Beauveria sp. Bp-01 and recombinant strain thereof and application

Abstract

This invention belongs to the field of microbial technology, specifically disclosing a strain of *Beauveria bassiana* Bp-01, its recombinant strain, and its applications. The *Beauveria bassiana* Bp-01 provided by this invention, with the preservation number CGMCC No. 42442, exhibits strong pathogenicity against the red turpentine beetle and can be used as an excellent biocontrol strain. To further enhance its insecticidal virulence and infection rate, this invention utilizes genetic engineering techniques to successfully construct a recombinant strain Bp, with the preservation number CGMCC No. 42333, by overexpressing the venom protein SERCA in *Beauveria bassiana* Bp-01. Verification showed that compared to the wild-type strain, this recombinant strain infects *Red turpentine beetle* larvae faster, has a higher lethality, and significantly enhanced virulence, further demonstrating stronger infection and colonization capabilities. This invention can be applied to the preparation of biocontrol agents to achieve efficient control of the forest pest, the red turpentine beetle.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Beauveria bassiana Bp-01 and its recombinant strain and applications. Background Technology

[0002] Red turpentine beetle ( Dendroctonus valens *LeConte* is a major forest pest originating in North America and invasive in my country. It belongs to the genus *LeConte* of the family Scolytidae in the order Coleoptera. Dendroctonus The red turpentine beetle (Pterocarya stenoptera) is an extremely destructive invasive borer, second only to the pine wilt nematode in its harmfulness, and is considered one of the most devastating forest pests. This insect primarily damages pine species such as Pinus tabuliformis, Pinus armandii, and Pinus bungeana, with particularly severe damage to fresh stumps and harvested timber. According to relevant statistics, the invasion of the red turpentine beetle has caused the death of nearly ten million healthy trees in my country.

[0003] Currently, the control of the red turpentine beetle relies mainly on chemical and biological control methods. However, the long-term and extensive use of chemical agents can easily cause environmental pollution and lead to pesticide resistance in pests. Therefore, biological control has become an important research direction for the green control of this insect. Among them, entomopathogenic fungi are one of the most promising biological control resources for the red turpentine beetle. Therefore, it is urgent to seek or construct new, environmentally friendly, and highly effective biocontrol strains for the red turpentine beetle. Summary of the Invention

[0004] In view of this, the present invention has creatively discovered a pathogenic fungus with high lethality against the red turpentine beetle—Beauveria bassiana Bp-01. Furthermore, using this as the starting strain, the recombinant strain Bp was prepared by overexpressing the venom protein SERCA, which targets host cell immunity, resulting in a strain that is more effective and has better lethality against the red turpentine beetle.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a strain of Beauveria bassiana (Beauveria bassiana). Beauveria pseudobassiana Bp-01, this strain was isolated from Kelegou Branch of Mulanweichang State-owned Forest Farm, Chengde City, Hebei Province in June 2022, and deposited at the China General Microbiological Culture Collection Center on January 27, 2026. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and its accession number is CGMCC No. 42442.

[0006] This invention, through isolation, screening, and activity verification from the habitat of the red turpentine beetle, creatively discovered a strain of Beauveria bassiana Bp-01 with lethal activity against the red turpentine beetle. Testing showed that this strain exhibited excellent pathogenicity against the larvae of the red turpentine beetle, with a median lethal time (LT).50 The treatment time is 4.88 days, which shows that it kills insects quickly and has strong pathogenicity. It has important development value and good application prospects in the field of biological control of red turpentine beetles.

[0007] Secondly, the present invention provides a recombinant Beauveria bassiana, which uses Beauveria bassiana Bp-01 as the starting strain and overexpresses the gene encoding the parasitic bee venom protein.

[0008] Preferably, the parasitic bee venom protein is SERCA, the venom protein of the tube-shaped bee.

[0009] Preferably, the amino acid sequence of the SERCA protein from the tubercle bee is shown in SEQ ID No. 1.

[0010] Preferably, the recombinant Beauveria bassiana contains the gene encoding the chit1 signal peptide of fungi.

[0011] In order to efficiently heterologously express the parasitic wasp venom protein SERCA, the gene encoding the original signal peptide of the venom protein is replaced with the gene encoding the chitase chit1 signal peptide of fungi.

