Bacillus thuringiensis binary toxin gene and application thereof in prevention and control of cotton bollworm

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a bacillus thuringiensis binary toxin gene and application thereof in cotton bollworm prevention and control. The Cry-Tpp binary toxin combination with inhibitory activity on lepidoptera insects is identified for the first time, gene resources with commercial potential are provided for development of novel microbial insecticides and insect-resistant transgenic crops, and a new direction is provided for enhancing the activity of the Cry toxoid, artificially modifying the Cry toxoid and solving the problem of insect resistance. The method is of great significance to deep development and development innovation of Bt resources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a binary toxin gene of Bacillus thuringiensis and application thereof in the prevention and treatment of cotton bollworms. BACKGROUND

[0002] Bacillus thuringiensis (Bt) is a gram-positive soil bacterium that can be isolated from soil, insect carcasses, fallen leaves, dust and sewage. It is of great concern due to its important role in microbial insecticides. It has high economic value for the prevention and control of many major lepidopteran agricultural pests such as cotton bollworm, Spodoptera exigua, armyworm, and small tiger. At the same time, it also has certain control effect on some pests of Diptera such as Aedes, Culex and some flies, and is widely used in various environments that need pest control in daily life. Bt is mostly obtained from agricultural products or by-products of agricultural products, and the synthesis of toxin proteins does not produce toxic and harmful pollutants, and has high value for environmental protection. In 1956, T. Angus found that the parasporal crystal protein is the main insecticidal component of Bt, which is an important marker for distinguishing Bt from other Bacillus. For example, Bt is very similar to Bacillus cereus, but Bt can generate parasporal crystal protein while forming spores. Studies have shown that during the formation of parasporal crystals, insecticidal crystal inclusion bodies are formed, mainly by insecticidal proteins such as Cry (Crystal protein) or Cyt (Crtolytic protein). Cry and Cyt toxins belong to a class of bacterial toxins called pore-forming toxins, which can dissolve in the alkaline environment of the lepidopteran gut, and then be rapidly processed into active fragments by midgut proteases. The activated toxin fragments bind to specific receptors on the brush border membrane of the midgut epithelial cells, and these membrane-bound toxins form ion channels or pores, leading to the destruction of the transmembrane potential of the midgut, resulting in the death of the insect. Since 1997, studies have shown that Bt has insecticidal activity against various insects (such as Homoptera, Lepidoptera, Orthoptera, etc.), as well as mites, nematodes and some primitive animals. Bt insecticidal proteins only act in the insect gut. When Bt toxin proteins are ingested by insects, they are cut off at the N- and C-termini under the action of alkaline proteases in the alkaline environment of the gut, making the Bt insecticidal proteins into soluble core active fragments. These active fragments bind to specific receptors on the insect midgut epithelial cells, causing pores to form in the cell membrane, ultimately leading to the death of the insect. Although Bt insecticidal toxins have limited use as a spray insecticide, Cry toxins have been introduced into transgenic crops, providing a more targeted and effective method for the control of agricultural pests, and greatly reducing the use of chemical insecticides.

[0003] However, insect populations can develop resistance to the toxin protein or the same strain used for a long time. There are many reports on the development of field resistance of insects to Bt toxin protein gene-modified crops. Most of the studies on the development of resistance of dipterans to Bt in the laboratory also show that if a single strain is used for a long time, mosquitoes can develop a resistant population in 3-4 generations. Therefore, in order to alleviate the problem of insect resistance, there is an increasing demand for new Bt proteins for both genetically modified crops in agriculture and green insecticides, and the economic significance of new toxin proteins in the agricultural ecosystem is increasingly highlighted. In order to solve this problem, scientists are constantly looking for new high-activity strains and Bt resources. ZJ192 is a new Bt strain isolated from the tropical rainforest in Hainan. Whole genome sequencing and open reading frame prediction were performed on it. The local blast results showed that there was a pair of new cry-like and tpp-like binary toxin genes in the strain. After cloning, expressing and analyzing the insecticidal activity of the toxin genes, the results showed that the Cry-Tpp binary toxin combination had specific activity against cotton bollworm. SUMMARY

[0004] The present application provides a pair of binary toxin genes Cry-like and Tpp-like, characterized in that the nucleotide sequences of the binary toxin genes are shown in SEQ ID NO1 and SEQ ID NO2, respectively.

