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

Abstract

The invention relates to "Bt gene, expression vector and engineering bacteria with high toxicity to Lepidoptera and Coleoptera insects", belonging to the technical field of biological control. Further, the present invention relates to the Bt crylBa3 gene sequence which is resistant to both Lepidoptera and Coleoptera pests, the crylBalcry3Aa gene combination method, and the broad host expression vector pGM1105. Furthermore, the present invention combines the Bt crylBa gene that is resistant to both Lepidoptera and Coleopteran pests with the cry3Aa gene that is active against Coleopteran pests to improve the toxicity of the gene to related pests; The host shuttle expression vector pGM1105 transformed the cry3Aa gene into the Bt17 strain containing the crylBa gene to achieve high-efficiency expression of the two genes. The toxicity to coleopteran pests produced by the combination of the two genes or their expression products involved in the present invention is stronger than that of similar natural strains and engineering strains found in the world at present, indicating that there is a difference between the two genes or their expression products. Significant synergy.

Description

Bt gene, expression vector and engineering bacteria highly virulent to Coleoptera and Lepidoptera insects

Technical field of the present invention:

The invention belongs to the technical field of biological control. Further, the present invention relates to a Bt gene, an expression vector and an engineering bacterium that are highly toxic to both Lepidoptera and Coleoptera pests. Furthermore, the present invention relates to the Bt cry1Ba3 gene sequence and the cry1Ba/cry3Aa gene combination method that are resistant to both Lepidoptera and Coleoptera pests to achieve high-efficiency expression of the two genes; it also relates to a broad host expression vector pGM1105.

Research background of the present invention:

Bacillus thuringiensis (Bt) can produce a crystal protein with insecticidal activity during the sporulation stage, which has been widely used in the control of agricultural, forestry, and sanitary pests.

In 1983, Krieg et al. (Krieg, A. et al., J. Appli. Entomology, 1983, 96: 500-508) isolated the first Bt strain (Bt. tenebrionis) highly virulent to coleopteran pests. In 1987, Herrnstadt (Herrnstadt, C. etc., Gene, 1987, 57 (1): 37-46) cloned the first active Bt insecticidal protein gene cry3Aa to coleopteran pests, used to control potato beetle (Leptinotarsa decernlineata (Say)). U.S. Ecogen Company (Sick, A. et al., Nucl. Acids Res., 1990.18, 1305; Entwistle, P.F. et al., An Environmental Biopesticide: Theory and Practice [M], Wiley Chichester Press, UK, 1993, 125-146) utilizes plasmid binding Bt engineering bacteria active on Lepidoptera and Coleoptera were constructed by means of cell engineering such as transfer.

Because a single gene encodes a protein, pests quickly develop resistance to the Cry3A protein (Whalon, M.E., etc., J.ofEconomic Entomology, 1993, 86 (2): 226-233; Nicholas, D., etc., Transgenic Plant and Insect Pests Biocontrol [M], John Wiley & Sons Press, USA, 1997, 1-18). In this way, due to the rapid generation of pest resistance, the service life of genetically modified products will be greatly shortened, resulting in huge waste. In 1993, Chang, C.Y.M. et al. (Chang, C.Y.M. et al., Appl. Environ. Micro. 1993, 59: 815-821.) reported the synergistic effect of different crystal proteins in Bt Israel subspecies on mosquito larvae.

Therefore, screening highly virulent protein combinations, and then carrying out gene combination, will be able to improve the insecticidal activity of Bt products and expand its insecticidal spectrum. More importantly, insecticidal proteins with different properties can effectively overcome or delay the emergence of insecticide resistance in pests, and have strong practicability.

Contents of the present invention:

Purpose of the present invention:

In view of the shortcomings of Bt preparations and transgenic products used in the world to prevent and control coleopteran pests, such as single gene species, easy resistance of pests, and narrow insecticidal spectrum, the present invention adopts two genes cry3Aa7 and cry1Ba3 with high toxicity to coleopterans Combining to improve virulence, and using cry1Ba3 gene to be active against lepidopteran pests, to expand the insecticidal spectrum of the product, so that it can be applied to transform microorganisms and plants, so that it can show toxicity to related pests, and overcome and delay pests Resistance to engineered bacteria and transgenic plants.

Further, in order to facilitate the study of the expression of the Bt insecticidal protein gene in different microbial recipient bacteria, the present invention constructs a vector that can freely shuttle among the three parental microorganisms, which overcomes the shortcomings of the existing vectors with a narrow host range, and through Insert the Btcry3Aa7 gene into it to complete a series of detection work.

