Application of FghnRNP G gene of Fusarium graminearum

Abstract

The present invention discloses an application of the FghnRNP G gene of Fusarium graminearum. The present invention obtains a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knocked out by knocking out the FghnRNP G gene in the Fusarium graminearum genome. Compared with the wild-type strain PH-1, the mycelial growth and spore production of the genetically engineered strain ΔFghnRNP G slightly decreased, and there was no significant difference in pathogenicity to wheat coleoptiles, but the yield of deoxynivalenol decreased significantly. The present invention also provides the application of the FghnRNP G gene as an inhibition target or a knockout target in inhibiting the synthesis of DON by Fusarium graminearum, which provides a new idea and research direction for the pollution prevention and control of deoxynivalenol in agricultural products.

Description

Technical Field

[0001] The invention relates to the field of agricultural product quality safety, and in particular to an application of a Fusarium graminearum FghnRNP G gene. Background Art

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a trichothecene fungal toxin produced by Fusarium graminearum and other fungi, which widely contaminates grains such as wheat, barley, oats, rye and corn and their products. DON can be transmitted through its C 12 / C 13 Epoxy groups interfere with the dissociation of ribosome aggregates in eukaryotic cells, hinder ribosome circulation, inhibit protein synthesis, and cause acute and chronic poisoning symptoms such as anorexia, nausea, vomiting, growth delay, reproductive disorders, anemia, and headaches, posing huge safety risks to human and animal health and has become one of the main threats to the quality and safety of my country's agricultural products.

[0003] At present, the prevention and control of DON in agricultural production mainly relies on chemical fungicides such as carbendazim. However, with the increase of drug resistance of Fusarium graminearum, the prevention and control effect is not stable, and there are a series of problems such as environmental pollution and introduction of new safety risks. It is urgent to develop new methods for the prevention and control of DON pollution. Summary of the invention

[0004] The present invention first provides a method for inhibiting the synthesis of DON by Fusarium graminearum, which comprises the following steps:

[0005] Knocking out the FghnRNP G gene in the genome of Fusarium graminearum to obtain a genetically engineered strain ΔFghnRNP G with the FghnRNP G gene knocked out;

[0006] Specifically, the present invention provides a method for inhibiting the synthesis of DON by Fusarium graminearum, comprising the following steps:

[0007] (1) Extracting genomic DNA of Fusarium graminearum;

[0008] (2) Using the genomic DNA of Fusarium graminearum as a template and based on the principle of homologous recombination, split-PCR was used to construct a knockout FghnRNP G gene fragment and PEG-mediated protoplast transformation was used to obtain the FghnRNP G knockout mutant;

[0009] (3) primers FghnRNP G-1F / FghnRNP G-2R and FghnRNP G-3F / FghnRNP G-4R were used to amplify the upstream and downstream fragments of FghnRNP G, 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;

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

[0011] (5) The fusion fragment was transferred into wild-type Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout;

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

[0013] FghnRNP G-1F: GCGGTATGCACATTGACGAC

[0014] FghnRNP G-2R: TTGACCTCCACTAGCTCCAGCCAAGCCATCATTGACGGAGCAAGGCA FghnRNPG-3F: GAATAGAGTAGATGCCGACCGCGGGTTTCGCCCCTTCTCATGGTCTA FghnRNP G-4R: GTCAGCAGCTGTTGGTTGTG

[0015] The primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene are as follows;

[0016] HPH-F:GGCTTGGCTGGAGCTAGTGGAGGTCAA

[0017] HPH-R:ATAGAGTAGATGCCGACCGCGGGTTC

[0018] HP-R: GTATTGACCGATTCCTTGCGGTCCGAA

[0019] PH-F: GATGTAGGAGGGCGTGGATATGTCCT

[0020] The sequence of the FghnRNP G gene is as described in SEQ ID NO.1; the protein encoded by the gene is as described in SEQ ID NO.2.