[0005] Another embodiment of the present application provides a pair of Cry-like and Tpp-like toxin proteins, characterized in that the amino acid sequences of the toxin proteins are shown in SEQ ID NO3 and SEQ ID NO4, respectively.

[0006] Another embodiment of the present application provides the use of the above-mentioned binary toxin genes or toxin proteins in the prevention and control of cotton bollworm.

[0007] Another embodiment of the present application provides an insecticidal pharmaceutical composition, characterized in that the pharmaceutical composition uses Cry-like and Tpp-like toxin proteins as effective components. The mass ratio of Cry-like and Tpp-like toxin proteins is 1-2:1-2, preferably 1:1. The pharmaceutical composition optionally further comprises other insecticidal active substances and / or pharmaceutically acceptable adjuvants.

[0008] Another embodiment of the present invention provides a method for controlling cotton bollworms, characterized in that the aforementioned binary toxin genes, Cry-like and Tpp-like, are used as genetic resources of insect-resistant transgenic crops for controlling cotton bollworms; or the Cry-like and Tpp-like toxin proteins are used as the main components of an insecticide for controlling cotton bollworms. The mass ratio of the Cry-like toxin proteins to the Tpp-like toxin proteins is 1-2:1-2, preferably 1:1.

[0009] Another embodiment of the present invention provides a strain B192-5 or B192-10 for producing Cry-like or Tpp-like toxin protein, characterized in that the preparation method of the strain comprises the following steps:

[0010] (1) Using seamless cloning technology, the cry-like or tpp-like toxin gene fragment was cloned into the E. coli-Bt shuttle expression vector pHT315-P1Ac and transformed into the cloning host cell DH5α competent cells to obtain the recombinant plasmid ZB192-5 or B192-10;

[0011] (2) transforming the recombinant plasmid B192-5 or B192-10 obtained in step (1) into the Dam and Dcm methylation-deficient strain JM110 to obtain a demethylation shuttle expression vector;

[0012] (3) After obtaining the demethylated recombinant vector extracted after transfer into JM110, the demethylated shuttle expression vector obtained in step (2) was transferred into the crystal-free mutant strain HD73 of Bacillus thuringiensis using the electroporation method. - In the experiment, the electroporator was set to 2200 kV and 200 Ω; strains B192-5 or B192-10 producing Cry-like or Tpp-like toxin proteins were obtained.

[0013] The cry-like or tpp-like toxin gene fragments in step (1) are preferably obtained by artificial synthesis based on their gene sequence information. The synthesis can be done by the individual or by a professional company. Synthesis of genes based on gene sequence information is a routine experimental method in the art.

[0014] Optionally, a verification step (4) is included: the transformed strain B192-5 or B192-10 is added to LB liquid culture medium without antibiotics and placed in a 37°C shaker for recovery for 3 hours. After centrifugation and resuspending the bacteria, 50 μl is spread on a plate containing erythromycin. The next day, a single colony is picked for PCR verification. If the verification is successful, the subsequent toxin gene expression is carried out.

[0015] Another embodiment of the present application provides the use of the above-mentioned bacterial strains B192-5 or B192-10 in the preparation of the Cry-like or Tpp-like toxin protein as described in the present application.

[0016] Compared with the prior art, the present application has the advantages that: the present application first identifies a Cry-Tpp binary toxin combination having inhibitory activity on Lepidoptera insects, provides a gene resource with commercialization potential for the development of new microbial insecticides and insect-resistant transgenic crops, and provides a new direction for how to enhance the activity of Cry-like toxins, artificial modification of Cry-like toxins and insect resistance problems, which has important significance for the in-depth development and development of Bt resources. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a prediction map of the phylogenetic tree, motif and conserved domain of Cry-like toxin system, wherein Figure 1 A is the phylogenetic tree between Cry-like toxin and the discovered toxin; Figure 1 B is the conserved motif of Cry-like toxin and the discovered toxin predicted by MEME; Figure 1 C is the conserved domain of Cry-like toxin and the known toxin predicted by NCBI Batch CD.

[0018] Figure 2 is a prediction map of the phylogenetic tree, motif and conserved domain of Tpp-like toxin system, wherein Figure 2 A is the phylogenetic tree between Tpp-like toxin and the discovered toxin; Figure 2 B is the conserved motif of Tpp-like toxin and the discovered toxin predicted by MEME; Figure 2 C is the conserved domain of Tpp-like toxin and the known toxin predicted by NCBI Batch CD.