Technical scheme of the present invention:

1. Cloning of cry3Aa gene in Bt22 strain

According to Narva (Narva, K.E., etc., EP0462721 A2, 1991, 8) and other methods, plasmid DNA was extracted from Bt22 strain, plasmid DNA was completely digested with HindIII enzyme, and Southern hybridization was carried out. The probe was obtained by amplifying plasmid DNA with cry3Aa gene-specific primers , which is 1.38kb. Primers are:

5'CGAACAATCGAAGTGAACATGATAC

3'CATCTGTTGTTTCTGGAGGCAAT

Labeled with 32p; the hybridization method was carried out according to the "Molecular Cloning Experiment Guide" to obtain the hybridization result (see Figure 1). It was found that there was a hybridization signal at 3kb, and the 3.0kb plasmid DNA HindIII fragment was recovered from the gel, purified and ligated with pBluescript SK(+), transformed into Escherichia coli JM107, and detected by PCR with cry3A gene-specific primers. Positive recombinant plasmid pBY33 (Figure 2, Figure 3). The recombinant plasmid contains a 3.0 kb cry3A gene fragment. Three recombinant plasmids, pBY33-5, pBY33-6 and pBY-16 were obtained by subcloning them, respectively containing 0.72, 1.6 and 0.675kb foreign gene fragments. DNA sequence determination was completed by Beijing Liuhetong Bioengineering Company. The gene was named cry3Aa7 by the International Nomenclature Committee of Btδ Endotoxin Genes.

2. Cloning cry1Ba gene from Bt17 strain

Extract the plasmid DNA of the Bt17 strain, partially digest it with Sau3A I enzyme, recover the 2-7kb DNA fragment from the gel, after purification, connect it with the pBluescript SK(+) vector digested with BamHI and treated with alkaline phosphatase, and transform the large intestine After bacillus JM107, use cry1Ba gene-specific primer Sun1Ba5/3 pair to perform PCR amplification to screen positive transformants. The 5' and 3' end primers are:

Forward primer (Forward primer) TCCTGCAGTTGACTTCAAATAGG;

Reverse primer (Reverse primer) CAGTCGACTCATCCGATAAACACGCCAC’.

The recombinant plasmid pHT3-66 was screened, which contained the full-length DNA fragment of the cry1Ba gene. DNA sequence analysis shows that the gene contains 3687bp, consists of 1229 amino acids, and has a molecular weight of 139.5kDa. The 1055th base gene is different from the known cry1Ba1 gene, and the amino acid encoded by it has also changed to arginine, that is, the amino acid sequence in the Bt17 strain is different from the known gene. The gene was named cry1Ba3 by the International Nomenclature Committee of Btδ Endotoxin Genes.

3. Construction of Shuttle Vector

The plasmid pHT315 (6.5kb) containing the Bt replicon was cut with AatII, and the blunt end was filled with Klenow enzyme; the plasmid pUCP19 was double-digested with StuI and StiI to obtain a 1.2kb plasmid DNA fragment containing the Pseudomonas replicon. The Klenow large fragment was filled up, the two DNAs were connected, transformed into Escherichia coli JM107, and the recombinant plasmid of 7.7 kb was screened, and pGM1105 (7.7 kb) was obtained after enzyme digestion and identification, which were respectively transformed into Pseudomonas fluorescens P303 strains (RifR, Nad) and Bt wild strains Bt17, Bt22, Btk crystal-free mutant BE20 (transformation methods such as Lereclus, Lereclus, D., etc., FEMS Microbiology Letters, 1989, 60: 211-217), all can grow positive transformants, and extract All kinds of transformant plasmids were subjected to enzyme digestion analysis, which proved that the vector pGM1105 could shuttle among the three bacteria and be inherited stably, with a stability greater than 90%.

4. Construction of expression vector

The cry3Aa7 gene (3.0kb) was cloned into the HindIII site of pGM1105, transformed Escherichia coli JM107, and the recombinant plasmid pLF31105 (10.7kb) containing the cry3Aa gene was screened out, and after it was transformed into Escherichia coli SCS110 bacterial strain, the plasmid was extracted, and these plasmids were Transform Pseudomonas fluorescens P303 strain and Bt wild strain Bt17 (containing crylBa3 gene) respectively, and perform protein detection respectively. SDS-PAGE analysis shows that cry3A gene can normally express 67kDa protein in both P303 and Bt17, and its introduction does not affect Bt17 The crylBa3 gene expresses a 140kDa protein. The cry3A gene was introduced into Bt17 to obtain a transformant. After various molecular tests, it was proved that the transformation was successful, and the transformant was named as the engineering bacterium BiotIII-I.

5. Cultivation and observation of engineered bacteria

Using GT medium, 1/2 LB medium and Z's medium (self-made in this laboratory), cultivate BiotIII-I engineering bacteria respectively, the results of scanning electron microscopy and optical microscope observations show that there are square and double cones in BiotIII-I. There were two types of crystals in body shape (see Figure 18), demonstrating the co-expression of the two genes.

6. Determination of insecticidal biological activity

The test insects were:

Lepidoptera pests - diamondback moth (Plutella xylostella);

Coleoptera pests - elm blue leaf beetle (Pyrrhalta aenescens),

Potato beetle (Leplinotarsa decernlineata).

Beneficial effects of the present invention:

The present invention combines the Bt cry1Ba gene that is resistant to both Lepidoptera and Coleopteran pests and the cry3Aa gene that is active against Coleopteran pests; the cry3Aa gene can be transformed and introduced by using the wide-host shuttle expression vector pGM1105 constructed by the present invention In the Bt17 strain containing the cry1Ba gene, the two genes were highly expressed. The combination of these two gene expression products is more virulent to coleopteran pests than the similar natural strains and engineering strains found in the world, indicating that there is a significant synergistic effect between the two gene expression products.

