A recombinant Beauveria bassiana, its preparation method and application

Abstract

This invention discloses a recombinant Beauveria bassiana, its preparation method, and its applications. Firstly, this invention discloses a recombinant Beauveria bassiana capable of expressing parasitic bee venom proteins. It further discloses the preparation method of the recombinant Beauveria bassiana and its application in insecticidal applications. Based on the codon preference of Beauveria bassiana, this invention optimizes the gene sequence of parasitic bee venom proteins, expands the resource library of virulence factors for biocontrol, and integrates this sequence into Beauveria bassiana to obtain recombinant Beauveria bassiana, which can efficiently express parasitic bee venom proteins, overcoming the shortcomings of slow onset and weak toxicity of traditional biocontrol bacteria. Furthermore, the preparation method of the recombinant Beauveria bassiana is simple, and the obtained recombinant Beauveria bassiana is very stable, with continuous expression of exogenous virulence factors.

Description

Technical Field

[0001] This invention relates to the field of biological control technology. Specifically, it relates to a recombinant Beauveria bassiana, its preparation method, and its application. Background Technology

[0002] Parasitic wasps are important natural enemies for controlling many pests in agricultural and forestry ecosystems. Their venom proteins are naturally occurring biological pesticide reservoirs, showing great promise for application in the biological control of pests. The venom proteins of parasitic wasps have the following advantages: First, they are highly efficient at killing insects. Through long-term co-evolution between parasitic wasps and their host pests, under the pressure of natural selection, certain venom proteins of parasitic wasps have formed highly effective toxic factors, and hosts are unlikely to develop resistance. Second, they are highly specific. Unlike the venom of bees, wasps, and other insects, the venom proteins of parasitic wasps are completely harmless to humans and other higher animals, and do not harm beneficial insects in the ecosystem such as bees, butterflies, and dragonflies; they only have a significant lethal effect on the host pests. Finally, they are environmentally friendly. Wasp venom originates from insects, does not pollute the environment, and is biodegradable. The venom of parasitic wasps can also be used as an synergist in the biological control of pests. Using the venom of parasitic wasps to treat insect pathogenic fungi, insect baculoviruses, and microsporidia significantly increased the sensitivity of host pests to these biopesticides, resulting in better insecticidal effects. *Microplitis mediator* is a parasitic wasp that targets lepidopteran larvae, including more than forty important agricultural pests such as the cotton bollworm (*Helicoverpa armigera*), armyworm (*Pseudaletia separata*), and cabbage cutworm (*Mamestra brassicae*). Field releases in Xinjiang and Hebei provinces have yielded good biological control results. Recently, using a combination of transcriptomics and proteomics, the main components of the *Microplitis mediator* venom protein were analyzed, and a snake venom-like metalloproteinase, MmVRF1, was identified. Intra-host injection of extremely small doses (1×10⁻⁶) yielded significant results. -4 The recombinant MmVRF1 protein (ng) can directly kill host larvae. However, parasitic wasps are small in size, making it difficult to obtain venom from them and apply it on a large scale.

[0003] Beauveria bassiana is an entomopathogenic fungus whose hosts include over 750 species of insects belonging to 149 families and 15 orders, and 13 species of ticks and mites belonging to 6 families. It is a commonly used biocontrol agent with advantages such as a broad insecticidal spectrum, ease of large-scale production, and environmental friendliness. However, the application of Beauveria bassiana in biological control still has some drawbacks, primarily its slow onset of insecticidal action and inconsistent control efficacy of different strains.

[0004] Therefore, combining the advantages and disadvantages of Beauveria bassiana and parasitic wasps, and utilizing modern molecular biology techniques to develop novel, highly efficient, low-toxicity, green, and stable insecticides to meet national development needs, is of profound significance. Summary of the Invention

[0005] The first objective of this invention is to provide a recombinant Beauveria bassiana that can efficiently express the parasitic bee venom protein MmVRF1, overcoming the shortcomings of Beauveria bassiana, such as slow onset of action and weak toxicity.

[0006] A second objective of this invention is to provide the application of the above-mentioned recombinant Beauveria bassiana.

[0007] The third objective of this invention is to provide a method for preparing the above-mentioned recombinant Beauveria bassiana, which is simple to operate and produces a very stable Beauveria bassiana with sustainable expression of exogenous virulence factors.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a recombinant Beauveria bassiana that can express parasitic bee venom protein.

[0010] For example, the recombinant Beauveria bassiana integrates an exogenous gene encoding a parasitic bee venom protein, wherein the gene encoding the parasitic bee venom protein is any of the following nucleotide sequences:

[0011] a1) The nucleotide sequence of SEQ ID NO.1 from position 67 to 1449;

[0012] b1) The nucleotide sequence of SEQ ID NO.2 from position 67 to 1449;

[0013] c1) The complementary sequence of a1) or b1) above;

[0014] d1) Polynucleotides derived from the nucleotide sequences of SEQ ID NO.1 or SEQ ID NO.2 due to genetic code degeneracy.

[0015] Preferably, in the recombinant Beauveria bassiana, an endogenous gene encoding the Beauveria bassiana chitase chit1 signal peptide is integrated before the exogenous gene encoding parasitic bee venom protein, wherein the gene encoding the Beauveria bassiana chitase chit1 signal peptide has any of the following nucleotide sequences:

[0016] a2) The nucleotide sequence of SEQ ID NO.3;

[0017] b2) The complementary sequence of a2) above; or

[0018] c2) Polynucleotides derived from the nucleotides in SEQ ID NO.3 due to the degeneracy of the genetic code.