[0019] Figure 3Figure 1 is a cloning map of cry-like and tpp-like toxin genes, Figure A and Figure B are agarose gel electrophoresis maps of gradient PCR amplification of cry-like and tpp-like toxin gene fragments, respectively, Figure C is a linearized PCR amplification of pHT315-P1 Ac, different annealing temperatures are set from left to right, the gradient is 65℃, 64.3℃, 63℃, 61.1℃, 58.8℃, 56.9℃, 55.7℃, 55℃, Figure D and Figure E are PCR results of single colonies picked from the competent DH5α transformed with cry-like and tpp-like homologous recombination products, respectively, Figure F and Figure G are verification results of double enzyme digestion of recombinant plasmids extracted from randomly selected cry-like and tpp-like PCR positive clones, the marker molecular weight size used is from top to bottom, 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp.

[0020] Figure 4 Figure 2 is an expression map of cry-like and tpp-like toxins, Figure A and Figure B are oil microscope observation results of spore crystal mixtures picked from plates cultured for 3 days after pHT315-P1 Ac-cry-like and pHT315-P1 Ac-tpp-like transformed HD73, respectively, red label C is parasporal crystal, S is spore; Figure C is the observation result of pHT315-P1 Ac empty vector transformed HD73 - Figure 3 is a SDS-PAGE result map of expression in LB liquid medium for 4 days, Figure D is the SDS-PAGE result map of expression in LB liquid medium for 4 days, Figure C and Figure D are total proteins collected from the first day to the fourth day of expression from left to right. - Figure 3 is a SDS-PAGE result map of expression in LB liquid medium for 4 days, Figure D is the SDS-PAGE result map of expression in LB liquid medium for 4 days, Figure C and Figure D are total proteins collected from the first day to the fourth day of expression from left to right.

[0021] Figure 5Figure 1 is a graph of qualitative bioactivity assay of B192-5 and B192-10 crystal mixtures against cotton bollworm, where panel A is a graph of bioassay results of B192-5 crystal mixture incorporated into diet at a final concentration of 100 pg / g against cotton bollworm; panel B is B192-10; panel C is pHT315-P1Ac-HD73-; panel D is B192-5 and B192-10 mixed at a ratio of 1 : 1 (each at a final concentration of 100 pg / g) after bioassay results; panel E shows the number of individuals of cotton bollworms whose growth was inhibited on the 5th day, with each sample divided into 6 groups, each group having 6 test insects; panel F shows the statistical data of the size of cotton bollworms in each group on the 5th day. Pairwise t-test was used for comparison between groups, and the significance level is shown as follows: ** means p < 0.005, **** means p < 0.0001.

[0022] Figure 6 Figure 1 is a graph of qualitative bioactivity assay of B192-5 and B192-10 crystal mixtures against cotton bollworm, where panel A is a graph of bioassay results of B192-5 crystal mixture incorporated into diet at a final concentration of 100 pg / g against cotton bollworm; panel B is B192-10; panel C is pHT315-P1Ac-HD73-; panel D is B192-5 and B192-10 mixed at a ratio of 1 : 1 (each at a final concentration of 100 pg / g) after bioassay results; panel E shows the number of individuals of cotton bollworms whose growth was inhibited on the 5th day, with each sample divided into 6 groups, each group having 6 test insects; panel F shows the statistical data of the size of cotton bollworms in each group on the 5th day. Pairwise t-test was used for comparison between groups, and the significance level is shown as follows: ** means p < 0.005, **** means p < 0.0001.

[0023] Figure 7 Figure 1 is a graph of qualitative bioactivity assay of B192-5 and B192-10 crystal mixtures against cotton bollworm, where panel A is a graph of bioassay results of B192-5 crystal mixture incorporated into diet at a final concentration of 100 pg / g against cotton bollworm; panel B is B192-10; panel C is pHT315-P1Ac-HD73-; panel D is B192-5 and B192-10 mixed at a ratio of 1 : 1 (each at a final concentration of 100 pg / g) after bioassay results; panel E shows the number of individuals of cotton bollworms whose growth was inhibited on the 5th day, with each sample divided into 6 groups, each group having 6 test insects; panel F shows the statistical data of the size of cotton bollworms in each group on the 5th day. Pairwise t-test was used for comparison between groups, and the significance level is shown as follows: ** means p < 0.005, **** means p < 0.0001. DETAILED DESCRIPTION