[0021] The present invention also provides a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knocked out, prepared by the above method;

[0022] The ability of the genetically engineered strain ΔFghnRNP G to synthesize DON is greatly reduced compared with the wild strain PH-1, but has little or no effect on the infection ability of wheat.

[0023] The present invention also provides the use of FghnRNP G gene as an inhibition target in inhibiting the synthesis of DON by Fusarium graminearum;

[0024] In other words, the application of FghnRNP G gene as a knockout target in inhibiting DON synthesis by Fusarium graminearum;

[0025] That is to say, the application of the Fusarium graminearum FghnRNP G gene provided by the present invention is to inhibit the translation of the FghnRNP G gene in Fusarium graminearum or the expression of its protein, thereby achieving the inhibition of DON synthesis by Fusarium graminearum.

[0026] The present invention also provides a method for reducing the synthesis of DON toxin by Fusarium graminearum, comprising the step of reducing or preventing the expression of the FghnRNP G gene of Fusarium graminearum or the protein encoded by the FghnRNP G gene.

[0027] In a preferred embodiment, the method for reducing or eliminating the expression of the Fusarium graminearum FghnRNP G gene or the protein encoded by the FghnRNP G gene comprises: mutating or knocking out the entire or partial sequence of the FghnRNP G gene; or constructing an interference vector to interfere with the expression of the FghnRNP G gene; or using a gene silencing system to silence the expression of the FghnRNP G gene.

[0028] The application of the Fusarium graminearum FghnRNP G gene provided by the present invention has important significance for effectively preventing and controlling DON toxin pollution caused by Fusarium graminearum by deeply understanding the molecular mechanism of toxin production of Fusarium graminearum and mining and identifying key genes in the DON biosynthesis process of Fusarium graminearum.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The present invention provides an application of the gene FghnRNP G that affects the biosynthesis of DON toxin in Fusarium graminearum, i.e., for inhibiting the synthesis of DON in Fusarium graminearum;

[0031] (2) The present invention provides a new method for preventing and controlling the biosynthesis of DON toxin in Fusarium graminearum, that is, inhibiting the ability of Fusarium graminearum to produce DON toxin by inhibiting the expression of FghnRNP G gene of Fusarium graminearum or the protein encoded by FghnRNP G gene;

[0032] (3) The present invention provides a gene target for preventing and controlling DON pollution. Agents that can disrupt the expression, shearing, and expression, modification, localization, or activity of the gene and its encoded protein can effectively prevent and control DON pollution caused by Fusarium graminearum.

[0033] (4) The homologous genes and homologous proteins of the FghnRNP G gene of Fusarium graminearum in other pathogenic fungi can be used as candidate targets. The new antibacterial agents developed by targeting this gene or protein can destroy the normal expression and function of this gene or protein, thereby effectively preventing and controlling DON pollution caused by fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Construction and verification of FghnRNP G gene knockout mutant

[0035] (A) Principle of gene knockout;

[0036] (B) PCR verification of knockout mutants

[0037] Among them, 1: Primer FghnRNP G-5F / FghnRNP G-6R, 2: Primer FghnRNP G-7F / HPH-

[0038] Down, 3: Primer HPH-Up / FghnRNP G-8R, 4: Primer HPH-F / HPH-R

[0039] Figure 2 Effects of FghnRNP G gene knockout on colony morphology and conidia

[0040] (A) Colony morphology;

[0041] (B) Conidia morphology;

[0042] (C) Growth rate. Different lowercase letters at the same time point indicate significant differences (P<0.05)

[0043] Figure 3 Effects of FghnRNP G gene knockout on pathogenicity

[0044] (A) Symptoms of the disease;

[0045] (B) Lesion length, the same lowercase letters indicate no significant difference (P ≥ 0.05)

[0046] Figure 4 Effects of FghnRNP G gene knockout on DON biosynthesis

[0047] (A) PDA culture medium;

[0048] (B) Rice medium;

[0049] Different lowercase letters for the same index indicate significant differences (P<0.05)

[0050] Figure 5 Effect of FghnRNP G gene knockout on the relative expression of TRI gene;

[0051] Different lowercase letters for the same index indicate significant differences (P<0.05) DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below in conjunction with specific embodiments, but it should be understood that the following embodiments are only used to illustrate the present invention rather than to limit the scope of the present invention.