[0019] The parasitic bee venom protein of this invention is specifically the venom protein of *Bretschneidera sinensis*. The genes encoding the parasitic bee venom protein and the gene encoding the chit1 signal peptide of *Beauveria bassiana* can be synthesized artificially or amplified using PCR technology. This invention removes and replaces the signal peptide of the parasitic bee venom protein with the chit1 signal peptide of *Beauveria bassiana*. Specifically, it integrates an endogenous gene encoding the chit1 signal peptide of *Beauveria bassiana* before the gene encoding the parasitic bee venom protein, enabling the recombinant *Beauveria bassiana* to heterologously secrete and express the parasitic bee venom protein.

[0020] For example, the recombinant Beauveria bassiana of the present invention has the accession number CGMCC NO.23043 and is classified as Beauveria bassiana. It was deposited at the China General Microbiological Culture Collection Center (address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​July 14, 2021.

[0021] In a second aspect, the present invention provides an insecticide comprising the above-mentioned recombinant Beauveria bassiana, or its descendants, or its conidia, or its mycelium, or any combination thereof.

[0022] Preferably, the insecticide further includes a pesticide-acceptable carrier; the pesticide-acceptable carrier may be one or more of the following: mica powder, light calcium carbonate, clay, talc, kaolin, diatomaceous earth, attapulgite, bentonite, sepiolite, urea, potassium chloride, sodium sulfate, ammonium sulfate, sodium nitrate, ammonium nitrate, and ammonium chloride.

[0023] In a specific embodiment of the present invention, the insecticide is used to control cotton bollworm and / or fall armyworm.

[0024] The application of the aforementioned recombinant Beauveria bassiana, or its descendants, or its conidia or mycelium, or any combination thereof, in the preparation of insecticides is also within the scope of protection of this invention.

[0025] Preferably, the insecticide of the present invention may also contain other insecticidal active ingredients. Exemplary examples include one or more of the following: metamizole, pyrethroids, carbamates, neonicotinoids, sodium channel blockers, insecticidal macrocyclic lactones, γ-aminobutyric acid (GABA) antagonists, chlorfluazurons, and juvenile hormone mimics.

[0026] Thirdly, the present invention provides a method for preparing recombinant Beauveria bassiana, comprising the following steps: operably introducing the gene encoding the chit1 signal peptide of Beauveria bassiana and the gene encoding parasitic bee venom protein into Beauveria bassiana.

[0027] Preferably, the introduction is performed via Agrobacterium-mediated transformation.

[0028] In a specific embodiment of the present invention, the method for preparing the recombinant Beauveria bassiana is as follows:

[0029] The genes encoding the chit1 signal peptide of Beauveria bassiana and the gene encoding parasitic bee venom protein were ligated into an expression vector and introduced into Beauveria bassiana via Agrobacterium-mediated transformation. This integrated the genes encoding the chit1 signal peptide of Beauveria bassiana and the gene encoding parasitic bee venom protein into the chromosome of Beauveria bassiana, resulting in an engineered strain of Beauveria bassiana capable of expressing parasitic bee venom protein, namely recombinant Beauveria bassiana.

[0030] In a preferred embodiment of the present invention, the method for preparing the recombinant Beauveria bassiana is as follows:

[0031] The gene encoding the chitase chit1 signal peptide of Beauveria bassiana (Bbsp) and the gene encoding parasitic bee venom protein (VRF1) were ligated into plasmid pBARGPE1 to obtain pBARGPE1-Bbsp-VRF1-FLAG. This yielded a cassette "gpdA Promotor-Bbsp-VRF1-FLAG-trpC Terminator" (abbreviated as gBVft) containing the fungal overexpression promoter gpdA Promotor and the terminator trpC Terminator. The gpdA Promotor was then used to initiate the expression of the exogenous parasitic bee venom protein gene.

[0032] gBVft was ligated to plasmid pPK2-Bar to obtain pPK2-Bar-gBVft;

[0033] pPK2-Bar-gBVft was introduced into Beauveria bassiana via Agrobacterium-mediated transformation to obtain a recombinant Beauveria bassiana engineered strain capable of expressing parasitic bee venom proteins.

[0034] The present invention also provides a method for killing insects, comprising the steps of applying the above-mentioned recombinant Beauveria bassiana, or the offspring of the recombinant Beauveria bassiana, or the conidia produced by it, or the mycelium produced by it, or any combination thereof, or the above-mentioned insecticide, or the recombinant Beauveria bassiana prepared by the above-mentioned preparation method.

[0035] Preferably, the application includes spraying the recombinant Beauveria bassiana of the present invention onto crops, such as corn, wheat, etc.

[0036] The recombinant Beauveria bassiana engineered strain of this invention exhibits a 1.67-fold reduction in the median lethal dose against cotton bollworm compared to the wild-type strain (experimental dose gradient: 1×10⁻⁶). 2 1×10 3 1×10 4 (per spore), shortening the lethal time by 1.55 times (at an experimental dose of 1×10⁻⁶ spores). 4 Under the condition of 1 spores, the insecticidal efficacy is significantly improved.

[0037] The beneficial effects of this invention are as follows:

[0038] 1. This invention optimizes the gene sequence of parasitic bee venom protein based on the codon preference of Beauveria bassiana, expands the resource library of virulence factors for biological control, and integrates this sequence into Beauveria bassiana to obtain recombinant Beauveria bassiana, which can efficiently express parasitic bee venom protein, overcoming the shortcomings of slow onset and weak toxicity of biocontrol bacteria.

