tccgcgaagc tttccgtctc 1260 catcccgtcg ccgccttcaa cctcccccac gtggcacttt ctgacacaac cgtcgccgga 1320 tatcacatcc ctaaaggaag tcaagtcctt cttagccgat atgggctggg ccgtaaccca 1380 aaagtttggg ccgacccact ttgctttaaa ccggagagac atctcaacga atgctccgaa 1440 gttactttga ccgagaacga tctccggttt atctcgttca gtaccgggaa aagaggttgt 1500 gcggctccgg cgctaggaac ggcgttgacc acgatgatgc tcgcgagact tcttcaaggt 1560 ttcacttgga agctacctga gaatgagaca cgtgtcgagc tgatggagtc tagtcacgat 1620 atgtttctgg ctaaaccgtt ggttatggtc ggtgacctta gattgccgga gcatctctac 1680 ccgacggtga agtgagatga gacgacgccg tatatatttt atgaaactac ttttatataa 1740 tcgcccaacc aagtttggtc aattccggtt accagaagat aattggtcaa attgtgaaca 1800 aacttgtgtg ttggtttctt ggttcttttt gggacacttg aattgtgtct cctttacctc 1860 ttcttttgtt gttttcaata aaaactttta ttaccatttc aaaaaaaaaa aaaaaa 1916 <210> 56 <211> 1974 <212> DNA <213> Arabidopsis (Arabidopsis thaliana) <400> 56 atgaacactt ttacctcaaa ctcttcggat ctcactacca ctgcaaccga aacatcgtcc 60 tttagcacct tgtatctcct ctcaacactt caagcttttg tggctataac cttagtgatg 120 ctactcaaga aattgatgac ggatcccaac aaaaagaaac cgtatctgcc accgggtccc 180 acaggatggc cgatcattgg aatgattccg acgatgctaa agagccggcc cgttttccgg 240 tggctccaca gcatcatgaa gcagctcaat actgagatag catgcgtgaa gttaggaaac 300 actcatgtga tcaccgtcac gtgccctaag atagcacgtg agatactcaa gcaacaagac 360 gctctcttcg cgtcgaggcc tttaacttac gctcagaaga tcctctctaa cggctacaaa 420 acctgcgtga tcactccctt tggtgaccaa ttcaagaaaa tgaggaaagt tgtgatgacg 480 gaactcgtat gtccagcgag acacaggtgg ctccaccaga agagatcaga agaaaacgat 540 catttaaccg cttgggtata caacatggtt aagaactcgg gctctgtcga tttccggttc 600 atgactaggc attactgtgg aaatgcaatc aagaagctta tgttcgggac gagaacgttc 660 tctaagaaca ctgcacctga cggtggaccc accgtagaag atgtagagca catggaagca 720 atgtttgaag cattagggtt taccttcgct ttttgcatct ctgattatct gccgatgctc 780 actggacttg atcttaacgg tcacgagaag attatgagag aatcaagtgc gattatggac 840 aagtatcatg acccaatcat cgacgagagg atcaagatgt ggagagaagg aaagagaact 900 caaatcgaag attttcttga tattttcatc tctatcaaag acgaacaagg caacccattg 960 cttaccgccg atgaaatcaa acccaccatt aaggtattta tcacgttcct ttcatataag 1020 gtttcgatcg taaaaatatc aaaagaacaa tttttgttaa attttatttg agaaagcatg 1080 catatcaaat ttatttacac atactaacat tttgattcat aaaacattta taaaagaaga 1140 aagaaacatt ttgtggtaaa agttgattag ttacaatatt tgtttttttt ttgctaaaca 1200 tgggctactt ttttgtttgt ctcttttgat tactttggtc aaagacagat gcatgcaact 1260 taattgtatt tatttttatg ttatacaaaa attaaagatc caaaattaat aaaagctggt 1320 atatatgttt ataatgaata ggagcttgta atggcggcgc cagacaatcc atcaaacgcc 1380 gtggaatggg ccatggcgga gatggtgaac aaaccggaga ttctccgtaa agcaatggaa 1440 gagatcgaca gagtcgtcgg gaaagagaga ctcgttcaag aatccgacat cccaaaacta 1500 aactacgtca aagctatcct ccgcgaagct ttccgtctcc atcccgtcgc cgccttcaac 1560 ctcccccacg tggcactttc tgacacaacc gtcgccggat atcacatccc taaaggaagt 1620 caagtccttc ttagccgata tgggctgggc cgtaacccaa aagtttgggc cgacccactt 1680 tgctttaaac cggagagaca tctcaacgaa tgctccgaag ttactttgac cgagaacgat 1740 ctccggttta tctcgttcag taccgggaaa agaggttgtg cggctccggc gctaggaacg 1800 gcgttgacca cgatgatgct cgcgagactt cttcaaggtt tcacttggaa gctacctgag 1860 aatgagacac gtgtcgagct gatggagtct agtcacgata tgtttctggc taaaccgttg 1920 gttatggtcg gtgaccttag attgccggag catctctacc cgacggtgaa gtga 1974 <210> 57 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer T7 <400> 57 aatacgactc actatag 17 <210> 58 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer EST3 <400> 58 gctaggatcc atgttgtata cccaag 26 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer EST6 <400> 59 cgggcccgtt ttccggtggc 20 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer EST7A <400> 60 ggtcaccaaa gggagtgatc acgc 24 <210> 61 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 5 * natural * righteousness <400> 61 atcgtcagtc gaccatatga acacttttac ctcaaactct tcgg 44 <210> 62 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 5 * cow * righteousness <400> 62 