[0053] The gene synthesis, primer synthesis and sequencing in the following examples were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0054] 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.

[0055] The nucleotide sequence of the Fusarium graminearum FghnRNP G gene in the embodiment is shown in SEQ ID NO.1:

[0056] SEQ ID NO.1:

[0057] tttattcaaa tgtgaatcca tccctctcag ttcactcacc aactcaaatc aactcatccacacaacaacc tttacataca acctgcacat aataccaaca atggcctctt acacacatag ccgaagtgcggtgagagctc agcgacgaga ctcgtcatca tcctcatctt cacacacaat gtcaaaccgc tcaagaccacgtcttctacg ctcaagcgca actgagcctt caatcaccgt ctccaatgct actggaaact catttgctggtcccgagtcg ccacacagag caactgatct tctcaaccgt gttgccgtct attctccgtc tcccgagcaagcctccaccc cagcggcaat cccacgccat aacagaccca tgtccatctg ccgtgatcac cacgagcactctccttccag cagccctgga agccaatccg ccgtcatgtc cgactctccc tctggttact actttagctttcccagcttt gaggagtgga accaggacga catcaccaag gacaatgatg attgagcggt ggcggaaagagatggtgagg ggatcgatta tcaagacctc aaactgaaaa aacgcacaca acaacatcac tccaataacaacaacattca ttcacccaac gcccgctaga ctcgtaacga gatagcattc actggctttt ccctggcggctcaaagaaga gccagcgaca gcgaaagcca caaaaaaccc cgatggaagc agcaaacgca tcgcccaccaataacatatc aacgccaacc tcttactgta ttgagatgag ccgagaatga gattgtggcg ggctgaatgcgctctgggac taaggtttat tgttgggtgg atctggattc acggcggaaa agggcttctt ggtagaagagaagcgacgaa cgcttttgaagccaaggcta tgcacgatga atgaggaata gctgcactac actccccctcaaaaaaggtg gagttgaaag gaaaccaaca agagtctggg tgaagcttaa aagcccattg actacaaaaaacacagaaac gctttagatc catctggaca aagcctacga aaaacagaaa aaaagaagga aaacagagtcaggaaaaacg gaaaaacagt caaaaaagaa acagacacat cgtgtgtata gcatggcaac tcatcagttaaatctcatag cctatataaa tcatagaaca gttaccaaga tatagaaaaa aatcatataa tgaca

[0058] The amino acid sequence of Fusarium graminearum FghnRNP G protein is shown in SEQ ID NO.2:

[0059] SEQ ID NO.2:

[0060] MASYTHSRSAVRAQRRDSSSSSSHTMSNRSRPRLLRSSATEPSITVSNATGNSFAGPESPHRATDLLNRVAVYSPSPEQASTPAAIPRHNRPMSICRDHHEHSPSSSSPGSQSAVMSDSPSGYYFSFPSFEEWNQDDITKDNDD

[0061] Example 1: Construction and verification of genetically engineered strain ΔFghnRNP G knockout FghnRNP G

[0062] (1) Extraction of genomic DNA of Fusarium graminearum strain PH-1: The wild-type Fusarium graminearum strain PH-1 (Fusarium graminearum PH-1 / NRRL 31084, American Type Culture Collection, No. ATCC MYA-4620) was inoculated on PDA medium (200 g / L potato, 20 g / L glucose, 20 g / L agar) and activated for 3 d. Then, holes were punched at the edge of the colony with a 7 mm diameter puncher. The bacterial cake was inverted and inoculated on a PDA culture dish and cultured in the dark at 28°C for 7 d. The hyphae were collected and the genomic DNA was extracted using a DNA kit (Shanghai Sangon Biotechnology Co., Ltd.).

