Beauveria bassiana engineering strain co-expressing two exogenous virulence proteins, construction method and application thereof

Abstract

The application discloses an engineered Beauveria bassiana strain co-expressing two exogenous virulence proteins and a construction method and application thereof. The engineered strain takes the Beauveria bassiana strain mo01 with high efficient intestinal invasion ability on anopheles as a receptor strain, and introduces the expression cassettes of trypsin-regulated inhibitor BbDPAR and Bt toxin protein BbCry10Aa or BbCry11Aa through agrobacterium-mediated transformation to obtain the engineered strain co-expressing two types of exogenous virulence proteins. The engineered strain can significantly enhance the intestinal invasion and lethal effect on Stegomyia after oral intake. The application provides a new technical scheme for developing a new type of engineered fungal insecticide.

Description

Technical Field

[0001] This invention relates to the fields of microbial genetic engineering and biological control of insect pathogenic fungi, and particularly to an engineered strain of Beauveria bassiana with co-expression of dual exogenous virulence proteins, its construction method, and its application in mosquito-borne biological control. Background Technology

[0002] Mosquito-borne diseases such as malaria continue to threaten global public health, and the long-term use of existing chemical pesticides has led to increased resistance and environmental problems. Entomopathogenic fungi are an important class of biocontrol agents, among which *Beauveria bassiana* can infect hosts through its endothelial cells; however, its application in mosquito control still faces challenges such as slow lethality, insufficient environmental adaptability, and unstable field efficacy. Recent studies have shown that some entomopathogenic fungi can establish infection through the gut, but research on highly efficient gut-infecting strains and their systemic applications in mosquito systems is still lacking. Therefore, developing novel, green mosquito control products based on gut infection mechanisms is of great significance. Summary of the Invention

[0003] In their research, the inventors discovered that the known Beauveria bassiana strain mo01 (i.e., ARSEF252 strain, see Lai Y, Zheng W, Zheng Y, et al. Unveiling a novel entrygate: Insect foregut as an alternative infection route for fungalentomopathogens[J]. The Innovation, 2024, 5(4): 100644.) can establish infection in the foregut of Anopheles mosquitoes after oral administration, and exhibits a significant dose-dependent intestinal infection ability in Anopheles mosquitoes, especially Anopheles skrjei. Furthermore, the strain not only causes death in Anopheles mosquitoes, but also produces sublethal effects such as inhibiting blood-feeding, affecting progeny development, and inhibiting the development of Plasmodium in Anopheles mosquitoes.

[0004] Based on the above findings, this invention provides a novel application of the Beauveria bassiana strain mo01 in the preparation of biological control agents for mosquito control. These biological control agents include, but are not limited to, insecticides.

[0005] Preferably, the strain mo01 is used in the form of conidia.

[0006] Furthermore, the present invention provides the application of the strain mo01 in the preparation of oral biological control agents (such as oral insecticides) that can be ingested by mosquitoes.

[0007] The biological control agent targets mosquitoes of the order Diptera, specifically Anopheles. In a specific embodiment, the Anopheles genus is Anopheles stearnii.

[0008] Furthermore, the biocontrol agent comprises an effective dose of conidia of Beauveria bassiana strain mo01, and a carrier or excipient acceptable for agricultural or sanitary insecticides. It is preferably formulated into a dosage form that can be ingested orally by mosquitoes (especially larvae or adult mosquitoes), such as: conidial suspension, wettable powder, granules, sugar solution, or attractant formulation.

[0009] Furthermore, the effective dose is a lethal dose or a sublethal dose.

[0010] This invention also provides a method for controlling mosquitoes, characterized by comprising: applying conidia of *Beauveria bassiana* strain mo01, or a biocontrol agent containing conidia of said strain mo01, to mosquito breeding grounds or feeding areas in a form that can be ingested orally by mosquitoes. Specifically, the following two control methods based on different mechanisms of action can be provided as needed: (A) Applying an effective lethal dose of the conidia of the strain mo01 or its biocontrol agent to mosquito breeding grounds or feeding grounds in a form that can be ingested orally by mosquitoes, so that after being ingested orally by mosquitoes, it can quickly establish infection in the intestines and cause death, thereby achieving a rapid decrease in population density in a short period of time.

[0011] (B) Apply an effective sublethal dose of the mo01 strain conidia or its biocontrol agent in the same manner. This method does not aim for rapid lethality, but rather sustainably suppresses mosquito population size and disease transmission efficiency from multiple dimensions, including behavior, reproduction, and population growth, by inducing sublethal effects such as inhibiting blood-feeding, inhibiting malaria parasite development, and delaying progeny development.

[0012] In practice, mosquitoes are typically orally ingested with the conidia or their biocontrol agents for 24 hours.

[0013] To further accelerate the mortality rate of target mosquitoes, this invention also provides a method for combined mosquito control, including the synergistic application of the following two infection pathways: (a) Ingesting conidia of Beauveria bassiana strain mo01 orally in mosquitoes; (b) Allow mosquitoes to come into contact with the conidia of Beauveria bassiana strain mo01 via their body surface.

[0014] This combined "internal and external attack" approach can significantly accelerate the infection process and increase mortality, thereby reducing control loopholes caused by behavioral differences. It is particularly suitable for resistance management and emergency epidemic control. The application of Beauveria bassiana strain mo01 in the preparation of biocontrol agents or public health preparations for blocking or reducing mosquito-borne diseases.

[0015] This invention also discovered that while the naturally occurring Beauveria bassiana can infect Anopheles mosquitoes via the gut, its lethality still has room for improvement. This invention specifically modifies the strain for gut infection, constructing a mo01 dual-plasmid engineered strain that expresses DPAR and either Cry10Aa or Cry11Aa. This engineered strain can infect Anopheles strideri mosquitoes via oral intestinal infection, establishing early infection in the foregut and improving mosquito-killing efficacy.

[0016] The dual plasmid co-transformation method involves the construction of two independent vectors, Agrobacterium-mediated transformation, and sequential transformation and screening in a mo01 background.

[0017] Engineered strains can be used to prepare insecticides and apply them to mosquito control.

[0018] This invention provides an engineered strain of Beauveria bassiana with co-expression of two exogenous virulence proteins (hereinafter referred to as the dual-virulence engineered strain), using Beauveria bassiana ( Beauveria bassiana mo01 is the recipient strain, simultaneously expressing BbDPAR and SP-BbCry10Aa proteins, or simultaneously expressing BbDPAR and SP-BbCry11Aa proteins. Specifically, the engineered strain simultaneously expressing BbDPAR and SP-BbCry10Aa proteins is designated mo01::DPAR / Cry10Aa, and the engineered strain simultaneously expressing BbDPAR and SP-BbCry11Aa proteins is designated mo01::DPAR / Cry11Aa.

[0019] The amino acid sequence of the BbDPAR protein is shown in SEQ ID NO. 8. The nucleotide sequence encoding the BbDPAR protein is shown in SEQ ID NO. 7.

