An overexpression vector for improving insecticidal toxicity of beaverellic fungus, an engineering strain, and application and method thereof

Abstract

The present application relates to the field of genetic engineering and agricultural pest biotechnology, and discloses an overexpression vector for improving the insecticidal toxicity of Beauveria bassiana, an engineering strain and application and method thereof. The insecticidal toxicity of the Beauveria bassiana is improved by overexpressing a short peptide HaTK3 gene of a fast excitatory peptide of a cotton bollworm in the Beauveria bassiana. The amino acid sequence of the HaTK3 gene is shown as SEQ ID NO. 2. The present application introduces an insect-derived neuro-peptide HaTK3 with immunosuppressive function into the Beauveria bassiana for overexpression, suppresses the immune response of a host, promotes the colonization and proliferation of the fungus in the host, improves the fungal toxicity from the perspective of host immune regulation, and thus significantly improves the insecticidal efficiency and shortens the death time.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and agricultural pest biotechnology, and more specifically, to an overexpression vector, engineered strain, and application and method for enhancing the insecticidal toxicity of Beauveria bassiana. Background Technology

[0002] Pests are a significant biological factor affecting agricultural production, accounting for approximately 18%–20% of global crop losses annually due to arthropod damage. While long-term reliance on chemical pesticides has controlled pests to some extent, their excessive use has led to increasingly prominent problems such as environmental pollution, pesticide residues, and pest resistance. Therefore, developing efficient, safe, and environmentally friendly biological control technologies has become an important direction for sustainable agricultural development. Insect pathogenic fungi, as a type of microbial resource that can directly infect and kill insects, have been widely developed as biopesticides due to their high safety and environmental friendliness. Among them, *Beauveria bassiana* (…) is… Beauveria bassiana (This is the most widely used method.)

[0003] However, fungal insecticides generally suffer from slow killing speed and poor environmental stability in practical applications, severely limiting their widespread use. The main reason is that entomopathogenic fungi need to overcome multiple defense mechanisms of the host's immune system during infection. Insects can resist pathogen infection through hemocyte phagocytosis, phenoloxidase (PO) cascade reactions, and Toll signaling pathway-mediated expression of antimicrobial peptides. Existing research has shown that enhancing the host's negative immune regulation or interfering with its immune processes can effectively improve the colonization ability and pathogenicity of fungi in the host. Therefore, screening and utilizing key molecules that can inhibit the host's immune response for fungal genetic modification is an effective strategy to enhance their insecticidal activity.

[0004] Insect neuropeptides are a class of important signaling molecules that regulate insect growth, development, metabolism, and immune responses, exhibiting high efficiency and multifunctionality. Previous studies have shown that introducing neurotoxins or neuropeptides into entomopathogenic fungi can significantly enhance their virulence. However, the application of immunosuppressive neuropeptides in fungal modification remains relatively limited.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] To address the problems of slow insecticidal speed and insufficient toxicity in existing insect-pathogenic fungal insecticides, and to improve the colonization ability and pathogenicity of fungi in the host, thereby enhancing insecticidal activity, this invention provides an overexpression vector, engineered strain, and its application and method for enhancing the insecticidal toxicity of Beauveria bassiana. For the first time, the insect-derived neuropeptide HaTK3, which has immunosuppressive function, is introduced into Beauveria bassiana for overexpression. From the perspective of host immune regulation, by inhibiting the host immune response, it promotes the colonization and proliferation of fungi in the host, enhances the insecticidal toxicity of fungi, and thus significantly enhances the lethality of fungi against pests and shortens the lethal time.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for improving the insecticidal toxicity of Beauveria bassiana by overexpressing the HaTK3 gene, a fast-acting peptide of cotton bollworm, in Beauveria bassiana, thereby improving the insecticidal toxicity of Beauveria bassiana. The amino acid sequence of the HaTK3 gene is shown in SEQ ID NO.2.

