Application of fusarium graminearum FgSCP43 gene

Abstract

The invention relates to a method for knocking out Fusarium graminearum FgSCP43 gene, and a mutant strain delta FgSCP43 with the FgSCP43 gene knocked out, which is obtained by the method. The FgSCP43 gene is knocked out from the fusarium graminearum, so that the spore yield of the fusarium graminearum is reduced, meanwhile, the pathogenicity to wheat coleoptiles and wheat ears is remarkably reduced, and the FgSCP43 plays an important role in the pathogenic process of the fusarium graminearum. The invention provides a new thought and direction for preventing and treating wheat scab.

Description

Technical Field

[0001] The present invention relates to the field of crop prevention and control, and in particular to the application of the FgSCP43 gene of Fusarium graminearum. Background Art

[0002] Fusarium head blight (FHB) is the most serious and frequent fungal disease during wheat planting, and it occurs in wheat-producing areas around the world. In recent years, due to climate warming and changes in farming methods such as returning straw to the field and crop rotation, wheat head blight has frequently become a disaster in my country, causing huge economic losses to wheat production. Fusarium graminearum is the main pathogen that causes head blight. After infecting wheat, it not only seriously affects wheat yield and quality, but also produces a variety of fungal toxins such as deoxynivalenol (DON) and zearalenone (ZEN), which accumulate in large quantities on infected wheat ears, seriously threatening the quality and safety of agricultural products and the health of humans and animals.

[0003] In 2020, the Ministry of Agriculture and Rural Affairs listed wheat fusarium head blight as the first batch of Class I crop pests and diseases. Strengthening the prevention and control of fusarium head blight is of great significance to ensuring the quality and safety of my country's grain production and agricultural products. Due to the lack of wheat varieties that are immune to fusarium head blight, chemical fungicides are still mainly used to prevent and control fusarium head blight in agricultural production. However, with the increase of resistance of the pathogen (Fusarium graminearum), the prevention and control efficiency has gradually decreased.

[0004] Therefore, it is necessary to conduct in-depth research on the pathogenicity mechanism of Fusarium graminearum and to discover and identify key pathogenic genes. Summary of the invention

[0005] The present invention firstly provides an application of the FgSCP43 gene of Fusarium graminearum, knocks out the FgSCP43 gene of Fusarium graminearum to reduce the pathogenicity of Fusarium graminearum to wheat, and provides a new target and direction for the prevention and treatment of wheat fusarium head blight.

[0006] The gene FgSCP43 that regulates the pathogenicity of Fusarium graminearum has a gene sequence as described in SEQ ID NO.1; and the protein encoded by the gene is described in SEQ ID NO.2.

[0007] The present invention also provides a method for knocking out the FgSCP43 gene of Fusarium graminearum, which specifically comprises the following steps:

[0008] (1) Extracting genomic DNA of Fusarium graminearum PH-1;

[0009] (2) using primers FgSCP43-1F / FgSCP43-2R and FgSCP43-3F / FGSCP43-4R to amplify the upstream and downstream fragments of FgSCP43, respectively, and using HPH-F, HP-R and PH-F, HPH-R to amplify the hygromycin phosphotransferase gene (HPH) to obtain H1 and H2 fragments;

[0010] (3) using the split-PCR method with primers FgSCP43-1F and HP-R to fuse the upstream fragment of FgSCP43 with the H1 fragment, and using primers FgSCP43-4R and PH-F to fuse the downstream fragment of FgSCP43 with the H2 fragment, to obtain fusion fragments respectively;

[0011] (4) The fusion fragment was transferred into Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a mutant strain ΔFgSCP43 with FgSCP43 gene knockout;

[0012] The primers for amplifying the upstream and downstream fragments are as follows:

[0013] FgSCP43-1F (SEQ ID NO.3): AGTGCCCTTGATGCAAACAA

[0014] FgSCP43-2R (SEQ ID NO.4):

[0015] TTGACCTCCACTAGCTCCAGCCAAGCCCATGTATGGCTTGCTCCCCA

[0016] FgSCP43-3F (SEQ ID NO.5):

[0017] GAATAGAGTAGATGCCGACCGCGGGTTGGTCAAGATGGCGTGGGATT

[0018] FgSCP43-4R(SEQ ID NO.6):ACTGGGTCTGCGACAATGAG

[0019] The primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene are as follows;

