CN1181203C - Bt gene, expression vector and engineering bacteria highly virulent to Lepidoptera and Coleoptera insects - Google Patents

Abstract

The present invention relates to a Bt gene with high toxicity on lepidoptera and coleoptera insects, an expression carrier and an engineering bacterium, which belongs to the technical field of biological prevention and treatment. Further, the present invention relates to a Bt cryBa3 gene sequence with resistance properties on lepidoptera and coleoptera pests, a crylBalcry 3Aa gene combination mode and a wide host expression carrier pGM1105. Further, the Bt crylBa gene with resistance properties on lepidoptera and coleoptera pests is combined with a cry 3Aa gene with the activity on the coleoptera pests so as to enhance the toxicity of the genes on relevant pests. The cry 3Aa gene is converted and led into a Bt17 strain containing cryBa genes by utilizing the wide host shuttle expression carrier pGM1105, and the wide shuttle expression carrier pGM1105 is constructed by the present invention so as to realize the high-efficiency expression of two kinds of genes. The toxicity of the two kinds of genes of the present invention or the combination of the expression products on the coleoptera pests is stronger than that of the same class natural strains and engineering strains internationally discovered at present to show that the two kinds of genes or the expression products have obvious synergistic effect.

Description

Bt gene, expression vector and engineering bacteria highly virulent to Lepidoptera and Coleoptera 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 (Leptinotarsadecernlineata (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 Bt engineering bacteria active to Lepidoptera and Coleoptera were constructed by combining cell engineering methods such as transfer.

Because a single gene encodes a protein, pests quickly develop resistance to the Cry3A protein (Whalon, M.E., etc., J.of Economic 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 methods such as Narva (Narva, K.E., EP0462721 A2, 1991, 8), extract plasmid DNA from Bt22 bacterial strain (this bacterial strain comes from the biotechnology laboratory of Institute of Plant Protection, Chinese Academy of Sciences, can provide to the public), complete with HindIII enzyme Plasmid DNA was digested, and Southern hybridization was performed. The probe was obtained by amplifying the plasmid DNA with cry3Aa gene-specific primers, and its size was 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 has a total of 2983 nucleotides, of which the coding region is 1932bp (see SEQ ID NO 1). The gene was named cry3Aa7 by the International Nomenclature Committee of Btδ Endotoxin Genes.

2. Cloning cry1Ba gene from Bt17 strain

Extract the Bt17 bacterial strain (this bacterial strain comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Sciences, which can be provided to the public) plasmid DNA, carry out partial digestion with Sau3A I enzyme, reclaim the 2-7kb DNA fragment from the gel, and purify it with The pBluescript SK(+) vector digested with BamHI and treated with alkaline phosphatase was ligated and transformed into Escherichia coli JM107, followed by PCR amplification with cry1Ba gene-specific primer Sun1Ba5/3 pair 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 (this plasmid comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Sciences and can be provided to the public) was cut with AatII, and the blunt end was filled with Klenow enzyme; Restriction digestion of plasmid pUCP19 (this plasmid comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Sciences, which is available to the public), obtained a 1.2kb plasmid DNA fragment containing the Pseudomonas replicon, filled in with the Klenow large fragment, and this 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 by enzyme digestion, which was transformed into Pseudomonas fluorescens P303 strain (RifR, Nad) and Bt wild strain Bt17, respectively. Bt22, Btk crystal-free mutant BE20 (transformation method by Lereclus, etc., Lereclus, D. et al., FEMS Microbiology Letters, 1989, 60: 211-217), can grow positive transformants, and extract various transformant plasmids for enzyme digestion The analysis proved that the vector pGM1105 can shuttle among the three bacteria and be inherited stably, and the stability is 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 cry1Ba3 gene) respectively, and perform protein detection respectively. SDS-PAGE analysis shows that cry3A gene can express 67kDa protein normally in both P303 and Bt17, and its introduction does not affect Bt17 The cry1Ba3 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