[0008] The applicant's research found that the cotton bollworm tachykinin HaTK3 has the function of significantly inhibiting the insect immune response. Therefore, the applicant creatively proposed to introduce the insect-derived neuropeptide HaTK3 with immunosuppressive function into Beauveria bassiana for overexpression. By inhibiting the host immune response, the applicant promoted the colonization and proliferation of fungi in the host, thereby enhancing the virulence of fungi from the perspective of host immune regulation, thus significantly improving the insecticidal efficiency and shortening the lethal time.

[0009] In one specific embodiment, the nucleotide sequence of the HaTK3 gene is shown in SEQ ID NO.1.

[0010] In one specific embodiment, the nucleotide sequence of the HaTK3 gene, after codon optimization, has the nucleic acid sequence shown in SEQ ID No. 3, and is used for expression in Beauveria bassiana.

[0011] In one specific implementation, the following steps are included: (1) Obtain the coding sequence of the cotton bollworm tachykinin HaTK3, fuse the chitinase signal peptide sequence of Beauveria bassiana at its N-terminus, and synthesize a recombinant gene fragment; (2) Construct a fungal expression vector containing the HaTK3 coding sequence; (3) The expression vector was introduced into Beauveria bassiana to obtain a recombinant strain with HaTK3 overexpression; (4) The recombinant strains were screened and molecularly identified to obtain genetically stable transgenic strains with enhanced insecticidal toxicity.

[0012] Secondly, the present invention provides a secretory overexpression vector for the cotton bollworm fast kinin peptide HaTK3, which enhances the insecticidal toxicity of Beauveria bassiana. The overexpression vector is a recombinant expression vector pK2-bar-Pb3-BbChiP-SP-HaTK3. The recombinant expression vector pK2-bar-Pb3-BbChiP-SP-HaTK3 was obtained by fusing the nucleotide sequence of the chitinase signal peptide BbChiP-SP from Beauveria bassiana with the nucleotide sequence of HaTK3 to obtain the recombinant fragment BbChiP-SP-HaTK3, which was then linked to the constitutive promoter Pb3 of the glyceraldehyde-3-phosphate dehydrogenase gene, and then inserted into the expression vector pK2-bar containing the fungal selection marker glufosinate resistance gene bar after enzyme digestion and ligation.

[0013] Thirdly, the present invention provides a method for constructing a secretory overexpression vector of the cotton bollworm tachykinin short peptide HaTK3, comprising the following steps: (1) Obtaining the BbChiP-SP-HaTK3 recombinant fragment: 1) The BbChiP-SP was spliced ​​with the codon-optimized HaTK3 nucleic acid sequence, and EcoRI restriction sites were introduced at both ends to artificially synthesize the pUC57-BbChiP-SP-HaTK3 plasmid; 2) Design primers HaTK3_F / R (sequence as shown in the instruction manual), use the synthesized plasmid as a template for PCR amplification, and obtain the BbChiP-SP-HaTK3 recombinant fragment by gel recovery; (2) Obtaining the Pb3 promoter fragment: Using Beauveria bassiana Bb0062 genomic DNA as a template, PCR amplification was performed using specific primers Pb3_F / R, and the Pb3 promoter fragment was obtained by gel recovery. (3) Obtaining the Pb3-BbChiP-SP-HaTK3 recombinant fragment: Using Pb3 and BbChiP-SP-HaTK3 fragments as templates, fusion amplification was performed using overlap extension PCR technology, and sequences homologous to the EcoRI site of the pK2-bar vector were introduced at both ends to obtain the Pb3-BbChiP-SP-HaTK3 recombinant fragment. (4) Construction of recombinant plasmids: The above recombinant fragments were ligated into the EcoRI-linearized pK2-bar vector using homologous recombination, and the recombinant plasmid pK2-Pb3-BbChiP-SP-HaTK3 was obtained by sequencing verification.

[0014] Fourthly, the present invention provides a transgenic Beauveria bassiana strain that overexpresses the cotton bollworm fast-kine short peptide HaTK3. The overexpression vector is introduced into the wild-type strain of Beauveria bassiana using an Agrobacterium tumefaciens AGL1-mediated genetic transformation method, and the engineered strain Bb::HaTK3 with high expression of HaTK3 is obtained by RT-qPCR screening.