[0020] HPH-F(SEQ ID NO.11):GGCTTGGCTGGAGCTAGTGGAGGTCAA

[0021] HPH-R(SEQ ID NO.12):AACCCGCGGTCGGCATCTACTCTATTC

[0022] HP-R(SEQ ID NO.15):GTATTGACCGATTCCTTGCGGTCCGAA

[0023] PH-F (SEQ ID NO. 16): GATGTAGGAGGGCGTGGATATGTCCT

[0024] The present invention also provides a mutant ΔFgSCP43 with the FgSCP43 gene of Fusarium graminearum knocked out prepared by the above method. The mutant ΔFgSCP43 is obtained by knocking out the FgSCP43 gene from the genome of the wild-type Fusarium graminearum strain PH-1 based on the principle of homologous recombination and a protoplast transformation method mediated by polyethylene glycol (PEG).

[0025] The mutant ΔFgSCP43 has the following characteristics:

[0026] (1) Compared with the wild-type strain PH-1, the mutant ΔFgSCP43 strain had no significant difference in growth;

[0027] (2) Compared with the wild-type strain PH-1, the spore yield of the mutant ΔFgSCP43 strain was significantly reduced;

[0028] (3) Compared with the wild-type strain PH-1, the DON production of the mutant ΔFgSCP43 was not significantly different;

[0029] (4) Compared with the wild-type strain PH-1, the mutant ΔFgSCP43 had significantly reduced pathogenicity to wheat coleoptiles and ears.

[0030] The present invention also provides the use of the FgSCP43 gene as an inhibition target or a knockout target in reducing the pathogenicity of Fusarium graminearum to wheat and preventing and controlling wheat scab;

[0031] That is to say, the application of the Fusarium graminearum FgSCP43 gene provided by the present invention is to reduce the pathogenicity of Fusarium graminearum to wheat and prevent and control wheat fusarium head blight by inhibiting the translation of the FgSCP43 gene in Fusarium graminearum or the expression of its protein.

[0032] The present invention also provides a method for reducing the pathogenicity of Fusarium graminearum to wheat, comprising the step of reducing or preventing the expression of the FgSCP43 gene of Fusarium graminearum or the protein encoded by the FgSCP43 gene.

[0033] In a preferred embodiment, the method for reducing or eliminating the expression of the Fusarium graminearum FgSCP43 gene or the protein encoded by the FgSCP43 gene includes: mutating or knocking out the entire or partial sequence of the FgSCP43 gene; or constructing an interference vector to interfere with the expression of the FgSCP43 gene; or using a gene silencing system to silence the expression of the FgSCP43 gene.

[0034] The application of the Fusarium graminearum FgSCP43 gene provided by the present invention is of great significance for effectively preventing and controlling wheat fusarium graminearum-induced fusarium head blight by deeply understanding the pathogenic mechanism of Fusarium graminearum and mining and identifying key genes in the process of Fusarium graminearum infecting wheat.

[0035] The present invention is beneficial in that:

[0036] (1) The present invention provides an application of the gene FgSCP43 for preventing and controlling wheat fusarium head blight, that is, by knocking out the FgSCP43 gene, the pathogenicity of Fusarium graminearum to wheat is reduced, thereby preventing and controlling wheat fusarium head blight caused by Fusarium graminearum;

[0037] (2) The present invention provides a new method for preventing and controlling wheat fusarium head blight, which inhibits the pathogenicity of Fusarium graminearum to wheat by inhibiting the expression of the FgSCP43 gene of Fusarium graminearum or the protein encoded by the FgSCP43 gene;

[0038] (3) The present invention provides a gene target for preventing and controlling wheat fusarium head blight, and the agent that destroys the expression, shearing, and expression, modification, location or activity of the gene and its encoded protein can effectively prevent and control wheat fusarium head blight caused by Fusarium graminearum;

[0039] (4) The homologous genes and homologous proteins of the FgSCP43 gene of Fusarium graminearum in other pathogenic fungi can be used as candidate targets. The new antibacterial agents developed by targeting the homologous genes or proteins can destroy the normal expression and function of the homologous genes or proteins, thereby effectively preventing and controlling wheat fusarium head blight caused by pathogenic fungi.