Furthermore, the cry1Ba3 gene sequence, cry1Ba/cry3Aa gene combination method, and broad host expression vector pGM1105 involved in the present invention are also unique to the present invention.

Description of drawings:

Figure 1 shows the results of Southern Blotting of the Bt22 plasmid.

Lanes 1, 2, 3, 4, and 5 represent cry3Aa1.38kb, λDNA/HindIII, Bt22 plasmid/PstI, Bt22 plasmid/HindIII, and Bt22 plasmid, respectively.

Fig. 2 is the identification map of Bt22 and Bt17cry genes.

Among them, lanes 1, 2, 3, 4, 5, and 6 represent cry3Aa 1.38kb PCR product, 1.38kb/EcoRI, pUC DNA mixture (1116,883,692,501,489,404,331,242,190,147 , 111, 110bps), λDNA/EcoO130I, cry1Ba1.6kb PCR product, 1.6kb/Bgl II.

Figure 3 is a restriction analysis of the recombinant plasmid pBY33 (6.0 kb).

Wherein, lanes 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 a flowchart of the subcloning of the cry3Aa gene.

Fig. 5 shows the results of digestion analysis of recombinant plasmids of three subclones of cry3Aa7 gene.

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

Figure 6 is the result of Southern hybridization of Bt17 plasmid DNA.

Among them, lanes 1, 2, 3, 4, 5, and 6 represent UV17 plasmad DNA/BamHI, UV17 plasmad DNA/SstI, UV17 plasmad DNA/SalI, UV17 plasmad DNA, λDNA/EcoO130I, cry1Ba1.6kb, respectively.

Fig. 7 is a flow chart of Bt17 plasmid DNA library construction.

Figure 8 is the result of partial digestion of Bt17 plasmid DNA.

Among them, lanes 1-7 represent the digestion results of different enzyme amounts, and lane M represents λDNA/Eco130I.

Fig. 9 is restriction analysis of recombinant plasmid of cry1Ba gene.

Wherein, Lanes 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 the PCR detection of the cry1Ba gene recombinant plasmid.

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

Fig. 11 is a flow chart of pGM1105 vector construction.

Figure 12 shows the results of digestion of pUCP19 and pHT315.

Wherein, lanes 1, 2, 3, M ₁ and M ₂ represent pUCP19/HindIII, pUCP19/SfiI+StuI, pHT315/AatII, λ/EcoO130I, 1kb gradients, respectively.

Figure 13 shows the results of pGM1105 (7.7kb) digestion.

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

Figure 14 shows the enzyme digestion analysis of the recombinant plasmid pLF31105.

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

Fig. 15 shows pLF31105 transformation of Pf, Bt, E.coil plasmids and analysis of restriction enzyme digestion.

Wherein, lanes 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.

Fig. 16 is the result of PCR analysis of cry3Aa gene in each transformant.

Among them, lanes 1, 2, and 3 are PCR products, which are respectively pLF31105 (BE20), pLF31105 (Pf), and pLF31105 (E.coli); lanes 4, 5, and 6 are PCR products/EcoRI, which are respectively pLF31105 (BE20), pLF31105(Pf), pLF31105(E.coli); Lanes M ₁ and M ₂ represent pUC Mix and λ/EcoO130I, respectively.

Fig. 17 is the expression result of cry3Aa gene of Bt engineering bacteria (1/2LB medium).

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

Figure 18 is the scanning electron microscope result of bivalent genetically engineered bacterial protein crystals.

Among them, 18a, 18b, and 18c represent BiotIII205, BiotIII-I, and Bt22, respectively.

Specific embodiments of the present invention:

Examples of the present invention are described below. It should be noted that the embodiments of the present invention are only illustrative but not limiting to the present invention.

It should be pointed out that although the combination and expression of the two genes in Bt have been described in detail in the examples, this does not mean that the gene combination of the present invention is limited to the transformation and production of the above-mentioned pests with resistance to the above-mentioned pests. Sexual Bt engineering bacteria.

Therefore, use the combination of cry1B and cry3 genes described in the present invention to improve the virulence and resistance to pests, and expand the scope of insecticidal, use the broad host expression vector described in the present invention, with the knowledge of those of ordinary skill in the art Any method introduced into any microorganism, plant or its tissue or cell, as well as the microorganisms, plants obtained therefrom having any insect-resistant activity, and the seeds, hybrids and transgenic offspring of such plant progeny are included in within the scope of the claims of the present invention.

