Gene FgPHM1 for regulating synthesis and pathogenicity of fusarium graminearum toxin DON and application thereof

Abstract

The invention discloses a gene FgPHM1 for regulating synthesis and pathogenicity of fusarium graminearum toxin DON and application of the gene FgPHM1, and belongs to the field of plant disease prevention and control. A novel gene FgPHM1 for regulating and controlling synthesis and pathogenicity of toxin DON is found in plant pathogenic fungus fusarium graminearum, and the nucleotide sequence of the gene FgPHM1 is as shown in SEQ ID NO. 1. Compared with a wild strain PH-1, the FgPHM1 gene knockout mutant has the advantages that the colony diameter of the mutant is reduced, the synthesis of the toxin DON is remarkably reduced, and the pathogenicity to wheat ears is lost, so that the FgPHM1 gene plays an important role in regulating and controlling the synthesis and pathopoiesis processes of the fusarium graminearum toxin DON. The invention provides important resources and research directions for prevention and control of pollution of toxin DON in agricultural products and prevention and control of wheat scab by using the FgPHM1 gene.

Description

Technical Field

[0001] The present invention belongs to the field of plant disease prevention and control, and specifically relates to a gene that regulates the synthesis and pathogenicity of Fusarium graminearum toxin DON FgPHM1 and its applications. Background Art

[0002] Wheat is one of the most important grain crops in my country. During the cultivation and production process, it will be infected by the plant pathogenic fungus Fusarium graminearum, causing fusarium headblight disease, resulting in reduced wheat yield and quality (Chen Ah, Islam T, MaZ-h (2022) An integrated pest management program for managing fusarium headblightdisease in cereals. J Integr Agr 21: 3434-3444.). During the infection of the host, Fusarium graminearum will produce a variety of mycotoxins such as deoxynivalenol (DON) and its derivatives, which will contaminate grain crops and remain in the food chain for a long time, causing acute poisoning such as vomiting, abdominal pain, and dizziness, which seriously threatens the health of humans and other animals (Sumarah MW (2022) The Deoxynivalenol Challenge. J AgricFood Chem 70: 9619-9624.).

[0003] The green ecological prevention and control of wheat fusarium fusarium rust and the pollution prevention and control of toxin DON in wheat agricultural products have important economic value. Therefore, the exploration and identification of key genes for the synthesis, infection and pathogenicity of Fusarium graminearum toxin DON can provide drug targets for the prevention and control of wheat fusarium fusarium rust, which has important scientific significance and application value. Summary of the invention

[0004] The purpose of the present invention is to provide a gene that regulates the synthesis and pathogenicity of Fusarium graminearum toxin DON FgPHM1 and its application, the gene deletion Δ Fgphm1 The mutant not only affects the growth of vegetative hyphae of Fusarium graminearum, but also reduces the production of conidia and the synthesis of toxin DON. More importantly, it basically loses its pathogenicity to wheat ears, indicating that FgPHM1 It is a critical gene for regulating the synthesis of toxin DON and pathogenicity of wheat ears in Fusarium graminearum. This gene is of great significance to the green ecological control of Fusarium graminearum, and is also an important gene resource for studying the synthesis and pathogenic mechanism of toxin DON in Fusarium graminearum.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: A gene regulating DON synthesis and pathogenicity of Fusarium graminearum FgPHM1 , the gene FgPHM1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1. FgPHM1 The amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

[0006] A sort of FgPHM1 Knockout mutant Δ Fgphm1 , the mutant ΔFgphm1 Knockout using genetic engineering FgPHM1 Gene.

[0007] Furthermore, the mutant Δ Fgphm1 Compared with Fusarium graminearum PH-1 The strain's toxin DON synthesis decreased significantly, the growth of vegetative hyphae decreased significantly, the conidia production decreased significantly, and its pathogenicity to wheat ears was basically lost.

[0008] Furthermore, the mutant Δ Fgphm1 DON toxin synthesis gene FgTRI4 , FgTRI6 , FgTRI10 and FgTRI12 The expression level was significantly downregulated.

[0009] The present invention also provides the above-mentioned gene for regulating the synthesis of Fusarium graminearum toxin DON FgPHM1 Application in preventing and controlling DON contamination in wheat agricultural products.

[0010] The present invention also provides the above-mentioned gene for regulating the pathogenicity of Fusarium graminearum FgPHM1 Application in the pathogenic mechanism of Fusarium graminearum infection of wheat ears.

[0011] A method for reducing the synthesis of DON by Fusarium graminearum, comprising: FgPHM1 Gene or the gene FgPHM1 The encoded protein expression is reduced or not expressed.