[0024] I. Strains, Plasmids, Main Reagents and Primers

[0025] The Bacillus thuringiensis ZJ192 of the present application is isolated from the original forest soil sample in Hainan Island by the inventors, and the strain information and genome information thereof are submitted to the GenBank database of NCBI (BioProject: Processed, PRJNA1260999: Bacillus thuringiensis strain: ZJ192 Genome sequencing (TaxID: 1428). Locus Tag Prefixes: ACQUW6 (SAMN48409181), BioSample: Processed, Successfully loaded SAMN48409181: ZJ192 (TaxID: 1428), Genomes: Processing (Details), PGAP file JBNSYV000000000). According to the morphological characteristics and 18S rDNA sequence comparison, it is identified as Bacillus thuringiensis. The strain has been preserved by the applicant in the Key Laboratory of Tropical Island Ecology of Hainan Normal University, and the applicant promises to provide the Bacillus thuringiensis ZJ192 of the present application to the public for 20 years from the application date. In addition, the DNA of the Bacillus thuringiensis ZJ192 can be synthesized by the person skilled in the art or a professional company according to the genome information disclosed in NCBI. Escherichia coli DH5α and JM110, pHT315-P1Ac E. coli-Bt shuttle expression vector (pHT315 is an expression vector, then the promoter sequence of cry1Ac gene is inserted to form the vector pHT315-P1Ac E. coli-Bt shuttle expression vector for expressing the target gene in the present patent, and the gene sequence thereof is SEQ ID NO5), Bacillus thuringiensis subsp. kurstaki acrystalliferous mutant HD73 - All are known materials, which can be obtained by commercial means and preserved by the laboratory.

[0026] Table 1 Strain and plasmid information table

[0027]

[0028]

[0029] II. Gene cloning, expression, analysis and activity test method

[0030] 2.1 Gene cloning

[0031] The open reading frame sequences of the genes (SEQ ID NO 1 and 2) were obtained according to the genome information of the Bt strain ZJ192, and cry-like and tpp-like toxin gene seamless cloning primers were designed, with the 5' end of the primers designed to be homologous to the vector pHT315-P1Ac homologous arm (Table 2, lowercase part).

[0032] The synthesis method of the cry-like and tpp-like toxin genes described in the present application is: one is obtained by PCR amplification; and the other is obtained by artificial synthesis by oneself or by a professional company according to the gene sequence information.

[0033] PCR: According to the genome information of the Bt strain ZJ192, a professional company was invited to synthesize the DNA or total DNA of the ZJ192 strain was obtained from the applicant, and a large amount of target fragment was obtained by Pfu high-fidelity PCR amplification. The target gene amplified by PCR was purified. The plasmid of the expression vector was subjected to PCR linearization treatment, and the cry-like and tpp-like toxin gene fragments were respectively cloned into the E. coli-Bt shuttle expression vector pHT315-P1Ac by using the seamless cloning technology, and the recombinant plasmids ZB192-5 and B192-10 were obtained by being introduced into the cloning host cell DH5α competent cells.

[0034] Artificial synthesis: According to the sequence information (SEQ ID NO 1 and 2) of the cry-like and tpp-like toxin genes, a professional company was invited to synthesize the cry-like and tpp-like toxin gene fragments; the cry-like and tpp-like toxin gene fragments were respectively cloned into the E. coli-Bt shuttle expression vector pHT315-P1Ac by using the seamless cloning technology, and the recombinant plasmids ZB192-5 and B192-10 were obtained by being introduced into the cloning host cell DH5α competent cells.

[0035] The correctness of the construction of the expression vector was verified by PCR, double enzyme digestion, sequencing and other methods; the positive transformants were selected and sent to a professional company for sequencing; the DNA sequence analysis was performed by using the Vector NTI Suite 11 software package of Invitrogen.

[0036] Table 2 primer information

[0037]

[0038]

[0039] 2.2 Bt crystal-free mutant strain HD73- Expression of toxin genes

[0040] The successfully constructed B192-5 and B192-10 recombinant plasmids were extracted in small amounts, and then transformed into Dam and Dcm methylation-deficient strain JM110 to obtain demethylated shuttle expression vectors. After obtaining the demethylated recombinant vectors extracted after being transformed into JM110, the recombinant plasmids were transformed into the crystal-free mutant strain HD73 of Bacillus thuringiensis by using the electroporation method - , and the electroporation instrument was set at 2200 kV and 200Ω. After transformation, LB liquid medium without antibiotics was added and placed in a 37°C shaker for recovery for 3 h. After centrifugation and resuspension of the bacterial cells, 50 μl was coated on an erythromycin-containing plate. The next day, single colonies were picked for PCR verification. Verification was successful, and the subsequent expression of toxin genes was carried out.