[0015] Fifthly, the present invention provides the application of the cotton bollworm fast kinin short peptide HaTK3 gene and the overexpression vector in improving the insecticidal toxicity of Beauveria bassiana.

[0016] In a sixth aspect, the present invention provides the application of the cotton bollworm fast-kinin short peptide HaTK3 gene, the overexpression vector, or the engineered strain of Beauveria bassiana in the preparation of fungal insecticides.

[0017] In a seventh aspect, the present invention provides a fungal insecticide comprising a short peptide of HaTK3 encoded by the HaTK3 gene, the overexpression vector, and / or the engineered strain of Beauveria bassiana.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides an overexpression vector, engineered strain and its application and method for improving the insecticidal toxicity of Beauveria bassiana. By introducing the insect-derived neuropeptide HaTK3 with immunosuppressive function into Beauveria bassiana for overexpression, the fungus colonizes and proliferates in the host by inhibiting the host immune response, thereby improving the toxicity of the fungus from the perspective of host immune regulation, thus significantly improving the insecticidal efficiency and shortening the lethal time. 2. The present invention provides an overexpression vector, engineered strain, and its application and method for enhancing the insecticidal toxicity of Beauveria bassiana. It utilizes genetic engineering technology to improve the insecticidal toxicity of Beauveria bassiana. Compared with traditional toxicity enhancement methods, it has the advantages of novel mechanism of action and strong targeting, providing a new technical path for the genetic improvement of insect pathogenic fungi. 3. The embodiments of the present invention provide an overexpression vector, engineered strain, and its application and method for enhancing the insecticidal toxicity of Beauveria bassiana. The obtained transgenic strain of Beauveria bassiana has significantly enhanced toxicity against cotton bollworm larvae, and also significantly enhanced lethality against lepidopteran pests such as the wax moth and the beet armyworm; thus improving the application effect of Beauveria bassiana in the biological control of agricultural pests. 4. The present invention provides an overexpression vector, engineered strain, and its application and method for enhancing the insecticidal toxicity of Beauveria bassiana. This invention helps to improve the application effect of fungal insecticides, thereby reducing the use of chemical pesticides and promoting the development of green agriculture. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The following are the results of the construction of the HaTK3 overexpression vector and the screening of HaTK3 high-expression transgenic strains provided in the embodiments of the present invention: (A) Schematic diagram of the construction of the HaTK3 overexpression vector; (B) Transgenic strains with the highest HaTK3 gene expression level were screened using the RT-qPCR method.

[0021] Figure 2 The results of virulence determination of WT and Bb::HaTK3 strains against cotton bollworm provided in the embodiments of the present invention; (A–C) Survival curves of WT and Bb::HaTK3 strains after infection with cotton bollworm under different concentrations of conidial suspension; (D) At 1×10 8 Under conidia / mL spore suspension conditions, the half-lethal time (LT) of fungi 50 (E) The median lethal concentration (LD50) of WT and Bb::HaTK3 strains. 50 ).

[0022] Figure 3 The effects of the WT and Bb::HaTK3 strains provided in the embodiments of the present invention on the immunity of cotton bollworm.

[0023] Figure 4 The results of toxicity tests of WT and Bb::HaTK3 strains against the greater wax moth and the beet armyworm provided in the embodiments of the present invention; (A–B) Survival curves and median lethal time (LT) of greater wax moth larvae after infection with WT and Bb::HaTK3 strains. 50 Survival curves and median lethal time (LT) of Spodoptera litura larvae after infection with (C–D)WT and Bb::HaTK3 strains. 50 ). Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0025] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods have not been specifically described in order to avoid obscuring the invention.