[0020] The amino acid sequence of the SP-BbCry10Aa protein is shown in SEQ ID NO.10. The nucleotide sequence encoding the SP-BbCry10Aa protein is shown in SEQ ID NO.9.

[0021] The amino acid sequence of the SP-BbCry11Aa protein is shown in SEQ ID NO.12. The nucleotide sequence encoding the SP-Cry11Aa protein is shown in SEQ ID NO.11.

[0022] A method for constructing the dual-virulence engineered strain includes the following steps: (1) Constructing pSurGFP- Pgpd -myc- TtrpCIntermediate vector: pSurGFP was used as the vector backbone and double-digested with SpeI-HF and EcoRV-HF; mo01 genomic DNA was used as a template and amplified using BbPgpd-F2 and BbPgpd-R2 primers. Pgpd Promoter, obtain fragment A; using pBarGFP as template, amplify with TtrpC-F and TtrpC-R as primers. TtrpC Terminator, obtain fragment B; perform homologous recombination of fragments A and B with the linearized pSurGFP vector to obtain pSurGFP- Pgpd - TtrpC Synthesize a Myc-tag double-stranded DNA fragment with SpeI sticky ends and EcoRV blunt ends, and insert pSurGFP- Pgpd - TtrpC Between the SpeI and EcoRV sites, the intermediate vector pSurGFP- was obtained. Pgpd -myc- TtrpC ; (2) Constructing Cry toxin recombinant plasmids: Using the PUC57-SP-Cry10Aa or PUC57-SP-Cry11Aa synthetic plasmids as templates, the SP-BbCry10Aa or SP-BbCry11Aa fragments were amplified using PUC57-F / R primers; The amplification product was combined with pSurGFP- enzymes double-digested with SpeI-HF and EcoRV-HF. Pgpd -myc- TtrpC Vector ligation to obtain pSurGFP- Pgpd -SP-Cry10Aa-myc- TtrpC or pSurGFP- Pgpd -SP-Cry11Aa-myc- TtrpC Bidirectional sequencing was performed using pSur-ov-F / R primers to verify the sequence accuracy. (3) Construction of DPAR recombinant plasmid: Using the PUC57-SP-DPAR synthetic plasmid as a template, the SP-BbDPAR fragment was amplified using PUC57-F / R primers; the amplified product was then combined with pSurGFP- which had been double-digested with SpeI-HF and EcoRV-HF. Pgpd -myc- TtrpC Vector ligation to obtain pSurGFP- Pgpd -SP-DPAR-myc- TtrpC Bidirectional sequencing was performed using pSur-ov-F / R primers to verify sequence accuracy; pBarGFP was used as the vector backbone, and after single digestion with EcoRV-HF, homologous recombination was used to insert the vector from pSurGFP-Pgpd -SP-DPAR-myc- TtrpC Amplified in the vector Pgpd- SP-DPAR-myc- TtrpC Sequence, obtain pBarGFP- Pgpd -SP-DPAR-myc- TtrpC ; (4) Agrobacterium-mediated transformation and screening: The pSurGFP- obtained in step (2) was transformed and screened. Pgpd -SP-Cry10Aa-myc- TtrpC The plasmid was transferred into Agrobacterium, and Beauveria bassiana mo01 conidia were infected by Agrobacterium-mediated transformation. A single-factor expression strain mo01 / Pgpd:Cry10Aa containing the Cry toxin expression unit was obtained through screening. The pBarGFP- obtained in step (3) was then... Pgpd -SP-DPAR-myc- TtrpC The plasmid was transferred into Agrobacterium, and the single-factor expression strain mo01 / Pgpd:Cry10Aa was infected by Agrobacterium-mediated transformation. The dual-virulence engineered strains mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa, which simultaneously contain Cry toxin expression units and DPAR expression units, were screened to obtain them. The above Pgpd The nucleotide sequence of the promoter is shown in SEQ ID NO.1; the nucleotide sequence of the SP signal peptide is shown in SEQ ID NO.2; the nucleotide sequence of the SP-BbCry10Aa gene is shown in SEQ ID NO.9; the nucleotide sequence of the SP-BbCry11Aa gene is shown in SEQ ID NO.11; the nucleotide sequence of the BbDPAR gene is shown in SEQ ID NO.7; the nucleotide sequence of the Myc-tag is shown in SEQ ID NO.4; TtrpC The nucleotide sequence of the terminator is shown in SEQ ID NO.6.

[0023] Preferably, the Agrobacterium is Agrobacterium tumefaciens, such as Agrobacterium AGL-1.

[0024] Furthermore, the induction medium used in the Agrobacterium-mediated method is IMAS medium, and the screening medium is M-100 solid medium containing Sur and Bar resistance.

[0025] Further, the steps for screening and verifying the obtained dual-virulence engineered strains include: extracting total RNA from the strains and performing qRT-PCR detection using Pgpd-DPAR-qF / R and BbCry10Aa-qF / R primers to confirm that both the DPAR and Cry10Aa genes were successfully transcribed; or performing qRT-PCR detection using Pgpd-DPAR-qF / R and BbCry11Aa-qF / R primers to confirm that both the DPAR and Cry11Aa genes were successfully transcribed.

[0026] This invention provides the application of the dual-virulence engineered strains mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa in the preparation of biological control agents for mosquito control.

[0027] Furthermore, the application is to prepare a biocontrol agent that can be ingested orally by mosquitoes.

[0028] Furthermore, the application is to prepare a biocontrol agent for inhibiting the blood-sucking behavior of mosquitoes and / or inhibiting the development of Plasmodium in mosquitoes.

[0029] Furthermore, the biocontrol agent comprises an effective dose of conidia of the dual-virulence engineered strains mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa, and a carrier or excipient acceptable for agricultural or sanitary pesticides.

[0030] Furthermore, the formulation of the biological control agent that can be ingested orally by mosquitoes is selected from conidial suspensions, wettable powders, granules, sugar solutions, or attractant formulations.

[0031] Furthermore, the mosquitoes are insects belonging to the family Culicidae in the order Diptera.

[0032] A method for controlling mosquitoes includes: applying conidia of the bivirulent engineered strain mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa, or a biocontrol agent containing conidia of the bivirulent engineered strain mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa, to mosquito breeding grounds or feeding areas in a form that can be ingested orally by mosquitoes.

[0033] A method for joint control of mosquitoes, characterized by the synergistic application of the following two infection pathways: (a) Ingesting mosquitoes via oral ingestion of conidia from the engineered strains mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa; (b) Inducing mosquitoes to contact conidia of the double-virulence engineered strain mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa via their body surface.