Adopt GT culture medium (recipe: liquid GT: add 10ml 0.8% CaCl ₂ per liter, 10ml G-Tris Salts (0.025% FeSO ₄ .7H ₂ O, 0.05% CuSO ₄ .5H ₂ O, 0.05% ZnSO ₄ . 7H ₂ O, 0.5% MnSO ₄ .H ₂ O, 2.0% MgSO ₄ ), 10ml 20% (NH ₄ ) ₂ SO ₄ , 10ml 5% K ₂ HPO ₄ , 10ml 20% Glucose, 50ml 1M Tris-HCl, pH7 .5, 1.5g Yeast extract, pH7.4; solid GT: add 1.3% agar to liquid GT), 1/2LB medium (formula: liquid LB: Tryptone 1%, Yeast extract 0.5%, NaCl 1%, pH 7.0; solid LB: add 1.3% agar to the liquid medium) and Z's medium (the formulation is peptone 1%, yeast powder 0.2%, soluble starch 0.3%, glucose 0.2%, K ₂ HPO ₄ 0.1%, KH ₂ PO ₄ 0.1%, CaCO ₃ 0.2%), respectively cultivate BiotIII-I engineering bacteria, scanning electron microscope and optical microscope observation results, in BiotIII-I, there are two kinds of crystals of square and bipyramid (see Figure 18), Co-expression of both genes was demonstrated.

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 cry3Aal.38kb, λDNA/HindIII, Bt22 plasmid/PstI, Bt22 plasmid/HindIII, and Bt22 plasmid, respectively.

Figure 2 is the identification map of Bt22 and Bt17 cry genes.

Wherein, lanes 1, 2, 3, 4, 5, 6 represent cry3Aal.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.

Fig. 3 is the restriction analysis of recombinant plasmid pBY33 (6.0kb);

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.

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

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

Among them, lanes 1, 2, 3, 4, 5, and 6 represent UV17plasmid DNA/BamHI, UV17plasmid DNA/SstI, UV17plasmidDNA/SalI, UV17plasmid DNA, λDNA/EcoO130I, cry1Ba 1.6kb, 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/EcoO130I.

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/EcoO130I, respectively.

Figure 10 is the PCR detection of the cry1Ba gene recombinant plasmid.

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/HindHI, 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 and M represent pLF31105(BE20), pLF31105(Pf), pLF31105(E.coli), pLF31105EcoRI, 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, respectively pLF31105 (BE20), pLF31105 (Pf), pLF31105 (E.coli); lanes 4, 5, and 6 are PCR products/EcoRI, respectively pLF31105 (BE20), pLF31105 (Pf), pLF31105(E.coli): Lanes M ₁ and M ₂ represent pUC Mix and λ/EcoO130I, respectively.

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

Wherein, lanes 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, BioIII-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 Sau3AI 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 into Escherichia coli JM107 Afterwards, PCR amplification was performed with the cry1Ba gene-specific primer Sun1Ba5/3 pair, 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, centrifuge to collect the bacteria at 4°C, add 1/2 volume of 0.1M MgCl ₂ in ice bath for 5min, centrifuge to collect the bacteria, add 1 /2 volume of 0.05M CaCl 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

Adopt GT substratum, 1/2LB substratum and Z's substratum, cultivate BiotIII-I engineering bacterium respectively, electron scanning microscope and optical microscope observation result, in BiotIII-I, there are two kinds of crystals of square and bipyramid (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 sample Beijing test insect (sensitive) Hainan test insect (resistance) 48 hours 96 hours 48 hours 96 hours Total number of insects Number of dead insects Corrected Mortality % Number of dead insects Corrected Mortality % Total number of insects Number of dead insects Corrected Mortality % Number of dead insects Corrected Mortality % Bt17BiotIII-ICK 20X60X180X540X162020X60X180X540X1620 313128303029302929 3028221629272220 96.7790.3278.5053.3396.6793.1073.3368.97 31312622302928253 10010092.0470.2510010092.5684.6210.34 3628282730303133293429 341797829191764 94.4360.7132.1425.9326.6796.6761.2951.5220.6711.76 36262118183029211394 10091.7271.0061.3453.6010092.5257.8236.0014.7113.7

      Table 2. Results of the insecticidal effect of engineered bacteria on the elm blue leaf beetle (96 hours)

Sample Treatment Duplicates Total Insects Dead Insects Mortality % Corrected Mortality %

1/2LB training 2 35 0 0 0

base

Bt22 10X 2 30 13 43.33 43.33

Bt17 10X 2 30 11 36.67 36.67

BiotIII-I 10X 2 27 19 63.33 63.33

Table 3 The results of the synergistic effect of engineering bacteria on the 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) The results of engineered bacteria killing potato beetles (see 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 to potato beetle insecticidal assay results (96 hours survey results)