[0026] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. Moreover, those skilled in the art will understand that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Example 1 Construction of Bb::HaTK3 overexpression vector and acquisition of transgenic strains (1) Obtaining and amplifying the Pb3-BbChiP-SP-HaTK3 fusion gene fragment The BbChiP-SP was spliced ​​with a codon-optimized HaTK3 nucleic acid sequence, and EcoRI restriction sites were introduced at both ends to artificially synthesize the pUC57-BbChiP-SP-HaTK3 plasmid. Using the synthesized pUC57-BbChiP-SP-HaTK3 recombinant plasmid as a template and HaTK3_F / R as primers, the BbChiP-SP-HaTK3 recombinant fragment was amplified. Using Bb0062 genomic DNA as a template and Pb3_F / R as primers, the Pb3 promoter fragment was amplified. Using the BbChiP-SP-HaTK3 recombinant fragment and the Pb3 promoter fragment as a mixed template and Pb3-HaTK3_F / R as primers, the Pb3-BbChiP-SP-HaTK3 fusion gene fragment was amplified by overlap extension PCR.

[0028] The nucleic acid sequence of HaTK3 is SEQ ID No. 1: ATGCCTAAGAGAAAGGCTCAGATGGGCTTCTTCGGTATGCGAGGCAAGAAGTGGACTGATTAA The amino acid sequence of HaTK3 is SEQ ID No. 2: MPKRKAQMGFFGMRGKKWTD* The codon-optimized nucleic acid sequence of HaTK3, SEQ ID No. 3: ATGCCCAAGCGCAAGGCCCAGATGGGCTTCTTCGGCATGCGCGGCAAGAAGTGGACCGACTAA.

[0029] The nucleotide sequence of HaTK3_F is as follows: ATGGCTCCTTTTCTTCAAACC The nucleotide sequence of HaTK3_R is as follows: TTAGTCGGTCCACTTCTTGCCG The Pb3-HaTK3_F nucleotide sequence is as follows: CTTTTAATCAATAACAGAATTCATGGCTCCTTTTCTTCAAACC The Pb3-HaTK3_R nucleotide sequence is as follows: TTCTGTCGACACTAGTGAATTCTTAGTCGGTCCACTTCTTGCCG The PCR amplification system consisted of 25 μL, including 12.5 μL of 2 × Phanta Max Buffer, 0.5 μL of dNTP Mix (10 mMeach), 0.5 μL each of forward and reverse primers, and 50 ng of template DNA. The remainder was made up with ddH2O. The high-fidelity enzyme used was Phanta Max Super-Fidelity DNA Polymerase (P505, Vazyme).

[0030] PCR amplification conditions were as follows: pre-denaturation at 95℃ for 3 min; 35 cycles of 95℃ for 15 s, 57℃ for 15 s, and 72℃ (1 kb / 30 s); extension at 72℃ for 5 min. The PCR product was purified using a DNA gel extraction kit to obtain the target fragment.

[0031] (2) Construction of HaTK3 overexpression vector Eco The linearized vector pK2-bar, digested with RI restriction endonuclease, served as the backbone vector. The Pb3-BbChiP-SP-HaTK3 fusion DNA fragment was ligated into the vector using homologous recombination.

[0032] The ligation system consisted of 10 μL of 5 × CE II Buffer, 200 ng of vector DNA, 80 ng of insert DNA, 1 μL of Exnase II, and the remainder was made up with ddH2O. After mixing, the mixture was incubated at 37°C for 30 min.

[0033] The ligation product was used to transform *E. coli* DH5α. The transformed bacterial culture was evenly spread on LK solid medium plates containing kanamycin. Single colonies were picked, verified by colony PCR, and then propagated in LK liquid medium. Plasmid DNA was extracted to obtain the correct recombinant expression vector pK2-bar-Pb3-BbChiP-SP-HaTK3.

[0034] (3) Agrobacterium-mediated genetic transformation of Beauveria bassiana The recombinant vector was introduced into Agrobacterium tumefaciens AGL1 via electroporation and genetic transformation of Beauveria bassiana was carried out according to the method of Fang et al. (2004).

[0035] The specific steps are as follows: A single colony of *Agrobacterium* that had been verified to be positive was inoculated into 5 mL of YCK liquid medium and cultured overnight with shaking at 28°C and 200 rpm. Then, 100 μL of the bacterial culture was inoculated into 25 mL of YCK medium and cultured until OD (Organic Dysplasia) was reached. 660 The value should be 0.7-0.9. Centrifuge 7 mL of bacterial culture (5000 rpm, 5 min), discard the supernatant, and resuspend in 7 mL of 1M liquid medium containing 200 mM acetylsyl syringone (AS) and 5 mM glucose (protected from light). According to (10 × OD... 660 Mix 1.5 mL of IM medium with 1.5 mL of bacterial suspension and incubate at 28°C in the dark with shaking for 6 h. Simultaneously, prepare a Beauveria bassiana spore suspension: scrape spores cultured for 14 days, treat with 0.5% Tween-80, filter, count, and adjust the concentration to approximately 5 × 10⁻⁶. 4 conidia / mL.