[0012] The recombinant Beauveria bassiana constructed in this invention is named recombinant strain Bp and deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42333.

[0013] Thirdly, the present invention provides a method for preparing recombinant Beauveria bassiana, the method comprising the following steps: replacing the coding gene of the signal peptide of parasitic bee venom protein with the coding gene of the chitase chit1 signal peptide of fungi, fusing it with the coding gene of parasitic bee venom protein, and introducing it into Beauveria bassiana Bp-01 to obtain recombinant Beauveria bassiana.

[0014] Preferably, the parasitic bee venom protein is SERCA, the venom protein of *Euphorbia kanssima*, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 2.

[0015] Preferably, the nucleotide sequence of the gene encoding the fungal chitase chit1 signal peptide is shown in SEQ ID No. 3.

[0016] Fourthly, the present invention provides the application of the above-mentioned Beauveria bassiana Bp-01 or the recombinant Beauveria bassiana in the biological control of red turpentine beetles.

[0017] Fifthly, the present invention provides a biocontrol agent comprising the above-mentioned Beauveria bassiana Bp-01 or the recombinant Beauveria bassiana.

[0018] The biocontrol agent provided by this invention can be used to control the major forest pest, the red turpentine beetle. It not only has high pathogenicity and precise targeting against the red turpentine beetle, but also has the advantages of being environmentally friendly, highly safe, and not easily inducing pesticide resistance in pests, and has important application value.

[0019] The *Beauveria bassiana* Bp-01 strain provided by this invention exhibits strong pathogenicity against *B. rubrum*, making it an excellent biocontrol strain. To further enhance the insecticidal virulence and infection rate of the strain, this invention utilizes genetic engineering techniques to combine the host cell-targeting venom protein SERCA with the advantages of biocontrol bacteria, such as ease of cultivation and large-scale production. By overexpressing the venom protein SERCA in *Beauveria bassiana* Bp-01, a recombinant strain Bp was successfully constructed. Verification showed that this recombinant strain can efficiently and stably express the exogenous venom protein SERCA. Bioassay results indicate that, compared to the wild-type strain Bp-01, the recombinant strain Bp infects *B. rubrum* larvae faster, has a higher lethality, and exhibits significantly enhanced virulence. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the phylogenetic tree of strain 01 in Example 1 of the present invention; Figure 2 This is a microscopic image of strain 01 in Example 1 of the present invention; Figure 3 This is a colony morphology diagram of strain 01 in Example 1 of the present invention; Figure 4 This is the colony growth rate curve of Beauveria bassiana Bp-01 in Example 1 of the present invention; Figure 5 This is a comparison chart of survival curves of different groups of red turpentine beetles in Example 1 of the present invention; Figure 6 The results of the proteomic analysis of the tubercle bee in Example 2 of this invention; Figure 7 This is a phylogenetic tree and protein domain diagram of the venom protein SERCA in Example 2 of the present invention; Figure 8 The results of Western blot analysis of the expression and purification of the venom protein SERCA in Example 2 of this invention; Figure 9This is a schematic diagram of the plasmid map of the recombinant plasmid pPK2-Bar-EGFP-gBSmt in Example 2 of the present invention; Figure 10 The PCR verification results are for the 11 transformants in Example 2 of this invention; Figure 11 This is an electrophoresis diagram showing the DNA and RNA detection of Beauveria bassiana Bp-01 and recombinant strain Bp in Example 2 of this invention; wherein... Figure 11 A represents the electrophoresis pattern for DNA verification, with primers SERCA-F / R; Figure 11 B represents the electrophoresis image for RNA validation, using the internal control primer RT-β-tubulin-F / R; Figure 11 C represents the electrophoresis pattern for RNA validation, using the internal control primer RT-SERCA-F / R; Figure 12 The results of Western blot analysis to determine whether the recombinant strain Bp in Example 2 of this invention expresses the venom protein SERCA. Figure 13 This is a colony morphology diagram of the recombinant strain Bp in Example 2 of the present invention; Figure 14 The above are statistical results of colony growth rate, sporulation yield, and spore germination rate of *Beauveria bassiana* Bp-01 and recombinant strain Bp in Example 2 of this invention; wherein, Figure 14 A represents the statistical results of the colony growth rate of the two strains. Figure 14 B represents the sporulation results of the two strains. Figure 14 C represents the statistical results of spore germination rates for the two strains; Figure 15 This is a comparison of the survival curves of different groups of red liptinous bark beetles in the effect examples of this invention; Figure 16 This is a typical example of a red turpentine beetle that died after 8 days of infection with two strains, as shown in the efficacy examples of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0024] The specific information of the primer sequences used in this invention is shown in Table 1.