[0012] The present invention is beneficial in that: The present invention will FgPHM1 After the gene was successfully knocked out from the wild-type strain PH-1 of Fusarium graminearum, the FgPHM1 The colony diameter of the gene knockout mutant was reduced, the synthesis of toxin DON was significantly reduced, and the pathogenicity to wheat ears was basically lost. The above results show that FgPHM1 The gene mainly regulates the synthesis of Fusarium graminearum toxin DON and the pathogenicity of wheat ears, and plays an important role in the synthesis of Fusarium graminearum toxin DON and the prevention and control of wheat fusarium fusarium ergot. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Fusarium graminearum FgPHM1 Schematic diagram of the homologous replacement principle of gene knockout (1A) and Southern hybridization results (1B); Xho I (X) was used to digest the genomic DNA of the wild type and mutants. A 5.9 kb band was obtained for the wild type PH-1 strain and a 5.9 kb band was obtained for the mutant Δ Fgphm1 A band of 4.9 kb was obtained, which was consistent with the predicted band size.

[0014] Figure 2 For wild-type PH-1, FgPHM1 Gene deletion mutant (Δ Fgphm1 ) and complement strain Δ Fgphm1-C Colony morphology (2A) and colony diameter (2B) in complete medium (CM), starch yeast medium (SYM) and minimal medium (MM). Different letters (a, b) in the figure indicate significant differences ( P <0.05).

[0015] Figure 3 is Δ Fgphm1 Conidia yield of mutants. Different letters (a, b) indicate significant differences ( P <0.05).

[0016] Figure 4 FgPhm1 regulates the synthesis of the toxin DON. FgPHM1 Gene deletion significantly reduces the production of toxin DON (4A), and the genes related to DON toxin synthesis in the mutant FgTRI4 , FgTRI6 , FgTRI10 , FgTRI12 The expression of α-glucose was significantly downregulated (4B). Different letters (a, b) in the figure indicate significant differences ( P <0.05); ** indicates extremely significant difference, ( P <0.01).

[0017] Figure 5 Subcellular localization of FgPhm1-GFP fusion protein in conidia, germ tubes and hyphae of Fusarium graminearum.

[0018] Figure 6 for FgPHM1 Gene deletion basically lost the pathogenicity of Fusarium graminearum to wheat ears (6A) and the corresponding pathogenicity index (6B). Different letters (a, b) in the figure indicate significant differences ( P <0.05). DETAILED DESCRIPTION

[0019] The formula and preparation method of some culture media used in the examples are as follows: Complete medium (CM) / L: sucrose 10 g, yeast extract 6 g, acid hydrolyzed casein 6 g, agar powder 20 g, double distilled water to 1 L.

[0020] Starch yeast medium (SYM) / L: sucrose 3 g, soluble starch 10 g, yeast powder 2 g, agar powder 18 g, double distilled water to 1 L.

[0021] Basic medium (MM) / L: glucose 10 g, NaNO3 6 g, KCl 0.52 g, KH2PO4 1.52 g, MgSO4·7H2O 0.312 g, VB1 0.01 g, agar powder 20 g, trace element stock solution 1 mL, pH adjusted to 6.5, and double distilled water to 1 L.

[0022] Toxigenic induction medium (TBI) / L: FeSO4·7H2O 0.01 g, MgSO4·7H2O 0.05 g, KCl 0.5 g, KH2PO41 g / L, arginine 0.0871 g, sucrose 30 g, trace element stock solution 200 µL, pH adjusted to 6.5, and dilute to 1 L with double distilled water.

[0023] Sodium carboxymethyl cellulose sporulation medium (CMC) / L: yeast extract 1 g, NH4NO3 1 g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, sodium carboxymethyl cellulose 15 g, double distilled water to 1 L.

[0024] The above culture media were all sterilized by high pressure at 121°C for 20 min.

[0025] In this patent, the wild type strain of Fusarium graminearum is PH-1, and Fusarium graminearum FgPHM1 Genes are italicized FgPHM1 The protein is expressed as FgPhm1. FgPHM1 Δ Fgphm1 Indicates that the complement strain is Δ Fgphm1-C The present invention first knocks out the phenol monooxygenase family gene and identifies a mutant Δ that regulates the pathogenicity of Fusarium graminearum. Fgphm1 The corresponding gene is FgPHM1 , its nucleotide sequence is SEQ ID NO. 1, and the encoded protein amino acid sequence is SEQ ID NO. 2.