atcgtcagtc gaccatatgg ctctgttatt agcagttttt acatcgtcct ttagcacctt 60 gtatctcc 68 <210> 63 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer 3 * end * Antisense <400> 63 actgctagaa ttcgacgtca ttacttcacc gtcgggtaga gatgc 45 <210> 64 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer CYP79B2.2 <400> 64 ggaattcatg aacactttta cctca 25 <210> 65 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer B2SB <400> 65 ttgtctagat cacttcaccg tcgggta 27 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer B2AF <400> 66 ggcctcgaga tgaacacttt tacctca 27 <210> 67 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer B2AB <400> 67 ttggaattcc ttcaccgtcg ggtagag 27 <210> 68 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer XbaI <400> 68 gtaccatcta gattcatgtt tgtgtataga g 31 <210> 69 <211> 2361 <212> DNA <213> Arabidopsis (Arabidopsis thaliana) <400> 69 gaattcattg atctggtctt gctaaaaact ttaaaattga tgagttcaac atcttcaaat 60 gcatgataac gggtccaacg gaaattgact tttttttcat gctcctgata tataataata 120 tctaacgatt acgggttcca ctaattgtca ttactcatta acattcctat ttaaaagttg 180 tgatagtttt agggttttac gtagtcgtgt catatagcga ttaactacgt acttgtagat 240 ttatcaatta cttctgttgt ttacgagaac ctaaaaaaaa gaagcagatg cctagtttat 300 agagcacgtg tactgtcttg aaaacttagg taggttggta aggttaccaa aagaccttaa 360 aggaatataa agttactaat taacttaagt aaagttggta ttgcttatat attgcaaagt 420 attacaaacc aatcccctct gtatattgtt ttaaaccata gattttttta caattaagtt 480 tatgatcaat caattatttc accatttcta ttaaattatg taaaaagaaa aggatatata 540 tatatatata taattaaata agaataaatc aaaataccga aattttttat tatccattct 600 ttgtggacat cgcccctaat atataaaaaa aaaaaacttt cgtataactg atttatattt 660 ttttgtaaaa acttaaagga agcctaagaa atatcttgtg atatttttga caaaatgtgg 720 tatatatctt tttataatat catttataaa gaaaatattg attacatggt gaaaaacatt 780 ttgctagcga tcaacaaaat taaataggca catgttaact gatctcatac gaccttgaaa 840 ttttaatctt tgtgtcgaga gaccgatctt tatgcaaatt atgaaactac acatggttta 900 tgcacggaag atcacattgc atgtatacca tattataaac caaaaatgat caagaagaag 960 gcgaaaacat ttgggtaaat tttaaatttc gatcatgcga ttttttagct catcatcaac 1020 agacaagaaa ctatcttttg tactgtaaat actaaataca aaataaaatc ttcatcattt 1080 tttgcatgtg tcaaataaat tacgcgaact tttttttttt atcgactatt aatagagaaa 1140 cctgttttat ttgccttgat ttggaaaaat ggagaaattg acttaagact tagtctcggt 1200 cacatcggca acaacggagc ttaaacggcg tccgcaacat ggaaactcaa gccacgaatc 1260 tgatatattg actatagaag tagtaagtaa ctttgactcg tcccacatca gtttcaattt 1320 ccacgagggt atttggcagg tgaactctct acgtacccaa aacataatgg ctattttatt 1380 tcataactga tatttagcaa ttaattattc gtccttttta aaccaatttc tatagttggg 1440 aaaataatca atttttacac tttcaatgta tacgttacag attttttttt attagtcatg 1500 cacatatttt caatttttac actttcaatg taaacaatcg attcttaatt gttaaaaata 1560 ggtttacgta aggaattaaa gatttgttta aaatatgttc cggccggtct aataatttac 1620 ttgacgttaa tttcttaaac acttttagat aggaggcttt gtttatccca aatgattttg 1680 taccactgcg acaatactag ctagacataa aatgttaata aatttttatt aagtaatata 1740 atcgaagtat tagatcaatg tagtagacag ttaggttaac taaaacaaga gtaaacactt 1800 ttttttttct tttcaggata ggtaaaacaa atttcacact attttgcgta tttccttaaa 1860 tttgttgttc gttttctcag caaagatgaa tattttgttt catagtaatt cacaagtata 1920 aactcgccag aactcctcaa acagtgaaat ataatatagc ttttaactgt ttttcggctg 1980 gaccgggttt ttaagtgcat atataacacg aggaattttg gcaggtcacc aacaaaactt 2040 ttaaaaatat taaaaattcc catcaagaat agaaattaat aaacaatgat atctctaata 2100 atatagatat tttgaaacgt taggaataat cgtaataatg ttcaacgttg gtggtggtac 2160 tcaagatgga ccctccctcc cacattttcc tcactccttc gtaagtcctt tccacgcata 2220 agggtattat agtcatttca cataaactaa cgactactag acttgtatat aaataggaag 2280 gtgaagctct ctctttatcc atgcagagac aacagaaacc acaacaaaaa ctttgagtcc 2340 tcttcttctc tatacacaaa c 2361 <210> 70 <211> 540 <212> PRT <213> turnip (Brassica napus) <400> 70 Met Asn Thr Phe Thr Ser Asn Ser Ser Asp Leu Thr Ser Thr Thr Thr 151015 Gln Thr Ser Pro Phe Ser Asn Met Tyr Leu Leu Thr Thr Leu Gln Ala ...