Sample Treatment Repeated Number of Insects Surviving Insects Control Efficiency % Analysis of Variance

Bt2321 1X 3 636 51 91.38 BCb

Bt22 1X 3 730 42 93.97 Bb

Bt17 1X 3 692 260 59.26 Dd

BiotIII-I 1X 3 631 5 99.15 Aa

                                                                                                                                                                        , 

Chemicals 1500X 3 760 0 100 Aa

H ₂ O 3 671 626 - -

Note: The chemical drug is "Baode" produced by Bayer Company in Germany

Attachment: DNA sequence and protein sequence involved in the present invention

(1) SEQ ID NO 1 (the nucleotide sequence of the cry3Aa7 gene, wherein the underlined part is the coding region 1932bp):

AAGCTTAATT AAAGATAATA TCTTTGAATT GTAACGCCCC TCAAAAGTAA 60

AAAAGAATAC GTTATATAGA AATATGTTTG AACCTTCTTC AGATTACAAA 120

CGGACTCTAC CTCAAATGCT TATCTAACTA TAGAATGACA TACAAGCACA 180

TTTGAAAATA TAACTACCAA TGAACTTGTT CATGTGAATT ATCGCTGTAT 240

CAATTCAATA TATAATATGC CAATACATTG TTACAAGTAG AAATTAAGAC 300

GCCTTACTAT ACCTAACATG ATGTAGTATT AAATGAATAT GTAAATATAT 360

AAGCGACTTA TTTATAATCA TTACATATTTT TTCTATTGGA ATGATTAAGA 420

ATAGTGTATA AATTATTTAT CTTGAAAGGA GGGATGCCTA AAAACGAAGA 480

CATATATTTG CACCGTCTAA TGGATTTATG AAAAATCATT TTATCAGTTT 540

TATTATGATA AGAAAGGGAG GAAGAAAA AT GAATCCGAAC AATCGAAGTG 600

AATAAAAACT ACTGAAAATA ATGAGGTGCC AACTAACCAT GTTCAATATC 660

AACTCCAAAT CCAACACTAG AAGATTTAAA TTATAAAGAG TTTTTAAGAA 720

TAATAATACG GAAGCACTAG ATAGCTCTAC AACAAAAGAT GTCATTCAAA 780

CGTAGTAGGT GATCTCCTAG GCGTAGTAGG TTTCCCGTTT GGTGGAGCGC 840

TTATACAAAC TTTTTAAATA CTATTTGGCC AAGTGAAGAC CCGTGGAAGG 900

ACAAGTAGAA GCATTGATGG ATCAGAAAAT AGCTGATTAT GCAAAAAATA 960

AGAGTTACAG GGCCTTCAAA ATAATGTCGA AGATTATGTG AGTGCATTGA 1020

AAAAAAATCCT GTGAGTTCAC GAAATCCACA TAGCCAGGGG CGGATAAGAG 1080

TCAAGCAGAA AGTCATTTTC GTAATTCAAT GCCTTCGTTT GCAATTTCTG 1140

TCTATTTCTA ACAACATATG CACAAGCTGC CAACACACAT TTATTTTTAC 1200