[0026] To further elucidate a gene that regulates the synthesis and pathogenicity of DON, a toxin in Fusarium graminearum FgPHM1 And its application, for example: Example 1: Fusarium graminearum FgPHM1 Obtaining gene knockout and complementation strains First, the target fragments A (741 bp) and B (798 bp) were amplified from the genomic DNA of Fusarium graminearum PH-1 strain using the following primers FgPHM1-AF / FgPHM1-AR and FgPHM1-BF / FgPHM1-BR, respectively, and the hygromycin phosphotransferase gene fragments H1 (798 bp) and H2 (918 bp) were amplified from the plasmid containing the hygromycin gene using primers HYG-F / HY-R and YG-F / HYG-R, respectively. Then, the A fragment obtained above was connected to the H1 fragment, and the H2 fragment was connected to the B fragment by overlap extension gene amplification technology (SOE-PCR) to obtain the AH and HB fragments. Then, the protoplasts of the wild-type strain PH-1 were prepared by enzymatic hydrolysis, and the fragments AH and HB (about 2 μg each) were added to the prepared protoplasts at the same time, and then the protoplasts were transformed by polyethylene glycol PEG3350-mediated protoplast transformation. Place the plate containing the culture medium upside down in a 28-degree incubator for 3-5 days, pick single colony transformants, extract the genomic DNA of each transformant, and use the following primers FgPHM1-OF / FgPHM1-OR and FgPHM1-UA / H853 to verify the candidate transformants, and further use Southern hybridization to verify the strain with single copy insertion. The principle is as follows Figure 1 A, the experimental results show that Δ Fgphm1-2 and Δ Fgphm1-4 For the correct single copy insertion mutant ( Figure 1 B) can be used for subsequent phenotypic determination.

[0027] Acquisition of complementation strain: The complementation fragment was amplified from the genomic DNA of the wild-type PH-1 strain using the following primers FgPHM1-CF / FgPHM1-CR and ligated to the pKNTG2 vector. The sequenced vector was transferred into Δ Fgphm1 The mutant protoplasts were used to obtain the complemented strain Δ Fgphm1-C .

[0028] The primer sequences used are: FgPHM1-AF: 5'-CATTTGCCTGTCCATTTCC-3'; FgPHM1-AR: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCGATCACGGACGACCTCTGC-3'; FgPHM1-BF: 5'-GAATAGAGTAGATGCCGACCGCGGGTTCCATCATGCTGGTGCTTT-3'; FgPHM1-BR: 5'-AATCCTGAGTGGCGTATCT-3'; FgPHM1-OF: 5'-TCCATACATTCTCCTCGTTC-3'; FgPHM1-OR: 5'-TCGCTCCTCTCGTAACTC-3'; FgPHM1-UA:5'-AATCCCACATTGTTTCATCT-3'; H853: 5'-GACAGACGTCGCGGTGAGTT-3'; HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3'; HY-R: 5'-GTATTGACCGATTCCTTGCGGTCCGAA-3'; YG-F: 5'-GATGTAGGAGGGCGTGGATATGTCCT-3'; HYG-R: 5'-AACCCGCGGTCGGCATCTACTCTATTC-3'; FgPHM1-CF: 5'-AGGGAACAAAAGCTGGGTACCAGATACCCAACGAGCAAAGG-3'; FgPHM1-CR: 5'-GCCGCCGCCGCCGCCAAGCTTCTGCGTTGGGATCTGCATAA-3'.

[0029] Example 2: Δ Fgphm1 Colony growth analysis of deletion mutants To observe FgPHM1 After the gene deletion, whether it affects the mycelial growth of Fusarium graminearum, wild-type PH-1, FgPHM1 Deletion mutant (Δ Fgphm1 ) and complement strains (Δ Fgphm1-C ) were transferred to complete medium (CM), starch yeast medium (SYM) and minimal medium (MM), respectively, and cultured inverted at 28°C for 3 days. The colony morphology was observed and the colony diameter was measured. Figure 2 AB shows the difference between wild-type strain PH-1 and complemented strain Δ Fgphm1-C Compared with Δ Fgphm1 The colony diameter of the mutant was significantly smaller and the aerial hyphae became thinner, indicating that FgPHM1 The gene deletion significantly affected the colony growth and aerial hyphae development of Fusarium graminearum.

[0030] Example 3: Δ Fgphm1Conidia yield analysis of deletion mutants For analysis FgPhm1 Whether to regulate the production of conidia of Fusarium graminearum, wild-type PH-1, Δ Fgphm1 Mutant and complement strain Δ Fgphm1-C The strains were transferred to CMC liquid conidia medium and placed in a shaker at 28°C and 180 rpm for continuous culture for 3, 6, and 9 days. The conidia yield of each strain was then counted on days 3, 6, and 9. Figure 3 As shown, the mutant Δ Fgphm1 At 3 days of culture, the conidia production was very low. As the culture time increased, the conidia production increased slightly, but was significantly lower than that of the wild type and complemented strains, indicating that FgPHM1 It plays a vital role in the formation of conidia of Fusarium graminearum.