[0040] The application of the Fusarium graminearum FgSCP43 gene provided by the present invention will help develop new fungicides, formulate effective strategies for the prevention and control of fusarium head blight, and ensure the quality and safety of grain production and agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Construction of FgSCP43 gene knockout mutant

[0042] Among them, 1F, 2R, 3F, 4R, 5F, 6R, 7F, and 8R refer to primers FgSCP43-1F, FgSCP43-2R, FgSCP43-3F, FgSCP43-4R, FgSCP43-5F, FgSCP43-6R, FgSCP43-7F, and FgSCP43-8R, respectively.

[0043] Figure 2 Validation of FgSCP43 knockout mutants

[0044] 1: Primer FgSCP43-5F / FgSCP43-6R,

[0045] 2: Primer FgSCP43-7F / HPH-Down,

[0046] 3: Primer HPH-Up / FgSCP43-8R,

[0047] 4: Primer HPH-F / HPH-R

[0048] Figure 3 Effects of FgSCP43 gene knockout on colony morphology and conidia

[0049] (A) Colony morphology; (B) Conidia morphology; (C) Growth rate

[0050] Figure 4 Effects of FgSCP43 gene knockout on DON biosynthesis

[0051] (A) PDA culture medium, (B) TBI culture medium, the same lowercase letters indicate no significant difference (P ≥ 0.05)

[0052] Figure 5 Effects of FgSCP43 gene knockout on pathogenicity

[0053] (A) Symptoms of coleoptile disease, (B) Length of coleoptile lesions, (C) Symptoms of wheat ear disease, (D) Disease incidence of wheat ear, different lowercase letters indicate significant differences (P<0.05) DETAILED DESCRIPTION

[0054] The gene synthesis, primer synthesis and sequencing in the following examples were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0055] The PEG-mediated protoplast transformation method used in the following examples is the method disclosed in Wang G, Wang C, Hou R, et al. The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum. PLoS One. 2012; 7(5): e38324.

[0056] The nucleotide sequence of the Fusarium graminearum FgSCP43 gene in the embodiment is shown in SEQ ID NO.1:

[0057] SEQ ID NO.1:

[0058] ATGGCTGCCTTTGTAGCAGCTACCCCTGCCCCCAAGGGCTGCACGCCTGGAACCTACTCGTGTATGGCCGACCTCAAGGGGCTGGCAGGTCTGCAACGTCGACCGCACCTGGGTGTTCGCCGGTGCTTGTCCTCCAAAGACGGCTTGCCTGTTCAACAAGCAGAATGGTTCGCCGTACTGCGTGCCCCCTGGCTTCCACTTC

[0059] The amino acid sequence of the Fusarium graminearum FgSCP43 protein is shown in SEQ ID NO.2:

[0060] SEQ ID NO.2:

[0061] MAAFVAATPAPKGCTPGTYSCMADLKGWQVCNVDRTWVFAGACPPKTACLFN KQNGSPYCVPPGFHF

[0062] Unless otherwise specified, the materials and instruments used in the following examples are all common commercially available products.

[0063] Example 1 Knockout of the FgSCP43 gene of Fusarium graminearum and construction of the knockout mutant ΔFgSCP43

[0064] (1) Obtaining gene knockout fusion fragments

[0065] The wild-type Fusarium graminearum strain PH-1 (Fusarium graminearum PH-1 / NRRL 31084, American Type Culture Collection Center, No. ATCC MYA-4620) was inoculated into PDA medium (potato 200g / L, glucose 20g / L, agar 20g / L) and cultured in the dark at 28°C for 7 days. Mycelium was collected and genomic DNA was extracted using a DNA kit (Shanghai Shenggong Biotechnology Co., Ltd.). Using genomic DNA as a template, according to the principle of homologous recombination ( Figure 1 ), and the FgSCP43 gene knockout fragment was constructed by split-PCR technology.

[0066] Primers FgSCP43-1F and FgSCP43-2R were used to amplify the upstream fragment of the FgSCP43 gene, while primers FgSCP43-3F and FgSCP43-4R were used to amplify the downstream fragment. In addition, primers HPH-F and HP-R, PH-F and HPH-R were used to amplify the hygromycin phosphotransferase gene, respectively, to obtain H1 and H2 fragments.