[0025] Table 1 Primer Sequences

[0026] Example 1 This embodiment provides the identification and preservation of Beauveria bassiana Bp-01, which was isolated from the Kelegou Branch of the Mulanweichang State-owned Forest Farm in Chengde City, Hebei Province. Through comprehensive analysis of cell morphology, ITS gene sequence, and other data, strain 01 was identified as Beauveria bassiana (Bp-01). Beauveria pseudobassiana Therefore, the strain was named Beauveria bassiana Bp-01.

[0027] I. Isolation of pathogenic bacteria Using tweezers, pick up a small amount of collected tunnel borer debris, shake it to scatter each sample onto PDA medium, and prepare three replicate plates using the same medium. Incubate at 25°C. Once the mycelium grows on the plates, inoculate it onto new plates for purification culture.

[0028] II. Identification of Fungi After culturing the purified fungi on a larger scale, cell suspensions or spore suspensions were prepared, and the lethality of different fungi against the larvae of the red turpentine beetle was preliminarily determined. Preliminary experiments showed that strain O1 could kill the larvae of the red turpentine beetle. Therefore, further identification and research are needed.

[0029] 2.1 Molecular identification DNA was extracted and purified from the strain using a fungal genomic DNA kit, and PCR amplification was performed using the universal fungal primers ITS1 / ITS4. The PCR reaction volume was 25 μL, specifically including: 5 μL 5×Q5 Reacton Buffer, 0.25 μL Q5 High-Fidelity DNA Ploymmerase, 5 μL 5×Q5 High GC Enhance, 3 μL DNA template, 1 μL each of forward and reverse primers, and 9.75 μL Nuclease-Free Water. The amplification program was: 98℃ pre-denaturation for 2 min; 98℃ for 20 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles; final extension at 72℃ for 10 min, followed by incubation at 4℃. After agarose gel electrophoresis, the PCR products were sent to Beijing Liuhe Huada Genomics Co., Ltd. to determine the ITS gene sequence and construct a phylogenetic tree for species identification. Simultaneously, the samples were stored in PDA medium containing 25% glycerol at -80℃.

[0030] The ITS gene sequence of purified strain 01 is shown in SEQ ID No. 4.

[0031] The determined gene sequences were aligned using BLAST, and relevant sequences were selected to construct a phylogenetic tree. The phylogenetic tree of strain 01 is shown below. Figure 1 As shown.

[0032] Figure 1 The results showed that strain 01 was Beauveria bassiana (Beauveria bassiana). Beauveria pseudobassiana ).

[0033] 2.2 Morphological identification Strain 01 was classified and identified based on its cultural characteristics, hyphae, and conidia morphology. The morphology of colony hyphae, conidia, and conidiophores of strain 01 was observed and photographed under an optical microscope. The microscopic image of strain 01 is shown below. Figure 2 As shown.

[0034] Depend on Figure 2 It can be seen that the mycelium of this strain is smooth and septate. The conidia are elliptical, with a smooth surface, pointed at one end and blunt at the other.

[0035] Strain 01 was grown in PDA medium for 30 days, and its colony morphology was observed. The colony morphology diagram is shown below. Figure 3 As shown. By Figure 3 It can be seen that the front of the colony is white, slightly yellowish in the middle half, and has fuzz. The center of the colony is slightly raised and the edge is irregular. The color of the back of the colony changes from light to dark from the edge to the center. The colony changes from yellowish-brown to blackish-brown in the middle third. The culture medium is purplish-red.

[0036] Based on comprehensive analysis of the strain's morphology, ITS gene sequence, and the constructed developmental tree, strain 01 was identified as Beauveria bassiana (Beauveria bassiana). Beauveria pseudobassiana Therefore, the strain was named *Beauveria bassiana* Bp-01. It was deposited on January 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42442.

