Highly-virulent metarhizium robertsii specifically modified for acrididae pests and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of agrochemicals, and in particular to highly-virulent metarhizium robertsii specifically modified for acrididae pests and a preparation method therefor and a use thereof. A protein comprises: a protein as shown in SEQ ID NO: 1, and / or a fusion protein thereof, and / or a homologous protein thereof. A toxin protein or a coding gene thereof is a key virulence factor of metarhizium acridum causing high virulence to locusts, and is only toxic to the acrididae pests and does not affect other insects. A recombinant entomopathogenic fungus effectively solves the disadvantage of original broad-spectrum entomopathogenic fungi having insufficient virulence to acrididae pests, and is also highly virulent to the acrididae pests while effectively controlling and preventing various other pests, thereby improving the pest prevention and control efficiency, achieving the effects of multi-purpose use with a single fungus and use cost saving, and not causing food and ecological safety issues.

Description

Highly toxic Metarhizium Robertsii modified specifically for locust pests, and preparation method and application thereof Technical Field

[0001] The present application relates to the technical field of pesticides, and in particular to a highly toxic Metarhizium Robertsii fungus modified specifically for Acrididae pests, and a preparation method and application thereof. Background Art

[0002] my country, a major agricultural country, consumes approximately 1.8 million tons of chemical pesticides annually, ranking first in the world. Excessive use of chemical pesticides can cause organic pollution in the soil and seriously endanger food safety. Furthermore, the misuse of chemical pesticides can lead to increased pest resistance, resulting in the embarrassing situation of being unable to combat pests with pesticides. Therefore, reducing chemical pesticide use is one of the primary challenges facing my country's progress towards becoming an agricultural powerhouse.

[0003] Entomopathogenic fungi are a widely used class of living biological pesticides that are environmentally friendly and difficult for pests to develop resistance to. Therefore, entomopathogenic fungal preparations used in conjunction with chemical pesticides hold great promise for future application. Currently, widely used entomopathogenic fungal preparations include Beauveria bassiana, Metarhizium anisopliae, M. anisopliae Robertsii, and M. anisopliae. Beauveria bassiana, M. anisopliae, and M. anisopliae Robertsii can infect and kill over 200 species of agricultural pests, while M. anisopliae's high toxicity specifically targets Acrididae pests.

[0004] At present, the green muscardine products on the market cannot achieve both extensiveness and high efficiency. Broad-spectrum green muscardine preparations show high toxicity to insects such as Lepidoptera, Diptera and Coleoptera, but are less toxic to locust pests (such as migratory locusts, desert locusts and small locusts). For example: Studies have shown that the median lethal time (LT50) of Roberts green muscardine preparations to Anopheles (Diptera), Pea Aphids (Hemiptera), Tenebrio molitor (Coleoptera), Greater Wax Moth (Lepidoptera) is 100%. 50 ) were 3.2±0.1 days, 5.11±0.23 days, 5.5±0.79 days, and 5.917±0.261 days, respectively, but the median lethal time for locusts was as high as 8.60±0.15 days. Although the locust green muscardine preparation can effectively kill various types of locust pests, it is only effective against locusts and cannot take into account other pests. If the two are used at the same time, the cost of use will increase, and it will also lead to interspecific competition between different fungi, weakening the control effect on pests.

[0005] Genetic engineering methods for modifying the genomes of entomopathogenic fungi are a mainstream approach to improving the insecticidal efficacy of entomopathogenic agents. For example, researchers have introduced a scorpion (Androctonus australis) neurotoxin gene into the genome of Metarhizium anisopliae and achieved stable expression, significantly enhancing the insecticidal efficacy of Metarhizium anisopliae. Other researchers have also expressed the β-neuropeptide (β-NP) toxin gene from the fire ant (Solenopsis invicta) in Beauveria bassiana, significantly increasing its insecticidal efficacy. While these genetic modification techniques enhance the pathogenicity of these strains to pests, because these toxins are also harmful to humans and animals, their application can lead to genetic and ecological contamination, raising food and environmental safety concerns.