Cloning of cry3Aa gene in embodiment 1, Bt22 bacterial strain

According to Narva (Narva, K.E., etc., EP0462721 A2, 1991, 8) and other methods, plasmid DNA was extracted from Bt22 strain, plasmid DNA was completely digested with HindIII enzyme, and Southern hybridization was carried out. The probe was obtained by amplifying plasmid DNA with cry3Aa gene-specific primers , is 1.38kb (see Figure 2 for identification results). Primers are:

5'CGAACAATCGAAGTGAACATGATAC

3'CATCTGTTGTTTCTGGAGGCAAT

Labeled with 32P; the hybridization method was carried out according to the "Molecular Cloning Experiment Guide" to obtain the hybridization result (see Figure 1). It was found that there was a hybridization signal at 3kb, and the 3.0kb plasmid DNA HindIII fragment was recovered from the gel, purified and ligated with pBluescript SK(+), transformed into Escherichia coli JM107, and detected by PCR with cry3A gene-specific primers. Positive recombinant plasmid pBY33 (Figure 3, Figure 5). The recombinant plasmid contains a 3.0 kb cry3A gene fragment. It was subcloned to obtain three recombinant plasmids, pBY33-5, pBY33-6, and pBY-16, respectively containing foreign gene fragments of 0.72, 1.6, and 0.675 kb (see Figure 4 for the subcloning flow chart). DNA sequence determination was completed by Beijing Liuhetong Bioengineering Company. The gene was named cry3Aa7 by the International Nomenclature Committee of Btδ Endotoxin Genes.

Cloning cry1Ba gene in embodiment 2, Bt17 bacterial strain

The nuclear identification and localization of the cry1Ba gene in the Bt17 strain showed that the gene was located on a large plasmid. The identification results are shown in Figure 2, and the results of Southern hybridization are shown in Figure 6. Extract the plasmid DNA of the Bt17 strain, partially digest it with Sau3A I enzyme, recover the 2-7kb DNA fragment from the gel, after purification, connect it with the pBluescript SK(+) vector digested with BamHI and treated with alkaline phosphatase, and transform the large intestine After bacillus JM107, PCR amplification was performed with cry1Ba gene-specific primer Sun1Ba5/3, and positive transformants were screened (see Figures 7 and 8 for the construction of the Bt17 plasmid DNA library). The 5' and 3' end primers are:

Forward primer (Forward primer) TCCTGCAGTTGACTTCAAATAGG;

Reverse primer (Reverse primer) CAGTCGACTCATCCGATAAACACGCCAC'.

The recombinant plasmid pHT3-66 was screened, which contained the full-length DNA fragment of the cry1Ba gene (results shown in Figures 9 and 10). DNA sequence analysis shows that the gene contains 3687bp, consists of 1229 amino acids, and has a molecular weight of 139.5kDa. The 1055th base gene is different from the known cry1Ba1 gene, and the amino acid encoded by it has also changed to arginine, that is, the amino acid sequence in the Bt17 strain is different from the known gene. The gene was named cry1Ba3 by the International Nomenclature Committee of Btδ Endotoxin Genes. The gene sequence is shown in SEQ ID NO 2.

Embodiment 3, the construction of shuttle carrier

The plasmid pHT315 (6.5kb) containing the Bt replicon was cut with AatII, and the blunt end was filled with Klenow enzyme; the plasmid pUCP19 was double-digested with StuI and StiI to obtain a 1.2kb plasmid DNA fragment containing the Pseudomonas replicon. The Klenow large fragment was blunted, the two DNAs were connected, transformed into Escherichia coli JM107, the 7.7kb recombinant plasmid was screened, and pGM1105 (7.7kb) was obtained after enzyme digestion (see Figures 11, 12, and 13 for the analysis results). It was transformed into Pseudomonas fluorescens P303 strain (with resistance to rifampicin Rif and nalidixic acid Nad) and Bt wild strains Bt17, Bt22, Btk crystal-free mutant BE20 (Lereclus etc. Transformation methods, Lereclus, D. et al., FEMS Microbiology Letters, 1989, 60:211-217).

The transformation method of Pseudomonas is as follows: 28°C, 220r/pm, culture the strain in LB medium overnight, inoculate with 1% the next day, and the culture conditions remain unchanged. When the OD ₆₀₀ is 0.5, take the cultured Pseudomonas bacteria liquid and put it in ice bath, collect the bacteria by centrifugation at 4°C, add 1/2 volume of 0.1M MgCl ₂ in ice bath for 5 minutes, collect the bacteria by centrifugation, add 1 /2 volume of 0.05MCaCl ₂ in ice bath for 30 min, centrifuge to collect the bacteria, and add 1/10 volume of the above CaCl ₂ for later use. The rest is the same as E. coli.

Both of the above two bacteria can grow positive transformants, and various transformant plasmids were extracted for enzyme digestion analysis, which proved that the vector pGM1105 can shuttle among the three bacteria and be genetically stable, with a stability greater than 90%.

Embodiment 4, the construction of expression vector

The cry3Aa7 gene (3.0kb) was cloned into the HindIII site of pGM1105, transformed into Escherichia coli JM107, and the recombinant plasmid pLF31105 (10.7kb) containing the cry3Aa gene was screened out (see Figure 14); after it was transformed into the Escherichia coli SCS110 strain, extracted Plasmids were transformed into Pseudomonas fluorescens P303 bacterial strain and Bt wild bacterial strain Bt17 (containing cry1Ba3 gene) respectively (analysis and identification results are shown in Figures 15 and 16); protein detection was carried out respectively, and SDS-PAGE analysis showed that cry3A gene was present in P303 and Bt17 can normally express 67kDa protein, and its introduction does not affect the expression of 140kDa protein in cry1Ba3 gene in Bt17. The cry3A gene was introduced into Bt17 to obtain a transformant. After various molecular tests, it was proved that the transformation was successful, and the transformant was named engineering bacteria BiotIII-I (see Figure 17 for protein analysis results).