[0034] The beneficial effects of this invention are as follows: (1) The first discovery of the highly efficient intestinal infection application of the known Beauveria bassiana strain mo01 in Anopheles skrünensis; (2) This strain can establish early infection in the foregut of Anopheles mosquitoes, breaking through the traditional surface infection mode; (3) In addition to being directly lethal, this strain also has a sublethal effect of inhibiting blood-sucking and inhibiting the development of Plasmodium. (4) The strain is suitable for developing novel green mosquito-borne fungal insecticides; (5) The lethality of mo01 infecting Anopheles skeletalis mosquitoes via the gut was enhanced by the synergistic expression of two exogenous virulence proteins. (6) The engineered strain is a targeted modification based on the intestinal infection scenario and has a clear application direction; (7) This invention provides a carrier tool, engineered strain and methodological basis for the development of engineered fungal insecticides. Attached Figure Description

[0035] Figure 1 Beauveria bassiana foregut conidia attachment and germination: (A) Confocal micrograph of mo01 hyphae infecting the mosquito gut, with black vertical lines distinguishing the foregut, anterior midgut, and posterior midgut, scale bar = 200 μm; (B) Local magnification of mo01 hyphae penetrating the foregut and infecting the host in Figure A, scale bar = 20 μm. GFP: mo01-GFP hyphae (green); DAPI: cell nucleus (pink).

[0036] Figure 2 Survival curves of mosquitoes infected with Beauveria bassiana at different conidial concentrations.

[0037] Figure 3 Survival curves of mosquitoes infected with Beauveria bassiana via both external and oral routes.

[0038] Figure 4Effects of sublethal infection on mosquito behavior and Plasmodium development: (A) Female Anopheles mosquitoes infected with low concentrations of mo01 in the intestine were starved and then provided with blood-feeding by anesthetized mice. The blood-feeding rate of Anopheles mosquitoes 5 minutes after feeding; (B) Effects of Beauveria bassiana intestinal infection on the growth and development of Anopheles mosquito larvae: Dissecting microscopic photograph of Anopheles mosquito larvae (left) scale bar = 500 μm; Quantitative analysis of the effect of intestinal infection with mo01 on the body length of Anopheles mosquito progeny larvae (right); (C) Effects of Beauveria bassiana intestinal infection on the development of Plasmodium oocysts in the midgut of Anopheles mosquitoes: Fluorescence microscopic photograph of Plasmodium oocyst development in the midgut of Anopheles mosquitoes (left), RFP: Plasmodium oocysts (red), scale bar = 200 μm; Comparison of the number of Plasmodium oocysts in the midgut of Anopheles mosquitoes after intestinal infection with mo01 (right).

[0039] Figure 5 Construction of engineered bacteria and comparison of survival curves (A) Construction of a dual exogenous virulence protein co-expression vector. P gpd : Glyceraldehyde-3-phosphate dehydrogenase promoter; SP: signal peptide; Myc-tag: c-Myc epitope tag; T trpC : Tryptophan synthase terminator; DPAR: trypsin regulatory inhibitor; Cry10Aa / Cry11Aa: Bt toxin protein; (B) Survival curve of Anopheles mosquito infected with engineered bacteria mo01::DPAR / Cry10Aa; (C) Survival curve of Anopheles mosquito infected with engineered bacteria mo01::DPAR / Cry11Aa. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1: Determination of the intestinal infection efficacy of strain mo01 against Anopheles stearothermia Test insect: Anopheles skeweri ( Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0042] To investigate the infection process of the mo01 strain in mosquitoes, *Anopheles stearothermia* (mo01) was introduced orally. A. stephensi Infect its conidia.

[0043] The mo01 strain was cultured on SDAY medium for 14 ± 4 days. Conidia and hyphae were collected using 0.01% Triton X-100 sterile water. After thorough vortexing, the hyphae were filtered through a sterilized pipette tip plugged with glass wool to collect a conidia suspension. The concentration was adjusted using a hemocytometer and prepared fresh for immediate use. The conidia of the mo01 strain were also suspended in 0.01% Triton X-100 solution to prepare a 1×10⁻⁶ solution.8 The stock solution with a concentration of conidia / mL was mixed with a 10% sucrose solution at a volume ratio of 1:1 to obtain a final concentration of 5×10⁻⁶. 7 A conidia / mL conidial suspension was used to wet cotton balls for mosquitoes to feed on and become infected.

[0044] In this embodiment, strain mo01 was orally administered 5 × 10⁻⁶ 7 Conidia / mL conidial suspension, 24 hours after feeding, 3–4 days old A. stephensi Conidia attachment, germination, and penetration of the intestinal wall can be observed in the foregut. Figure 1 (A–B) Using green fluorescent protein (GFP) labeling, clear hyphal structures extending from the germination point were visible; at the same time, 4′,6-diamidinyl-2-phenylindole (DAPI) staining results clarified the location of the cell nuclei inside the hyphae, showing that these hyphae had penetrated the intestinal wall epithelial cell layer.

[0045] Example 2: Effect of different conidial concentrations of strain mo01 on Anopheles mosquitoes To investigate whether infection efficiency is affected by inoculum dosage, a conidium concentration gradient of 1×10⁻⁶ was set up. 5 1×10 6 1×10 7 1×10 8 conidia / mL.

[0046] Test insect: Anopheles skeweri ( Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0047] Experimental method: The mo01 strain conidial suspension (prepared with 0.01% Triton X-100) was mixed with a 10% sucrose solution at a volume ratio of 1:1 using a serial dilution method to obtain a final concentration of 1×10⁻⁶. 5 1×10 6 1×10 7 1×10 8 A mixture of conidia / mL was used. Cotton balls soaked in this mixture were then fed to mosquitoes for infection. Infection lasted for 24 hours, after which the cotton balls were replaced with clean cotton balls containing 10% sucrose. From day 1 post-infection, the survival status of mosquitoes in each group was observed and recorded daily until all mosquitoes died, and the median time to death (LT) was calculated. 50 .

[0048] Multi-concentration gradient experiments showed that as the concentration of the conidial suspension increased from 1×10⁻⁶, the concentration of the conidial suspension increased. 5 conidia / mL increased to 1×10 7The efficacy of conidia / mL and mo01 in intestinal infection of Anopheles mosquitoes showed a significant upward trend, exhibiting a clear dose-dependent effect. When the concentration reached 1×10⁻⁶, the efficacy increased significantly. 7 When conidia / mL, the infection effect tends to saturate. Figure 2 The time-dose-mortality model was used to estimate the time to death (LT). 50 The results are shown in Table 1.

[0049] Table 1. Comparison of virulence of strain mo01 at different conidial concentrations Example 3: Comparison of toxicity of strain mo01 and control strain against Anopheles störeni mosquitoes Test insect: Anopheles skeweri ( Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0050] Experimental method: 1×10 8 A suspension of conidia of each strain (prepared with 0.01% Triton X-100) at a concentration of conidia / mL was mixed with a 10% sucrose solution at a volume ratio of 1:1 to obtain a final concentration of 5 × 10⁻⁶. 7 A mixture of conidia / mL was used. Cotton balls soaked in this mixture were then fed to mosquitoes for infection. Infection lasted for 24 hours, after which the cotton balls were replaced with clean cotton balls containing 10% sucrose. From day 1 post-infection, the survival status of mosquitoes in each group was observed and recorded daily until all mosquitoes died, and the median time to death (LT) was calculated. 50 .