[0041] 2.3 Expression of toxin genes in crystal-free mutant strains

[0042] The correct Bt strains B192-5 and B192-10 containing recombinant plasmids were verified and incubated at 28°C and 200 rpm on LB plates for 4 d. Microscopic samples were prepared, and spores and parasporal crystals were observed under a microscope (oil immersion, 1000x). The overnight activated B192-5 and B192-10 bacterial liquid was added to fresh 100 ml LB medium at a ratio of 1:100, and incubated at 28°C and 220 rpm until the sporulation stage (4 d). The bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 5 min, the supernatant was removed, and the precipitate was suspended in 10 mL of 1x PBS buffer. The cells were broken by ultrasonic treatment (20 KHz, 10 s:10 s) for 10 min. Then, the precipitate was collected by centrifugation at 4°C and 12000 rpm for 10 min, and resuspended in 5 ml of 1x PBS buffer. Finally, the supernatant was separated from the precipitate by centrifugation at 4°C and 8000 rpm for 5 min, and the bacterial cells after washing the precipitate twice were resuspended in 10 mL of PBS.

[0043] 2.4 SDS-PAGE electrophoresis analysis

[0044] Take 20 μL of prepared Bt toxin protein and 5 μL of 5x SDS-PAGE Sample Buffer, 100 ℃ boiling water for 5 min, then 12000 rpm centrifugation for 5 min, then 7 μL of 0.1 mg / ml BSA is used as a comparison. At a voltage of 85V, use Mini-Protein II (BioRad, USA) to electrophorese for about 60 min, then at a voltage of 110V, electrophorese for about 90 min. After taking the gel, use Coomassie Brilliant Blue R250 staining solution to stain for 30 min at 60 rpm, then use the destaining solution to destain multiple times until the protein band is clear. Use Bio-Rad GS-800 Calibrated Densitometer scanning system to photograph the protein gel, and use Quantity One software to analyze the protein band image and its concentration.

[0045] 2.5 Biological activity assay

[0046] Take the collected expression bacteria and add a certain amount of 50 mM Na2CO3 solution with pH 10.5 to suspend, and incubate at 37℃ water bath for 1 h. After incubation, separate the dissolved protein at 12000 rpm for 10 min. For lepidoptera insects, add a certain amount of toxin protein to 60 g of prepared artificial feed to a concentration of 100 μg / g, mix and roll to a non-particle state, then divide into 25 mL size sauce boxes and flatten, divide 6 boxes of feed; Place at room temperature for 3 h to remove excess moisture in the feed. Use a hair brush to transfer the test larvae into the sauce box, 6 heads per box. Place in a 25℃ light incubator, with a light cycle of 12:12; relative humidity is 65%. Observe daily and record the number of dead and live insects for 7 days, calculate the mortality rate; use GraphPad prism10 for analysis, complete the preliminary biological activity assay.

[0047] III. Results and analysis

[0048] 3.1 ZJ192 binary toxin sequence analysis

[0049] Bacillus ZJ192 was isolated from the original forest soil sample in Hainan Island by the laboratory. The culture of LB culture of the bacteria for 4 days was found to have strong inhibitory activity on cotton bollworm by preliminary qualitative biological activity.

[0050] To further explore the toxin type of the strain, the whole genome of the strain was sequenced and the genome sketch was obtained (submitted to NCBI). Local blast comparison was performed on all predicted open reading frames, and it was found that the open reading frames N63_4 (SEQ ID NO2) and N63_5 (SEQ ID NO1) of the strain had the highest amino acid sequence identity with the currently known toxins Tpp49Aa1 and Cry54Aa2, respectively. These two toxins were temporarily named Tpp-like and Cry-like, wherein the sequence similarity of Tpp-like with Tpp49Aa1 was 31%, and the sequence similarity of Cry-like with Cry54Aa2 was 28%.