[0039] 2. The preparation method of the recombinant Beauveria bassiana of the present invention is simple to operate, and the obtained recombinant Beauveria bassiana is very stable, and exogenous virulence factors can be continuously expressed. Attached Figure Description

[0040] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0041] Figure 1 This diagram illustrates the use of parasitic wasp venom to enhance the virulence of biocontrol bacteria. A shows an anatomical diagram of the parasitic cotton bollworm by the *Rhododendron simsii* wasp and its venom gland-related organs. B shows a diagram of the construction of a vector using *Agrobacterium* transformation to construct engineered bacteria and enhance their virulence.

[0042] Figure 2 Electrophoresis results for constructing the pBARGPE1-Bbsp-VRF1-FLAG vector: A shows the band of the chitase chit1 signal peptide amplified using primer Bbsp-F / R with Beauveria bassiana genomic DNA as a template (represented as "Bbsp" in the figure); B shows the band of the venom protein (excluding its own signal peptide) amplified using primer VRF1-F / R with cDNA from the venom gland of Brachymys lateralis as a template (represented as "VRF1" in the figure); C shows the band of the linearized plasmid pBARGPE1 (represented as "linearized pBARGPE1"), which was linearized using primer L-pBARGPE1-F / R with plasmid pBARGPE1 as a template.

[0043] Figure 3 This is a schematic diagram of the structure of the carrier pBARGPE1-Bbsp-VRF1-FLAG containing the FLAG tag.

[0044] Figure 4 Electrophoresis results for constructing the pPK2-Bar-VRF1-FLAG vector fragment; where A is the band of linearized pPK2-Bar, which was linearized using primer L-pPK2-F / R as a template (represented as "linearized pPK2-Bar" in the figure); B is the band of gBVft carrying the adapter, which was amplified using primer O-gBVft-F / R as a template of pBARGPE1-Bbsp-VRF1-FLAG (represented as "gBVft" in the figure).

[0045] Figure 5 This is a schematic diagram of the structure of the fungal transformation vector pPK2-Bar-gBVft.

[0046] Figure 6 1 / 4 SDAY medium (PPT) + Chloramphenicol + The results of screening transformants.

[0047] Figure 7 The electrophoresis results of positive transformants were used to verify PCR.

[0048] Figure 8 The results of Western blot analysis of the MmVRF1 protein secreted by the lateral groove of *Beauveria bassiana* venom in positive transformants are shown; where 1-8 are different positive transformants.

[0049] Figure 9 To Figure 7 Results of sporulation detection and comparison of the three positive transformants with the deepest middle band (Bb-VRF1-1,2,7) and wild-type Beauveria bassiana ARSEF 2860 (abbreviated as Bb2860).

[0050] Figure 10 The results of germination rate detection and comparison of three positive transformants (Bb-VRF1-1,2,7) and wild-type Beauveria bassiana ARSEF 2860 (abbreviated as Bb2860) are presented.

[0051] Figure 11The results show the detection and comparison of colony expansion rates of three positive transformants (Bb-VRF1-1,2,7) and wild-type Beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb2860).

[0052] Figure 12 The colony morphology of wild-type Beauveria bassiana ARSEF 2860 (Bb2860) and Bb-VRF1 transformed with the parasitic bee venom protein expression gene are displayed on three culture media (①Czapek–Dox Medium; ②1 / 4 SDAY Medium containing only 1 / 4 of the component; ③PDA Potato Dextrose Agar Medium).

[0053] Figure 13 The results show the insecticidal efficiency of the immersion method against third-instar cotton bollworms.

[0054] Figure 14 The results show the insecticidal efficiency of the injection method against 5th instar cotton bollworms.

[0055] Figure 15 The results show the insecticidal efficiency of the injection method against the 6th instar fall armyworm.

[0056] Figure 16 The results show the insecticidal efficiency of the third instar diamondback moth determined by the immersion method.

[0057] Figure 17 Symptoms of test insects infected with Bb-VRF1: A is bollworm, B is fall armyworm, and C is diamondback moth. Detailed Implementation

[0058] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0061] The gene sequence encoding the venom protein of the red-sulcus parasitic wasp in the following embodiments is shown in SEQ ID NO.2. The gene sequence of the parasitic wasp venom protein was optimized according to the codon bias of Beauveria bassiana, as shown in SEQ ID NO.1.

[0062] The primers used in the following examples are shown in Table 1:

[0063] Table 1 Primer sequences

[0064]

[0065]

[0066] Note: 1. Primers starting with L are linearized primers; 2. Primers starting with O are primers with a connector, which contains an overlapping region. The underlined part is the overlapping region.

[0067] 3. The FLAG tag is contained within the underscores of L-pBARGPE1-R and O-VRF1-R, and is introduced upon completion of the recombinant cloning. Specifically, the FLAG tag sequence is 5'-GATTACAAGGACGACGATGACAAG-3'.

[0068] The underlined part of O-VRF1-R is 5'- GTCGTCCTTGTAATC -3', whose inverse complementary sequence 5'-GATTACAAGGACGAC-3' is the 1-15bp end of the FLAG tag 5'.

[0069] The underlined portion 5'-GACGACGATGACAAG-3' of L-pBARGPE1-R is the 1-15bp 3' end of the FLAG tag. During recombination cloning, the two can recombine at complementary sequences to form the FLAG tag.

[0070] The present invention will now be described in detail with reference to specific embodiments, which are intended to understand rather than limit the invention.

[0071] Example 1: Preparation of Recombinant Beauveria bassiana

[0072] A schematic diagram illustrating how the *Rhododendron lateral sulci* parasitic wasp uses venom to regulate its host, the cotton bollworm. Figure 1 As shown in Figure A, the parasitic wasp venom protein is secreted by the venom glands, stored in the venom sac, and injected into the host through the ovipositor during parasitism.