[0067] The reagents used in the split-PCR for constructing the FgSCP43 gene knockout fragment were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The reaction system is shown in Table 1 below, and the reaction procedure is shown in Table 2 below:

[0068] Table 1 PCR reaction system

[0069]

[0070] Table 2 PCR reaction program

[0071]

[0072] The split-PCR method was used to fuse the upstream fragment of FgSCP43 with the H1 fragment using primers FgSCP43-1F and HP-R, and the downstream fragment of FgSCP43 with the H2 fragment using primers FgSCP43-4R and PH-F to obtain the fusion fragments L1H1 and L2H2, respectively.

[0073] The reagents used for split-PCR of the fusion fragment were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The reaction system is shown in Table 3 below, and the reaction procedure is shown in Table 4 below:

[0074] Table 3 PCR reaction system

[0075]

[0076] Table 4 PCR reaction conditions

[0077]

[0078] (2) Construction of the gene knockout mutant ΔFgSCP43

[0079] The activated wild-type strain PH-1 was inoculated into CMC medium (carboxymethyl cellulose 15 g / L, NH 4 NO 3 10g / L, MgSO 4 7H 2 O 0.5 g / L, KH2PO4 1 g / L, yeast extract 1 g / L), cultured at 25°C and 175 r / min for 4 days, spores were collected by filtration through a double-layer microcloth (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and transferred to YEPD liquid medium (yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, pH 6.0). After culturing at 25°C and 175 r / min for 12 hours, mycelium was collected and mixed with protoplast buffer (Driselase 250 mg, Lysing enzyme 50 mg, Chitnase 0.5 mg, 1.2 mol / L KCl (Sigma-Aldrich (Shanghai) Trading Co., Ltd.)), and cultured at 30°C and 90 r / min for another 2 hours. Miracloth was used to filter the protoplasts, and the protoplasts were washed with 1.2 mol / L KCl. 50 mL STC buffer (100 g sucrose, 50 mL 0.5 mol / L Tris-HCl (pH 8.0), and CaCl 2 ·2H 2 O 3.6755g, water 450mL) suspended the precipitate to obtain protoplasts.

[0080] The fusion fragments L1H1 and L2H2 were fully mixed with the protoplasts, 1.25 mL of 40% PTC buffer (PEG8000 40 g, STC buffer 100 mL) was added, mixed evenly, and allowed to stand at room temperature for 20 minutes. Then, TB3 medium (yeast extract 3 g / L, acid hydrolyzed casein 3 g / L, sucrose 200 g / L) containing 50 μg / mL of ampicillin (Shanghai Shenggong Biotechnology Co., Ltd.) was added, and the transformants were cultured overnight at room temperature. Preliminary screening was performed by hygromycin to obtain the knockout strain ΔFgSCP43.

[0081] The genomic DNA of the knockout strain ΔFgSCP43 was extracted, and the target gene was detected with FgSCP43-5F / FgSCP43-6R, the upstream recombinant fragment was detected with FgSCP43-7F / HPH-Down, the downstream recombinant fragment was detected with HPH-Up / FgSCP43-8R, and the hygromycin gene was detected with HPH-F / HPH-R. Figure 2It can be seen that the knockout strain ΔFgSCP43 had no band when amplified with FgSCP43-5F / FgSCP43-6R, but had bands when amplified with FgSCP43-7F / HPH-Down, HPH-Up / FgSCP43-8R and HPH-F / HPH-R, respectively, while the wild strain PH1 was just the opposite, proving that the target gene FgSCP43 was knocked out.

[0082] The primer sequences used in the above steps are shown in Table 5:

[0083] Table 5 Primers used in the construction and verification of the gene knockout mutant ΔFgSCP43

[0084]

[0085]

[0086] Example 2: Mycelial growth and conidia determination of the mutant strain ΔFgSCP43 of Fusarium graminearum (1) Colony growth rate and morphological observation

[0087] The activated wild-type strain PH-1 and mutant strain ΔFgSCP43 were inoculated into PDA medium and cultured in the dark at 25°C. The colony diameter was recorded and photographed every day using the cross method. Three replicates were set for each strain.

[0088] The results show (see Figure 3 A, Table 6), compared with the wild-type strain PH-1, the colony morphology and growth rate of the mutant strain ΔFgSCP43 had no significant differences, indicating that the FgSCP43 gene is not involved in regulating the hyphal growth of Fusarium graminearum.