TCAAATTTAT GGAGAAGAAT GGGGATACGA AAAAGAAGAT ATTGCTGAAT 1260

ACAACTAAAA CTTACGCAAG AATATACTGA CCATTGTGTC AAATGGTATA 1320

AGATAAATTA AGAGGTTCAT CTTATGAATC TTGGGTAAAC TTTAACCGTT 1380

GATGACATTA ACAGTATTAG ATTTAATTGC ACTATTTCCA TTGTATGATG 1440

CCCAAAAGAA GTTAAAACCG AATTAACAAG AGACGTTTTTA ACAGATCCAA 1500

CAACAACCTT AGGGGCTATG GAACAACCTT CTCTAATATA GAAAATTATA 1560

ACATCTATTT GACTATCTGC ATAGAATTCA ATTTCACACG CGGTTCCAAC 1620

TGGAAATGAC TCTTTCAATT ATTGGTCCGG TAATTATGTT TCAACTAGAC 1680

ATCAAATGAT ATAATCACAT CTCCATTCTA TGGAAATAAA TCCAGTGAAC 1740

TTTAGAATTT AATGGAGAAA AAGTCTATAG AGCCGTAGCA AATACAAATC 1800

GCCGTCCGCT GTATATTCAG GTGTTACAAA AGTGGAATTT AGCCAATATA 1860

AGATGAAGCA AGTACACAAA CGTACGACTC AAAAAGAAAT GTTGGCGCGG 1920

TTCTATCGAT CAATTGCCTC CAGAAACAAC AGATGAACCT CTAGAAAAGG 1980

TCAACTCAAT TATGTAATGT GCTTTTTAAT GCAGGGTAGT AGAGGAACAA 2040

AACTTGGACA CATAAAAGTG TAGACTTTTT TAACATGATT GATTCGAAAA 2100

ACTTCCGTTA GTAAAGGCAT ATAAGTTACA ATCTGGTGCT TCCGTTGTCG 2160

GTTTACAGGA GGAGATATCA TTCAATGCAC AGAAAATGGA AGTGCGGCAA 2220

TACACCGGAT GTGTCGTACT CTCAAAAATA TCGAGCTAGA ATTCATTATG 2280

TCAGATAACA TTTACACTCA GTTTAGACGG GGCACCATTT AATCAATACT 2340

AACGATAAAT AAAGGAGACA CATTAACGTA TAATTCATTT AATTTAGCAA 2400

ACCATTCGAA TTATCAGGGA ATAACTTACA AATAGGCGTC ACAGGATTAA 2460

TAAAGTTTTATATAGACAAAAA TTGAATTTAT TCCAGTGAAT TAA ATTAACT 2520

GAAGTAGTGA CCATCTATGA TAGTAAGCAA AGGATAAAAA AATGAGTTCA 2580

AACATAGTGT TCTTCAACTT TCGCTTTTTG AAGGTAGATG AAGAACACTA 2640

CAAAATGAAG GAAGTTTTAA ATATGTAATC ATTTAAAGGG AACAATGAAA 2700

AGTCATTATC TATAACAAAA TAACATTTTT ATATAGCCAG AAATGAATTA 2760

CTTTTCTAAA TTGACGTTTT TCTAAACGTT CTATAGCTTC AAGACGCTTA 2820

TATTTGTATA CAGAGCTGTT GTTTCCATCG AGTTATGTCC CATTTGATTC 2880

CAAGATCTTT ATTTTCGTTA TAATGATTGG TTGCATAAGT ATGGCGTAAT 2940

TTTTCTTTTC ATCAAAAGCC CTCGTGTATT TCTCTGTAAG CTT 2983

(2) SEQ ID NO 2.1 (nucleotide sequence of cry1Ba3 gene)