[0037] 1.3 Colony growth rate and sporulation rate One plate of cultured *Beauveria bassiana* Bp-01 was punched with a 6mm punch and inoculated onto PDA medium. Six replicates were performed, and the plates were incubated at 25°C. Colony diameter and sporulation were measured every 5 days. The colony growth rate curve is shown below. Figure 4 As shown in Table 2, the sporulation yield was determined.

[0038] Table 2. Results of sporulation of Beauveria bassiana Bp-01 at different time points

[0039] Measurements showed that strain 01 exhibited a rapid increase in average colony diameter, reaching 4.44 cm after 20 days. The average sporulation on PDA medium was 2.23 × 10⁻⁶ on day 5. 7pcs / cm 2 As the culture time increased, the sporulation rate continuously increased, reaching an average of 2.32 × 10⁻⁶ on day 10. 8 pcs / cm 2 .

[0040] 1.4 Pathogenicity analysis of red turpentine beetle larvae Spore suspensions of *Beauveria bassiana* Bp-01 and *Beauveria bassiana* ACCC30107 were prepared using an appropriate amount of 0.05% Tween-20 aqueous solution. The suspensions were vortexed for 3 min, filtered three times with sterile absorbent cotton, diluted with sterile water, and observed and counted under a microscope using a hemocytometer. The spore concentration was calculated and diluted to 3 × 10⁻⁶. 7 The mortality rate against *C. rubrum* larvae was determined by measuring the number of insects per mL. The specific method is as follows: Ninety 2nd-3rd instar larvae of the red turpentine beetle, all of uniform size, were randomly divided into two groups of 45 larvae each. They were placed in a tea strainer and soaked in spore suspensions of two fungi for 30 seconds each. After soaking, they were removed and placed on sterile filter paper to absorb excess moisture. They were then transferred to 35mm culture dishes containing pre-packaged artificial feed, with the bottom covered with sterile, water-sprayed filter paper for humidity control. After treatment, they were placed in an artificial climate chamber (temperature 25±1℃, humidity ≥75%). Infection and mortality rates of the red turpentine beetle were recorded daily until all larvae died. The corrected mortality rate and stunted larvae rate were calculated, and survival curves were plotted. A comparison of survival curves for different groups of red turpentine beetles is shown in the figure below. Figure 5 As shown.

[0041] like Figure 5 As shown, larvae of the red turpentine beetle infected with either *Beauveria bassiana* Bp-01 or *Beauveria bassiana* ACCC30107 began to die on day 3, and all larvae in both groups died 12 days after infection. This indicates that both *Beauveria bassiana* Bp-01 and *Beauveria bassiana* ACCC30107 are lethal to *Beauveria bassiana* larvae, and there is no significant difference between the two.

[0042] Example 2 This invention provides a method for preparing recombinant Beauveria bassiana, the method comprising the following steps: 1. Annotation and screening of parasitic bee venom proteins The female adults of Sclerodermus guani were frozen with liquid nitrogen and ground on ice, then placed into a 1.5 mL centrifuge tube containing 600 μL of Trizol reagent to extract total RNA. Non-reference transcriptome sequencing was performed with 3 biological replicates set. The female adults of Sclerodermus guani were dissected in phosphate buffer (PBS, pH 7.4) under a stereomicroscope. Their venom glands and non-venom gland tissues were distinguished, washed several times in PBS respectively and transferred into 1.5 mL centrifuge tubes, and the centrifuge tubes were always placed in liquid nitrogen during the dissection process. 3 biological replicates were set for both the venom gland and non-venom gland tissues respectively for proteome sequencing. Based on the results of non-reference transcriptome and proteome sequencing, BLASTP alignment was performed in the NCBI database to identify venom protein-related genes and conduct functional annotation. The analysis results of the proteome of Sclerodermus guani are as Figure 6 shown. As Figure 6 can be seen, 4 kinds of venom proteins are highly specifically expressed in the venom gland.

[0043] Furthermore, through homologous sequence alignment analysis, it was found that the venom protein SERCA could specifically target the cellular immunity of the host. Among them, the phylogenetic tree and schematic diagram of the protein domain of the venom protein SERCA are as Figure 7 shown, and the amino acid sequence of the venom protein SERCA is as shown in SEQ ID No.1.