Table 2, engineering bacteria lead % Jiao Zheng Si Wang to Yu fleautiauxia armata desinsection Zuo Yong result (96 Xiao Shi) Yang Pin processing Chong negative total columns Si and lead %1/2B Pei Yang base 2 35 00 0B22 10X 2 30 13 4333 433B17 10X 2 30 11 3,667 366 BiotIII-I 10X 2 27 19 6,333 633

Table 3 engineering bacteria is to elm fleautiauxia armata insecticidal synergistic exercising result (96 hours)

Sample	Handle	Dead borer population	Borer population alive	Corrected mortality (%)
Sample	Handle	Dead borer population	Borer population alive	Corrected mortality (%)	????CK	????1	????1	????16	????2.94
????2	????0	????17				????1	????1	????16
????2	????0	????17	????0∶100	????1		????5	????10	????37.93
????2	????6	????8		????1		????5	????10
????2	????6	????8		????20∶80	????1	????9	????8		????52.94
????2	????9	????8			????1	????9	????8
????2	????9	????8	????40∶60		????1	????8	????8	????53.13
????2	????9	????7			????1	????8	????8
????2	????9	????7		????50∶50	????1	????8	????7		????68.75
????2	????14	????3			????1	????8	????7
????2	????14	????3	????60∶40		????1	????13	????4	????72.73
????2	????11	????5			????1	????13	????4
????2	????11	????5		????80∶20	????1	????12	????5		????67.65
????2	????11	????6			????1	????12	????5
????2	????11	????6	????100∶0		????1	????5	????12	????55.88
????2	????14	????3			????1	????5	????12

Sample ligand compared with the beginning concentration: cry3Aa7 albumen (Bt22) content and cry1Ba3 albumen (Bt17) content are 0.5mg/mL

(3) engineering bacteria is to colorado potato bug desinsection result (referring to table 4):

The result shows that Bt17 (cry1Ba) bacterial strain has mesogenic to colorado potato bug; Bt22 (cry3Aa) and foreign project bacterium Bt2321 have high virulence, reach 93.97% and 91.38% respectively; The engineering bacteria BiotIII-I virulence of bivalent gene combination is the strongest, and prevention effect reaches 99.15%, and is suitable with chemical pesticide.Prove absolutely that bivalent gene has remarkable role in synergy.