[0051] Post-infection A. stephensi When the animals were kept under suitable conditions, the time to lethality (LT) was estimated using a time-dose-mortality model. 50 The results are shown in Table 2.

[0052] Table 2. Comparison of toxicity of strain mo01 and control strain against Anopheles störeni. Example 4: Co-infection effect of strain mo01 To comprehensively evaluate the infectivity of strain mo01, this example compares the virulence differences under three treatment methods: enteric infection alone, skin contact infection alone, and combined infection.

[0053] Test insect: Anopheles skeweri ( Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0054] Sugary meal: 1×10 8A suspension of conidial spores of strain mo01 (prepared with 0.01% Triton X-100) at a concentration of conidia / mL was mixed with a 10% sucrose solution at a volume ratio of 1:1 to obtain a final concentration of 5 × 10⁻⁶. 7 A mixture of conidia / mL was used to wet cotton balls, which were then fed to mosquitoes for infection.

[0055] Oral infection group: at a final concentration of 5 × 10⁻⁶ 7 A conidia / mL suspension of *mo01* strain conidia (prepared with 0.01% Triton X-100) was used on 3–4 day old *mo01* strains. A. stephensi Oral feeding for 24 hours. Topical infection group: 5×10 7 A suspension of conidia of *Conidia mol01* strain (mL / L) was placed in a spray bottle and sprayed evenly onto 3–4 day old *Conidia* strains placed ventrally upwards on ice after CO2 anesthesia. A. stephensi Mosquitoes were first infected lesions on their bodies and then transferred to clean rearing cups. The combined infection group (oral and topical infection) was given a sugar meal containing the same concentration of conidia immediately after oral infection, 24 hours later. The control group received the same sugar meal as the oral infection group, prepared with 0.01% Triton X-100 solution without conidia. All other treatments were the same as the oral infection group. After 24 hours of treatment, all groups were replaced with clean cotton balls containing 10% sucrose. From day 1 post-infection, the survival status of mosquitoes in each group was observed and recorded daily until all mosquitoes died, and the time to lethality (LT) was calculated. 50 .

[0056] In this embodiment, both oral and topical contact infection are performed simultaneously, LT 50 The mortality rate was shortened to 4 days, but reached 100% within 7 days. Figure 3 ).

[0057] Examples 2-4 were derived from three independent, replicated batches. Therefore, at the same concentration (5 × 10⁻⁶), 7 At conidia / mL), LT infection with single oral infection by strain mo01 was... 50 The values ​​fluctuated somewhat between different experimental batches. This fluctuation is a common variation in insect bioassays due to the difficulty in completely standardizing the physiological state of the tested insects and environmental conditions. Nevertheless, each independent experiment consistently demonstrated that strain mo01 possesses highly efficient oral infection capabilities against Anopheles stearothermiae, with its LT... 50 All values ​​are within the usable range (3.5-6.0 days).

[0058] Example 5: Sublethal effect assessment of strain mo01 Test insect: Anopheles skeweri ( Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0059] The tested malaria parasite was Plasmodium berghei ( Plasmodium berghei ).

[0060] Experimental animals: 6-8 week old male ICR mice, used for blood feeding, maintenance and proliferation of Plasmodium berghei.

[0061] Sublethal infection treatment: The sublethal concentration of strain mo01 against Anopheles steggii was selected. In this experiment, 1×10⁻⁶ was used. 6 Treatment was performed at a concentration of conidia / mL. Control group: treated with a 0.01% Triton X-100 solution that did not contain conidia.

[0062] Intestinal infection: Oral infection was used (as in Example 3), and the sugary meal was removed 24 hours after infection.

[0063] Female Anopheles mosquitoes infected with sublethal concentrations of their intestinal tract were starved and then provided with blood from anesthetized mice. Five minutes after feeding, the blood-feeding rate was measured as (number of engorged mosquitoes / total number of tested mosquitoes) × 100%. Starting from day 7 post-infection, the successfully fed female mosquitoes were reared in a 27°C incubator for 3 days, then transferred to 12-well plates with moistened filter paper for individual oviposition. After oviposition, water was added for continued rearing. Larvae were hatched and reared under standard conditions, and the hatching rate, larval development time (number of days from hatching to pupation), and other parameters were recorded. Adult emergence rate. Infection rate 1×10 6 The blood absorption rate results for conidia / mL are as follows: Figure 4 As shown in Figure A.

[0064] The larval development time of mosquito offspring in the treatment group was significantly prolonged by 2-3 days (p<0.05), and the larval body length was significantly shortened, while the number of eggs laid per batch and the hatching rate were not significantly affected. The prolonged development time implies a reduced population growth rate and increased larval exposure to predators and other risks in the environment. This sublethal effect on larvae is not a simple delay, but a combination of developmental retardation and physical weakness (significantly prolonged development time and significantly reduced individual body size during the same period). These results indicate that sublethal doses of mo01 intestinal infection not only affect adult mosquito feeding behavior but also have adverse effects on offspring development, thus exerting a sustained suppressive effect at the population level.

[0065] Malaria parasite infection: On the 3rd day after intestinal infection, malaria parasite infection was performed. Mice infected with malaria parasites were anesthetized and placed on mosquito breeding cups for feeding. After 5 minutes of feeding, engorged mosquitoes were removed to ensure that all mosquitoes were exposed to the same dose of malaria parasites. After 7 days of rearing in a 19°C incubator, surviving mosquitoes were randomly collected from the treatment and control groups, and the number of malaria parasite oocysts was recorded.

[0066] In this embodiment, at a sublethal dose of infection, 1×10 6 Treatment with conidia / mL reduced hemoseptic rate by 20% and prolonged offspring development by 2–3 days; in Plasmodium oocyst development experiments, 1×10⁻⁶ mL of conidia / mL treatment ... 5 Treatment with conidia / mL reduced the number of Plasmodium oocysts from an average of 246 to 175, 1×10 6 The conidia / mL treatment further reduced it to 110 ( Figure 4 A–C).

[0067] Example 6: Construction of engineered strains This invention employs Agrobacterium-mediated fungal transformation to construct a dual-virulence engineered strain co-expressing a trypsin regulatory inhibitor (BbDPAR) and Bt toxin proteins (BbCry10Aa / BbCry11Aa). To promote the secretory expression of exogenous virulence proteins, SP signal peptide sequences were fused to the 5′ ends of the coding sequences of BbDPAR, BbCry10Aa, and BbCry11Aa, respectively, to construct SP-BbDPAR, SP-BbCry10Aa, and SP-BbCry11Aa expression units; the nucleotide sequence of the SP signal peptide is shown in SEQ ID NO.2. The key plasmids used in the experiment and their functions are shown in Table 10. All plasmids were constructed based on pSurGFP and pBarGFP. The pSurGFP and pBarGFP plasmids were previously constructed by the applicant's laboratory; the specific construction methods can be found in the following literature: [1]Lai Y, Zheng W, Zheng Y, et al. Unveiling a novel entry gate:Insect foregut as an alternative infection route for fungal entomopathogens[J]. The Innovation, 2024, 5(4): 100644. The specific primers used and their functions are shown in Table 11.