Embodiment 5, cultivation, observation and detection of engineering bacteria

Using GT medium, 1/2LB medium and Z's medium (developed by our laboratory), we cultivated BiotIII-I engineering bacteria respectively, and observed the results with scanning electron microscope and optical microscope, and there were square and double cones in BiotIII-I. There were two types of crystals in body shape (see Figure 18), demonstrating the co-expression of the two genes.

Embodiment 6, engineered bacterium is to several kinds of insect toxicity assays

Test insects:

Lepidoptera pests - second instar larvae of diamondback moth (Plutella xylostella);

Coleoptera pests—second to fourth instar larvae of Pyrrhalta aenescens;

Coleopteran pests - the second to fourth instar larvae of the potato beetle (Leplinotarsa decernlineata).

Insecticidal bioassay method:

Plutella xylostella: 96-hour investigation results using cabbage leaf weighing and then dipping method;

  Elm blue leaf beetle: using fresh elm leaves soaking method, 96 hours of investigation results;

Potato beetle: results of a 96-hour survey using a foliar spray.

(1) Engineering bacterium is to the insecticidal result of diamondback moth of different populations (referring to table 1):

The poisonous effect of the engineering bacteria BiotIII-I of the bivalent gene combination is comparable to that of the starting strain Bt17 to Plutella xylostella at different dilution multiples. For example, the 180-fold diluted sample, the BiotIII-I corrected mortality (CM) is 92.56%, and the 540-fold CM It is 84.62%; and it still has a strong poisonous effect on the Bt diamondback moth population from Hainan, with a 180-fold CM of 57.82%, while the bacterial strain BtC005 used in production has only 36% of the 180-fold CM of this resistant population.

(2) engineering bacterium is to the insecticidal result of Ulmus blue leaf beetle (referring to table 2, table 3):

From Table 2, it was found that among these tested samples, the insecticidal effects CM of engineering bacteria BiotIII-I, Bt22, and Bt17 were 63.33%, 43.33%, and 36.67%, respectively. The engineered bacteria BiotIII-I with bivalent gene combination was the most virulent, stronger than the two original strains Bt22 and Bt17.

According to the results in Table 3, when the two gene expression products of cry1Ba and cry3A (protein concentrations were 0.5 mg/mL) were mixed according to the ratio of 50:50 and 40:60, the insecticidal toxicity was the highest, and the CM was 68.75% and 68.75% respectively. 72.73%; it proves that the synergistic effect of these two proteins is remarkable.

      Table 1. Determination results of engineered bacteria on different populations of diamondback moth

Beijing test insect (sensitive) Hainan test insect (resistant) sample

48 hours 96 hours 48 hours 96 hours

Correction Dead Correction Dead Correction Dead Total number of dead insects Number of dead insects Number of dead insects Total number of dead insects Number of dead insects

Differential rate % Destry % Destry % Destry % 20x 31 30 96.77 31 100 36 34 94.43 36 10060K 31 28 9032 31 10 28 6071 26 9172180x 22 7850 26 9204 9 3214 2100540x 5333 22702527 7 7 7 2593 18 61341620x 30 8 2667 18 536320x 30 29 30 10 30 29 30060x 27 9310 29 10 319 625252180x 30 22 7333 33 17 51525782540 8462629x29626136262613626261362613626136261362626261367161362613367161362613671362636713626367136261367161336261362613626136263613626362613626133613626136261362616 147CK 29 3 1034 29 4 137

Table 2. The results of engineering bacteria on elm blue leaf pesticides (96 hours) Sample treatment repeat total number of total number of number of number of number of number of correction rate of correct mortality % 1/2B medium 2 35 0 0 0b22 10x 2 30 13 433B17 10x 2 30 11 3667 366BiotIII-I 10X 2 27 19 6333 633

Table 3 Results of synergistic effect of engineered bacteria on elm blue leaf beetle (96 hours) sample deal with Number of dead insects Number of live insects Adjusted mortality rate (%) CK 1 1 16 2.94 2 0 17 0:100 1 5 10 37.93 2 6 8 20:80 1 9 8 52.94 2 9 8 40:60 1 8 8 53.13 2 9 7 50:50 1 8 7 68.75 2 14 3 60:40 1 13 4 72.73 2 11 5 80:20 1 12 5 67.65 2 11 6 100:0 1 5 12 55.88 2 14 3

Sample ratio initial concentration: cry3Aa7 protein (Bt22) content and cry1Ba3 protein (Bt17) content are both 0.5mg/mL

(3) engineering bacterium is to potato beetle insecticidal result (referring to table 4):

The results showed that Bt17 (cry1Ba) strain had moderate toxicity to potato beetle; Bt22 (cry3Aa) had high toxicity to foreign engineering bacteria Bt2321, reaching 93.97% and 91.38% respectively; Strong, the control effect reaches 99.15%, which is equivalent to chemical pesticides. It fully demonstrates that bivalent genes have a significant synergistic effect.