Table 4 engineering bacteria is processed the multiple Zong borer population survival of Chong borer population control Xiao to colorado potato bug desinsection measurement result (96 Xiao Shi investigation result) Yang Pin and is led % variance analysis Bt2321 1X 3 636 51 91.38 BCbBt22 1X 3 730 42 93.97 BbBt17 1X 3 692 260 59.26 DdBiotIII-I 1X 3 631 5 99.15 Aa

10X 3 548 129 74.77 Cc chemical drug 1500X 3 760 0 100 AaH ₂O 3 671 626--to annotate: chemical drug is " protects " that German Bayer company produces

Appendix: The invention relates to DNA sequences and protein sequences (1) SEQ ID NO 1 (cry3Aa7 nucleotide sequence, wherein the underlined part of the coding region of 1932bp): AAGCTTAATT AAAGATAATA TCTTTGAATT GTAACGCCCC TCAAAAGTAA GAACTACAAA 60 AAAAGAATAC GTTATATAGA AATATGTTTG AACCTTCTTC AGATTACAAA TATATTCGGA 120 CGGACTCTAC CTCAAATGCT TATCTAACTA TAGAATGACA TACAAGCACA ACCTTGAAAA 180 TTTGAAAATA TAACTACCAA TGAACTTGTT CATGTGAATT ATCGCTGTAT TTAATTTTCT 240 CAATTCAATA TATAATATGC CAATACATTG TTACAAGTAG AAATTAAGAC ACCCTTGATA 300 GCCTTACTAT ACCTAACATG ATGTAGTATT AAATGAATAT GTAAATATAT TTATGATAAG 360 AAGCGACTTA TTTATAATCA TTACATATTT TTCTATTGGA ATGATTAAGA TTCCAATAGA 420 ATAGTGTATA AATTATTTAT CTTGAAAGGA GGGATGCCTA AAAACGAAGA ACATTAAAAA 480 CATATATTTG CACCGTCTAA TGGATTTATG AAAAATCATT TTATCAGTTT GAAAATTATG 540 TATTATGATA AGAAAGGGAG GAAGAAAAAT GAATCCGAAC AATCGAAGTG AACATGATAC 600 AATAAAAACT ACTGAAAATA ATGAGGTGCC AACTAACCAT GTTCAATATC CTTTAGCGGA 660 AACTCCAAAT CCAACACTAG AAGATTTAAA TTATAAAGAG TTTTTAAGAA TGACTGCAGA 720 TAATAATACG GAAGCACTAG ATAGCTCTAC AACAAAAGAT GTCATTCAAA AAGGCATTTC 780 CGTAGTAGGT GATCTCCTAG GCGTAGTAGG TTTCCCGTTT GGTGGAGCGC TTGTTTCGTT 840 TTATACAAAC TTTTTAAATA CTATTTGGCC AAGTGAAGAC CCGTGGAAGG CTTTTATGGA 900 ACAAGTAGAA GCATTGATGG ATCAGAAAAT AGCTGATTAT GCAAAAAATA AAGCTCTTGC 960 AGAGTTACAG GGCCTTCAAA ATAATGTCGA AGATTATGTG AGTGCATTGA GTTCATGGCA 1020 AAAAAATCCT GTGAGTTCAC GAAATCCACA TAGCCAGGGG CGGATAAGAG AGCTGTTTTC 1080 TCAAGCAGAA AGTCATTTTC GTAATTCAAT GCCTTCGTTT GCAATTTCTG GATACGAGGT 1140 TCTATTTCTA ACAACATATG CACAAGCTGC CAACACACAT TTATTTTTAC TAAAAGACGC 1200 TCAAATTTAT GGAGAAGAAT GGGGATACGA AAAAGAAGAT ATTGCTGAAT TTTATAAAAG 1260 ACAACTAAAA CTTACGCAAG AATATACTGA CCATTGTGTC AAATGGTATA ATGTTGGATT 1320 AGATAAATTA AGAGGTTCAT CTTATGAATC TTGGGTAAAC TTTAACCGTT ATCGCAGAGA 1380 GATGACATTA ACAGTATTAG ATTTAATTGC ACTATTTCCA TTGTATGATG TTCGGCTATA 1440 CCCAAAAGAA GTTAAAACCG AATTAACAAG AGACGTTTTA ACAGATCCAA TTGTCGGAGT 1500 CAACAACCTT AGGGGCTATG GAACAACCTT CTCTAATATA GAAAATTATA TTCGAAAACC 1560 ACATCTATTT GACTATCTGC ATAGAATTCA ATTTCACACG CGGTTCCAAC CAGGATATTA 1620 TGGAAATGAC TCTTTCAATT ATTGGTCCGG TAATTATGTT TCAACTAGAC CAAGCATAGG 1680 ATCAAATGAT ATAATCACAT CTCCATTCTA TGGAAATAAA TCCAGTGAAC CTGTACAAAA 1740 TTTAGAATTT AATGGAGAAA AAGTCTATAG AGCCGTAGCA AATACAAATC TTGCGGTCTG 1800 GCCGTCCGCT GTATATTCAG GTGTTACAAA AGTGGAATTT AGCCAATATA ATGATCAAAC 1860 AGATGAAGCA AGTACACAAA CGTACGACTC