[0089] Table 6 Growth rate and conidia production of FgSCP43 gene knockout mutant strain ΔFgSCP43

[0090]

[0091] Different superscript letters indicate significant differences (P<0.05)

[0092] (2) Conidia yield determination and morphological observation

[0093] The activated wild-type strain PH-1 and mutant strain ΔFgSCP43 were inoculated into CMC medium, shaken at 25°C and 150 r / min for 3 days, and conidia were collected by filtration with sterile filter cloth. The conidia were counted and the conidia production was calculated using a hemocytometer. Three replicates were set for each strain.

[0094] The results show (see Figure 3B, Table 6), the mutant strain ΔFgSCP43 can produce sickle-shaped conidia normally. Compared with the wild-type strain PH-1, the size, shape and number of septa of ΔFgSCP43 spores were not significantly different, but the conidia production was significantly reduced by about 43.4%. Therefore, the FgSCP43 gene may play a role in the conidial development of Fusarium graminearum.

[0095] Example 3: DON production analysis of the mutant strain ΔFgSCP43 of Fusarium graminearum

[0096] The activated wild-type strain PH-1 and mutant strain ΔFgSCP43 were inoculated into PDA medium and TBI medium, respectively. PDA medium was cultured at 25°C in the dark for 7 days, and TBI medium was cultured at 25°C and 150r / min in a shaker for 3 days. After the PDA culture, the culture was placed in a 50°C oven to dry, and crushed and mixed. Subsequently, 2g of the sample was accurately weighed into a 50mL centrifuge tube, 10mL of acetonitrile-water (84:16, V / V) was added, vortexed for 1 minute, and ultrasonically extracted for 1 hour after soaking for 5 minutes. After the extraction was completed, centrifuged at 4000r / min for 10 minutes, 5mL of the supernatant was taken, and dried under nitrogen at 40°C. Then, the residue was dissolved with 1mL of 5mmol / L ammonium acetate solution-methanol (80:20, V / V), vortexed for 30 seconds, and filtered through a 0.22μm filter membrane, and determined by UPLC-MS / MS. After TBI culture, the culture medium was filtered through a 0.22 μm filter membrane, appropriately diluted and filtered through a 0.22 μm filter membrane, and then measured by UPLC-MS / MS. Each treatment was repeated 5 times.

[0097] The UPLC-MS / MS measurement conditions are shown in Table 7:

[0098] Table 7 UPLC-MS / MS chromatographic conditions

[0099]

[0100] Mass spectrometry conditions: electrospray ionization (ESI) positive ion mode was used; the nebulizer gas and auxiliary gas were both high-purity air, and the collision gas was high-purity nitrogen; the specific parameters were as follows: nebulizer gas pressure 50 psi; auxiliary gas pressure 50 psi; nebulizer temperature 500.0℃; spray voltage 5 500V; spray voltage curtain gas pressure 35 psi; collision gas pressure 8 psi; DON was quantified using the multiple reaction monitoring mode: parent ion m / z 297.3; quantitative daughter ion m / z 203.0, collision energy 38 eV; qualitative daughter ion m / z 175.1, collision energy 28 eV.

[0101] The results showed that (see Figure 4A, B), in PDA medium and TBI medium, the DON production in the mutant strain ΔFgSCP43 was not significantly different from that in the wild type PH-1. Therefore, the FgSCP43 gene is not involved in regulating the biosynthesis of DON in Fusarium graminearum.

[0102] Example 4: Determination of virulence of the mutant strain ΔFgSCP43 of Fusarium graminearum

[0103] Coleoptile inoculation method: Sterilize the surface of wheat seeds ("Jimai 22") with 75% ethanol for 5 minutes, place them in a culture dish covered with filter paper containing water, and culture them at room temperature for 3 days to accelerate germination. Then cut off the tip of the coleoptile by 2-3 mm. 2 Sterile filter papers of different sizes were immersed in the prepared spore suspensions of wild-type strain PH-1 and knockout mutant strain ΔFgSCP43 of Fusarium graminearum (concentration of about 2×10 5 Each strain was inoculated into 15 wheat seedlings, and sterile water was used as a control. The seedlings were cultured in a 25°C light incubator with 95% relative humidity and alternating light and dark (12h / 12h) for 7 days, and the length of pathogenicity was measured.