[0006] Meanwhile, unmodified broad-spectrum Metarhizium and Beauveria bassiana preparations have minimal impact on some Hymenoptera pollinating insects (such as honeybees and bumblebees). However, genetically engineered, highly virulent strains often lack host selectivity, increasing their pathogenicity to both agricultural pests and pollinators. Consequently, a reduction in pollinators is also detrimental to the growth of some crops and, in turn, affects yields.

[0007] Summary of the Invention

[0008] In view of this, the present invention provides a highly toxic Metarhizium Robertsii fungus specifically modified for locust pests, as well as its preparation method and application. This protein is a key virulence factor that causes the high pathogenicity of Metarhizium locust to locusts. It is toxic only to locust pests and does not affect other insects. The recombinant insect pathogenic fungus of the present application can achieve the effect of multiple uses of one fungus and save usage costs without causing food and ecological safety issues.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a use of a protein in the preparation of a biopesticide, wherein the protein comprises at least one of the following proteins:

[0011] (11) protein represented by SEQ ID NO: 1;

[0012] (12) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1;

[0013] (13) Proteins with the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in SEQ ID NO: 1;

[0014] (14) A protein that is at least 75% identical to the protein shown in SEQ ID NO: 1 and has the same function.

[0015] In a specific embodiment of the present invention, the above protein is used to control Acrididae pests.

[0016] In the embodiments of the present invention, the Acrididae pest belongs to the class Insecta, order Orthoptera, superfamily Acridoidea, commonly known as locusts or grasshoppers, such as migratory locusts (such as the East Asian migratory locust, the Asian migratory locust, and the Tibetan migratory locust), desert locusts, small car locusts, earth locusts, rice locusts, cotton locusts, and bamboo locusts.

[0017] In a second aspect, the present invention provides a use of a gene in the preparation of a biopesticide, wherein the gene comprises at least one of the following genes:

[0018] (21) The gene shown in SEQ ID NO: 2;

[0019] (22) a complementary sequence, a degenerate sequence, or a homologous sequence of the gene shown in SEQ ID NO: 2, wherein the homologous sequence is a sequence having at least 75% identity with the gene shown in SEQ ID NO: 2;

[0020] (23) A sequence that hybridizes with the gene represented by SEQ ID NO: 2 under stringent conditions or a complementary sequence thereof.

[0021] "Identity" refers to sequence similarity to a natural nucleic acid sequence or amino acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0022] The above-mentioned "at least 75%" is, for example, any value among 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or any value within the range consisting of any two of the above values.

[0023] In the application of the protein or gene of the present invention in the preparation of a biopesticide, the biopesticide is a biopesticide for at least preventing and controlling Acrididae pests.

[0024] In a third aspect, the present invention provides a biomaterial for expressing the above-mentioned gene, wherein the biomaterial comprises at least one of an expression cassette, a vector, a recombinant cell, and a recombinant microorganism.

[0025] In an embodiment of the present invention, the vector includes at least one of a plasmid, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and a linear polynucleotide.

[0026] In an embodiment of the present invention, recombinant cells include plant cells and / or animal cells that are not used for the purpose of reproduction.

[0027] In an embodiment of the present invention, the recombinant microorganism includes at least one of fungi, bacteria, and algae.

[0028] In an embodiment of the present invention, the fungus includes at least one of yeast, Aspergillus niger, and Trichoderma reesei.

[0029] In an embodiment of the present invention, the bacteria include at least one of Agrobacterium, Escherichia coli, Bacillus, Pseudomonas, and lactic acid bacteria.

[0030] In an embodiment of the present invention, the algae include at least one of Cyanobacteria, Synechococcus, Synechococcus elongatus, Synechocystis, Anabaena, Chlamydomonas, and Chlamydomonas reinhardtii.

[0031] In a fourth aspect, the present invention provides a recombinant entomopathogenic fungus comprising the above-mentioned gene and / or the above-mentioned biological material.