Table 4 Engineering bacteria on the results of potato beetle pesticides (96 -hour survey results) Sample treatment Repeat the total number of total insects for the number of bumozoids. -I 1X 3 631 5 99.15 Aa

10X 3 548 129 74.77 Cc chemical medicine 1500X 3 760 0 100 AaH ₂ O 3 671 626 - -Note: The chemical medicine is "Baode" produced by Bayer Company in Germany

Attachment: DNA sequence and protein sequence involved in the present invention (1) SEQ ID NO 1 (nucleotide sequence of cry3Aa7 gene, wherein the underlined part is the coding region 1932bp): AAGCTTAATT AAAGATAATA TCTTTGAATT GTAACGCCCC TCAAAAGTAA GAACTACAAA 60AAAAGAATAC GTTATATAGA AATATGTTTG AACCTTCTTC AGATTACAAA TATATCCGGAACT 1 CTCAAATGCT TATCTAACTA TAGAATGACA TACAAGCACA ACCTTGAAAA 180TTTGAAAATA TAACTACCAA TGAACTTGTT CATGTGAATT ATCGCTGTAT TTAATTTTCT 240CAATTCAATA TATAATATGC CAATACATTG TTACAAGTAG AAATTAAGAC ACCCTTGATA 300GCCTTACTAT ACCTAACATG ATGTAGTATT AAATGAATAT GTAAATATAT TTATGATAAG 360AAGCGACTTA TTTATAATCA TTACATATTT TTCTATTGGA ATGATTAAGA TTCCAATAGA 420ATAGTGTATA AATTATTTAT CTTGAAAGGA GGGATGCCTA AAAACGAAGA ACATTAAAAA 480CATATATTTG CACCGTCTAA TGGATTTATG AAAAATCATT TTATCAGTTT GAAAATTATG 540TATTATGATA AGAAAGGGAG GAAGAAAA AT 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 TAA ATTAACT AGAAAGTAAA 2520GAAGTAGTGA CCATCTATGA TAGTAAGCAA AGGATAAAAA AATGAGTTCA TAAAATGAAT 2580AACATAGTGT TCTTCAACTT TCGCTTTTTG AAGGTAGATG AAGAACACTA TTTTTATTTT 2640CAAAATGAAG GAAGTTTTAA ATATGTAATC ATTTAAAGGG AACAATGAAA GTAGGAAATA 2700AGTCATTATC TATAACAAAA TAACATTTTT ATATAGCCAG AAATGAATTA TAATATTAAT 2760CTTTTCTAAA TTGACGTTTT TCTAAACGTT CTATAGCTTC AAGACGCTTA GAATCATCAA 2820TATTTGTATA CAGAGCTGTT GTTTCCATCG AGTTATGTCC CATTTGATTC GCTAATAGAA 2880CAAGATCTTT ATTTTCGTTA TAATGATTGG TTGCATAAGT ATGGCGTAAT TTATGAGGGC 2940TTTTCTTTTC ATCAAAAGCC CTCGTGTATT TCTCTGTAAG CTT 2983(2)SEQ ID NO 2(cry1Ba3基因的核苷酸序列和其编码的蛋白质的氨基酸序列)1 TTGACTTCAAATAGGAAAAATGAGAATGAAATTATAAATGCTGTATCGAATCATTCCGCA 601 L T S N R K N E N E I I N A V S N H S A 2061 CAAATGGATCTATTACCAGATGCTCGTATTGAGGATAGCTTGTGTATAGCCGAGGGGAAC 12021 Q M D L L P D A R I E D S L C I A E G N 40121 AATATCGATCCATTTGTTAGCGCATCAACAGTCCAAACGGGTATTAACATAGCTGGTAGA 18041 N I D P F V S A S T V Q T G I N I A G R 60181 ATACTAGGCGTATTGGGCGTACCGTTTGCTGGACAACTAGCTAGTTTTTATAGTTTTCTT 24061 I L G V L G V P F A G Q L A S F Y S F L 80241 GTTGGTGAATTATGGCCCCGCGGCAGAGATCAGTGGGAAATTTTCCTAGAACATGTCGAA 30081 V G E L W P R G R D Q W E I F L E H V E 100301 CAACTTATAAATCAACAAATAACAGAAAATGCTAGGAATACGGCTCTTGCTCGATTACAA 360101 Q L I N Q Q I T E N A R N T A L A R L Q 120361 GGTTTAGGAGATTCCTTCAGAGCCTATCAACAGTCACTTGAAGATTGGCTAGAAAACCGT 420121 G L G D S F R A Y Q Q S L E D W L E N R 140421 GATGATGCAAGAACGAGAAGTGTTCTTTATACCCAATATATAGCTTTAGAACTTGATTTT 480141 D D A R T R S V L Y T Q Y I A L E L D F 160481 CTTAATGCGATGCCGCTTTTCGCAATTAGAAACCAAGAAGTTCCATTATTGATGGTATAT 540161 L N A M P L F A I R N Q E V P L L M V Y 180541 GCTCAAGCTGCAAATTTACACCTATTATTATTGAGAGATGCCTCTCTTTTTGGTAGTGAA 600181 A Q A A N L H L L L L R D A S L F G S E 200601 TTTGGGCTTACATCGCAGGAAATTCAACGCTATTATGAGCGCCAAGTGGAACGAACGAGA 