[0051] To further determine the phylogenetic relationship between Cry-like and Tpp-like toxins and currently discovered toxins, multiple sequence alignment was performed using the ClustalW program in MEGA 11 software, and the construction of the phylogenetic tree was performed using the Neighbor-Joining (NJ) method, with a bootstrap repetition value of 1000. The results showed that the Cry-like toxin was far from the evolutionary relationship with most of the currently known toxins, and was closest to Cry48Aa1, one of the binary toxins of the target Diptera Figure 1 A). Cry48Aa1 has a C-terminal domain that may be involved in toxin crystallization in addition to the three N-terminal domains that have the main insecticidal function, and the protein size is about 130 kDa, while Cry-like does not have a C-terminal crystallization domain, and the protein size is about 74 kDa. The evolutionary relationship between Tpp-like toxin and the currently discovered Tpp toxin is also far, and the closest is the binary toxin Tpp1Aa / Tpp2Aa (formerly BinA / BinB) in Lysinibacillus sphaericus.

[0052] Conserved domain (CD) and conserved motif prediction were performed on the amino acid sequences encoded by the cry-like and tpp-like toxin genes using InterPro Scan (https: / / www.ebi.ac.uk / interpro / search / sequence) and MEME (https: / / meme-suite.org / meme / tools / meme).

[0053] The conserved motif prediction results showed that the Cry-like toxin had a conserved motif like other main Cry family proteins, indicating that the Cry-like toxin may have similar protein functions to other Cry family members, such as receptor binding or cell membrane perforationFigure 1 -B). The conserved motifs of Tpp-like toxins are mainly similar to those of Tpp49, Tpp35, Tpp1, Tpp2, and Tpp36. Current research results indicate that Tpp49, Tpp1, and Tpp2 target Diptera, and Tpp35 targets Coleoptera. Therefore, Tpp-like toxins that are evolutionarily closely related to these toxin families are likely to target similar insects.

[0054] The prediction results of the conserved domains showed that Cry-like toxins have a typical 3D-Cry toxin-like structure, containing the conserved domains Endotoxin_N (pfam: PF03945) and Endotoxin_C (pfam: PF03944) ( Figure 1 -C). Tpp-like toxins have the same domain structure as other Tpp family toxins, namely, they contain the Ricin B-like lectins superfamily domain (SUPERFAMILY: SSF50370) and the Toxin_10 domain (Pfam: PF05431) ( Figure 2 -C).

[0055] Based on the evolutionary relationship, conserved motifs, and conserved domains with known toxins, the Cry-like and Tpp-like toxins in Bacillus ZJ192 are likely to function as a novel binary toxin pair that exerts insecticidal function.

[0056] 3.2 ZJ192 binary toxin gene cloning

[0057] The full-length PCR amplification primers for the toxin gene were prepared based on the open reading frames of cry-like and tpp-like genes (Table 2). First, a gradient annealing temperature was set to find the optimal annealing temperature for the two full-length primers of the toxin gene. Agarose gel electrophoresis analysis showed that the optimal annealing temperature for the cry-like amplification primers was 55°C ( Figure 3 -A), the optimal annealing temperature of the tpp-like amplification primer is 65°C ( Figure 3 -B). After amplifying the target gene in large quantities using the optimal annealing temperature, purify the product and combine the purified fragment (or cry-like and tpp-like genes artificially synthesized by professional companies) with the PCR linearized pHT315-P1Ac shuttle expression vector ( Figure 3 -C) for seamless cloning. Colony PCR results showed that the connection efficiency of the two genes was close to 100% ( Figure 3-D). Three positive colonies were randomly selected for shake flask amplification and plasmid extraction for BamH I and EcoR I double enzyme digestion analysis. The results showed that the cry-like recombinant vector produced a 1200 bp fragment after double enzyme digestion, and the tpp-like recombinant vector produced 1300 bp and 900 bp fragments. Since there are enzyme digestion sites in both genes, and the predicted size is consistent with the enzyme digestion product, it indicates that the two genes have been successfully connected into the shuttle expression vector.

[0058] 3.3 Expression of binary toxin genes

[0059] The recombinant plasmids pHT315-P1Ac-cry-like and pHT315-P1Ac-tpp-like introduced into the methylation-deficient strain JM110 were extracted, and the demethylated plasmids were obtained. After using electroporation, they were transformed into the non-crystal mutant strain HD73 of Bacillus thuringiensis to obtain recombinant protein expression strains B192-5 and B192-10, respectively. The strains were inoculated on solid LB plates and incubated at 28°C for 4 days. The recombinant strains that had entered the spore stage were observed under an oil immersion lens. -

[0060] The results showed that B192-5 (Cry-like) and B192-10 (Tpp-like) could both produce parasporal crystals, and B192-5 produced round parasporal crystals Figure 4 -A). Currently, the Cry toxins that can form round parasporal crystals mainly target dipteran insects, such as Cry4Aa and Cry4Ba from Bacillus thuringiensis subsp. israelensis and other strains such as Cry30Fa and Cry19Aa. Unlike other types of Tpp family toxins, Tpp-like toxin forms a kind of small parasporal crystal similar to an elongated rice grain Figure 4 -B). This crystal shape is different from the currently known Tpp toxins, such as Tpp49Aa1 which forms a bipyramidal crystal.