AAAAAGAAAT GTTGGCGCGG TCAGCTGGGA 1920 TTCTATCGAT CAATTGCCTC CAGAAACAAC AGATGAACCT CTAGAAAAGG GATATAGCCA 1980 TCAACTCAAT TATGTAATGT GCTTTTTAAT GCAGGGTAGT AGAGGAACAA TCCCAGTGTT 2040 AACTTGGACA CATAAAAGTG TAGACTTTTT TAACATGATT GATTCGAAAA AAATTACACA 2100 ACTTCCGTTA GTAAAGGCAT ATAAGTTACA ATCTGGTGCT TCCGTTGTCG CAGGTCCTAG 2160 GTTTACAGGA GGAGATATCA TTCAATGCAC AGAAAATGGA AGTGCGGCAA CTATTTACGT 2220 TACACCGGAT GTGTCGTACT CTCAAAAATA TCGAGCTAGA ATTCATTATG CTTCTACATC 2280 TCAGATAACA TTTACACTCA GTTTAGACGG GGCACCATTT AATCAATACT ATTTCGATAA 2340 AACGATAAAT AAAGGAGACA CATTAACGTA TAATTCATTT AATTTAGCAA GTTTCAGCAC 2400 ACCATTCGAA TTATCAGGGA ATAACTTACA AATAGGCGTC ACAGGATTAA GTGCTGGAGA 2460 TAAAGTTTAT ATAGACAAAA TTGAATTTAT TCCAGTGAAT TAAATTAACT AGAAAGTAAA 2520 GAAGTAGTGA CCATCTATGA TAGTAAGCAA AGGATAAAAA AATGAGTTCA TAAAATGAAT 2580 AACATAGTGT TCTTCAACTT TCGCTTTTTG AAGGTAGATG AAGAACACTA TTTTTATTTT 2640 CAAAATGAAG GAAGTTTTAA ATATGTAATC ATTTAAAGGG AACAATGAAA GTAGGAAATA 2700 AGTCATTATC TATAACAAAA TAACATTTTT ATATAGCCAG AAATGAATTA TAATATTAAT 2760 CTTTTCTAAA TTGACGTTTT TCTAAACGTT CTATAGCTTC AAGACGCTTA GAATCATCAA 2820 TATTTGTATA CAGAGCTGTT GTTTCCATCG AGTTATGTCC CATTTGATTC GCTAATAGAA 2880 CAAGATCTTT ATTTTCGTTA TAATGATTGG TTGCATAAGT ATGGCGTAAT TTATGAGGGC 2940 TTTTCTTTTC ATCAAAAGCC CTCGTGTATT TCTCTGTAAG CTT 2983 (2) SEQ ID NO 2 (cry1Ba3 nucleotide sequence and its encoded amino acid sequence of the protein) 1 TTGACTTCAAATAGGAAAAATGAGAATGAAATTATAAATGCTGTATCGAATCATTCCGCA 60 1 L T S N R K N E N E I I N A V S N H S A 20 61 CAAATGGATC TATTACCAGATGCTCGTATTGAGGATAGCTTGTGTATAGCCGAGGGGAAC 120 21 Q M D L L P D A R I E D S L C I A E G N 40 121 AATATCGATC CATTTGTTAGCGCATCAACAGTCCAAACGGGTATTAACATAGCTGGTAGA 180 41 N I D P F V S A S T V Q T G I N I A G R 60 181 ATACTAGGCG TATTGGGCGTACCGTTTGCTGGACAACTAGCTAGTTTTTATAGTTTTCTT 240 61 I L G V L G V P F A G Q L A S F Y S F L 80 241 GTTGGTGAAT TATGGCCCCGCGGCAGAGATCAGTGGGAAATTTTCCTAGAACATGTCGAA 300 81 V G E L W P R G R D Q W E I F L E H V E 100 301 CAACTTATAA ATCAACAAATAACAGAAAATGCTAGGAATACGGCTCTTGCTCGATTACAA 360 101 Q L I N Q Q I T E N A R N T A L A R L Q 120 361 GGTTTAGGAG ATTCCTTCAGAGCCTATCAACAGTCACTTGAAGATTGGCTAGAAAACCGT 420 121 G L G D S F R A Y Q Q S L E D W L E N R 140 421 GATGATGCAA GAACGAGAAGTGTTCTTTATACCCAATATATAGCTTTAGAACTTGATTTT 480 141 D D A R T R S V L Y T Q Y I A L E L D F 160 481 CTTAATGCGA TGCCGCTTTTCGCAATTAGAAACCAAGAAGTTCCATTATTGATGGTATAT 540 161 L N A M P L F A I R N Q E V P L L M V Y 180 541 GCTCAAGCTG CAAATTTACACCTATTATTATTGAGAGATGCCTCTCTTTTTGGTAGTGAA 600 181 A Q A A N L H L L L L R D A S L F G S E 200 601 TTTGGGCTTA CATCGCAGGAAATTCAACGCTATTATGAGCGCCAAGTGGAACGAACGAGA 660 201 F G L T S Q E I Q R Y Y E R Q V E R T R 220 661 GATTATTCCG ACTATTGCGTAGAATGGTATAATACAGGTCTAAATAGCTTGAGAGGGACA 720 221 D Y S D Y C V E W Y N T G L N S L R G T 240 721 AATGCCGCAA GTTGGGTACGGTATAATCAATTCCGTAGAGATCTAACGTTAGGAGTATTA 780 241 N A A S W V R Y N Q F R R D L T L G V L 260 