[0036] Agrobacterium bacterial suspension and spore suspension were mixed at a 1:1 ratio and evenly spread on IM solid plates lined with sterile filter membranes. The mixture was then incubated at 26°C for 48 h. Subsequently, the filter membranes were transferred to CZA medium containing antibiotic resistance and incubated for 5–7 days to screen for transformants.

[0037] (4) Screening of transgenic strains with high expression of HaTK3 Seven transgenic strains that were positive by mycelial PCR were randomly selected and inoculated into 1 / 4 SDB liquid medium for culture. WT strain was used as a control. After culturing at 26℃ and 200 rpm for 3 days, bacterial cells were collected, RNA was extracted, and cDNA was obtained through reverse transcription.

[0038] HaTK3 expression levels were detected by RT-qPCR in a 10 μL reaction volume, comprising 5 μL of 2×SYBR Mix, 0.5 μL each of primers (Q_HaTK3_F / R: TCTTCAAACCAGCCTCGCGC / GGTCCACTTCTTGCCGCGCAT), and 4 μL of template cDNA. Bbactin was used as an internal reference gene.

[0039] Experimental results show that: [the following is a description of a successful construction project] Figure 1 A shows the HaTK3 overexpression vector. HaTK3 expression was not detected in wild-type Beauveria bassiana (Bb-WT), but it was detected in all 7 transgenic strains, with strain 3 showing the highest expression level. This strain was named Bb::HaTK3 and designated as the strain for subsequent experiments. Figure 1 B). This indicates that a transgenic strain with high expression of HaTK3 was successfully screened.

[0040] Example 2 Virulence assays of WT and Bb::HaTK3 strains The virulence of WT and Bb::HaTK3 strains was determined using the third instar larvae of cotton bollworm, the final instar larvae of large wax moth, and the third instar larvae of beet armyworm, respectively, through the body wall infection inoculation method. The specific method is as follows: Conidia of *Beauveria bassiana* cultured on PDA solid medium for 14 days were scraped into a 50 mL centrifuge tube containing 20 mL of sterile Tween-80 solution. The tube was shaken thoroughly to ensure uniform dispersion of the spores. The tube was then filtered through sterile double-layer lens paper to remove mycelia and other impurities. After mixing, the spore concentration was determined using a hemocytometer. The tube was then diluted with Tween-80 solution to different concentrations, and 20 mL spore suspensions were prepared and transferred to sterile wide-mouth bottles for later use.

[0041] When using cotton bollworm larvae as test insects, three concentration gradients of spore suspension were set up (2 × 10⁻⁶). 7 1 × 10 8 and 5 × 10 8 (conidia / mL) For each treatment, 24 third-instar cotton bollworm larvae were placed in a spore suspension and gently agitated for about 15 seconds to ensure the larvae were fully and evenly contacted with the spore suspension. Each treatment was performed in triplicate. After treatment, excess spore suspension was filtered through a mesh screen, and the larvae were dried with absorbent paper. The larvae were then divided into 24-well rearing plates for further rearing, and their survival was observed and recorded regularly.

[0042] When using the larvae of the large wax borer as test insects, the concentration of the spore suspension was set at 1 × 10⁻⁶. 7 For each treatment, 33 final-instar larvae were selected and placed in a spore suspension. The suspension was gently agitated for about 15 seconds to ensure full contact between the larvae and the spore suspension. Three biological replicates were set up. After treatment, excess spore suspension was filtered off and surface moisture was aspirated. The larvae were then aliquoted into disposable 150 mm diameter culture dishes for further rearing, and their survival status was recorded regularly.