[0032] In an embodiment of the present invention, the recombinant entomopathogenic fungi include broad-spectrum Metarhizium and / or Beauveria bassiana.

[0033] In an embodiment of the present invention, the broad-spectrum Metarhizium includes one or both of Metarhizium robertsii and Metarhizium anisopliae.

[0034] In an embodiment of the present invention, the broad-spectrum green muscardine has a wide insecticidal spectrum and can infect more than 200 pests of the Lepidoptera, Coleoptera, Homoptera, Hemiptera, Orthoptera and Diptera.

[0035] In a fifth aspect, the present invention provides the use of the above-mentioned recombinant entomopathogenic fungi in the preparation of biopesticides.

[0036] In a sixth aspect, the present invention provides a method for preparing the above-mentioned recombinant entomopathogenic fungus, comprising: introducing the above-mentioned gene and / or the above-mentioned biological material into the entomopathogenic fungus to obtain the recombinant entomopathogenic fungus.

[0037] In the embodiments provided by the present invention, the method of introducing the above-mentioned gene into the entomopathogenic fungus includes: connecting the above-mentioned gene to a vector to obtain a recombinant vector, transferring the recombinant vector into Agrobacterium, and transferring it into the entomopathogenic fungus using Agrobacterium-mediated method.

[0038] In addition to the above-mentioned method for preparing recombinant entomopathogenic fungi, other conventional methods can also be used to introduce the above-mentioned genes into entomopathogenic fungi, such as protoplast transformation, gene editing technology, etc.

[0039] In a seventh aspect, the present invention provides a biopesticide comprising the above-mentioned recombinant entomopathogenic fungus.

[0040] In the embodiments of the present invention, the biopesticide may also include other types of biopesticides. Other types of biopesticides recognized by those skilled in the art are within the scope of protection of the present invention and are not limited by the present invention.

[0041] In an eighth aspect, the present invention provides an insecticidal composition comprising the above-mentioned recombinant entomopathogenic fungus and a chemical pesticide.

[0042] In an embodiment of the present invention, the biopesticide or insecticide composition may further include pesticide-acceptable excipients.

[0043] In an embodiment of the present invention, the biopesticide or insecticide composition is selected from the following dosage forms: solution, emulsion, wettable powder, granular wettable powder, suspension, dust, foam, paste, tablet, granule, aerosol, natural agent impregnated with active compound, synthetic agent impregnated with active compound, microcapsule, seed coating agent, preparation equipped with combustion device, cold mist, hot mist, etc.

[0044] In an embodiment of the present invention, the pesticide-acceptable excipients include one or more of the following: fillers, surfactants, binders, colorants, and the like.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. Through omics analysis combined with functional research, this application discovered a unique toxin protein encoding gene in Metarhizium arbuscular mycoides, which is the key virulence factor that causes the high pathogenicity of Metarhizium arbuscular mycoides to locusts. The protein encoded by this gene is only toxic to Acrididae pests and does not affect other insects.

[0047] 2. After integrating the toxin protein encoding gene into a broad-spectrum entomopathogenic fungus (such as Metarhizium Robertsii), the present application successfully constructed a recombinant entomopathogenic fungus strain capable of producing a large amount of locust toxin, effectively solving the shortcoming of the original broad-spectrum entomopathogenic fungus's insufficient pathogenicity to locust pests, so that it can effectively prevent and control a variety of other pests while also having high toxicity to locust pests, thereby improving the efficiency of pest control, achieving the effect of multiple uses of one fungus and saving usage costs.

[0048] 3. The protein in this application itself is a virulence factor of Metarhizium anisopliae, which is only effective against locust pests and will not affect humans and other animals (such as poultry, livestock, etc.). Therefore, the recombinant strain prepared by the present invention will not cause food and ecological safety problems and can be used safely.