[0063] (2) Using the genomic DNA of Fusarium graminearum strain PH-1 as a template, based on the principle of homologous recombination ( Figure 1 A), the FghnRNP G knockout mutant was obtained by split-PCR to construct a knockout FghnRNP G gene fragment and PEG-mediated protoplast transformation;

[0064] (3) primers FghnRNP G-1F / FghnRNP G-2R and FghnRNP G-3F / FghnRNP G-4R were used to amplify the upstream and downstream fragments of FghnRNP G, 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;

[0065] The reagents used in PCR were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The reaction system is as follows:

[0066]

[0067] The PCR program is as follows:

[0068]

[0069]

[0070] (4) using the split-PCR method with primers FghnRNP G-1F and HP-R to fuse the upstream fragment of FghnRNP G with the H1 fragment, and using primers FghnRNP G-4R and PH-F to fuse the downstream fragment of FghnRNP G with the H2 fragment, to obtain fusion fragments respectively;

[0071] The reagents used in PCR were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The reaction system is as follows:

[0072]

[0073] The PCR program is as follows:

[0074]

[0075] (5) The fusion fragment was transferred into wild-type Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout;

[0076] The wild-type strain PH-1 was activated on a PDA plate for 3 days, and a hole was punched at the edge of the colony with a 7 mm diameter puncher. The bacterial cake was inoculated into CMC medium (carboxymethyl cellulose 15 g / L, NH4NO3 10 g / L, MgSO4·7H2O0.5 g / L, KH2PO4 1 g / L, yeast extract 1 g / L), and cultured at 25°C and 175 r / min for 4 days. The spores were collected by filtration through a double-layer microcloth (Sigma-Aldrich (Shanghai) Trading Co., Ltd.). After centrifugation at 25°C and 5000 r / min for 5 minutes, the supernatant was poured out and the precipitate was transferred to YEPD liquid medium (yeast powder 10 g, peptone 20 g, glucose 20 g, distilled water 1000 mL, pH 6.0) and cultured at 25°C and 175 r / min for 12 hours. Filter the culture medium with sterile microcloth and collect the mycelia, mix them at a ratio of 0.5 g mycelia / 10 mL 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 culture them at 30°C, 90 r / min for 2 h. Filter the released protoplasts with miracloth into a triangular flask, rinse with 1.2 mol / L KCl, and centrifuge at 25°C, 4 000 r / min for 5 min. Pour off the supernatant, add 50 mL STC buffer (sucrose 100 g, 0.5 mol / L Tris-Cl (PH8.0) 50 mL, CaCl2·2H2O 3.6755 g, pure water 450 mL) to suspend the precipitate, and centrifuge at 25°C, 4 000 r / min for 5 min. Pour off the supernatant, add 600 μl STC buffer and gently suspend to obtain protoplasts.

[0077] The fusion DNA fragments obtained in (4) (5-10 μg each) were added to a centrifuge tube containing 200 μL protoplasts and mixed evenly, and then allowed to stand at room temperature for 20 min. 1.25 mL of 40% PTC buffer (PEG8000 40 g, STC buffer 100 mL) was added, and the mixture was mixed evenly by inversion, and allowed to stand at room temperature for 20 min. 5 mL of TB3 medium (Yeast Extract 3 g / L, acid hydrolyzed casein 3 g / L, sucrose 200 g / L) containing 50 μg / mL ampicillin was added, and the transformants were cultured overnight at room temperature, and the knockout strains were obtained by preliminary screening using hygromycin.