[0061] Liquid LB was used for shake flask fermentation of B192-5 and B192-10, and bacterial liquid was collected from the first day to the fourth day. After centrifugation, the bacterial cells were washed and prepared into SDS-PAGE samples for the preparation of parasporal crystal mixtures. The results showed that B192-5 and B192-10 - similarly Figure 4 -C), B192-5 and B192-10 had the same protein bands on the first day, and B192-5 began to express the target protein on the second day, with a size of about 75 kDa, which is the same as the predicted size of Cry-like toxin Figure 4 ​-D, Line 1-4), the expression level was stable on the third day. B192-10 expressed in large amount on the second day, and the size was about 45 kDa, which was the same as the predicted size of Tpp-like toxin Figure 4 -D, Line 5-8). The target protein of B192-10 began to degrade on the third day, which indicated that the parasporal crystal formed by Tpp-like toxin was not stable and was easily degraded by endogenous proteases in the bacteria.

[0062] 3.4 Determination of synergistic insecticidal activity of Cry-Tpp binary toxin

[0063] The B192-5 and B192-10 were fermented in large amount, and the spore-crystal mixture was collected. The target protein in the spore-crystal mixture of B192-5 and B192-10 was quantified using the optical density analysis method with gradient concentration of BSA as the concentration reference. The spore-crystal mixture was mixed with the insect feed in a certain proportion so that the concentration of the target protein in the feed was 100 μg / g. The qualitative bioactivity determination results showed that B192-5 and B192-10 had no bioactivity on all the tested insects, including Plutella xylostella, Spodoptera exigua, Helicoverpa armigera and Aedes aegypti. Since the spore-crystal mixture of the original bacteria had activity on Helicoverpa armigera, the spore-crystal mixture of B192-5 and B192-10 was added to the insect feed in a mass ratio of 1:1. The results showed that when Cry-like and Tpp-like toxins were used together, strong inhibitory activity was produced on Helicoverpa armigera Figure 5 ), but still had no activity on Plutella xylostella, Spodoptera exigua and Aedes aegypti, that is, the target of the synergistic action of Cry-like and Tpp-like was very specific.

[0064] 3.5 Prediction of three-dimensional structure of Cry-like and Tpp-like toxins

[0065] The protein three-dimensional structures of Cry-like and Tpp-like toxins were predicted using Alphafold3 (https: / / alphafoldserver.com / ). The results showed that Cry-like toxins have a pyramid-shaped structure like other typical 3D-Cry toxins, which is composed of three structurally independent functional units, Endotoxin_N, Endotoxin_M, and Endotoxin_C. The Endotoxin_N domain is composed of 8-9 alpha helices, which are speculated to be involved in cell membrane perforation function, and the Endotoxin_M and Endotoxin_C domains are composed of beta sheets, which are speculated to be involved in receptor recognition process. By comparing the three-dimensional structures of Cry48Aa1 with close evolutionary relationship and Cry2Aa1 and Cry54Aa1 with distant evolutionary relationship, it was found that Cry-like and Cry48Aa1 were similar in Endotoxin_N and Endotoxin_C, and they might have similar functions. The Lectin-like domain and Toxin_10 domain of Tpp-like toxin were significantly different. Combined with amino acid sequence differences and phylogenetic relationships, Tpp-like might have different targets from the known Tpp toxins.

[0066] IV. DISCUSSION

[0067] Bt is one of the most successful biopesticides, and has been commercially used for more than 90 years. The biggest feature of this strain is that it produces a spore crystal composed of proteins and insecticidal crystal inclusion bodies, mainly formed by Cry or Cyt toxins, during the late growth stage. These protein crystals can specifically kill insects of Diptera, Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, and Orthoptera, and are biodegradable and environmentally friendly, with broad development prospects. In this study, we discovered new binary toxin genes cry-like and tpp-like in Bt strain ZJ192, and performed cloning and expression analysis. The successfully expressed binary toxin proteins were subjected to bioactivity determination, and the results showed that these toxins showed excellent specific inhibitory activity against cotton bollworm, with an inhibition rate of up to 95%.