[0049] 4. Since the protein of the present application is only effective against Acrididae pests, the recombinant strain prepared by the present invention will not have a substantial impact on pollinating insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows the results of screening for toxin protein-encoding genes in Metarhizium anisopliae (MAC) that can be upregulated in migratory locusts; Figure 1-1 shows the gene expression intensity of Metarhizium anisopliae (MAA) in migratory locusts; Figure 1-2 shows the gene expression intensity of Metarhizium anisopliae (MAC) in migratory locusts; Log2 (foldchange) is a commonly used metric in bioinformatics, which is used to measure the difference between the gene expression intensity of a sample and the expression intensity of the corresponding control sample;

[0051] Figure 2 is a plasmid construction map; Genomic DNA is translated as genomic DNA; bar cassette is translated as bar gene, which is a resistance gene;

[0052] Figure 3 shows the purification results of the protein encoded by MAC_08119;

[0053] Figure 4 shows the toxicity of wild-type Metarhizium anisopliae strain (MAC-WT) and Metarhizium anisopliae strain lacking MAC_08119 (ΔMAC_08119) to locusts;

[0054] Figure 5 shows the toxicity of the wild-type Metarhizium anisopliae strain (MAC-WT), the Metarhizium anisopliae strain lacking MAC_08119 (ΔMAC_08119), and the Metarhizium anisopliae strain lacking MAC_08119 after injection of purified protein (ΔMAC_08119+purified protein) to locusts;

[0055] Figure 6 shows the toxicity of Metarhizium anisopliae overexpressing the MAC_08119 gene (OE: MAC_08119) to locusts;

[0056] Figure 7 shows the relative mRNA expression levels of the MAC_08119 gene in wild-type Metarhizium Robertsii (MAA-WT) and recombinant Metarhizium Robertsii (OE: MAC_08119), where * indicates a significant difference between the two groups, i.e., P < 0.05;

[0057] Figure 8 shows the toxicity of wild-type Metarhizium Robertsii (MAA-WT) and recombinant Metarhizium Robertsii strain (OE: MAC_08119) to locusts; P < 0.01 indicates that the difference between the two groups is extremely significant;

[0058] Figure 9 shows the toxicity of wild-type Metarhizium anisopliae (MAA-WT) and recombinant Metarhizium anisopliae strain (OE: MAC_08119) to Tenebrio molitor.

[0059] Figure 10 shows the toxicity of wild-type Metarhizium Robertsii (MAA-WT) and recombinant Metarhizium Robertsii strain (OE: MAC_08119) to bumblebees. DETAILED DESCRIPTION

[0060] The present invention discloses a highly toxic Roberts' green muscardine specifically modified for locust pests, and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0061] The reagents, instruments, strains or biological materials used in the present invention can all be obtained through commercial channels.

[0062] The present invention will be further described below in conjunction with the embodiments:

[0063] Example 1 Identification of specific virulence factors of Metarhizium anisopliae

[0064] (1) Screening of genes encoding potential virulence factors

[0065] A transcriptome-wide search identified toxin protein-encoding genes in Metarhizium anisopliae (MAC) that are upregulated in migratory locusts. The 37 candidate genes identified were then compared with homologous genes in Metarhizium robertsii (MAA) to further identify toxin protein-encoding genes specific to Metarhizium anisopliae. After two rounds of screening, MAC_08119 was identified as a key candidate gene (Figure 1). Among them, MAA_00392 from Metarhizium robertsii is a homologous gene of MAC_08119 in Metarhizium anisopliae.

[0066] The MAC_08119 protein sequence (SEQ ID NO: 1) is as follows:

[0067] The MAC_08119 gene sequence (SEQ ID NO: 2) is as follows:

[0068] (2) Functional verification of virulence factors

[0069] The virulence of the protein encoded by MAC_08119 to the host was verified from both positive and negative aspects through knockout and overexpression techniques. The principles of knockout and overexpression are both chromosomal homologous replacement, which is currently a very mature technology used for genetic manipulation of Metarhizium. The specific methods are as follows:

[0070] (a) Construction of MAC_08119 knockout and overexpression plasmids.