[0043] When using beet armyworm larvae as test insects, the concentration of the spore suspension was also 1 × 10⁻⁶. 7 For each treatment, 24 third-instar larvae were placed in a spore suspension and gently agitated for about 15 seconds to ensure even contact between the larvae and the suspension. Three biological replicates were performed. After treatment, excess spore suspension was filtered off and surface moisture was aspirated. The larvae were then aliquoted into 24-well rearing plates for further rearing, and their survival was observed and recorded regularly. For data containing only two groups, Student's t-test was used to analyze the significance of differences. * indicates a significant difference (*). P <0.05), ** indicates a highly significant difference ( P <0.01).

[0044] The above results indicate that, when using cotton bollworm larvae as test insects, the survival rate of insects infected with Bb::HaTK3 was significantly lower than that of those infected with WT at different spore suspension concentrations; simultaneously, the half-lethal time (LT) of Bb::HaTK3 was significantly shorter. 50 Significantly lower than WT, at 1× 10 8 The maximum shortening was 26.17% at conidia / mL. P <0.01)( Figure 2 AD). Furthermore, the median lethal concentration (LD50) of the Bb::HaTK3 strain against cotton bollworms... 50 The value was also significantly reduced, decreasing by 90.52% compared to WT. P <0.01)( Figure 2 E), indicating that the virulence of the Bb::HaTK3 strain against the cotton bollworm was significantly enhanced.

[0045] When using the larvae of the large wax moth as test insects, the median lethal time of Bb::HaTK3 was significantly shortened by 49.73% compared to WT (P<0.01). Figure 4 AB); In Spodoptera litura larvae, the median lethal time of Bb::HaTK3 was also significantly shortened by 23.22% compared to WT (P<0.01). Figure 4 CD). The above results indicate that the Bb::HaTK3 strain also exhibits significantly enhanced viral virulence against the larvae of the wax moth and the beet armyworm.

[0046] Example 3 Determination of the effects of WT and Bb::HaTK3 strains on the immune response of cotton bollworm RNA was extracted from whole cotton bollworms infected with WT and Bb::HaTK3 for 96 h using the TRIzol method, and cDNA was synthesized using an RT reverse transcription kit. Subsequently, the expression levels of immune-related genes in the insects were detected using a real-time PCR system (Bio-Rad), with HaEF-1α as an internal reference gene.

[0047] Simultaneously, hemolymph samples from *Botrytis cinerea* were collected at different time points after WT and Bb::HaTK3 treatment. The number of *Botrytis cinerea* cells in the hemolymph was counted under a regular microscope. Each treatment was performed in triplicate. For data analysis, Student's t-test was used to analyze the significance of differences between the two groups (*). P <0.05,** P <0.01); comparisons of three or more groups were performed using one-way ANOVA combined with Tukey's multiple comparison test, with different lowercase letters indicating statistical significance. P <0.05).

[0048] The results showed that, compared with WT infection, the expression levels of immune-related genes in cotton bollworms infected with Bb::HaTK3 were significantly reduced, with a decrease of 1.28-2.89 times. P <0.05)( Figure 3 A). Furthermore, the results of bacterial count detection in hemolymph showed that the Bb::HaTK3 infection group was significantly higher than the WT group at all time points, with an increase of 2.85-6.02 times (P<0.01). Figure 3 B).

[0049] The above results indicate that the Bb::HaTK3 strain can significantly inhibit the expression of immune-related genes in cotton bollworms, thereby promoting their proliferation in hemolymph, increasing the number of insect cells, and ultimately leading to a significant enhancement of the strain's virulence.

[0050] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for improving the insecticidal toxicity of Beauveria bassiana, characterized in that, The insecticidal toxicity of Beauveria bassiana was enhanced by overexpressing the HaTK3 gene, a fast-acting peptide of cotton bollworm, in Beauveria bassiana. The amino acid sequence of the HaTK3 gene is shown in SEQ ID NO.

2.

2. The method for improving the insecticidal toxicity of Beauveria bassiana according to claim 1, characterized in that, The nucleotide sequence of the HaTK3 gene is shown in SEQ ID NO.

1.