[0068] Based on pSurGFP, pSurGFP- was obtained. Pgpd -myc- TtrpCVector: pSurGFP was digested with SpeI-HF and EcoRV-HF (purchased from NEB); using 100g DNA as a template, it was amplified using BbPgpd-F2 and BbPgpd-R2 primers. Pgpd The formula is overlap-A-Bbgpd promoter-SpeI-GACT-EcoRV; using pBarGFP as a template and TtrpC-F and TtrpC-R as primers for amplification. TtrpC The three fragments, namely SpeI-GACT-EcoRV-TtrpC-ATC-overlap, were cloned and recombined using the ClonExpress MultiS OneStep Cloning Kit (purchased from Vazyme) in a one-step process to form pSurGFP- Pgpd - TtrpC A Myc-tag is inserted between SpeI and EcoRV. Two primers are designed and annealed to form a double strand, with one end being a sticky SpeI end and the other an EcoRV blunt end. This is then ligated to the vector double-digested product to form pSurGFP- Pgpd -myc- TtrpC .

[0069] Subsequently, SP-BbCry10Aa (nucleotide sequence as shown in SEQ ID NO. 9), SP-BbCry11Aa (gene nucleotide sequence as shown in SEQ ID NO. 11), or SP-BbDPAR were amplified using PUC57-F / R primers, and then homologously recombinated into pSurGFP- enzymes digested with SpeI-HF and EcoRV-HF (purchased from NEB) using the ClonExpress MultiS One Step Cloning Kit (purchased from Vazyme). Pgpd -myc- TtrpC On the vector, the single-factor expression vector pSurGFP- was obtained. Pgpd -SP-Cry10Aa-myc- TtrpC pSurGFP- Pgpd- SP-Cry11Aa-myc- TtrpC and pSurGFP- Pgpd- SP-DPAR-myc- TtrpC The inserted fragment in the recombinant plasmid was verified by bidirectional sequencing (using pSur-ov-F / R primers) to ensure sequence correctness.

[0070] pSurGFP- was transformed using Agrobacterium-mediated transformation. Pgpd -SP-Cry10Aa-myc- TtrpC and pSurGFP- Pgpd-SP-Cry11Aa-myc- TtrpC The correct plasmids were transformed into strain mo01 to obtain single-factor expression strains mo01 / Pgpd:Cry10Aa and mo01 / Pgpd:Cry11Aa expressing the BbCry10Aa / BbCry11Aa proteins. The specific steps included plasmid transformation and Agrobacterium-mediated fungal transformation.

[0071] plasmid transformation pSurGFP- Pgpd -SP-Cry10Aa-myc- TtrpC and pSurGFP- Pgpd -SP-Cry11Aa-myc- TtrpC Introduce Agrobacterium separately: 1) Take AGL-1 Agrobacterium competent cells (purchased from Vidi Biotechnology) out of -80 ℃, quickly transfer them to ice, and let them stand for 5 min until they thaw.

[0072] 2) Add the target plasmid, gently tap the bottom of the 1.5 mL EP tube to mix, and let stand on ice for 30 min.

[0073] 3) Heat shock in a 37 ℃ water bath for 5 min, then quickly transfer to liquid nitrogen and let stand for 5 min, avoiding shaking.

[0074] 4) Add 700 uL of antibiotic-free liquid LB medium to a 1.5 mL EP tube and incubate at 28 ℃ and 200 rpm for 3 h.

[0075] 5) Centrifuge at 6000 rpm for 1 min to collect the bacterial cells, keep about 100 μL of supernatant, gently pipette to resuspend the bacterial cells, and spread evenly on LB agar plates containing the corresponding antibiotics.

[0076] 6) Invert the plate and place it in a 27°C incubator for 2 days.

[0077] 7) Use the above-mentioned validation primer pSur-ov-F / R to perform colony PCR validation (2× Taq Master Mix (DyePlus) purchased from Vazyme) to screen out positive colonies.

[0078] Agrobacterium-mediated fungal transformation: 1) A single AGL-1 positive colony containing the target plasmid was inoculated into 4 mL of LB solid medium (Table 3) containing 50 μg / mL kanamycin (purchased from Yisheng Biotechnology) and 60 μg / mL carbapenem antibiotics (purchased from Shaoxin Biotechnology). The culture was incubated overnight at 28℃ and 200 rpm for 16-20 h.

[0079] 2) Measure the OD of the bacterial solution the next day. 600 , making OD 600 Dilute to 0.6-0.8. In a 50 mL centrifuge tube, dilute the bacterial culture with IMAS solid medium prepared according to 2.5×MM Salt for Indunction Medium (IM) (Table 4) (Table 5) to OD. 600 The concentration was reduced to 0.15, and the mixture was incubated at 28 °C and 200 rpm for 4-6 h to allow the OD to reach a certain level. 600 Up to 0.6-0.8.

[0080] 3) During bacterial culture, prepare a suspension of Beauveria bassiana mo01 spores. After counting with a hemocytometer, dilute the spore suspension to 5 × 10⁻⁶ with IMAS solution. 5 Units / mL are available for use.

[0081] 4) OD 600 Mix 100 μL each of the bacterial culture at a growth rate of 0.6-0.8 and the diluted spore suspension, spread evenly on IMAS solid medium (Table 5) lined with sterile black filter paper, wrap the plate with aluminum foil to protect it from light, and incubate in a constant temperature incubator at 27 ℃ for 2 days in the dark.

[0082] 5) After 48 h, the black filter paper was transferred with sterile forceps to M-100 solid medium containing the corresponding fungal resistance (addition of Sur resistance in plasmid, addition of Bar resistance in plasmid) and Cefo 400 ug / mL (Cefotaxime Na Salt thiazomycin, purchased from LABLEAD) (Table 8) (the preparation system of M-100 Salt Solution in M-100 solid medium is shown in Table 7, and the preparation system of M-100 trace element solution in M-100 Salt Solution is shown in Table 6). The plate was wrapped with aluminum foil and incubated in a constant temperature incubator at 27 ℃ for another day in the dark.

[0083] 6) The next day, cover the black filter paper with M-100 medium containing the corresponding fungal resistance and Cefo 400 μg / mL (Table 8). After the medium solidifies, wrap it with aluminum foil and incubate in a constant temperature incubator at 27 ℃ in the dark.