781 GATCTAGTGG CACTATTCCCAAGCTATGACACTCGCACTTATCCAATAAATACGAGTGCT 840 261 D L V A L F P S Y D T R T Y P I N T S A 280 841 CAGTTAACAA GAGAAGTTTATACAGACGCAATTGGAGCAACAGGGGTAAATATGGCAAGT 900 281 Q L T R E V Y T D A I G A T G V N M A S 300 901 ATGAATTGGT ATAATAATAATGCACCTTCGTTCTCTGCCATAGAGGCTGCGGCTATCCGA 960 301 M N W Y N N N A P S F S A I E A A A I R 320 961 AGCCCGCATC TACTTGATTTTCTAGAACAACTTACAATTTTTAGCGCTTCATCACGATGG 1020 321 S P H L L D F L E Q L T I F S A S S R W 340 1021 AGTAATACTAGGCATATGACTTATTGGCGGGGGCGCACGATTCAATCTCGGCCAATAGGA 1080 341 S N T R H M T Y W R G R T I Q S R P I G 360 1081 GGCGGATTAAATACCTCAACGCATGGGGCTACCAATACTTCTATTAATCCTGTAACATTA 1140 361 G G L N T S T H G A T N T S I N P V T L 380 1141 CGGTTCGCATCTCGAGACGTTTATAGGACTGAATCATATGCAGGAGTGCTTCTATGGGGA 1200 381 R F A S R D V Y R T E S Y A G V L L W G 400 1201 ATTTACCTTGAACCTATTCATGGTGTCCCTACTGTTAGGTTTAATTTTACGAACCCTCAG 1260 401 I Y L E P I H G V P T V R F N F T N P Q 420 1261 AATATTTCTGATAGAGGTACCGCTAACTATAGTCAACCTTATGAGTCACCTGGGCTTCAA 1320 421 N I S D R G T A N Y S Q P Y E S P G L Q 440 1321 TTAAAAGATTCAGAAACTGAATTACCACCAGAAACAACAGAACGACCAAATTATGAATCT 1380 441 L K D S E T E L P P E T T E R P N Y E S 460 1381 TACAGTCACAGGTTATCTCATATAGGTATAATTTTACAATCCAGGGTGAATGTACCGGTA 1440 461 Y S H R L S H I G I I L Q S R V N V P V 480 1441 TATTCTTGGACGCATCGTAGTGCAGATCGTACGAATACGATTGGACCAAATAGAATCACC 1500 481 Y S W T H R S A D R T N T I G P N R I T 500 1501 CAAATCCCAATGGTAAAAGCATCCGAACTTCCTCAAGGTACCACTGTTGTTAGAGGACCA 1560 501 Q I P M V K A S E L P Q G T T V V R G P 520 1561 GGATTTACTGGTGGGGATATTCTTCGAAGAACGAATACTGGTGGATTTGGACCGATAAGA 1620 521 G F T G G D I L R R T N T G G F G P I R 540 1621 GTAACTGTTAACGGACCATTAACACAAAGATATCGTATAGGATTCCGCTATGCTTCAACT 1680 541 V T V N G P L T Q R Y R I G F R Y A S T 560 1681 GTAGATTTTGATTTCTTTGTATCACGTGGAGGTACTACTGTAAATAATTTTAGATTCCTA 1740 561 V D F D F F V S R G G T T V N N F R F L 580 1741 CGTACAATGAACAGTGGAGACGAACTAAAATACGGAAATTTTGTGAGACGTGCTTTTACT 1800 581 R T M N S G D E L K Y G N F V R R A F T 600 1801 ACACCTTTTACTTTTACACAAATTCAAGATATAATTCGAACGTCTATTCAAGGCCTTAGT 1860 601 T P F T F T Q I Q D I I R T S I Q G L S 620 1861 GGAAATGGGGAAGTGTATATAGATAAAATTGAAATTATTCCAGTTACTGCAACCTTCGAA 1920 621 G N G E V Y I D K I E I I P V T A T F E 640 1921 GCAGAATATGATTTAGAAAGAGCGCAAGAGGCGGTGAATGCTCTGTTTACTAATACGAAT 1980 641 A E Y D L E R A Q E A V N A L F T N T N 660 1981 CCAAGAAGATTGAAAACAGATGTGACAGATTATCATATTGATCAAGTATCCAATTTAGTG 2040 661 P R R L K T D V T D Y H I D Q V S N L V 680 2041 GCGTGTTTATCGGATGAATTCTGCTTGGATGAAAAGAGAGAATTACTTGAGAAAGTGAAA 2100 681 A C L S D E F C L D E K R E L L E K V K 700 2101 TATGCGAAACGACTCAGTGATGAAAGAAACTTACTCCAAGATCCAAACTTCACATCCATC 2160 701 Y A K R L S D E R N L L Q D P N F T S I 720 2161 AATAAGCAACCAGACTTCATATCTACTAATGAGCAATCGAATTTCACATCTATCCATGAA 2220 721 N K Q P D F I S T N E Q S N F T S I H E 740 2221 CAATCTGAACATGGATGGTGGGGAAGTGAGAACATTACCATCCAGGAAGGAAATGACGTA 