[0073] A schematic diagram of recombinant Beauveria bassiana is shown below. Figure 1 As shown in Figure B, the specific steps are as follows:

[0074] I. Carrier Construction

[0075] 1.1 Construction of pBARGPE1-Bbsp-VRF1-FLAG

[0076] Using genomic DNA extracted from *Beauveria bassiana* ARSEF 2860 as a template, PCR amplification was performed using primers Bbsp-F / R to obtain the gene fragment of the chitinase chit1 signal peptide (nucleotide sequence shown in SEQ ID NO. 3). Because *Beauveria bassiana* exhibits weak codon bias and low gene expression levels, sequence optimization only required adjustments to rare codons. Using cDNA extracted from the venom glands of female *Beauveria bassiana* var. *medium* (nucleotide sequence shown in SEQ ID NO. 2) two days after emergence as a template, point mutations were performed according to the instructions of the Mut Express II Fast Mutagenesis Kit V2 (Vazyme, C214-01). Based on the codon preference of *Beauveria bassiana*, the rare codons CGC at positions 916-918 of SEQ ID NO. 2 were replaced with CGT (i.e., the optimized gene sequence of *Beauveria bassiana* venom protein is the nucleotide sequence shown in SEQ ID NO. 1). PCR amplification was performed using primers VRF1-F / R to obtain a gene fragment containing the optimized *Beauveria bassiana* venom protein (excluding its own signal peptide) (nucleotide sequence shown in positions 67-1449 of SEQ ID NO. 1). Using plasmid pBARGPE1 as a template, it was linearized using primers L-pBARGPE1-F / R to obtain the linearized plasmid pBARGPE1.

[0077] The high-fidelity PCR reaction system (50 μL) is shown in Table 2. The high-fidelity PCR reaction conditions are: 98℃ pre-denaturation for 3 min; 98℃ for 30 sec, 55 or 60℃ for 30 sec, 68℃ 1 kb / min, cycle number 35; 68℃ for 3 min, 12℃ ∞.

[0078] Table 2 High-fidelity PCR reaction system (50 μL):

[0079] dNTP mixture (2.5 mM) 4μL Upstream primer (10 μM) 2μL Downstream primer (10 μM) 2μL template 5ng-100ng Primer STAR GXL DNA Polymerase 1μL <![CDATA[ddH2O]]> Up to 50 μL

[0080] The specificity of PCR products and band size were verified using agarose gel electrophoresis, and the results are as follows: Figure 2As shown, A is the band of the chitinase chit1 signal peptide amplified using primer Bbsp-F / R with Beauveria bassiana genomic DNA as a template (represented as "Bbsp" in the figure); B is the band of the venom protein (excluding its own signal peptide) amplified using primer VRF1-F / R with cDNA of the venom gland of Brachymys lateralis as a template (represented as "VRF1" in the figure); C is the band of the linearized plasmid pBARGPE1, which was linearized using primer L-pBARGPE1-F / R with plasmid pBARGPE1 as a template (represented as "linearized pBARGPE1" in the figure). After confirming the sequencing results were correct, PCR amplification was performed using primers O-Bbsp-F / R (with an overlapping region at the adapter) to obtain the gene fragment of the chitase chit1 signal peptide of Beauveria bassiana carrying the adapter. PCR amplification was performed using primers O-VRF1-F / R (with an overlapping region at the adapter) to obtain the gene fragment of the venom protein of Braconia medius (without its own signal peptide) carrying the adapter. The linearized plasmid pBARGPE1 was digested with Dpn I to obtain the digested linearized plasmid pBARGPE1 to eliminate the influence of the template strand.

[0081] The concentrations of each PCR product (i.e., the gene fragment of the chitase chit1 signal peptide from *Beauveria bassiana* carrying the adapter and the gene fragment of the venom protein from *Brachys erythrophagus lateralis* carrying the adapter (excluding its own signal peptide)) and the digestion product (i.e., the linearized plasmid pBARGPE1 after digestion) were determined using a micro-ultraviolet spectrophotometer. The ligation reaction system was prepared according to the instructions of the ClonExpressMultiS One Step Cloning Kit (Vazyme, catalog number C113-01) to construct the FLAG-tagged vector pBARGPE1-Bbsp-VRF1-FLAG (structural schematic shown in Figure 1). Figure 3 (as shown), thereby obtaining a cassette “gpdA Promotor-Bbsp-VRF1-FLAG-trpCTerminator” containing a fungal overexpression promoter and terminator, which can be used to construct engineered bacteria expressing exogenous virulence factors.

[0082] 1.2 Construction of the vector pPK2-Bar-gBVft

[0083] The cassette “gpdA Promotor-Bbsp-VRF1-FLAG-trpC Terminator” will be abbreviated as gBVft. Using the vector pBARGPE1-Bbsp-VRF1-FLAG as a template, PCR amplification was performed using primers O-gBVf-F / R with the adapter to obtain gBVft carrying the adapter. Using plasmid pPK2-Bar as a template, it was linearized using primers L-pPK2-F / R to obtain the linearized plasmid pPK2-Bar. Agarose gel electrophoresis was used to verify the specificity of the PCR products and the band size; the results are as follows. Figure 4 As shown, A is the band of linearized pPK2-Bar, which was linearized using the primer L-pPK2-F / R with pPK2-Bar as a template (represented as "linearized pPK2-Bar" in the figure); B is the band of gBVft carrying the adapter, which was amplified using the primer O-gBVft-F / R with pBARGPE1-Bbsp-VRF1-FLAG as a template (represented as "gBVft" in the figure).