[0084] 7) When small colonies appear on the plate, use a sterile toothpick to pick out the hyphae of a single Beauveria bassiana colony that has penetrated the surface of the M-100 medium (Table 8), and transfer it to a new M-100 solid medium (Table 8) containing the corresponding fungal resistance and Cefo 400 μg / mL for re-screening. Wrap the plate with aluminum foil and incubate in a constant temperature incubator at 27 ℃ in the dark for 2 days.

[0085] 8) Observe the fluorescence of the mycelium under a fluorescence microscope. Transfer the suspected transformants to SDAY plates using sterile toothpicks (Table 9) for subsequent verification and strain preservation. Use RT-qPCR technology to screen for the strain with the highest protein expression level (using primers BbCry10Aa-q / R and BbCry11Aa-q / R).

[0086] 9) Preservation of Beauveria bassiana strains mo01::Cry10Aa and mo01::Cry11Aa: Low-temperature cryopreservation was used. First, prepare sterile cryovials and add 1 mL of 25% sterile glycerol containing 0.02% Tween-20 (purchased from Beyotime). Scrape Beauveria bassiana conidia and hyphae from the SDAY plate (Table 9) into the sterile glycerol and suspend by shaking. After placing at -20 ℃ for one day, transfer to -80 ℃ for long-term storage.

[0087] Using Ov-mcISP-F / R primers, from pSurGFP- Pgpd- SP-DPAR-myc- TtrpC Amplification in vector Pgpd- SP-DPAR-myc- TtrpC The gene was then homologously recombinated with the pBarGFP vector digested with EcoRV-HF (purchased from NEB) using a ClonExpress MultiS One Step Cloning Kit (purchased from Vazyme) to obtain pBarGFP- Pgpd -SP-DPAR-myc- TtrpC ; for pBarGFP- Pgpd -SP-DPAR-myc- TtrpC The plasmid was validated by bidirectional sequencing (using pBarGFP EcoRV-F / R primers) to ensure sequence correctness. The validated pBarGFP- Pgpd -SP-DPAR-myc- TtrpC The vectors were transformed into mo01::Cry10Aa and mo01::Cry11Aa strains using Agrobacterium-mediated transformation (for specific transformation steps, refer to the plasmid transformation and Agrobacterium-mediated fungal transformation steps described above), respectively, to construct the final dual-virulence engineered strains mo01::DPAR / Cry10Aa and mo01::DPAR / Cry11Aa.

[0088] Use P gpdqRT-PCR analysis using primers -DPAR-qF / R, BbCry10Aa-qF / R, and BbCry11Aa-qF / R confirmed that both exogenous genes were successfully expressed at the transcriptional level, thus obtaining the dual-virulence engineered strains mo01::DPAR / Cry10Aa and mo01::DPAR / Cry11Aa.

[0089] Table 3. LB (Luria-Bertani medium) culture medium Table 4. 2.5×MM Salt for induction medium (IM) Table 5. IMAS (Induced Minimal Agar with Acetosyringone) Culture Medium Table 6. M-100 Trace Element Solution Culture Medium Table 7 M-100 Salt Solution Culture Medium Table 8 M-100 Culture Medium Table 9 SDAY Culture Medium The insecticide is used to infect Anopheles streptococci via the intestines after oral administration.

[0090] Table 10. Plasmids used in experiments Table 11 Primers used in the experiment Example 7: Virulence assay of engineered strains Test strains: dual-virulence engineered strains, single-factor expression strains (expressing only DPAR or only Cry10Aa / Cry11Aa), and wild-type control strain mo01. The construction method for the single-factor expression strain mo01 / Pgpd:DPAR is the same as that for mo01 / Pgpd:Cry10Aa or mo01 / Pgpd:Cry11Aa.

[0091] Test insect: Anopheles skeweri (Anopheles stephensi Adult insects, female individuals 3-4 days after emergence.

[0092] Experimental method: Conidia of each strain were suspended in 0.01% Triton X-100 solution to prepare a solution with a concentration of 1×10⁻⁶. 8 A conidia / mL conidial suspension was prepared. The conidial suspension was mixed with 10% sucrose at a 1:1 ratio to obtain a final concentration of 5 × 10⁻⁶. 7 A mixture of conidia / mL was prepared, and cotton balls were then soaked in the solution for mosquitoes to feed on and become infected. The infection lasted for 24 hours, after which the cotton balls were replaced with clean cotton balls containing 10% sucrose. From day 1 post-infection, the survival status of each group of mosquitoes was observed and recorded daily until all mosquitoes died, and the median time to death (LT) was calculated. 50 .

[0093] like Figure 5 As shown, the LT of the dual-virulence engineered strain mo01::DPAR / Cry10Aa constructed via Agrobacterium-mediated transformation... 50 The incubation period was 3.5 days, with an 89% mortality rate on day 8. The single-factor expression strain was mo01 / Pgpd:DPAR LT. 50 The incubation period was 4.5 days, with a 7-day mortality rate of 73%; mo01 / Pgpd:Cry10Aa LT 50 The survival time was 4.5 days, with a 7-day mortality rate of 75%. The dual-virulence engineered strain was significantly superior to the single-factor expression strain. The dual-virulence engineered strain mo01::DPAR / Cry11Aa LT 50 The mortality rate was 98% after 3 days and 7 days, with a single-factor control strain mo01 / Pgpd:DPAR LT. 50 The incubation period was 4.5 days, with a 7-day mortality rate of 73%; mo01 / Pgpd:Cry11Aa LT 50 The survival time was 4.5 days, and the mortality rate was 74% after 7 days. These results indicate that the dual-virulence engineered strain is significantly superior to the single-factor expression strain.