[0071] (a1) Construction of MAC_08119 knockout plasmid

[0072] The plasmid construction map is shown in Figure 2. Using wild-type MAC genome DNA as a template, the upstream and downstream sequences (i.e., left and right arms) of the gene MAC_08119 were amplified using primers "MAC_08119LF and MAC_08119LR" and primers "MAC_08119RF and MAC_08119RR", respectively. The specific primer sequences are as follows:

[0073] Table 1 Primer sequences

[0074] First, the PDHt-Bar plasmid was digested with restriction endonucleases EcoRI and BamHI. The left arm, amplified using primers MAC_08119LF and MAC_08119LR, was then ligated to PDHt-Bar using homologous recombination to create the plasmid PDHt-MAC_08119L-Bar. Sequencing confirmed the correct construction of the plasmid. Next, the PDHt-MAC_08119L-Bar plasmid was digested with restriction endonucleases XbaI and SacI, and the DNA sequence amplified using primers MAC_08119RF and MAC_08119RR was ligated to PDHt-MAC_08119L-Bar using homologous recombination to create the knockout plasmid PDHt-Bar-MAC_08119L / R. Sequencing also confirmed the correct construction of the plasmid.

[0075] (a2) Construction of MAC_08119 overexpression plasmid vector

[0076] First, using the wild-type MAC genome as a template, the DNA fragment of the MAC_08119 gene was amplified using primers PDHt-8119-F and PDHt-8119-R. The PDHt-Gpd-Bar plasmid was then digested with restriction endonucleases EcoRI and MunI. The digestion product was recovered and then recombined with the aforementioned DNA fragment using homologous recombination. Sequencing confirmed the correct construction of the plasmid, and the recombinant plasmid was named "PDHt-Gpd-MAC_08119-Bar." The primer sequences for PDHt-8119-F and PDHt-8119-R are as follows:

[0077] PDHt-8119-F (SEQ ID NO: 7):

[0078] PDHt-8119-R (SEQ ID NO: 8):

[0079] (b) Construction of fungal genetic transformation system mediated by Agrobacterium tumefaciens. The knockout vector or overexpression vector plasmid constructed above was transformed into Agrobacterium AGL-1. The positive Agrobacterium AGL-1 transformed strain identified by PCR was selected and shaken in LB liquid medium (containing 50 mg / mL kanamycin Kan) until the bacterial solution concentration OD 660 The OD value is 0.8-1.0. Collect the cells and dilute and resuspend them with an appropriate amount of IM+200μM acetosyringone (As) liquid medium. 660 The bacterial solution was cultured at 28°C in the dark until the OD 660 It is 0.5-0.7, for standby use.

[0080] (c) Preparation of M. anisopliae spore suspension: Scrape M. anisopliae conidia from 1 / 4SDAY medium cultured for 2 weeks and add them to 1.5 mL of sterile water containing 200 μM acetosyringone (As). Vortex and filter with sterile lens paper to remove hyphae. Collect the filtrate and adjust the concentration of M. anisopliae spore suspension to 3 × 10 6 conidia / mL, set aside.

[0081] (d) The above prepared Metarhizium anisopliae spore suspension was mixed with the above OD 660 Mix 0.5-0.7% AGL-1 bacterial suspension at a 1:1 ratio and spread 200 μL evenly on an IM medium plate. After 48 hours of incubation in the dark, wash with sterile water and spread the wash onto agar medium containing 500 μg / mL glufosinate and 300 μg / mL cefotaxime. Cultivate for 7-10 days until resistant colonies appear. Extract the fungal genome and verify transformants by PCR using specific primers.

[0082] (e) Virulence verification of knockout and overexpression of Metarhizium anisopliae. Spores of Metarhizium anisopliae with deletion or overexpression of MAC_08119 gene cultured on culture medium for 14 days were scraped and suspended in sterile water containing 0.05% Tween-80. After vortexing, the spores were collected by filtration with lens paper. The spores were counted under a microscope using a hemocytometer and the final concentration of the spore suspension was adjusted to 1×10 8 Conidia / mL. 5 μL of the spore suspension was dripped under the dorsal plate of East Asian migratory locust larvae. Wild-type Metarhizium anisopliae spores (MAC-WT) were used as a control. The number of dead locusts was recorded every 12 hours.