[0078] (6) The genomic DNA of the genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout was extracted, and FghnRNP G-5F / FghnRNP G-6R was used to detect whether the target gene was knocked out, FghnRNP G-7F / HPH-Down was used to detect the upstream recombinant fragment, HPH-Up / FghnRNP G-8R was used to detect the downstream recombinant fragment, and HPH-F / HPH-R was used to detect the hygromycin gene.

[0079] If there is no band in FghnRNP G-5F / FghnRNP G-6R, and there is a band in FghnRNP G-7F / HPH-Down, HPH-Up / FghnRNP G-8R and HPH-F / HPH-R, it proves that the target gene is knocked out. This example has successfully obtained a genetically engineered strain of FghnRNP G gene ΔFghnRNP G ( Figure 1 B).

[0080] The primer sequences used in the above steps (synthesized by Shanghai Shenggong Biotechnology Co., Ltd.) are shown in Table 1 below:

[0081] Table 1 Primers used in the construction and verification of the genetically engineered strain ΔFghnRNP G knockout

[0082]

[0083]

[0084] Example 2: Mycelial growth and conidia determination of genetically engineered Fusarium graminearum strain ΔFghnRNP G (1) Colony growth rate and morphological observation

[0085] The wild-type strain PH-1 and the genetically engineered strain ΔFghnRNP G were activated on PDA plates for 3 days, and holes were punched at the edge of the colonies with a 7 mm diameter puncher. The bacterial cakes were inoculated upside down on the PDA culture dishes and cultured in the dark at 25°C. The colony diameters were recorded and photographed every day using the cross method. Three replicates were set for each strain.

[0086] The results show that Figure 2 A, Table 2), compared with the wild-type strain PH-1, the colony morphology of the genetically engineered strain ΔFghnRNP G was not significantly different, but the strain growth rate was significantly slowed by 16.2%, and the colony diameter decreased by about 27.1% after 96 hours of culture. Therefore, the FghnRNP G gene is involved in regulating the hyphal growth of Fusarium graminearum.

[0087] Table 2 Growth rate and spore production of FghnRNP G gene knockout mutant strain ΔFghnRNP G

[0088]

[0089] Different superscript letters indicate significant differences (P<0.05)

[0090] (2) Conidia yield determination and morphological observation

[0091] Use a 7mm diameter cork puncher to punch holes at the edge of the colonies of the wild-type strain PH-1 and the genetically engineered strain ΔFghnRNP G activated for 3 days, take 3 pieces of bacterial cakes, transfer them to 50mL CMC medium (carboxymethyl cellulose 15g / L, NH4NO3 10g / L, MgSO4·7H2O 0.5g / L, KH2PO4 1g / L, yeast extract 1g / L), and shake at 25℃ and 150r / min for 3 days. Collect conidia by filtering with sterile filter cloth, and then count and calculate the conidia production with a hemocytometer. Set up 3 replicates for each strain. Pipette the conidia suspension, observe the conidia morphology under a microscope and take pictures.

[0092] The results show that Figure 2 B, Table 2), the genetically engineered strain ΔFghnRNP G can produce sickle-shaped conidia, and the number of septa per spore is 3-7; compared with the wild-type strain PH-1, the conidia production of the genetically engineered strain ΔFghnRNP G was reduced by about 43.4%, but there was no significant difference in spore size, shape and number of septa. Therefore, the decrease in conidia production may be caused by the slow growth of the mutant strain.

[0093] Example 3: Determination of virulence of genetically engineered Fusarium graminearum strain ΔFghnRNP G

[0094] Wheat (Jimai 22) seeds (Wuhan Aolaite Biotechnology Co., Ltd.) were disinfected with 75% ethanol for 5 min, rinsed with sterile water, placed in a culture dish covered with filter paper containing water, and cultured at room temperature for 3-5 days. Wheat with uniform growth was selected, and the tip of the wheat coleoptile was cut off by scissors at a length of 2-3 mm. 10 μL of 2×10 5 Spore suspensions of the wild-type strain PH-1 and the mutant strain ΔFghnRNP G at a concentration of spores / mL were dropped onto the incision and cultured in a moisturizing manner at 25°C in the dark for 7 days. The length of the necrotic lesions was counted and photographed, and sterile water was inoculated as a control treatment.