[0084] After confirming the sequencing results were correct, the linearized plasmid pPK2-Bar was digested with Dpn I to eliminate the influence of the template strand. The concentrations of each PCR product (gBVft carrying the adapter) and the digestion product (linearized plasmid pPK2-Bar) were measured using a micro-ultraviolet spectrophotometer. The ligation reaction system was prepared according to the instructions of the ClonExpress MultiS One Step Cloning Kit (Vazyme, catalog number C113-01) to construct the fungal expression vector pPK2-Bar-gBVft (structural schematic shown in Figure 1). Figure 5 (As shown).

[0085] II. Construction of engineered bacteria

[0086] A fungal transformation system was constructed using Agrobacterium tumefaciens mediated transformation (ATMT): plasmid pPK2-Bar-gBVft was transformed into AGL-1 Agrobacterium tumefaciens competent cells, and the cells were transfected onto LB agar plates (kana). + After confirming the bands were correct and the PCR product sequencing was accurate, AGL-1 *Agrobacterium tumefaciens* transformed with plasmid pPK2-Bar-gBVft was obtained. A culture of AGL-1 *Agrobacterium tumefaciens* transformed with plasmid pPK2-Bar-gBVft was prepared and co-cultured with a suspension of *Beauveria bassiana* conidia. The first and fastest-growing colonies were preferentially selected and transferred to 1 / 4 SDAY plates (PPT). + Chloramphenicol+ Perform two screenings (e.g.) Figure 6 (As shown). Four to seven days after the second screening, genomic DNA was extracted from the mycelium of the transformants and used as a template for PCR detection using primers gBVft-F / R. After confirming the bands by agarose gel electrophoresis and sequencing, positive transformants were identified.

[0087] The specific steps are as follows:

[0088] 2.1 Transformation of plasmids into AGL-1 Agrobacterium tumefaciens competent cells

[0089] 1) Take AGL-1 Agrobacterium tumefaciens competent cells stored at -70℃ and thaw them on ice.

[0090] 2) Under aseptic conditions, add 1 μg of plasmid pPK2-Bar-gBVft to a centrifuge tube containing AGL-1 Agrobacterium tumefaciens competent cells, mix gently, and incubate on ice for 5 min.

[0091] 3) Place the centrifuge tubes in liquid nitrogen for 5 minutes to freeze quickly, then quickly place them in a 37°C water bath for 5 minutes, followed by an ice bath for 5 minutes.

[0092] 4) Under aseptic conditions, add 800 μL of LB liquid medium (antibiotic-free), and incubate at 28°C and 150 rpm for 2-3 hours on a constant temperature shaker. Centrifuge at 5000 rpm for 1 minute, discard the supernatant, and collect the bacterial cells.

[0093] 5) Resuspend the bacterial cells in 100 μL of LB liquid medium (antibiotic-free), and spread an appropriate amount of the bacterial suspension onto LB solid medium (kanamycin). + Incubate on a plate at 28°C inverted for 2-3 days.

[0094] 6) Select a single colony of the transformant for colony PCR. After confirming that the band size is correct by electrophoresis, perform sequencing verification. If the sequencing verification is correct, AGL-1 Agrobacterium tumefaciens transformed with plasmid pPK2-Bar-gBVft is obtained. Prepare the bacterial culture as glycerol bacteria (15% glycerol) and store it at -80℃.

[0095] 2.2 Agrobacterium tumefaciens-mediated genetic transformation of Beauveria bassiana

[0096] 2.2.1 Preparation of Beauveria bassiana conidia suspension

[0097] 1) Scrape an appropriate amount of Beauveria bassiana ARSEF 2860 cells from a SDAY plate that has been cultured for about 10 days into 5 mL of sterile water containing 0.05% Tween-20 and vortex to disperse for 2-3 min.

[0098] 2) Take an appropriate amount of sterile glass wool and fill the bottom of a sterile syringe. Filter the suspension and collect it in a sterile centrifuge tube to remove mycelium. Centrifuge at 8000 rpm for 3 minutes, discard the supernatant, and collect the spores.

[0099] 3) Resuspend the spores in sterile water, centrifuge at 8000 rpm for 3 min, discard the supernatant, and collect the spores again.

[0100] 4) Resuspend the spores in IM liquid medium to obtain a Beauveria bassiana conidia suspension, and calculate the spore concentration using a hemocytometer.

[0101] 2.2.2 Transformation and Transformant Screening of Beauveria bassiana

[0102] 1) Add 3 mL of LB liquid medium (kanamycin) to AGL-1 Agrobacterium tumefaciens transformed with plasmid pPK2-Bar-gBVft. + Incubate at 28℃ and 200rpm for 16-24 hours with constant temperature shaking.

[0103] 2) Determine the OD of the bacterial culture 660 Afterwards, centrifuge at 5000 rpm for 3 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in 1M liquid medium and adjust the OD. 660 The culture was carried out at 0.15°C for 6 hours with constant temperature shaking at 28°C and 200 rpm to obtain AGL-1 Agrobacterium tumefaciens culture transformed with plasmid pPK2-Bar-gBVft.

[0104] 3) Take AGL-1 Agrobacterium tumefaciens bacterial suspension transformed with plasmid pPK2-Bar-gBVft and Beauveria bassiana conidia suspension (concentration 10). 4 Mix 100 μL of each (each cell / mL) thoroughly in a centrifuge tube to obtain a mixture.

[0105] 4) Spread the mixture onto IM solid culture medium plates lined with cellophane (the cellophane, which has been autoclaved, is moistened with sterile water and then laid flat on the culture medium), and incubate at 26°C for 48 hours.