[0044] The female adults of Sclerodermus guani were collected for the expression and purification of the venom protein SERCA, and the expression and purification effects were verified by Western blot experiment. The Western blot detection results of the expression and purification of the venom protein SERCA are as Figure 8 shown, where GST-SERCA represents the fusion protein constructed by the GST tag and the venom protein SERCA of Sclerodermus guani. As Figure 8 can be seen, the migration position of the GST-SERCA fusion protein band is slightly higher, presumably due to the conformational effect of the GST tag and the hydrophobicity of the protein SERCA itself, resulting in a reduced migration rate in SDS-PAGE. The theoretical molecular value of the GST-SERCA fusion protein is about 138.4 kDa, which is composed of the venom protein SERCA with a molecular weight of about 112.4 kDa and the 26 kDa GST tag together. Through Western blot detection, the venom protein SERCA of Sclerodermus guani was successfully expressed and purified.

[0045] 2. Construction of recombinant plasmid In order to express the venom protein SERCA in fungi, the present invention used the Agrobacterium vector pPK2-Bar-EGFP derived from the backbone plasmid pPK2 to transform Beauveria bassiana. Using ClonExpress ®Seamless cloning was performed using the UltraOne Step Cloning Kit (Vazyme, C115). First, the linearized vector PUC19 was obtained using the SpeI restriction endonuclease, and then cloning was performed according to ClonExpress... ® The UltraOne Step Cloning Kit instructions describe how to configure the ligation reaction system. The gene encoding the chit1 signal peptide of Beauveria bassiana ARSEF 2860 (nucleotide sequence shown in SEQ ID No. 3), the gene encoding the venom protein SERCA (nucleotide sequence shown in SEQ ID No. 2), and the gene encoding the fluorescent protein mCherry (nucleotide sequence shown in SEQ ID No. 5) of Bbsp (nucleotide sequence shown in SEQ ID No. 3), are ligated into plasmid PUC19 to obtain the recombinant plasmid PUC19-Bbsp-SERCA-mCherry.

[0046] The pPK2-Bar-EGFP vector was linearized using the SpeI restriction endonuclease. The target fragment Bbsp-SERCA-mCherry (BSm) was amplified using the recombinant plasmid PUC19-Bbsp-SERCA-mCherry as a template. ClonExpress was used to further refine the linearized pPK2-Bar-EGFP vector. ® The UltraOne Step Cloning Kit ligates the target fragment BSm, the eukaryotic promoter gpdA Promoter, and the terminator trpC Terminator into the linearized vector pPK2-Bar-EGFP to obtain the recombinant plasmid pPK2-Bar-EGFP-gBSmt. This plasmid is then transformed into *E. coli* DH5α competent cells, clones are picked, and the recombinant plasmid is amplified using primers QSERCA-F / R. This confirms the presence of the recombinant plasmid pPK2-Bar-EGFP-gBSmt expressing the venom protein SERCA. A schematic diagram of the plasmid pPK2-Bar-EGFP-gBSmt is shown below. Figure 9 As shown.

[0047] 3. Construction of recombinant strains Transformant preparation: Competent Agrobacterium tumefaciens cells were removed from a -80°C freezer and thawed on ice for 10 min. The competent cells were then uniformly mixed with 10 μL of recombinant plasmid pPK2-Bar-EGFP-gBSmt and added to a pre-chilled electroporation cuvette. The mixture was incubated on ice for 5 min and then electroporated at 2.5 kV. Immediately after electroporation, 900 μL of YEB liquid medium was added, transferred to centrifuge tubes, and the cells were thawed at 28°C and 150 rpm for 150 min. The cells were then centrifuged at 6000 rpm for 1 min to obtain bacterial cells, which were spread onto YEB solid medium containing the appropriate antibiotics and incubated at 28°C for 48-72 h. Transformants were picked and added to 5 mL of YEB liquid medium, and incubated overnight at 28°C and 220 rpm. Positive transformants were verified by PCR and stored for later use. In this invention, 11 transformants were selected for PCR verification. The PCR verification results are shown below. Figure 10 As shown. By Figure 10 It can be seen that transformants 1, 2, 3, 4, 5, and 6 are all the same size as the target slice and are positive transformants, which can be used for subsequent transformations.