[0094] sequence list SEQ ID NO.1 (P gpd gene) SEQ ID NO.2 (SP gene) ATGCGTGAACTTTCTTCGGTTCTCGCCCTTTCGGGCTTGCTGGCCCTGGCGTCGGCA SEQ ID NO.3(amino acid sequence of SP protein ) MRELSSVLALSGLLALASA SEQ ID NO.4 (Myc-tag gene) GAGCAGAAGCTCATCTCCGAAGAGGACCTG SEQ ID NO.5 ( amino acid sequence of Myc-tag protein) EQKLISEEDL SEQ ID NO.6 (T trpC terminator nucleotide sequence) AGTAGATGCCGACCGGGATCGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTCGGGTTTACCTCTTCCAGATACAGCTCAACTGCAATGCATTAATGCATTGGACCTCGCAACCCTAGTACGCCCTTCAGGCTCCGGCGAAGCAGAAGAATAGCTTAGCAGAGTCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCCCTG SEQ ID NO.7 ( nucleotide sequence of the BbDPAR fusion gene) ATGGCTCCTTTTCTTCAAACCAGCCTCGCGCTCCTTCCATTGTTGGCTTCCACCATGGTCAGCGCCTCGCCATTGGCGCCGCGAGCCGGCGACCCCGCCCGCGATCCTGCCCGCGACCCCGCTCGTGACCCCGCCCGCTAG SEQ ID NO.8 ( amino acid sequence of BbDPAR fusion protein ) MAPFLQTSLALLPLLASTMVSASPLAPRAGDPARDPARDPARDPAR SEQ ID NO.9 ( nucleotide sequence of the SP-Bb Cry10Aa gene) SEQ ID NO.10 (amino acid sequence of SP-BbCry10Aa fusion protein ) MRELSSVLALSGLLALASANPYQNKNEYEIFNAPSNGFSKSNNYSRYPLANKPNQPLKNTNYKDWLNVCQDNQQYGNNAGNFASSETIVGVSAGIIVVGTMLGAFAAPVLAAGIISFGTLLPIFWQGSDPANVWQDLLNIGGRPIQEIDKNIINVLTSIVTPIKNQLDKYQEFFDKWEPARTHANAKAVHDLFTTLEPIIDKDLDMLKNNASYRIPTLPAYAQIATWHLNLLKHAATYYNIWLQNQGINPSTFNSSNYYQGYLKRKIQEYTDYCIQTYNAGLTMIRTNTNATWNMYNTYRLEMTLTVLDLIAIFPNYDPEKYPIGVKSELIREVYTNVNSDTFRTITELENGLTRNPTLFTWINQGRFYTRNSRDILDPYDIFSFTGNQMAFTHTNDDRNIIWGAVHGNIISQDTSKVFPFYRNKPIDKVEIVRHREYSDIIYEMIFFSNSSEVFRYSSNSTIENNYKRTDSYMIPKQTWKNEEYGHTLSYIKTDNYIFSVVRERRRVAFSWTHTSVDFQNTIDLDNITQIHALKALKVSSDSKIVKGPGHTGGDLVILKDSMDFRVRFLKNVSRQYQVRIRYATNAPKTTVFLTGIDTISVELPSTTSRQNPNATDLTYADFGYVTFPRTVPNKTFEGEDTLLMTLYGTPNHSYNIYIDKIEFIPITQSVLDYTEKQNIEKTQKIVNDLFVNEQKLISEEDL SEQ ID NO.11 ( nucleotide sequence of the SP-Bb Cry11Aa gene): SEQ ID NO.12 ( amino acid sequence of the SP-BbCry11Aa fusion protein ): MRELSSVLALSGLLALASAEDSSLDTLSIVNETDFPLYNNYTEPTIAPALIAVAPIAQYLATAIGKWAAKAAFSKVLSLIFPGSQPATMEKVRTEVETLINQKLSQDRVNILNAEYRGIIEVSDVFDAYIKQPGFTPATAKGYFLNLSGAIIQRLPQFEVQTYEGVSIALFTQMCTLHLTLLKDGILAGSAWGFTQADVDSFIKLFNQKVLDYRTRLMRMYTEEFGRLCKVSLKDGLTFRNMCNLYVFPFAEAWSLMRYEGLKLQSSLSLWDYVGVSIPVNYNEWGGLVYKLLMGEVNQRLTTVKFNYSFTNEPADIPARENIRGVHPIYDPSSGLTGWIGNGRTNNFNFADNNGNEIMEVRTQTFYQNPNNEPIAPRDIINQILTAPAPADLFFKNADINVKFTQWFQSTLYGWNIKLGTQTVLSSRTGTIPPNYLAYDGYYIRAISACPRGVSLAYNHDLTTLTYNRIEYDSPTTENIIVGFAPDNTKDFYSKKSHYLSETNDSYVIPALQFAEVSDRSFLEDTPDQATDGSIKFARTFISNEAKYSIRLNTGFNTATRYKLIIRVRVPYRLPAGIRVQSQNSGNNRMLGSFTANANPEWVDFVTDAFTFNDLGITTSSTNALFSISSDSLNSGEEWYLSQLFLVKESAFTTQINPLLKEQKLISEEDL SEQ ID NO.13 (BbPgpd-F2 primer): TCGAGCTCGGTACCCGGGTGAGGCTG GTGAATATGACTAG SEQ ID NO.14 (BbPgpd-R2 primer): AGCGAGTTGCGACATTGTTCTTGATTA GAAAAGTGAGG SEQ ID NO.15(TtrpC-F primer): AAGGCCAGCGACTGAGATGAGGACT CCTCAGCTA SEQ ID NO.16(TtrpC-R primer): CTCTAGAGGATCCCCGGGAAGTGATG AGGGGACAAGTCT SEQ ID NO.17(PUC57-F primer): TAAAACGACGGCCAGAGAATTC SEQ ID NO.18(PUC57-R primer): AAGCTTGCATGCAGGCCTC SEQ ID NO.19(pSur-ov-F primer): TCACCAGAGAAGCTGCGC SEQ ID NO.20(pSur-ov-R primer): GGACAAATGAACGTATCTTATCGAG SEQ ID NO.21(Ov-mcISP-F primer): GTGTCTGTATTTCCGGATATCGGTTGGGTATGCTCCGGC SEQ ID NO.22(Ov-mcISP-R primer): ACCGACGGAATTGAGGATATcCAGGGCTGGTGACGGAATT SEQ ID NO.23(pBarGFP EcoRV-F primer): CCGGTCTTGCGATGATTATC SEQ ID NO.24(pBarGFP EcoRV-R primer): ATCATGTATGTAGTGGGTGTGC SEQ ID NO.25(BbCry10Aa-qF primer): GCCAAGACACCAGCAAAGTG SEQ ID NO.26(BbCry10Aa-qR primer): GGTGCTGTTGCTGCTGTAAC SEQ ID NO.27 (Pgpd-DPAR-qF primer): TCTCTCTTCCACCTCACCCT SEQ ID NO.28 (Pgpd-DPAR-qR primer): AACAATGGAAGGAGCGCGAG SEQ ID NO.29 (BbCry11Aa-qF primer): TTTAACACCGCGACCCGTTA SEQ ID NO.30 (BbCry11Aa-qR primer): GCCCAGGTCGTTAAAGGTGA

Claims

1. An engineered strain of *Beauveria bassiana* co-expressing dual exogenous virulence proteins, characterized in that, Beauveria bassiana (Beauveria bassiana) mo01 is the recipient strain, which simultaneously expresses BbDPAR protein and SP-BbCry10Aa protein, or simultaneously expresses BbDPAR protein and SP-BbCry11Aa protein; the amino acid sequence of the BbDPAR protein is shown in SEQ ID NO.8; the amino acid sequence of the SP-BbCry10Aa protein is shown in SEQ ID NO.10; and the amino acid sequence of the SP-BbCry11Aa protein is shown in SEQ ID NO.

12.