[0106] 5) Remove the cellophane from the IM solid medium and spread it evenly on 1 / 4 SDAY medium (containing 100 μg / mL glufosinate and 25 μg / mL chloramphenicol). Incubate at 26°C for about 5 days until resistant colonies appear.

[0107] 6) Perform secondary screening, picking resistant colonies and transferring them to 1 / 4 SDAY medium (containing 100 μg / mL glufosinate and 25 μg / mL chloramphenicol), incubating at 26℃ for approximately 5 days. Results are as follows... Figure 6 As shown.

[0108] 7) Select normally growing resistant colonies, extract genomic DNA, and perform PCR verification using primers Bbsp-F / VRF1-R. If the band size is correct, it is preliminarily determined to be a positive transformant.

[0109] PCR verification electrophoresis results are as follows Figure 7 The image shows the PCR product bands of eight positive transformants, each 1485 bp in size, consistent with the target band size. This preliminarily confirms that the pPK2-Bar-gBVft transformation into Beauveria bassiana yields positive transformants containing the gene sequence expressing parasitic bee venom protein (referred to as Beauveria bassiana positive transformants).

[0110] 3. Detection of MmVRF1, a protein from the venom of *Beauveria bassiana*, expressed in positive transformants.

[0111] Mycelia weighing approximately 0.1 g (wet weight) were scraped from 1 / 4 SDAY medium containing the corresponding Beauveria bassiana positive transformants. The mycelia were added to SDB liquid medium and incubated at 26°C with shaking at 200 rpm for 3 days. The supernatant was collected and precipitated with an equal volume of pre-cooled acetone at -20°C for 4 hours. After centrifugation at 12000 rpm for 5 minutes, the precipitate was dissolved in an appropriate amount of ddH2O and subjected to Western blot analysis to detect MmVRF1. The results are as follows: Figure 8 As shown, lanes 1-8 are the MmVRF1 bands secreted into the culture medium during the liquid culture of 8 Beauveria bassiana positive transformants.

[0112] Eight recombinant Beauveria bassiana engineered strains capable of expressing MmVRF1 were finally obtained. The first, second, and seventh strains with high MmVRF1 expression levels in the Western blot results were selected for further determination of growth characteristics.

[0113] 4. Determination of growth characteristics of recombinant Beauveria bassiana expressing MmVRF1

[0114] 4.1 Determination of sporulation rate of recombinant Beauveria bassiana expressing MmVRF1

[0115] Conidia of recombinant Beauveria bassiana expressing MmVRF1 were formulated into 1×10⁻⁶ spores. 7 A spore suspension of 1 spore / mL was dropped 1 μl into the center of a PDA solid medium plate and incubated upside down in a 26°C incubator for 14 days. Colonies were punched from the center to halfway point of the sporulation region using a 5 mm diameter punch and placed in 5 mL of sterile water containing 0.05% (v / v) Tween-20. The plates were vortexed for 2 min, and the sporulation yield was measured and calculated using a hemocytometer. The sporulation yield was then recalculated to the required medium area (cm²). 2The amount of spores produced was measured. The 1st, 2nd, and 7th strains with high VRF1 expression levels in the Western blot results were named Bb-VRF1-1, Bb-VRF1-2, and Bb-VRF1-7, respectively. Three PDA plates were selected for each strain to detect spore production. Wild-type *Beauveria bassiana* ARSEF 2860 (abbreviated as Bb2860) was used as a control for comparison. The results are as follows: Figure 9 As shown, only Bb-VRF1-2 had a significantly lower sporulation rate than Bb2860, while the sporulation rates of Bb-VRF1-1 and Bb-VRF1-7 were not significantly different from those of Bb2860.

[0116] 4.2 Spore germination rate determination

[0117] After the recombinant Beauveria bassiana strain expressing MmVRF1 produced spores, an appropriate amount of spores was selected and inoculated into SDB liquid medium. The medium was incubated at 26°C with shaking at 200 rpm for 24 hours, followed by microscopic examination. Three shake tubes were taken from each strain, and each tube was examined using a hemocytometer with three fields of view to calculate the germination rate. Results are as follows: Figure 10 As shown, only the spore germination rate of Bb-VRF1-2 was significantly lower than that of Bb2860, while the spore germination rates of Bb-VRF1-1 and Bb-VRF1-7 were not significantly different from those of Bb2860.

[0118] 4.3 Determination of colony growth rate

[0119] Three single colonies of recombinant Beauveria bassiana expressing MmVRF1 were selected and inoculated into PDA medium, with Beauveria bassiana (ARSEF 2860) serving as a control for comparison. Colony diameter was measured daily using calipers for 15 days. Colony growth rate (mm / d) = colony diameter / 15. Results are as follows: Figure 11 As shown, the colony growth rates of Bb-VRF1-1, Bb-VRF1-2, and Bb-VRF1-7 were not significantly different from those of Bb2860.

[0120] 4.4 Colony morphology observation

[0121] Three solid culture media were prepared: ① Czapek-Dox Medium (Coolaber, MM1010-250g); ② 1 / 4 SDAY Medium (Hopebio, HB0235-6) containing only 1 / 4 of the components; ③ Potato Dextrose Agar Medium (Coolaber, PM0520-250g). 1 μl of a spore suspension of Bb2860 and Bb-VRF1 was dropped into the center of an agar plate and incubated at 26°C for 20 days. The morphology of the colonies on the different media was observed. Figure 12 As shown, the colony morphology of Bb2860 and Bb-VRF1 was consistent on the three solid culture media, indicating that the introduction of parasitic bee venom protein did not affect the colony morphology of Bb-VRF1.