[0048] Preparation of spore suspension: In a clean bench, elute the conidia of *Beauveria bassiana* Bp-01 from PDA plates cultured for 10-15 days using sterile water containing 0.05% Twenn-20 to centrifuge tubes. Vortex for 3 min, filter with sterile absorbent cotton to remove hyphae, and collect the filtrate in new sterile centrifuge tubes. Repeat three times. Centrifuge at 12,000 rpm for 3 min, discard the supernatant, collect the spores, resuspend the spores in an appropriate amount of sterile water, count the spore count using a hemocytometer, and adjust the concentration to 1×10⁻⁶. 4 A spore suspension of conidia / mL.

[0049] Fungal transformation: Take 200 μL of the prepared positive transformants and add them to 3 mL of YEB liquid medium containing double antibiotics (the medium contains carbenicillin and kanamycin at a final concentration of 50 μg / mL each). Incubate overnight at 28°C and 200 rpm. Take 200 μL of the overnight culture and add another 3 mL of YEB liquid medium for secondary activation. Incubate overnight at 28°C and 200 rpm on a shaker to obtain activated bacterial culture. Pour the activated bacterial culture into a centrifuge tube, centrifuge at 7000 rpm for 3 min, discard the supernatant, collect the bacterial cells, wash twice with sterile water, and resuspend the bacterial cells in 15 mL of 1M liquid medium to allow OD to adjust. 600 Between 0.15 and 0.2 (OD in this invention) 600 (0.18), and cultured in the dark at 28℃ and 200rpm in a shaker until OD 600 The value was 0.6, and it was recorded as SERCA-Agrobacterium bacterial suspension.

[0050] Take 200 μL each of SERCA-Agrobacterium bacterial suspension and prepared spore suspension, mix thoroughly, and then take 100 μL to spread on IM solid medium. Incubate at 28°C for 48 h. Take slightly more resistant PDA medium (containing 600 μg / mL thiazomycin and 200 μg / mL glufosinate) than the lower co-cultured IM plate and cover it on the IM plate. Incubate at 25°C for 5-10 days until resistant colonies (suspected transformants) appear. Use an inoculation needle to pick a small amount from the edge of the suspected transformant and transfer it to the same resistant PDA medium for a second screening. Pick resistant colonies from the second screening plate, shake them with PDB liquid medium, and inoculate them onto PDA plates. Mark the corresponding positions on the back of the second screening plate.

[0051] After the spores had grown well on the PDA plates, the mycelia were used for genome extraction and PCR verification. After the bacteria were shaken in resistant PDB liquid medium (containing 200 μg / mL glufosinate), RNA was extracted and verified by RT-PCR with β-tublin as an internal control, resulting in a recombinant strain Bp expressing the venom protein SERCA.

[0052] DNA and RNA were extracted from *Beauveria bassiana* Bp-01 and the recombinant strain Bp, respectively, and amplified by PCR. The amplification products were then detected by agarose gel electrophoresis. The electrophoresis results of DNA and RNA detection in *Beauveria bassiana* Bp-01 and the recombinant strain Bp are shown below. Figure 11 As shown, where Figure 11 A represents the electrophoresis pattern for DNA verification, with primers SERCA-F / R; Figure 11 B represents the electrophoresis image for RNA validation, using the internal control primer RT-β-tubulin-F / R; Figure 11 C represents the electrophoresis image for RNA validation, using the internal reference primer RT-SERCA-F / R.

[0053] Depend on Figure 11 It can be seen from DNA and RNA verification that the recombinant strain Bp carries the gene that heterologously expresses the venom protein SERCA.

[0054] This invention further verifies, using Western blot experiments, whether the recombinant strain Bp can express the venom protein SERCA. The specific steps are as follows: Mycelia of either the recombinant strain Bp or *Beauveria bassiana* Bp-01 (wet weight approximately 0.1 g) were scraped from PDA solid medium. The mycelia were added to PDB liquid medium and incubated at 25°C with shaking at 200 rpm for 3 days. The mycelia were then collected. RIPA lysis buffer and PMSF solution were mixed at a ratio of 100:1 on ice. An appropriate amount of mycelia was transferred to a 1.5 mL centrifuge tube, and 700 μL of the mixture was added. The mixture was vortexed and incubated on ice for 30 min, vortexing every 10 min. After the reaction, the mixture was centrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was transferred to a new 1.5 mL centrifuge tube. The extracted protein was then detected by Western blot analysis. The results are shown below. Figure 12 As shown.