1 TTGACTTCAAATAGGAAAAATGAGAATGAAATTATAAATGCTGTATCGAATCATTCCGCA 60

61 CAAATGGATCTATTACCAGATGCTCGTATTGAGGATAGCTTGTGTATAGCCGAGGGGAAC 120

121 AATATCGATCCATTTGTTAGCGCATCAACAGTCCAAACGGGTATTAACATAGCTGGTAGA 180

181 ATACTAGGCGTATTGGGCGTACCGTTTGCTGGACAACTAGCTAGTTTTTATAGTTTTCTT 240

241 GTTGGTGAATTATGGCCCCGCGGCAGAGATCAGTGGGAAATTTTCCTAGAACATGTCGAA 300

301 CAACTTATAAATCAACAAATAACAGAAAATGCTAGGAATACGGCTCTTGCTCGATTACAA 360

361 GGTTTAGGAGATTCCTTCAGAGCCTATCAACAGTCACTTGAAGATTGGCTAGAAAACCGT 420

421 GATGATGCAAGAACGAGAAGTGTTTCTTATACCCAATATATAGCTTTAGAACTTGATTTT 480

481 CTTAATGCGATGCCGCTTTTCGCAATTAGAAACCAAGAAGTTCCATTATTGATGGTATAT 540

541 GCTCAAGCTGCAAATTTACACCTATTATTGAGAGATGCCTCTCTTTTTGGTAGTGAA 600

601 TTTGGGCTTACATCGCAGGAAATTCAACGCTATTATGAGCGCCAAGTGGAACGAACGAGA 660

661 GATTATTCCGACTATTGCGTAGAATGGTATAATACAGGTCTAAATAGCTTGAGAGGGACA 720

721 AATGCCGCAAGTTGGGTACGGTATAATCAATTCCGTAGAGATCTAACGTTAGGAGTATTA 780

781 GATCTAGTGGCACTATTTCCCAAGCTATGACACTCGCACTTATCCAATAAATACGAGTGCT 840

841 CAGTTAAACAAGAGAAGTTTATACAGACGCAATTGGAGCAACAGGGGTAAATATGGCAAGT 900

901 ATGAATTGGTATAATAATAATGCACCTTCGTTCTCTGCCATAGAGGCTGCGGCTATCCGA 960

961 AGCCCGCATCTACTTGATTTTCTAGAACAACTTACAATTTTTTAGCGCTTCATCACGATGG 1020

1021 AGTAATACTAGGCATATGACTTATTGGCGGGGGCGCACGATTCAATCTCGGCCAATAGGA 1080

1081 GGCGGATTAAATACCTCAACGCATGGGGCTACCAATACTTCTATTAATCCTGTAACATTA 1140

1141 CGGTTCGCATCTCGAGACGTTTATAGGACTGAATCATATGCAGGAGTGCTTCTATGGGGA 1200

1201 ATTTACCTTGAACCTATTCATGGTGTCCCTACTGTTAGGTTTAATTTTACGAACCCTCAG 1260

1261 AATATTTCTGATAGAGGTACCGCTAACTATAGTCAACCTTATGAGTCACCTGGGCTTCAA 1320

1321 TTAAAAAGATTCAGAAACTGAATTACCACCAGAAACAACAGAACGACCAAATTATGAATCT 1380

1381 TACAGTCACAGGTTATCTCATAGGTATAATTTTACAATCCAGGGTGAATGTACCGGTA 1440

1441 TATTCTTGGACGCATCGTAGTGCAGATCGTACGAATACGATTGGACCAAATAGAATCACC 1500

1501 CAAATCCCAATGGTAAAAGCATCCGAACTTCCTCAAGGTACCACTGTTGTTAGAGGACCA 1560

1561 GGATTTACTGGTGGGGATATTCTTCGAAGAACGAATACTGGTGGATTTGGACCGATAAGA 1620

1621 GTAACTGTTAACGGACCATTAACACAAAGATATCGTATAGGATTCCGCTATGCTTCAACT 1680

1681 GTAGATTTTGATTTCTTTGTATCACGTGGAGGTACTACTGTAAATAATTTTAGATTCCTA 1740

1741 CGTACAATGAACAGTGGAGACGAACTAAAATACGGAAATTTTGTGAGACGTGCTTTTACT 1800

1801 ACACCTTTTTACTTTTACACAAAATTCAAGATATAATTCGAACGTCTATTCAAGGCCTTAGT 1860

1861 GGAAATGGGGAAGTGTATATAGATAAAATTGAAATTATTCCAGTTACTGCAACCTTCGAA 1920