660201 F G L T S Q E I Q R Y Y E R Q V E R T R 220661 GATTATTCCGACTATTGCGTAGAATGGTATAATACAGGTCTAAATAGCTTGAGAGGGACA 720221 D Y S D Y C V E W Y N T G L N S L R G T 240721 AATGCCGCAAGTTGGGTACGGTATAATCAATTCCGTAGAGATCTAACGTTAGGAGTATTA 780241 N A A S W V R Y N Q F R R D L T L G V L 260781 GATCTAGTGGCACTATTCCCAAGCTATGACACTCGCACTTATCCAATAAATACGAGTGCT 840261 D L V A L F P S Y D T R T Y P I N T S A 280841 CAGTTAACAAGAGAAGTTTATACAGACGCAATTGGAGCAACAGGGGTAAATATGGCAAGT 900281 Q L T R E V Y T D A I G A T G V N M A S 300901 ATGAATTGGTATAATAATAATGCACCTTCGTTCTCTGCCATAGAGGCTGCGGCTATCCGA 960301 M N W Y N N N A P S F S A I E A A A I R 320 961 AGCCCGCATCTACTTGATTTTCTAGAACAACTTACAATTTTTAGCGCTTCATCACGATGG 1020321 S P H L L D F L E Q L T I F S A S S R W 3401021 AGTAATACTAGGCATATGACTTATTGGCGGGGGCGCACGATTCAATCTCGGCCAATAGGA 1080341 S N T R H M T Y W R G R T I Q S R P I G 3601081 GGCGGATTAAATACCTCAACGCATGGGGCTACCAATACTTCTATTAATCCTGTAACATTA 1140361 G G L N T S T H G A T N T S I N P V T L 3801141 CGGTTCGCATCTCGAGACGTTTATAGGACTGAATCATATGCAGGAGTGCTTCTATGGGGA 1200381 R F A S R D V Y R T E S Y A G V L L W G 4001201 ATTTACCTTGAACCTATTCATGGTGTCCCTACTGTTAGGTTTAATTTTACGAACCCTCAG 1260401 I Y L E P I H G V P T V R F N F T N P Q 4201261 AATATTTCTGATAGAGGTACCGCTAACTATAGTCAACCTTATGAGTCACCTGGGCTTCAA 1320421 N I S D R G T A N Y S Q P Y E S P G L Q 4401321 TTAAAAGATTCAGAAACTGAATTACCACCAGAAACAACAGAACGACCAAATTATGAATCT 1380441 L K D S E T E L P P E T T E R P N Y E S 4601381 TACAGTCACAGGTTATCTCATATAGGTATAATTTTACAATCCAGGGTGAATGTACCGGTA 1440461 Y S H R L S H I G I I L Q S R V N V P V 4801441 TATTCTTGGACGCATCGTAGTGCAGATCGTACGAATACGATTGGACCAAATAGAATCACC 1500481 Y S W T H R S A D R T N T I G P N R I T 5001501 CAAATCCCAATGGTAAAAGCATCCGAACTTCCTCAAGGTACCACTGTTGTTAGAGGACCA 1560501 Q I P M V K A S E L P Q G T T V V R G P 5201561 GGATTTACTGGTGGGGATATTCTTCGAAGAACGAATACTGGTGGATTTGGACCGATAAGA 1620521 G F T G G D I L R R T N T G G F G P I R 5401621 GTAACTGTTAACGGACCATTAACACAAAGATATCGTATAGGATTCCGCTATGCTTCAACT 1680541 V T V N G P L T Q R Y R I G F R Y A S T 5601681 GTAGATTTTGATTTCTTTGTATCACGTGGAGGTACTACTGTAAATAATTTTAGATTCCTA 1740561 V D F D F F V S R G G T T V N N F R F L 5801741 CGTACAATGAACAGTGGAGACGAACTAAAATACGGAAATTTTGTGAGACGTGCTTTTACT 1800581 R T M N S G D E L K Y G N F V R R A F T 6001801 ACACCTTTTACTTTTACACAAATTCAAGATATAATTCGAACGTCTATTCAAGGCCTTAGT 1860601 T P F T F T Q I Q D I I R T S I Q G L S 6201861 GGAAATGGGGAAGTGTATATAGATAAAATTGAAATTATTCCAGTTACTGCAACCTTCGAA 1920621 G N G E V Y I D K I E I I P V T A T F E 6401921 GCAGAATATGATTTAGAAAGAGCGCAAGAGGCGGTGAATGCTCTGTTTACTAATACGAAT 1980641 A E Y D L E R A Q E A V N A L F T N T N 6601981 CCAAGAAGATTGAAAACAGATGTGACAGATTATCATATTGATCAAGTATCCAATTTAGTG 2040661 P R R L K T D V T D Y H I D Q V S N L V 6802041 GCGTGTTTATCGGATGAATTCTGCTTGGATGAAAAGAGAGAATTACTTGAGAAAGTGAAA 2100681 A C L S D E F C L D E K R E L L E K V K 7002101 TATGCGAAACGACTCAGTGATGAAAGAAACTTACTCCAAGATCCAAACTTCACATCCATC 2160701 Y A K R L S D E R N L L Q D P N F T S I 7202161 AATAAGCAACCAGACTTCATATCTACTAATGAGCAATCGAATTTCACATCTATCCATGAA 2220721 N K Q P D F I S T