[0055] Depend on Figure 12 It can be seen that the recombinant strain Bp shows a specific band at the 100kDa~140kDa position, while the control strain Beauveria bassiana Bp-01 does not show this band, and the theoretical molecular weight of the venom protein SERCA is about 112.4kDa. Figure 12 This demonstrates that the recombinant strain Bp achieved heterologous expression of the venom protein SERCA.

[0056] 4. Morphological characteristics of recombinant strain Bp The recombinant strain Bp was grown in resistant PDA medium (containing 200 μg / mL glufosinate) for 10 days, and its colony morphology is shown in the figure below. Figure 13 As shown.

[0057] Depend on Figure 13 As can be seen, the colonies of the recombinant strain Bp are white on the front, with villi, a slightly raised center, and irregular edges. The color on the back of the colonies changes from light to dark from the edge to the center, and the culture medium is purplish-red, similar to the wild type. The hyphae of this fungus are smooth and septate.

[0058] Microscopic examination of the conidia revealed that the conidia of this recombinant strain were elliptical, with a smooth surface, pointed at one end and blunt at the other. Green fluorescence was observed in the colony hyphae and conidia under a fluorescence microscope.

[0059] 5. Determination of colony growth rate, sporulation rate, and germination rate of recombinant strain Bp Two pre-cultured *Beauveria bassiana* strains, Bp-01 and Bp, were taken and inoculated onto PDA medium using a 6 mm punch. Six replicates were performed, with the wild-type strain serving as a control. The cultures were incubated at 25°C. The diameter of the spores was measured and the sporulation yield was recorded every 5 days. Spores from the 10-day-old recombinant strain Bp or *Beauveria bassiana* strain Bp-01 were diluted with LB liquid medium to a spore concentration of 1×10⁻⁶. 6 Spore suspensions of *Beauveria bassiana* Bp-01 and recombinant strain Bp were cultured in triplicate at 25°C and 200 rpm for 14 h, and then examined under an optical microscope. 10 μL of each suspension was examined in eight fields of view using a hemocytometer to calculate the germination rate. Statistical results of colony growth rate, sporulation yield, and spore germination rate for *Beauveria bassiana* Bp-01 and recombinant strain Bp are as follows: Figure 14 As shown in the figure. The statistical results of the colony growth rates of the two strains are as follows: Figure 14 As shown in Figure A, the statistical results of sporulation yield of the two strains are as follows: Figure 14 As shown in B, the statistical results of spore germination rates for the two strains are as follows: Figure 14 As shown in C.

[0060] Depend on Figure 14 It can be seen that there are no significant differences in colony growth rate, sporulation amount and spore germination rate between the recombinant strain Bp and Beauveria bassiana Bp-01.

[0061] On November 28, 2025, the recombinant strain Bp was deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 42333.

[0062] Example 3 This invention provides a biocontrol agent, the preparation method of which includes the following steps: After expanding the culture of *Beauveria bassiana* Bp-01 provided in Example 1, washing with 0.05% Tween-20 and filtering to prepare a conidial suspension, and preparing a 3×10⁻⁶ solution. 7 A suspension of Beauveria bassiana Bp-01 spores at a concentration of conidia / mL is used as a biocontrol agent.

[0063] Example 4 This invention provides a biocontrol agent, the preparation method of which includes the following steps: After amplifying the recombinant strain Bp provided in Example 2, the spores are washed with 0.05% Tween-20 and filtered to prepare a conidial suspension, which is then prepared into a 3×10⁻⁶ solution. 7 A suspension of recombinant strain Bp spores at a concentration of conidia / mL is used as a biocontrol agent.