3. The method for improving the insecticidal toxicity of Beauveria bassiana according to claim 1, characterized in that, The nucleotide sequence of the HaTK3 gene, after codon optimization, is shown in SEQ ID No. 3, and is used for expression in Beauveria bassiana.

4. The method for improving the insecticidal toxicity of Beauveria bassiana according to claim 1, characterized in that, Includes the following steps: (1) Obtain the coding sequence of the cotton bollworm tachykinin HaTK3, fuse the chitinase signal peptide sequence of Beauveria bassiana at its N-terminus, and synthesize a recombinant gene fragment; (2) Construct a fungal expression vector containing the HaTK3 coding sequence; (3) The expression vector was introduced into Beauveria bassiana to obtain a recombinant strain with HaTK3 overexpression; (4) The recombinant strains were screened and molecularly identified to obtain genetically stable transgenic strains with enhanced insecticidal toxicity.

5. A secretory overexpression vector for the cotton bollworm rapid kinase short peptide HaTK3, which enhances the insecticidal toxicity of Beauveria bassiana, characterized in that, The overexpression vector is the recombinant expression vector pK2-bar-Pb3-BbChiP-SP-HaTK3; The recombinant expression vector pK2-bar-Pb3-BbChiP-SP-HaTK3 was obtained by fusing the nucleotide sequence of the chitinase signal peptide BbChiP-SP from Beauveria bassiana with the nucleotide sequence of HaTK3 to obtain the recombinant fragment BbChiP-SP-HaTK3, which was then linked to the constitutive promoter Pb3 of the glyceraldehyde-3-phosphate dehydrogenase gene, and then inserted into the expression vector pK2-bar containing the fungal selection marker glufosinate resistance gene bar after enzyme digestion and ligation.

6. The method for constructing the secretory overexpression vector of the cotton bollworm tachykinin short peptide HaTK3 as described in claim 5, characterized in that, Includes the following steps: (1) Obtaining the BbChiP-SP-HaTK3 recombinant fragment The BbChiP-SP was spliced ​​with the codon-optimized HaTK3 nucleic acid sequence, and restriction enzyme sites were introduced to obtain the pUC57-BbChiP-SP-HaTK3 plasmid. 2) PCR amplification was performed using the synthesized plasmid as a template to obtain the BbChiP-SP-HaTK3 recombinant fragment; (2) Obtaining the Pb3 promoter fragment Using Beauveria bassiana Bb0062 genomic DNA as a template, PCR amplification was performed to obtain the Pb3 promoter fragment; (3) Obtaining the Pb3-BbChiP-SP-HaTK3 recombinant fragment Using the Pb3 promoter fragment and the BbChiP-SP-HaTK3 recombinant fragment as templates, overlap extension PCR was used to perform fusion amplification to obtain the Pb3-BbChiP-SP-HaTK3 recombinant fragment. (4) Construction of recombinant plasmids The recombinant fragment was ligated into the pK2-bar vector via homologous recombination to obtain the recombinant plasmid pK2-Pb3-BbChiP-SP-HaTK3.

7. A transgenic Beauveria bassiana strain overexpressing the cotton bollworm tachykinin short peptide HaTK3, characterized in that, The overexpression vector described in claim 5 was introduced into the wild-type strain of Beauveria bassiana via Agrobacterium tumefaciens AGL1-mediated genetic transformation, and the engineered strain Bb::HaTK3 with high expression of HaTK3 was obtained by RT-qPCR screening.

8. The application of the cotton bollworm fast kinase short peptide HaTK3 gene as described in claim 1 and the overexpression vector as described in claim 5 in improving the insecticidal toxicity of Beauveria bassiana.

9. The application of the cotton bollworm fast kinin short peptide HaTK3 gene of claim 1, the overexpression vector of claim 5, or the engineered strain of Beauveria bassiana of claim 7 in the preparation of fungal insecticides.

10. A fungal insecticide, characterized in that, It includes the HaTK3 short peptide encoded by the HaTK3 gene as described in claim 1, the overexpression vector as described in claim 5, and / or the Beauveria bassiana engineered strain as described in claim 7.

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