[0083] (f) Purification of the MAC_08119 protein. The MAC_08119 protein was expressed and purified in vitro using an E. coli in vitro expression system (Figure 3). The purified protein was then injected into locusts to verify its virulence.

[0084] (g) The results showed that compared with the wild-type M. anisopliae strain (MAC-WT), the deletion of MAC_08119 (△MAC_08119) significantly inhibited the pathogenicity of M. anisopliae to locusts (Figure 4). Injection of the purified protein restored the virulence of the MAC_08119-deficient (△MAC_08119) strain to locusts (Figure 5). Finally, overexpression of this gene (OE:MAC_08119) in M. anisopliae significantly increased its pathogenicity to locusts (Figure 6). This suggests that the protein encoded by the MAC_08119 gene is a toxin protein secreted by M. anisopliae against its host locust.

[0085] Example 2 Genetic modification of Metarhizium Robertsii

[0086] (a) Construct an overexpression strain. The steps are the same as steps (a) to (d) in Example 1.

[0087] (b) Detection of MAC_08119 overexpression effect.

[0088] RNA was extracted from cultured recombinant M. anisopliae using the Kangwei Century Ultrapure RNA Nucleic Acid Extraction Kit. The extracted RNA was then converted to cDNA using the Takara PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time). Quantitative PCR analysis was performed using the Talent Fluorescence Quantification Detection Kit (SYBR Green) from Tiangen Biotechnology. The gpd gene of M. anisopliae was used as an internal reference to quantify the RNA expression of the MAC_08119 gene (OE: MAC_08119) in wild-type M. anisopliae (MAA-WT) and recombinant M. anisopliae.

[0089] As shown in Figure 7, the wild-type M. anisopliae (MAA-WT) does not have the M. anisopliae MAC_08119 gene, and the recombinant M. anisopliae (OE:MAC_08119) overexpresses the M. anisopliae MAC_08119 gene.

[0090] Example 3 Detection of the pathogenicity of recombinant Metarhizium Robertsii to various insects

[0091] This example selected migratory locusts and pollinating insects bumblebees and mealworms as research subjects to test the toxicity of the recombinant Metarhizium Robertsii prepared in Example 2. The toxicity test steps are as follows:

[0092] Spores of wild-type M. anisopliae (MAA-WT) or recombinant M. anisopliae (OE: MAC_08119) cultured on medium for 14 days were scraped and suspended in sterile water containing 0.05% Tween-80. After vortexing, the spores were collected by filtration through lens paper. The spores were counted under a microscope using a hemocytometer and the final concentration of the spore suspension was adjusted to 1×10 8 Conidia / mL, 5 μL of spore suspension was dripped under the dorsal plate of the fifth instar larvae of the gregarious East Asian migratory locust; the final concentration of the spore suspension was adjusted to 1×10 7 Spray evenly over the surfaces of bumblebees or mealworms using a 500 μg / mL conidia spray. Wild-type Metarhizium anisopliae (MAA-WT) was used as a control. The number of dead insects was recorded every 12 hours.

[0093] The results showed that the recombinant M. anisopliae strain (OE: MAC_08119) had a significantly higher lethality against migratory locusts than the wild-type strain (MAA-WT), with a half-lethal time approximately 3 days earlier than the wild-type strain (Figure 8). However, there was no significant increase in pathogenicity against mealworms (Figure 9) and bumblebees (Figure 10). Therefore, by introducing M. anisopliae virulence factors into M. anisopliae, the pathogenicity of M. anisopliae against locust pests can be effectively improved, compensating for its poor locust control effect. At the same time, its characteristic of minimal damage to pollinating insects is retained.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a protein in the preparation of a biological pesticide, characterized in that: The protein comprises at least one of the following proteins: (11) the protein represented by SEQ ID NO: 1; (12) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1; (13) A protein having the same function as the protein shown in SEQ ID NO: 1 obtained by substitution and / or deletion and / or addition of one or more amino acid residues; (14) A protein that is at least 75% identical to the protein shown in SEQ ID NO: 1 and has the same function.