[0122] Taking into account the expression level of parasitic bee venom protein, sporulation rate, germination rate, colony growth rate, and colony morphology, a recombinant Beauveria bassiana strain Bb-VRF1-1 (abbreviated as BbVRF01) expressing MmVRF1 was finally obtained with stable growth. The BbVRF01 strain was deposited on July 14, 2021, with the accession number CGMCC NO.23043, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing).

[0123] Example 2: Insecticidal efficiency determination of recombinant Beauveria bassiana expressing MmVRF1

[0124] The insecticidal efficiency was determined using the immersion method, as follows: Conidial suspensions of wild-type Beauveria bassiana ARSEF 2860 (Bb2860, hereinafter referred to as Bb2860) (control group) and recombinant Beauveria bassiana BbVRF01 (treatment group) (BbVRF01) were prepared, and the suspension concentration was adjusted to 1×10⁻⁶. 8 Spores / mL. Immerse the test insects in the suspension for 20 seconds, then remove them and blot off excess water with absorbent paper. Each treatment has three replicates, with 24 test insects per replicate. Place the test insects in an artificial climate chamber at 26℃, 80% relative humidity, and a photoperiod L:D = 14h:10h. Clean the rearing boxes regularly and change the feed as needed. Observe every 24 hours. Remove immobile insect carcasses and place them separately on a culture plate for further observation. Statistical analysis and survival curves are generated. Estimate the lethal time based on the data from different treatments.

[0125] The insecticidal efficiency was determined using an injection method, as follows: Conidial suspensions of wild-type *Beauveria bassiana* ARSEF 2860 (Bb2860) (control group) and recombinant *Beauveria bassiana* BbVRF01 (treatment group) were prepared, and the suspension concentration was adjusted to a suitable level. Using a microsyringe, 5 μl of the spore suspension was slowly injected into the base of the second pair of tarsi of test insects that had been anesthetized on ice. Three replicates were performed for each treatment, with 24 test insects per treatment. The injected test insects were placed in an artificial climate chamber at 26°C, 50% relative humidity, and a photoperiod L:D = 14h:10h. The rearing boxes were cleaned regularly, and the feed was changed promptly. Observations were conducted every 24 hours. Motionless insect carcasses were removed and placed separately on culture plates for further observation. Survival curves were statistically analyzed and plotted. Estimates of the time to lethality were calculated based on the data from different treatments.

[0126] I. Insecticidal efficacy determination against Helicoverpa armigera (cotton bollworm)

[0127] The third instar cotton bollworms on day 1 were infected using the immersion method. Wild-type *Beauveria bassiana* (ARSEF 2860, abbreviated as Bb2860) was used as the control group (Bb2860), and recombinant *Beauveria bassiana* (BbVRF01) was used as the treatment group (BbVRF01) for insecticidal efficiency determination. The spore suspension was adjusted to 1×10⁻⁶. 8 Spores / ml, each test worm was soaked for 20 seconds, then removed, fed normally, and subsequently observed. Survival curves are shown below. Figure 13 As shown, compared with the control group Bb2860, the survival curve of cotton bollworm in the treatment group BbVRF01 was steeper and the mortality rate was higher, indicating that the secretion of parasitic bee venom protein can enhance the lethality of Beauveria bassiana against cotton bollworm.

[0128] Fifth-instar cotton bollworms on day 1 were infected using an injection method. Wild-type *Beauveria bassiana* (ARSEF 2860, abbreviated as Bb2860) was used as the control group (Bb2860), and recombinant *Beauveria bassiana* (BbVRF01) was used as the treatment group (BbVRF01) for insecticidal efficiency determination. The spore suspension was adjusted to 5 × 10⁻⁶. 6 Spores / ml. Infection was performed by injecting 5 μl of suspension into each test worm using a microsyringe. Patients were fed normally and subsequently monitored. Survival curves are shown below. Figure 14 The symptoms and symptoms are as follows: Figure 17As shown in Figure A, compared with the control group Bb2860, the survival curve of cotton bollworm in the treatment group BbVRF01 was steeper and the mortality rate was higher, indicating that the secretion of parasitic bee venom protein can enhance the lethality of Beauveria bassiana against cotton bollworm.

[0129] II. Insecticidal efficacy determination against fall armyworm (Spodoptera frugiperda)

[0130] Fall armyworms in their 6th instar, day 1, were infected using an injection method. Wild-type *Beauveria bassiana* (ARSEF 2860, abbreviated as Bb2860) was used as the control group (Bb2860), and recombinant *Beauveria bassiana* (BbVRF01) was used as the treatment group (BbVRF01). Insecticidal efficacy was determined by adjusting the spore suspension concentration to 2 × 10⁻⁶. 6 Spores / ml. Infection was performed by injecting 5 μl of suspension into each test worm using a microsyringe. Patients were fed normally and subsequently monitored. Survival curves are shown below. Figure 15 As shown, the symptoms are as follows Figure 17 As shown in Figure B, compared with the control group Bb2860, the survival curve of fall armyworm in the treatment group BbVRF01 was steeper and the mortality rate was higher, indicating that the secretion of parasitic wasp venom protein can enhance the lethality of Beauveria bassiana against fall armyworm.