[0095] The results show that ( Figure 3 ), 7 days after inoculation of coleoptiles, both the wild-type strain PH-1 and the genetically engineered strain ΔFghnRNP G can cause obvious brown necrotic lesions in wheat coleoptiles, and there is no significant difference in the length of the lesions. Therefore, the FghnRNP G gene does not affect the pathogenicity of Fusarium graminearum.

[0096] Example 4: DON production analysis of genetically engineered Fusarium graminearum strain ΔFghnRNP G

[0097] The wild-type strain PH-1 and the genetically engineered strain ΔFghnRNP G were activated on PDA plates for 3 days, and holes were punched at the edge of the colony with a 7 mm diameter puncher. The bacterial cakes were inoculated into PDA medium and rice medium (50 g rice, 20 mL water) respectively. The PDA medium was cultured at 25°C in the dark for 7 days, and the rice medium was cultured at 25°C in the dark for 4 weeks.

[0098] After the culture was completed, the culture was dried in an oven at 50°C, crushed and mixed, and 2 g of the sample was accurately weighed in a 50 mL centrifuge tube, 10 mL of acetonitrile-water (84:16, V / V) was added, vortexed for 1 min, soaked for 5 min, ultrasonically extracted for 1 h, centrifuged at 4 000 r / min for 10 min, 5 mL of the supernatant was taken, dried at 40°C nitrogen, and the residue was dissolved in 1 mL of 5 mmol / L ammonium acetate solution-methanol (80:20, V / V), vortexed for 30 s, diluted appropriately, filtered through a 0.22 μm filter membrane, and UPLC-MS / MS was determined. Each treatment was repeated 5 times. The UPLC-MS / MS determination conditions are as follows:

[0099] Chromatographic conditions: Column: Agilent Poroshell 120EC-C 18 Chromatographic column (Agilent Technologies (China) Co., Ltd., 100mm×3.0mm, 2.7mm); mobile phase: mobile phase A is 5mmol / L ammonium acetate solution, mobile phase B is methanol; gradient elution program: 0-0.5min, 10% A, 90% B; 0.5-4min, 10%-90% A, 90%-10% B; 4-4.5min, 90% A, 10% B; 4.5-4.7min, 90%-10% A, 10%-90% B; 4.7-6min, 10% A, 90% B; flow rate 0.4mL / min; injection volume 3μL; column temperature 40℃.

[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 nebulizer gas pressure was 50 psi; the auxiliary gas pressure was 50 psi; the nebulizer temperature was 500.0℃; the spray voltage was 5 500 V; the spray voltage curtain gas pressure was 35 psi; the collision gas pressure was 8 psi; DON was quantified by 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 the DON production in the genetically engineered strain ΔFghnRNP G was significantly lower than that in the wild-type PH-1, and the DON production in PDA medium and rice medium decreased by 62.8% and 74.7%, respectively. Figure 4 A, B). Therefore, the FghnRNP G gene plays an important role in the biosynthesis of DON in Fusarium graminearum.

[0102] Example 5: Analysis of TRI gene expression in genetically engineered Fusarium graminearum strain ΔFghnRNP G

[0103] The TRI gene is the core gene cluster for the biosynthesis of trichothecene mycotoxins such as DON (Chen Y, Kistler HC, Ma Z. Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management. Annu Rev Phytopathol. 2019; 57: 15-39). Real-time fluorescence quantitative PCR (qRT-PCR) was further used to investigate the effect of FghnRNP G gene deletion on TRI gene expression.