1921 GCAGAATATGATTTAGAAAGAGCGCAAGAGGCGGTGAATGCTCTGTTTACTAATACGAAT 1980

1981 CCAAGAAGATTGAAAACAGATGTGACAGATTATTCATATTGATCAAGTATCCAATTTAGTG 2040

2041 GCGTGTTTATCGGATGAATTCTGCTTGGATGAAAAGAGAGAATTACTTGAGAAAGTGAAA 2100

2101 TATGCGAAACGACTCAGTGATGAAAGAAACTTACTCCAAGATCCAAACTTCACATCCATC 2160

2161 AATAAGCAACCAGACTTCATATCTACTAATGAGCAATCGAATTTCACATCTATCCATGAA 2220

2221 CAATCTGAACATGGATGGTGGGGAAGTGAGAACATTACCATCCAGGAAGGAAATGACGTA 2280

2281 TTTAAAGAGAATTACGTCACACTACCGGGTACTTTTAATGAGTGTTATCCGACGTATTTA 2340

2341 TATCAAAAAATAGGGGAGTCGGAATTAAAAGCTTATACTCGCTACCAATTAAGAGGTTAT 2400

2401 ATTGAAGATAGTCAAAGATTTAGAGATATATTTGATTCGTTATAATGCGAAACATGAAACA 2460

2461 TTGGATGTTCCAGGTACCGAGTCCCTATGGCCGCTTTCAGTTGAAAGCCCAATCGGAAGG 2520

2521 TGCGGAGAACCGAATCGATGCGCACCACATTTTGAATGGAATCCTGATCTAGATTGTTCC 2580

2581 TGCAGAGATGGAGAAAAATGTGCGCATCATTCCCATCATCATTTCTCTTTGGATATTGATGTT 2640

2641 GGATGCACAGACTTGCATGAGAATCTAGGCGTGTGGGTGGTATTCAAGATTAAGACGCAG 2700

2701 GAAGGTCATGCAAGACTAGGGAATCTGGAATTTATTGAAGAGAAACCATTATTAGGAGAA 2760

2761 GCACTGTCTCGTGTGAAGAGGGCAGAGAAAAAATGGAGAGACAAACGTGAAAAACTACAA 2820

2821 TTGGAAACAAAACGAGTATATACAGAGGCAAAAGAAGCTGTGGATGCTTTATTCGTAGAT 2880

2881 TCTCAATATGATAGATTACAAGCGGATACAAACATCGGCATGATTCATGCGGCAGATAAA 2940

2941 CTTGTTCATCGAATTCGAGAGGCGTATCTTTCAGAATTACCTGTTATTCCCAGGTGTAAAT 3000

3001 GCGGAAATTTTTGAAGAATTAGAAGGTCACATTATCACTGCAATCTCCTTATACGATGCG 3060

3061 AGAAATGTCGTTAAAAATGGTGATTTTAATAATGGATTAACATGTTGGAATGTAAAAGGG 3120

3121 CATGTAGATGTACAACAGAGCCATCATCGTTCTGACCTTGTTATCCCAGAATGGGAAGCA 3180

3181 GAAGTGTCACAAAGCAGTTCGCGTCTGTCCGGGGTGTGGCTATATCCTTCGTGTCACAGCG 3240

3241 TACAAAGAGGGATATGGAGAGGGCTGCGTAACGATCCATGAAATCGAGAACAATACAGAC 3300

3301 GAACTAAAATTTAAAAACCGTGAAGAAGAGGAAGTGTATCCAACGGATACAGGAACGTGT 3360

3361 AATGATTATACTGCACACCAAGGTACAGCTGGATGCGCAGATGCATGTAATTCCCGTAAT 3420

3421 GCTGGATATGAGGATGCATATGAAGTTGATACTACAGCATCTGTTAATTACAAACCGACT 3480

3481 TATGAAGAAGAAACGTATACAGATGTAAGAAGAGATAATCATTGTGAATATGACAGAGGG 3540

3541 TATGTCAATTATTCCACCAGTACCAGCTGGTTATGTGACAAAAAGAATTAGAATACTTCCCA 3600

3601 GAAACAGATACAGTATGGATTGAGATTGGAGAAACGGAAGGAAAGTTTATTGTAGATAGC 3660

3661 GTGGAATTACTCCTCATGGAAGAATAG 3687

SEQ ID NO 2.1 (amino acid sequence of protein encoded by cry1Ba3 gene)