[0131] III. Insecticidal efficacy determination against diamondback moth Plutella xylostella

[0132] Diamondback moths on day 1 of the third instar were infected using the immersion method. Wild-type Beauveria bassiana (ARSEF 2860, abbreviated as Bb2860) was used as the control group (Bb2860), and recombinant Beauveria bassiana (BbVRF01) was used as the treatment group (BbVRF01) for insecticidal efficiency determination. The spore suspension was adjusted to 1×10⁻⁶. 8 Spores / ml, each test worm was soaked for 20 seconds, then removed, fed normally, and subsequently observed. Survival curves are shown below. Figure 16 The symptoms and symptoms are as follows: Figure 17 As shown in Figure C, compared to the treatment group BbVRF01, the survival curve of the diamondback moth in the control group Bb2860 was steeper and the mortality rate was higher. This suggests that the diamondback moth may be less closely related to the fall armyworm than to the cotton bollworm, so the secretion of parasitic wasp venom protein could not enhance the lethality of Beauveria bassiana against the diamondback moth. This also indirectly proves that using parasitic wasp venom to enhance the virulence of biocontrol bacteria is less likely to miss the target and will not harm beneficial insects in the application environment.

[0133] IV. Wild-type Beauveria bassiana ARSEF 2860 (abbreviated as Bb2860) and engineered recombinant Beauveria bassiana (BbVRF01) against Helicoverpa armigera LD 50 With LT 50 Measurement

[0134] The lethality of Bb2860 and BbVRF01 against 5th instar cotton bollworms was further determined using an injection method at three concentration gradients. Based on the measured data, the lethality (LD50) was assessed using Probit regression. 50 With LT 50 Perform the calculations (using SPSS Ver. 21 software).

[0135] Table 3 shows the results of LLD (Low Damage) treatment of 5th instar cotton bollworms infected by injection using Bb2860. 50 The experimental data obtained are shown in Table 4. Table 4 presents the LD50 data obtained using BbVRF01 on 5th instar cotton bollworms infected via injection. 50 The experimental data obtained were used. The LD50 of Bb2860 against 5th instar cotton bollworms was calculated using Probit regression. 50 2.15×10 3 One spore, BbVRF01, LD50 against 5th instar cotton bollworm. 50 It is 1.29 × 10 3 One spore. LD of BbVRF01 50 It is 1.67 times lower than Bb-2860.

[0136] Table 5 shows the results of LT (Low Temperature) treatment using Bb2860 for 5th instar cotton bollworm infection via injection. 50 The experimental data obtained are shown in Table 6. Table 6 presents the results of LT assays using BbVRF01 on 5th instar cotton bollworms infected via injection. 50 The experimental data obtained were measured. Based on Probit regression calculations, the LT (Long-Term Intensity) of Bb2860 against 5th instar cotton bollworms was calculated. 50 The LT (Long-Term Inhibition) of BbVRF01 against 5th instar cotton bollworms was 5.327 days. 50 The value is 3.432. The LT value of BbVRF01 50 It is 1.55 times shorter than Bb-2860.

[0137] Table 3. LD50 of Bb2860 against bollworm 50 Measurement of experimental data

[0138]

[0139] Table 4. LD50 of BbVRF01 against bollworm 50 Measurement of experimental data

[0140]

[0141] Table 5. Bb2860 against bollworm LT 50 Measurement of experimental data

[0142]

[0143] Table 6. BbVRF01's effect on bollworm LT 50 Measurement of experimental data

[0144]

[0145] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A recombinant Beauveria bassiana, characterized in that, The recombinant Beauveria bassiana can express parasitic bee venom protein, and its preservation number is CGMCC NO.23043.

2. The recombinant Beauveria bassiana according to claim 1, characterized in that, The recombinant Beauveria bassiana is derived from Beauveria bassiana and integrates an exogenous gene encoding a parasitic bee venom protein. The Beauveria bassiana is a wild-type Beauveria bassiana. Beauveria bassiana ARSEF 2860, the gene encoding parasitic bee venom protein is the nucleotide sequence shown in positions 67-1449 of SEQ ID NO.

1.

3. The recombinant Beauveria bassiana according to claim 2, characterized in that, The recombinant Beauveria bassiana contains an endogenous gene encoding the chit1 signal peptide of Beauveria bassiana, which is integrated before the exogenous gene encoding parasitic bee venom protein. The gene encoding the chit1 signal peptide of Beauveria bassiana has the nucleotide sequence shown in SEQ ID NO.

3.

4. An insecticide, characterized in that, The insecticide includes any of the recombinant Beauveria bassiana as described in claims 1-3, or its progeny, or its conidia, or its mycelium, or any combination thereof.

5. The insecticide according to claim 4, characterized in that, It also includes pesticide-acceptable carriers.

6. The insecticide according to claim 4, characterized in that, The insecticide is used to control cotton bollworm and / or fall armyworm.

7. The use of any of the recombinant Beauveria bassiana according to claims 1-3, or the offspring of the recombinant Beauveria bassiana, or the conidia produced therefrom, or the mycelium produced therefrom, or any combination thereof, in the preparation of an insecticide.

8. A method for preparing recombinant Beauveria bassiana, characterized in that, The steps include: operatively introducing the gene encoding the chit1 signal peptide of Beauveria bassiana and the gene encoding parasitic bee venom protein into Beauveria bassiana. The gene encoding the parasitic bee venom protein is the nucleotide sequence shown in positions 67-1449 of SEQ ID NO.1; The gene encoding the chit1 signal peptide of Beauveria bassiana is the nucleotide sequence shown in SEQ ID NO.

3.

9. The preparation method according to claim 8, characterized in that, The introduction was performed using Agrobacterium-mediated transformation.

10. A method for killing insects, characterized in that, This includes the application of any of the recombinant Beauveria bassiana according to claims 1-3, or the offspring of the recombinant Beauveria bassiana, or the conidia produced therefrom or the mycelium produced therefrom, or any combination thereof, or the insecticide according to any of claims 4-6, or the recombinant Beauveria bassiana prepared by the preparation method according to claim 8 or 9.

Images