[0104] The wild-type strain PH-1 and the genetically engineered strain ΔFghnRNP G were inoculated into PDA medium, cultured at 25°C in the dark for 7 days, and the mycelia were collected. RNA was extracted using a kit (Beijing Quanshijin Biotechnology Co., Ltd.), cDNA was synthesized, and the expression of related TRI genes was detected. -ΔΔCt Methods The relative expression of TRI gene was calculated. The primers used (synthesized by Shanghai Shenggong Biotechnology Co., Ltd.) are shown in Table 3 below:

[0105] Table 3 Primers for detecting TRI gene expression

[0106]

[0107]

[0108] The qRT-PCR system is as follows:

[0109]

[0110] The qRT-PCR reaction procedure is as follows:

[0111]

[0112] Depend on Figure 5 It can be seen that compared with the wild-type strain PH-1, the relative expression levels of TRI4, TRI5, TRI6, TRI8, TRI10, TRI12 and TRI101 genes in the genetically engineered strain ΔFghnRNP G were significantly downregulated. Therefore, it can be shown that the FghnRNP G gene affects the biosynthesis of DON toxin in Fusarium graminearum by regulating the expression of TRI genes in Fusarium graminearum.

Claims

1. A method for inhibiting the synthesis of DON by Fusarium graminearum, characterized in that The method comprises the following steps: The method comprises the following steps: (1) extracting genomic DNA of Fusarium graminearum; and (2) Using the genomic DNA of Fusarium graminearum as a template, split-PCR was used to construct a knockout FghnRNP G gene fragment, and the FghnRNP G knockout mutant was obtained by PEG-mediated protoplast transformation; (3) Primers FghnRNP G-1F / FghnRNP G-2R and FghnRNP G-3F / FghnRNP G-4R were used to amplify the upstream and downstream fragments of FghnRNP G, respectively, and HPH-F / HP-R and PH-F / HPH-R were used to amplify the hygromycin phosphotransferase gene HPH to obtain the H1 and H2 fragments; (4) using the split-PCR method with primers FghnRNP G-1F and HP-R to fuse the upstream fragment of FghnRNP G with the H1 fragment, and using primers FghnRNP G-4R and PH-F to fuse the downstream fragment of FghnRNP G with the H2 fragment, to obtain fusion fragments respectively; (5) The fusion fragment was transferred into wild-type Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout; The primers for amplifying the upstream and downstream fragments are as follows: FghnRNP G-1F: GCGGTATGCACATTGACGAC FghnRNP G-2R: TTGACCTCCACTAGCTCCAGCCAAGCCATCATTGACGGAGCAAGGCA FghnRNP G-3F: GAATAGAGTAGATGCCGACCGCGGGTTTCGCCCCTTCTCATGGTCTA FghnRNP G-4R: GTCAGCAGCTGTTGGTTGTG The primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene are as follows: HPH-F:GGCTTGGCTGGAGCTAGTGGAGGTCAA HPH-R:ATAGAGTAGATGCCGACCGCGGGTTC HP-R: GTATTGACCGATTCCTTGCGGTCCGAA PH-F: GATGTAGGAGGGCGTGGATATGTCCT The sequence of the FghnRNP G gene is as described in SEQ ID NO.

1.