[0104] Wheat ear inoculation method: Conidia of wild-type PH-1 and knockout mutant strain ΔFgSCP43 of Fusarium graminearum were prepared (concentration of about 2×10 5 / mL). Wheat ears (Fumai 13) at the flowering stage were selected as inoculation objects. 20 μL of conidia suspension was inoculated in the middle and lower part of each ear. 15 ears of each strain were inoculated. The temperature was kept moist at 25℃ for 24-48 hours. The results were observed after 14 days, and the number of diseased grains per ear was counted to measure the pathogenicity.

[0105] The results show that ( Figure 5 ), after inoculation of coleoptiles, the mutant strain ΔFgSCP43 produced significantly fewer brown necrotic lesions in wheat coleoptiles than the wild-type strain PH-1, and the length of the lesions decreased by about 66.24%; after inoculation of wheat spikes, the mutant strain ΔFgSCP43 had significantly fewer diseased grains than the wild-type strain PH-1, with the incidence rate reduced by 70.05%. Therefore, the FgSCP43 gene plays a vital role in the pathogenicity of Fusarium graminearum.

Claims

1. A method for knocking out the FgSCP43 gene of Fusarium graminearum, characterized in that The steps include: (1) Extracting genomic DNA of Fusarium graminearum PH-1; (2) primers FgSCP43-1F / FgSCP43-2R and FgSCP43-3F / FGSCP43-4R were used to amplify the upstream and downstream fragments of FgSCP43, respectively, and HPH-F, HP-R and PH-F, HPH-R were used to amplify the hygromycin phosphotransferase gene HPH to obtain H1 and H2 fragments; (3) using the split-PCR method with primers FgSCP43-1F and HP-R to fuse the upstream fragment of FgSCP43 with the H1 fragment, and using primers FgSCP43-4R and PH-F to fuse the downstream fragment of FgSCP43 with the H2 fragment, to obtain fusion fragments respectively; (4) The fusion fragment was transferred into Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a mutant strain ΔFgSCP43 with FgSCP43 gene knockout; The primers for amplifying the upstream fragment and the downstream fragment are as follows: FgSCP43-1F is shown in SEQ ID NO.3, FgSCP43-2R is shown in SEQ ID NO.4, FgSCP43-3F is shown in SEQ ID NO.5, and FgSCP43-4R is shown in SEQ ID NO.6; Primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene, HPH-F is shown in SEQ ID NO.11, HPH-R is shown in SEQ ID NO.12, HP-R is shown in SEQ ID NO.15, and PH-F is shown in SEQ ID NO.

16.

2. The FgSCP43 gene knockout mutant strain ΔFgSCP43 prepared by the method of claim 1.

3. Application of FgSCP43 gene as an inhibition target or knockout target to reduce the pathogenicity of Fusarium graminearum to wheat or to prevent and control wheat fusarium head blight.

4. A method for reducing the pathogenicity of Fusarium graminearum to wheat, comprising the steps of knocking out the FgSCP43 gene of Fusarium graminearum or reducing or not expressing the expression of the protein encoded by the FgSCP43 gene.

5. The method for reducing the pathogenicity of Fusarium graminearum to wheat according to claim 4, wherein the Fusarium graminearum FgSCP43 gene knockout specifically comprises the following steps: (1) Extracting genomic DNA of Fusarium graminearum PH-1; (2) primers FgSCP43-1F / FgSCP43-2R and FgSCP43-3F / FGSCP43-4R were used to amplify the upstream and downstream fragments of FgSCP43, respectively, and HPH-F, HP-R and PH-F, HPH-R were used to amplify the hygromycin phosphotransferase gene HPH to obtain H1 and H2 fragments; (3) using the split-PCR method with primers FgSCP43-1F and HP-R to fuse the upstream fragment of FgSCP43 with the H1 fragment, and using primers FgSCP43-4R and PH-F to fuse the downstream fragment of FgSCP43 with the H2 fragment, to obtain fusion fragments respectively; (4) The fusion fragment was transferred into Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a mutant strain ΔFgSCP43 with FgSCP43 gene knockout; The primers for amplifying the upstream fragment and the downstream fragment are as follows: FgSCP43-1F is shown in SEQ ID NO.3, FgSCP43-2R is shown in SEQ ID NO.4, FgSCP43-3F is shown in SEQ ID NO.5, and FgSCP43-4R is shown in SEQ ID NO.6; Primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene, HPH-F is shown in SEQ ID NO.11, HPH-R is shown in SEQ ID NO.12, HP-R is shown in SEQ ID NO.15, and PH-F is shown in SEQ ID NO.16.

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