2280 741 Q S E H G W W G S E N I T I Q E G N D V 760 2281 TTTAAAGAGAATTACGTCACACTACCGGGTACTTTTAATGAGTGTTATCCGACGTATTTA 2340 761 F K E N Y V T L P G T F N E C Y P T Y L 780 2341 TATCAAAAAATAGGGGAGTCGGAATTAAAAGCTTATACTCGCTACCAATTAAGAGGTTAT 2400 781 Y Q K I G E S E L K A Y T R Y Q L R G Y 800 2401 ATTGAAGATAGTCAAGATTTAGAGATATATTTGATTCGTTATAATGCGAAACATGAAACA 2460 801 I E D S Q D L E I Y L I R Y N A K H E T 820 2461 TTGGATGTTCCAGGTACCGAGTCCCTATGGCCGCTTTCAGTTGAAAGCCCAATCGGAAGG 2520 821 L D V P G T E S L W P L S V E S P I G R 840 2521 TGCGGAGAACCGAATCGATGCGCACCACATTTTGAATGGAATCCTGATCTAGATTGTTCC 2580 841 C G E P N R C A P H F E W N P D L D C S 860 2581 TGCAGAGATGGAGAAAAATGTGCGCATCATTCCCATCATTTCTCTTTGGATATTGATGTT 2640 861 C R D G E K C A H H S H H F S L D I D V 880 2641 GGATGCACAGACTTGCATGAGAATCTAGGCGTGTGGGTGGTATTCAAGATTAAGACGCAG 2700 881 G C T D L H E N L G V W V V F K I K T Q 900 2701 GAAGGTCATGCAAGACTAGGGAATCTGGAATTTATTGAAGAGAAACCATTATTAGGAGAA 2760 901 E G H A R L G N L E F I E E K P L L G E 920 2761 GCACTGTCTCGTGTGAAGAGGGCAGAGAAAAAATGGAGAGACAAACGTGAAAAACTACAA 2820 921 A L S R V K R A E K K W R D K R E K L Q 940 2821 TTGGAAACAAAACGAGTATATACAGAGGCAAAAGAAGCTGTGGATGCTTTATTCGTAGAT 2880 941 L E T K R V Y T E A K E A V D A L F V D 960 2881 TCTCAATATGATAGATTACAAGCGGATACAAACATCGGCATGATTCATGCGGCAGATAAA 2940 961 S Q Y D R L Q A D T N I G M I H A A D K 980 2941 CTTGTTCATCGAATTCGAGAGGCGTATCTTTCAGAATTACCTGTTATCCCAGGTGTAAAT 3000 981 L V H R I R E A Y L S E L P V I P G V N 1000 3001 GCGGAAATTTTTGAAGAATTAGAAGGTCACATTATCACTGCAATCTCCTTATACGATGCG 3060 1001 A E I F E E L E G H I I T A I S L Y D A 1020 3061 AGAAATGTCGTTAAAAATGGTGATTTTAATAATGGATTAACATGTTGGAATGTAAAAGGG 3120 1021 R N V V K N G D F N N G L T C W N V K G 1040 3121 CATGTAGATGTACAACAGAGCCATCATCGTTCTGACCTTGTTATCCCAGAATGGGAAGCA 3180 1041 H V D V Q Q S H H R S D L V I P E W E A 1060 3181 GAAGTGTCACAAGCAGTTCGCGTCTGTCCGGGGTGTGGCTATATCCTTCGTGTCACAGCG 3240 1061 E V S Q A V R V C P G C G Y I L R V T A 1080 3241 TACAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCATGAAATCGAGAACAATACAGAC 3300 1081 Y K E G Y G E G C V T I H E I E N N T D 1100 3301 GAACTAAAATTTAAAAACCGTGAAGAAGAGGAAGTGTATCCAACGGATACAGGAACGTGT 3360 1101 E L K F K N R E E E E V Y P T D T G T C 1120 3361 AATGATTATACTGCACACCAAGGTACAGCTGGATGCGCAGATGCATGTAATTCCCGTAAT 3420 1121 N D Y T A H Q G T A G C A D A C N S R N 1140 3421 GCTGGATATGAGGATGCATATGAAGTTGATACTACAGCATCTGTTAATTACAAACCGACT 3480 1141 A G Y E D A Y E V D T T A S V N Y K P T 1160 3481 TATGAAGAAGAAACGTATACAGATGTAAGAAGAGATAATCATTGTGAATATGACAGAGGG 3540 1161 Y E E E T Y T D V R R D N H C E Y D R G 1180 3541 TATGTCAATTATCCACCAGTACCAGCTGGTTATGTGACAAAAGAATTAGAATACTTCCCA 3600 1181 Y V N Y P P V P A G Y V T K E L E Y F P 1200 3601 GAAACAGATACAGTATGGATTGAGATTGGAGAAACGGAAGGAAAGTTTATTGTAGATAGC 3660 1201 E T D T V W I E I G E T E G K F I V D S 1220 3661 GTGGAATTACTCCTCATGGAAGAATAG 3687 1221 V E L L L M E E * 1240 ...