2. Use of a gene in the preparation of a biological pesticide, characterized in that: The gene includes at least one of the following genes: (21) the gene shown in SEQ ID NO: 2; (22) a complementary sequence, a degenerate sequence or a homologous sequence of the gene shown in SEQ ID NO: 2, wherein the homologous sequence is a sequence having at least 75% identity with the gene shown in SEQ ID NO: 2; (23) A sequence that hybridizes with the gene represented by SEQ ID NO: 2 under stringent conditions or a complementary sequence thereof.

3. The use according to claim 1 or 2, characterized in that: The biological pesticide is a biological pesticide for controlling at least Acrididae pests.

4. A biomaterial, characterized in that: The biological material is used to express the gene according to claim 2, and the biological material includes at least one of an expression cassette, a vector, a recombinant cell, and a recombinant microorganism.

5. The biomaterial according to claim 4, characterized in that The vector comprises at least one of a plasmid, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and a linear polynucleotide; The recombinant cells include plant cells and / or animal cells that are not used for the purpose of reproduction; The recombinant microorganism includes at least one of fungi, bacteria, and algae; The fungus includes at least one of yeast, Aspergillus niger, and Trichoderma reesei; The bacteria include at least one of Agrobacterium, Escherichia coli, Bacillus, Pseudomonas, and lactic acid bacteria; The algae include at least one of cyanobacteria, Synechococcus, Synechococcus elongatus, Synechocystis, Anabaena, Chlamydomonas, and Chlamydomonas reinhardtii.

6. A recombinant insect pathogenic fungus, characterized in that: The recombinant insect pathogenic fungus comprises the gene according to claim 2 and / or the biological material according to any one of claims 4-5.

7. The recombinant insect pathogenic fungus according to claim 6, characterized in that The insect pathogenic fungi include broad-spectrum Metarhizium and / or Beauveria bassiana; The broad-spectrum Metarhizium includes one or two of Metarhizium Roberts and Metarhizium scarabensis.

8. Use of the recombinant insect pathogenic fungus according to claim 6 or 7 in the preparation of biological pesticides.

9. The method for preparing the recombinant insect pathogenic fungus according to claim 6 or 7, characterized in that: The gene according to claim 2 and / or the biological material according to any one of claims 4 to 5 are introduced into insect pathogenic fungi to obtain recombinant insect pathogenic fungi.

10. The preparation method according to claim 9, characterized in that: The method of introducing the gene of claim 2 into insect pathogenic fungi comprises: connecting the gene of claim 2 to a vector to obtain a recombinant vector, transferring the recombinant vector into Agrobacterium, and transferring the recombinant vector into the insect pathogenic fungi using the Agrobacterium-mediated method.

11. A biological pesticide, characterized in that: The biological pesticide comprises the recombinant insect pathogenic fungus according to claim 6 or 7.

12. The biopesticide according to claim 11, characterized in that: The biopesticides also include other types of biopesticides.

13. The biopesticide according to claim 11 or 12, characterized in that: The biopesticide also includes pesticide-acceptable adjuvants.

14. The biopesticide according to any one of claims 11 to 13, characterized in that The biological pesticide is selected from the following dosage forms: solution, emulsion, wettable powder, granular wettable powder, suspension, dust, foam, paste, tablet, granule, aerosol, natural agent impregnated with active compound, synthetic agent impregnated with active compound, microcapsule, seed coating agent, preparation equipped with combustion device, cold fog agent, hot fog agent.

15. An insecticidal composition, characterized in that: The insecticidal composition comprises the recombinant insect pathogenic fungus according to claim 6 or 7 and a chemical pesticide.

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