N E Q S N F T S I H E 7402221 CAATCTGAACATGGATGGTGGGGAAGTGAGAACATTACCATCCAGGAAGGAAATGACGTA 2280741 Q S E H G W W G S E N I T I Q E G N D V 7602281 TTTAAAGAGAATTACGTCACACTACCGGGTACTTTTAATGAGTGTTATCCGACGTATTTA 2340761 F K E N Y V T L P G T F N E C Y P T Y L 7802341 TATCAAAAAATAGGGGAGTCGGAATTAAAAGCTTATACTCGCTACCAATTAAGAGGTTAT 2400781 Y Q K I G E S E L K A Y T R Y Q L R G Y 8002401 ATTGAAGATAGTCAAGATTTAGAGATATATTTGATTCGTTATAATGCGAAACATGAAACA 2460801 I E D S Q D L E I Y L I R Y N A K H E T 8202461 TTGGATGTTCCAGGTACCGAGTCCCTATGGCCGCTTTCAGTTGAAAGCCCAATCGGAAGG 2520821 L D V P G T E S L W P L S V E S P I G R 8402521 TGCGGAGAACCGAATCGATGCGCACCACATTTTGAATGGAATCCTGATCTAGATTGTTCC 2580841 C G E P N R C A P H F E W N P D L D C S 8602581 TGCAGAGATGGAGAAAAATGTGCGCATCATTCCCATCATTTCTCTTTGGATATTGATGTT 2640861 C R D G E K C A H H S H H F S L D I D V 8802641 GGATGCACAGACTTGCATGAGAATCTAGGCGTGTGGGTGGTATTCAAGATTAAGACGCAG 2700881 G C T D L H E N L G V W V V F K I K T Q 9002701 GAAGGTCATGCAAGACTAGGGAATCTGGAATTTATTGAAGAGAAACCATTATTAGGAGAA 2760901 E G H A R L G N L E F I E E K P L L G E 9202761 GCACTGTCTCGTGTGAAGAGGGCAGAGAAAAAATGGAGAGACAAACGTGAAAAACTACAA 2820921 A L S R V K R A E K K W R D K R E K L Q 9402821 TTGGAAACAAAACGAGTATATACAGAGGCAAAAGAAGCTGTGGATGCTTTATTCGTAGAT 2880941 L E T K R V Y T E A K E A V D A L F V D 9602881 TCTCAATATGATAGATTACAAGCGGATACAAACATCGGCATGATTCATGCGGCAGATAAA 2940961 S Q Y D R L Q A D T N I G M I H A A D K 9802941 CTTGTTCATCGAATTCGAGAGGCGTATCTTTCAGAATTACCTGTTATCCCAGGTGTAAAT 3000981 L V H R I R E A Y L S E L P V I P G V N 10003001 GCGGAAATTTTTGAAGAATTAGAAGGTCACATTATCACTGCAATCTCCTTATACGATGCG 30601001 A E I F E E L E G H I I T A I S L Y D A 10203061 AGAAATGTCGTTAAAAATGGTGATTTTAATAATGGATTAACATGTTGGAATGTAAAAGGG 31201021 R N V V K N G D F N N G L T C W N V K G 10403121 CATGTAGATGTACAACAGAGCCATCATCGTTCTGACCTTGTTATCCCAGAATGGGAAGCA 31801041 H V D V Q Q S H H R S D L V I P E W E A 10603181 GAAGTGTCACAAGCAGTTCGCGTCTGTCCGGGGTGTGGCTATATCCTTCGTGTCACAGCG 32401061 E V S Q A V R V C P G C G Y I L R V T A 10803241 TACAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCATGAAATCGAGAACAATACAGAC 33001081 Y K E G Y G E G C V T I H E I E N N T D 11003301 GAACTAAAATTTAAAAACCGTGAAGAAGAGGAAGTGTATCCAACGGATACAGGAACGTGT 33601101 E L K F K N R E E E E V Y P T D T G T C 11203361 AATGATTATACTGCACACCAAGGTACAGCTGGATGCGCAGATGCATGTAATTCCCGTAAT 34201121 N D Y T A H Q G T A G C A D A C N S R N 11403421 GCTGGATATGAGGATGCATATGAAGTTGATACTACAGCATCTGTTAATTACAAACCGACT 34801141 A G Y E D A Y E V D T T A S V N Y K P T 11603481 TATGAAGAAGAAACGTATACAGATGTAAGAAGAGATAATCATTGTGAATATGACAGAGGG 35401161 Y E E E T Y T D V R R D N H C E Y D R G 11803541 TATGTCAATTATCCACCAGTACCAGCTGGTTATGTGACAAAAGAATTAGAATACTTCCCA 36001181 Y V N Y P P V P A G Y V T K E L E Y F P 12003601 GAAACAGATACAGTATGGATTGAGATTGGAGAAACGGAAGGAAAGTTTATTGTAGATAGC 36601201 E T D T V W I E I G E T E G K F I V D S 12203661 GTGGAATTACTCCTCATGGAAGAATAG 36871221 V E L L L M E E * 1240

Claims (15)

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.

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