1 L T S N R K N E N E I I I N A V S N H S A 20

21 Q M D L L P D A R I E D S L C I A E G N 40

41 N I D P F V S A S T V Q T G I N I A G R 60

61 I L G V L G V P F A G Q L A S F Y S F L 80

81 V G E L W P R G R D Q W E I F L E H V E 100

101 Q L I N Q Q I T E N A R N T A L A R L Q 120

121 G L G D S F R A Y Q Q S L E D W L E N R 140

141 D D A R T R S V L Y T Q Y I A L E L D F 160

161 L N A M P L F A I R N Q E V P L L M V Y 180

181 A Q A A N L H L L L L L R D A S L F G S E 200

201 F G L T S Q E I Q R Y Y E R Q V E R T R 220

221 D Y S D Y C V E W Y N T G L N S L R G T 240

241 N A A S W V R Y N Q F R R D L T L G V L 260

261 D L L V A L L F P S Y D T T R T Y P I N T S A 280

281 Q L L T R E E V Y T D A I G A T G V N M A S 300

301 M N W W Y N N N N A A P S F S A I E E A A A A A I I R 320

321 S P H L L L D F L E Q L T I I F S A S S S R W 340

341 S N T T R H M T Y W R G R T I Q S S R P I G 360

361 G G L N T T S T H G A T N T S I N P V T L 380

381 R F A S S R D V Y R R T E S Y A G V L L L W G 400

401 I Y Y L E P P I H G V P T V R F N F T N P P Q 420

421 N I S D R R G T A A N Y S Q P Y E S S P G L Q 440

441 L K D S E E T E L P P E T T E E R P N Y E E S 460

461 Y S S H H R L L S H I I G I I L Q S S R V N V V P V 480

481 Y S W T H H R S A A D R T N T I G P N R I T 500

501 Q I P P M V V K A S E L P Q G T T T V V V R G P 520

521 G F T T G G G D I L R R R T N T G G G F G P I R 540

541 V T V V N G P L T Q R Y R I G F R R Y A S T 560

561 V D D F D F F F V S R G T T T V N N N F R F F L 580

581 R T M N N S G D E E L K Y G N F V R R R A F T 600

601 T P F T T F T Q I Q D I I I R T S I Q G L S 620

621 G N G E E V Y I D K I E I I I P V T T A T F E 640

641 A E E Y D L L E R A A Q E A V N A L F T N T T N 660

661 P R R R L K T D V T T D D Y H I D Q V V S N L L V 680

681 A C L S D D E F C L D E K R E L L E K 700

701 Y A A K R L L S D E E R R N L L Q D P N F T S I 720

721 N K K Q P P D F I S T N E Q S N F T T S I H H E 740

741 Q S S E H G W W W G S E N I T I I Q E E G N D V 760

761 F K E E N Y V T T L P G T F N E E C Y P T T Y L 780

781 Y Q Q K K I G E S E L K A Y T R Y Q L R G Y Y 800

801 I E D S Q D L E I Y L I R R Y N A K H E E T 820

821 L D V V P G T E S L L W P L S V E S P I G R 840

841 C G E P N R C A P H F E W N P D L D C S 860

861 C R D G E K C C A H H H S H H H F S L L D I D V 880

881 G C C T D D L H E N L G V W W V V F K K I K T T Q 900

901 E G H A R L L G N L E F I E E E K P L L G E 920

921 A L L S R R V K R A E K K W W R D K R E K L Q 940

941 L E T K K R V Y T E A K E A A V D A L F V D 960

961 S Q Y D R L L Q A D T N I G M I H A A A D D K 980

981 L V H R I R E A Y L S E L P V I P G V N 1000

1001 A E I I F E E E L E G H I I I T A I S L Y D D A 1020

1021 R N V V V K N G D F F N N N G L T C W N V K G 1040

1041 H V D V Q Q Q S S H H H R S D L V I P E W E A 1060

1061 E V S Q A V R V C C P G C G G Y I L L R V T A 1080

1081 Y K E E G Y G E G C V T I H E I E E N N T D 1100

1101 E L K K F K N R E E E E E V Y P T D T G T C 1120

1121 N D Y T A H Q Q G T A G C A D A C N S R N 1140

1141 A G Y E E D A Y E V D T T T A S V N Y K K P T 1160

1161 Y E E E E T T Y T T D D V R R D N H C E E Y D R R G 1180

1181 Y V N Y P P V P A G Y V T K E L E Y F P 1200

1201 E T D T V W I E I G E T E G K F I V D S 1220

1221 V E L L L L M E E * 1240

Claims (3)

1. a Bt gene cry1Ba3 is characterized in that the gene has a nucleotide sequence as shown in SEQ ID NO 2.1, and is toxic to insects. 2. A protein active to insects, characterized in that the protein is encoded by the gene of claim 1, has an amino acid sequence as shown in SEQ ID NO 2.2, and is toxic to insects. 3. a method for using the cry1Ba3 gene of claim 1 in combination with the cry3Aa7 gene transformation microorganism or plant having the nucleotide sequence shown in SEQ ID NO 1, it is characterized in that the method can make the transformed microorganism or plant show resistance to Toxicity of insects, and overcome or delay insects' resistance to engineered bacteria or transgenic plants.

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