[0068] The binary toxins discovered so far include the Tpp1 / Tpp2 combination targeting Diptera, the Cry48 / Tpp49 combination, and the Gpp34 / Tpp35 combination targeting Coleoptera. No research has reported on the effect of Bacillus binary toxin combinations on Lepidoptera. The Cry-like and Tpp-like toxins identified in this study are new types of toxins according to the Bt classification rules, and the synergistic activity assay identified that they need to work together. This toxin combination can become a powerful candidate gene for cotton bollworm-resistant transgenic crop breeding and new microbial insecticides, and provides a new approach to solving the current Cry toxin resistance problem.

[0069] Today, many pests have developed varying degrees of resistance to the widely used Cry and Vip3 toxins in agriculture, which poses new challenges to crop control. The discovery and research of ZJ192 strain provides a new approach to solving this problem. The toxin proteins expressed by ZJ192 strain exhibit significant insecticidal activity against Plutella xylostella and Helicoverpa armigera, and their gene sequences have low similarity to existing Cry and Vip3 toxins, indicating their unique insecticidal mechanism and potential. This opens up a new way for the development of new transgenic crops, which can effectively delay the development of pest resistance. The research of ZJ192 strain not only enriches the existing insecticidal gene library, but also provides a theoretical basis for the development of new biological insecticides.

[0070] Future research can further explore the biological activity of ZJ192 on other pests such as Hemiptera, as well as its performance and stability in different ecosystems. Future research should further explore the application potential of the binary toxin in ZJ192, and combine with ecological and environmental protection strategies to promote the sustainable development of Bt biological insecticides. The mechanism of the novel insecticidal toxin combination acting on Lepidoptera insects needs further exploration, providing a good direction for the artificial modification and modification of the new generation of insecticidal toxins.

Claims

1. A pair of binary toxin genes, Cry-like and Tpp-like, characterized by The nucleotide sequences of the binary toxin genes are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.

2. A pair of Cry-like and Tpp-like toxin proteins, characterized by The amino acid sequences of the toxin proteins are shown in SEQ ID NO3 and SEQ ID NO4 respectively.

3. Use of the binary toxin gene according to claim 1 or the toxin protein according to claim 2 in controlling cotton bollworm.

4. An insecticide composition, characterized in that The pharmaceutical composition comprises the Cry-like and Tpp-like toxin proteins as claimed in claim 2 as active ingredients. The mass ratio of the Cry-like and Tpp-like toxin proteins in the pharmaceutical composition is 1-2:1-2, preferably 1:

1.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition may optionally further comprise other insecticide active substances and / or pesticide-acceptable adjuvants.

6. A method for controlling cotton bollworm, characterized in that The binary toxin genes Cry-like and Tpp-like described in claim 1 are used as genetic germplasm resources of insect-resistant transgenic crops for controlling cotton bollworm.

7. A method for controlling cotton bollworm, characterized in that The Cry-like and Tpp-like toxin proteins described in claim 2 are used as the main components of the insecticide for controlling cotton bollworm.

8. The method according to claim 7, characterized in that The mass ratio of the Cry-like and Tpp-like toxin proteins is 1-2:1-2, preferably 1:

1.

9. A strain B192-5 or B192-10 producing Cry-like or Tpp-like toxin protein, characterized in that The preparation method of the strain comprises the following steps: (1) Using seamless cloning technology, the cry-like or tpp-like toxin gene described in claim 1 is cloned into the E. coli-Bt shuttle expression vector pHT315-P1Ac, and then transferred into the cloning host cell DH5α competent cells to obtain the recombinant plasmid ZB192-5 or B192-10; (2) transforming the recombinant plasmid B192-5 or B192-10 obtained in step (1) into the Dam and Dcm methylation-deficient strain JM110 to obtain a demethylation shuttle expression vector; (3) After obtaining the demethylated recombinant vector extracted after transfer into JM110, the demethylated shuttle expression vector obtained in step (2) is transferred into the crystal-free mutant strain HD73- of Bacillus thuringiensis using the electroporation method, and the electroporation instrument is set to 2200 kV and 200 Ω; strains B192-5 or B192-10 producing Cry-like or Tpp-like toxin proteins can be obtained.

10. Use of the strain B192-5 or B192-10 according to claim 9 in the preparation of the Cry-like or Tpp-like toxin protein according to claim 2.

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