2. The genetically engineered strain ΔFghnRNP G with FghnRNP G gene knocked out prepared by the method of claim 1.

3. Application of FghnRNP G gene as an inhibition target or knockout target in inhibiting DON synthesis by Fusarium graminearum; knocking out the FghnRNP G gene in the Fusarium graminearum genome to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout; comprising the following steps: (1) Extracting genomic DNA of Fusarium graminearum; (2) Using the genomic DNA of Fusarium graminearum as a template, split-PCR was used to construct a knockout FghnRNP G gene fragment, and the FghnRNP G knockout mutant was obtained by PEG-mediated protoplast transformation; (3) Primers FghnRNP G-1F / FghnRNP G-2R and FghnRNP G-3F / FghnRNP G-4R were used to amplify the upstream and downstream fragments of FghnRNP G, respectively, and HPH-F / HP-R and PH-F / HPH-R were used to amplify the hygromycin phosphotransferase gene HPH to obtain the H1 and H2 fragments; (4) using the split-PCR method with primers FghnRNP G-1F and HP-R to fuse the upstream fragment of FghnRNP G with the H1 fragment, and using primers FghnRNP G-4R and PH-F to fuse the downstream fragment of FghnRNP G with the H2 fragment, to obtain fusion fragments respectively; (5) The fusion fragment was transferred into wild-type Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout; The primers for amplifying the upstream and downstream fragments are as follows: FghnRNP G-1F: GCGGTATGCACATTGACGAC FghnRNP G-2R: TTGACCTCCACTAGCTCCAGCCAAGCCATCATTGACGGAGCAAGGCA FghnRNP G-3F: GAATAGAGTAGATGCCGACCGCGGGTTTCGCCCCTTCTCATGGTCTA FghnRNP G-4R: GTCAGCAGCTGTTGGTTGTG The primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene are as follows: HPH-F:GGCTTGGCTGGAGCTAGTGGAGGTCAA HPH-R:ATAGAGTAGATGCCGACCGCGGGTTC HP-R: GTATTGACCGATTCCTTGCGGTCCGAA PH-F: GATGTAGGAGGGCGTGGATATGTCCT The sequence of the FghnRNP G gene is as described in SEQ ID NO.

1.

4. A method for reducing DON synthesis in Fusarium graminearum, comprising the steps of reducing or not expressing the expression of the FghnRNP G gene or the protein encoded by the FghnRNP G gene in the Fusarium graminearum genome; knocking out the FghnRNP G gene in the Fusarium graminearum genome to obtain a genetically engineered strain ΔFghnRNP G with the FghnRNP G gene knocked out; comprising the following steps: (1) Extracting genomic DNA of Fusarium graminearum; (2) Using the genomic DNA of Fusarium graminearum as a template, split-PCR was used to construct a knockout FghnRNP G gene fragment, and the FghnRNP G knockout mutant was obtained by PEG-mediated protoplast transformation; (3) Primers FghnRNP G-1F / FghnRNP G-2R and FghnRNP G-3F / FghnRNP G-4R were used to amplify the upstream and downstream fragments of FghnRNP G, respectively, and HPH-F / HP-R and PH-F / HPH-R were used to amplify the hygromycin phosphotransferase gene HPH to obtain the H1 and H2 fragments; (4) using the split-PCR method with primers FghnRNP G-1F and HP-R to fuse the upstream fragment of FghnRNP G with the H1 fragment, and using primers FghnRNP G-4R and PH-F to fuse the downstream fragment of FghnRNP G with the H2 fragment, to obtain fusion fragments respectively; (5) The fusion fragment was transferred into wild-type Fusarium graminearum PH-1 by PEG-mediated protoplast transformation, and hygromycin resistance was used for screening to obtain a genetically engineered strain ΔFghnRNP G with FghnRNP G gene knockout; The primers for amplifying the upstream and downstream fragments are as follows: FghnRNP G-1F: GCGGTATGCACATTGACGAC FghnRNP G-2R: TTGACCTCCACTAGCTCCAGCCAAGCCATCATTGACGGAGCAAGGCA FghnRNP G-3F: GAATAGAGTAGATGCCGACCGCGGGTTTCGCCCCTTCTCATGGTCTA FghnRNP G-4R: GTCAGCAGCTGTTGGTTGTG The primers for amplifying the H1 and H2 fragments of the hygromycin phosphotransferase gene are as follows: HPH-F:GGCTTGGCTGGAGCTAGTGGAGGTCAA HPH-R:ATAGAGTAGATGCCGACCGCGGGTTC HP-R: GTATTGACCGATTCCTTGCGGTCCGAA PH-F: GATGTAGGAGGGCGTGGATATGTCCT The sequence of the FghnRNP G gene is as described in SEQ ID NO.1.

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