Claims

1. the gene order of a Bt gene cry1Ba3 is characterized in that this sequence has the nucleotide sequence shown in SEQ ID NO 2, and coding has the protein of the aminoacid sequence shown in SEQ ID NO 2, and insect is had toxicity;

2. the gene order of claim 1, wherein said gene order comprises the partial sequence of this gene order;

3. the gene order of claim 1, wherein said gene order comprises the homologous sequence of this gene order;

4. the gene order of claim 1, wherein said insect is lepidopteran and coleopteron;

5. one kind has active proteinic aminoacid sequence to insect, it is characterized in that this proteinic aminoacid sequence is coded by the gene order of claim 1, has the aminoacid sequence shown in SEQ ID NO 2, and insect is had toxicity;

6. the aminoacid sequence of claim 5, wherein said aminoacid sequence comprises the partial sequence of this aminoacid sequence;

7. the aminoacid sequence of claim 5, wherein said aminoacid sequence comprises the homologous sequence of this aminoacid sequence;

8. the aminoacid sequence of claim 5, wherein said insect is lepidopteran and coleopteron;

9. the Genetic carrier that can shuttle back and forth between bacillus thuringiensis-intestinal bacteria-pseudomonas is characterized in that this shuttle expression carrier is to duplicate sub constructed by one from bacillus thuringiensis-colibacillary shuttle vectors and the plasmid DNA from pseudomonas;

10. expression vector, this expression vector of its feature is constructed by the hereditary shuttle vectors of claim 9 and the gene order shown in SEQID NO 1;

11. one kind transforms the method that the Bt cell obtains engineering bacteria, it is characterized in that this method application rights requires 10 expression vector;

12. the combination of gene cry1Ba and cry3Aa7 is characterized in that this combination can be applicable to transform microorganism and plant, makes it to show the toxicity to relevant insect, and overcomes or delay insect to engineering bacteria and the drug-fast generation of transgenic plant;

13. the combination of protein C ry1Ba and Cry3Aa7 is characterized in that this combination can be applicable to transform microorganism and plant, makes it to show the toxicity to relevant insect, and overcomes or delay insect to engineering bacteria and the drug-fast generation of transgenic plant;

14. a broad sense expression vector pGM1105 is characterized in that this carrier is according to claim 1,5,9,10,11 and make up, and can be used in multiple host living beings (cell);

15. an expression vector pLF31105 is characterized in that this carrier carries the gene order shown in SEQ ID NO 1.