2. A method for constructing the engineered strain of claim 1, comprising the following steps: (1) Construction of the intermediate vector pSurGFP-Pgpd-myc-TtrpC: pSurGFP was used as the vector backbone and double-digested with SpeI-HF and EcoRV-HF; using mo01 genomic DNA as a template, the Pgpd promoter was amplified with BbPgpd-F2 / -R2 primers to obtain fragment A; using pBarGFP as a template, the TtrpC terminator was amplified with TtrpC-F / R primers to obtain fragment B; fragment A and fragment B were homologously recombinated with the linearized pSurGFP vector to obtain pSurGFP-Pgpd-TtrpC; a Myc-tag double-stranded DNA fragment was synthesized with SpeI sticky ends and EcoRV blunt ends at both ends, and inserted between the SpeI and EcoRV sites of pSurGFP-Pgpd-TtrpC to obtain the intermediate vector pSurGFP-Pgpd-myc-TtrpC; (2) Constructing Cry toxin recombinant plasmids: Using the PUC57-SP-Cry10Aa or PUC57-SP-Cry11Aa synthetic plasmids as templates, the SP-BbCry10Aa or SP-BbCry11Aa fragments were amplified using PUC57-F / R primers; The amplified product was ligated into the pSurGFP-Pgpd-myc-TtrpC vector, which was double-digested with SpeI-HF and EcoRV-HF, to obtain pSurGFP-Pgpd-SP-Cry10Aa-myc-TtrpC or pSurGFP-Pgpd-SP-Cry11Aa-myc-TtrpC; bidirectional sequencing was performed using pSur-ov-F and pSur-ov-FR primers to verify the sequence accuracy. (3) Construction of DPAR recombinant plasmid: Using the PUC57-SP-DPAR synthetic plasmid as a template, the SP-BbDPAR fragment was amplified using PUC57-F / R primers; the amplified product was then combined with pSurGFP-digested with SpeI-HF and EcoRV-HF. Pgpd -myc- TtrpC Vector ligation to obtain pSurGFP- Pgpd -SP-DPAR-myc- TtrpC Bidirectional sequencing was performed using pSur-ov-F / R primers to verify the sequence accuracy. Using pBarGFP as the vector backbone, after single digestion with EcoRV-HF, the vector was inserted from pSurGFP- via homologous recombination. Pgpd -SP-DPAR-myc- TtrpC Amplified in the vector Pgpd- SP-DPAR-myc- TtrpC Sequence, obtain pBarGFP-Pgpd-SP-DPAR-myc-TtrpC; (4) Agrobacterium-mediated transformation and screening: The pSurGFP-Pgpd-SP-Cry10Aa-myc-TtrpC plasmid obtained in step (2) was transformed into Agrobacterium, and Beauveria bassiana mo01 conidia were infected by Agrobacterium-mediated transformation to screen for single-factor expression strain mo01 / Pgpd:Cry10Aa containing Cry toxin expression unit; The pBarGFP-Pgpd-SP-DPAR-myc-TtrpC plasmid obtained in step (3) was transformed into Agrobacterium, and the single-factor expression strain mo01 / Pgpd:Cry10Aa was infected by Agrobacterium-mediated transformation to screen for dual-virulence engineered strain mo01::DPAR / Cry10Aa or mo01::DPAR / Cry11Aa containing both Cry toxin expression unit and DPAR expression unit; The nucleotide sequence of the Pgpd promoter is shown in SEQ ID NO.1; the nucleotide sequence of the SP signal peptide is shown in SEQ ID NO.2; the nucleotide sequence of the SP-BbCry10Aa gene is shown in SEQ ID NO.9; the nucleotide sequence of the SP-BbCry11Aa gene is shown in SEQ ID NO.11; the nucleotide sequence of the BbDPAR gene is shown in SEQ ID NO.7; the nucleotide sequence of the Myc-tag is shown in SEQ ID NO.4; the nucleotide sequence of the TtrpC terminator is shown in SEQ ID NO.6; the nucleotide sequences of BbPgpd-F2 and BbPgpd-R2 are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively; the nucleotide sequences of TtrpC-F and TtrpC-R are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively; and the nucleotide sequences of PUC57-F and PUC57-R are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively. The nucleotide sequences of PUC57-F and PUC57-R are shown in SEQ ID NO.17 and SEQ ID NO.18, respectively; the nucleotide sequences of pSur-ov-F and pSur-ov-FR are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively.

3. The method according to claim 2, wherein the step of screening and validating the obtained dual-virulence engineered strain includes: Total RNA was extracted from the strain, and qRT-PCR was performed using primers Pgpd-DPAR-qF / R and BbCry10Aa-qF / R to confirm successful transcription of both the DPAR and Cry10Aa genes; or qRT-PCR was performed using primers Pgpd-DPAR-qF / R and BbCry11Aa-qF / R to confirm successful transcription of both the DPAR and Cry11Aa genes. Specifically, Pgpd-DPAR-qF / R contains the Pgpd-DPAR-qF primer with the nucleotide sequence shown in SEQ ID NO. 27 and the Pgpd-DPAR-qR primer with the sequence shown in SEQ ID NO. 28; BbCry10Aa-qF / R contains the BbCry10Aa-qF primer with the nucleotide sequence shown in SEQ ID NO. 25 and the BbCry10Aa-qF primer with the sequence shown in SEQ ID NO.

28. Primer NO.26 is the BbCry10Aa-qR primer; BbCry11Aa-qF / R contains the BbCry11Aa-qF primer with the nucleotide sequence shown in SEQ ID NO.29 and the BbCry11Aa-qR primer with the sequence shown in SEQ ID NO.

30.

4. The method according to claim 2, wherein the BbDPAR gene is obtained by amplification using the PUC57-SP-DPAR plasmid as a template and primers with sequences as shown in SEQ ID NO.21 (Ov-mcISP-F) and SEQ ID NO.22 (Ov-mcISP-R).

5. According to the method of claim 2, after the pBarGFP-Pgpd-SP-DPAR-myc-TtrpC recombinant plasmid is constructed, bidirectional sequencing is performed using primers with the sequence shown in SEQ ID NO.23 (pBarGFP EcoRV-F) and the sequence shown in SEQ ID NO.24 (pBarGFP EcoRV-R) to verify the sequence correctness.

6. The use of the engineered strain according to claim 1 in the preparation of a biological control agent for controlling mosquitoes.

7. The application according to claim 6, wherein the application is for preparing a biological control agent that can be ingested orally by mosquitoes.

8. The application according to claim 6, wherein the mosquito is an insect belonging to the family Culicidae in the order Diptera.

9. A method for controlling mosquitoes, comprising: The conidia of the engineered strain described in claim 1 or biocontrol agents containing them are applied to mosquito breeding grounds or feeding areas in a form that can be ingested orally by mosquitoes.

10. A method for joint control of mosquitoes, characterized in that, This includes the synergistic application of the following two infection pathways: (a) Ingesting conidia of the engineered strain of claim 1 orally by mosquitoes; (b) The mosquito comes into contact with the conidia of the engineered strain of claim 1 via its body surface.

Images