[0064] Example of effect This invention investigated the pathogenicity of the recombinant strain Bp against the larvae of the red turpentine beetle. The specific details are as follows: The concentration to be prepared is 3×10 7 The lethality of recombinant strain Bp spore suspension (conidia / mL) against the larvae of Beauveria bassiana was determined using *Beauveria bassiana* Bp-01 as a control. The specific method is as follows: Ninety 2nd-3rd instar larvae of the red turpentine beetle, all of uniform size, were selected and randomly divided into two groups. The larvae were placed in a tea strainer and incubated using 3×10 [presumably referring to a specific method or technique]. 7 After soaking larvae in a spore suspension of the recombinant strain Bp (conidia / mL) or a spore suspension of Beauveria bassiana Bp-01 for 30 seconds, the larvae were removed, excess water was absorbed using sterile filter paper, and they were transferred to 35mm culture dishes containing pre-prepared artificial feed. A layer of sterile water-sprayed filter paper was placed at the bottom of the culture dish to maintain humidity. The treated larvae were then placed in an artificial climate chamber (temperature 25±1℃, humidity ≥75%) for incubation. Infection and mortality of *Beauveria bassiana* were recorded daily until all larvae died. The corrected mortality rate and larval stunting rate were calculated, and survival curves were plotted. A comparison of survival curves for different groups of *Beauveria bassiana* is shown below. Figure 15 As shown in the image. A typical example of a *Red Turtle Beetle* that died 8 days after infection with both strains is shown in the image. Figure 16 As shown.

[0065] Depend on Figure 15 It was observed that from day 3 onwards, larvae of the lipid bark beetle infected with either *Beauveria bassiana* Bp-01 or the recombinant strain Bp began to die. During the observation period from day 3 to day 10, the survival rate of larvae in the *Beauveria bassiana* Bp-01 treatment group was significantly higher than that in the recombinant strain Bp treatment group. Seven days after infection with the recombinant strain Bp, all larvae died, compared to the *Beauveria bassiana* Bp-01 treatment group (LT...). 50 Compared to 4.88 days, its LT 50 The survival time was shortened to 3.69 days. Specifically, at 4 days after infection, the survival rate of *Beauveria bassiana* in the *Beauveria bassiana* Bp-01 treatment group was 78.0%, significantly higher than that in the recombinant strain Bp treatment group (survival rate 33.3%). At 5 days after infection, the survival rate of *Beauveria bassiana* in the *Beauveria bassiana* Bp-01 treatment group was 34.1%, also significantly higher than that in the recombinant strain Bp treatment group (survival rate 4.4%).

[0066] Depend on Figure 16 It can be seen that 8 days after infection by the two strains, the surface hyphae growth of the dead red turpentine beetle larvae in the recombinant strain Bp treatment group was significantly greater than that in the Beauveria bassiana Bp-01 treatment group.

[0067] In summary, both Beauveria bassiana Bp-01 and the recombinant strain Bp are lethal to the larvae of the red turpentine beetle, and the pathogenicity of the recombinant strain Bp is significantly higher than that of the wild-type strain Bp-01, with a faster insecticidal speed.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Beauveria paravasculorum (Bp-01), characterized by: Beauveria pseudobassiana Its accession number is CGMCC No.42442. ​ 2. A recombinant Beauveria sp. characterized in that: Using Beauveria bassiana Bp-01 as the starting strain, it overexpressed the gene encoding the parasitic bee venom protein.

3. The recombinant Beauveria sp. of claim 2, wherein: The parasitic bee venom protein is SERCA, the venom protein of the tubercle bee.

4. The recombinant Beauveria bassiana as described in claim 3, characterized in that: The amino acid sequence of the SERCA venom protein from the tubercle bee is shown in SEQ ID No.

1.

5. The recombinant Beauveria bassiana as described in any one of claims 2-4, characterized in that: The recombinant Beauveria bassiana contains the gene encoding the chit1 signal peptide of fungi.

6. A method for preparing recombinant Beauveria bassiana, characterized in that: The preparation method includes the following steps: replacing the coding gene of the signal peptide of parasitic bee venom protein with the coding gene of the chitase chit1 signal peptide of fungi, fusing it with the coding gene of parasitic bee venom protein, and introducing it into Beauveria bassiana Bp-01 to obtain recombinant Beauveria bassiana.

7. The method for preparing recombinant Beauveria bassiana as described in claim 6, characterized in that: The parasitic bee venom protein is SERCA, the venom protein of *Apis kusnezoffii*, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 2; and / or The nucleotide sequence of the gene encoding the chit1 signal peptide of the fungus is shown in SEQ ID No.

3.

8. The application of Beauveria bassiana Bp-01 as described in claim 1 or the recombinant Beauveria bassiana as described in any one of claims 2-5 in the biological control of red turpentine beetles.

9. A biocontrol agent, characterized in that: Includes Beauveria bassiana Bp-01 as described in claim 1 or recombinant Beauveria bassiana as described in any one of claims 2-5.

Images