[0031] Example 4: Fusarium graminearum Δ Fgphm1 Analysis of mutant toxin DON synthesis.

[0032] In order to study the role of FgPhm1 in the synthesis of toxin DON, wild-type PH-1, Δ Fgphm1 Mutant and complement strain Δ Fgphm1-C They were transferred to DON-induced TBI liquid culture medium and cultured at 28°C in the dark for 7 days. The culture solution was then filtered, and the culture solution and mycelium were collected separately. The culture solution was used for the detection of toxin DON, and the mycelium was dried and weighed for quantitative analysis of PH-1, Δ Fgphm1 and Δ Fgphm1-C DON production of the strain. Figure 4 As shown in A, compared with the wild type and complemented strains, Δ Fgphm1 The DON content of the mutant was significantly reduced, indicating that FgPhm1 is involved in the synthesis of DON toxin from Fusarium graminearum. To further determine whether FgPhm1 regulates the expression of key genes for DON toxin synthesis, PH-1, Δ Fgphm1 Mutant and complement strain Δ Fgphm1-C Transferred to TBI liquid culture medium, cultured at 28°C in the dark for 3 days. Collected hyphae, extracted RNA to detect genes related to DON toxin synthesis FgTRI4 , FgTRI6 , FgTRI10 and FgTRI12 The results showed that the expression levels of these genes were significantly reduced (4B), indicating that FgPhm1 regulates the expression of genes related to DON toxin synthesis.

[0033] The primers used were as follows: FgTR4-QF: 5'-ACCAGGTCCTCAGTCTTG-3'; FgTR4-QR:5'-TCGTTTGTGCTTGCCATAG-3'; FgTR6-QF: 5'-GCTACTCAGAATGCCCTCAG-3'; FgTR6-QR: 5'-CGCATGTTATCCACCCTGCTA-3'; FgTR10-QF: 5'-ATGATGATTGAGGATATGTTGT-3'; FgTR10-QR: 5'-GAGTGATGAGGTCTGGAA-3'; FgTR12-QF: 5'-GCTGTAACTGTCCCCAGCAT-3'; FgTR12-QR:5'-GTGAAGTTGCGACCGTACTC-3'; Tubulin-QF: 5'-GTCAGTGCGGTAACCAAATCGGT-3'; Tubulin-QR: 5'-CTCAGAGGTGCCGTTTAGAAACACC-3'.

[0034] Example 5: Subcellular localization of Fusarium graminearum FgPhm1 protein In order to observe where the FgPhm1 protein is localized in Fusarium graminearum, we transformed the complemented strain Δ Fgphm1-C The cells were inoculated in CMC liquid conidia medium and CM complete medium, and then the fluorescence signal of FgPhm1-GFP was observed. The results showed that at different developmental stages, such as vegetative hyphae, conidia, and conidial germination tubes, FgPhm1-GFP was mainly located in the cytoplasm in a crescent-shaped and ellipsoidal shape, and co-localized with the periplasmic marker protein FgNcbp2-mCherry, indicating that FgPhm1 was mainly located in the periplasm of the nucleus ( Figure 5 ).

[0035] Example 6: Fusarium graminearum Δ Fgphm1 Analysis of the pathogenicity of mutants to wheat ears To analyze whether FgPhm1 is involved in the pathogenicity of Fusarium graminearum, wild-type PH-1, Δ Fgphm1 Mutant and complement strain Δ Fgphm1-C Inoculate the middle of wheat ears at the flowering stage and mark the inoculation point with a marker ( Figure 6 A, black dots), bagged and kept moist for 7 days, and took photos and counted the incidence of different strains after 14 days. Figure 6 As shown in A, Δ Fgphm1The pathogenicity of the mutant to wheat ears was significantly reduced, with only slight lesions at the inoculation site that could not spread to adjacent grains, and the pathogenicity index was less than 1 ( Figure 6 B), while the wild type PH-1 and the complemented strain Δ Fgphm1-C The lesions on the inoculated wheat ears were very obvious and extended to the entire ear. The pathogenicity index was around 13, indicating that FgPhm1 was essential for Fusarium graminearum to infect wheat ears.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A gene that regulates the synthesis and pathogenicity of DON toxin from Fusarium graminearum FgPHM1 , characterized in that: The gene FgPHM1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The gene for regulating the synthesis and pathogenicity of Fusarium graminearum toxin DON according to claim 1 FgPHM1 , characterized in that: The gene FgPHM1 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A FgPHM1 Knockout mutant Δ Fgphm1 , characterized in that: The mutant Δ Fgphm1 Knockout using genetic engineering FgPHM1 Gene.

4. The mutant Δ according to claim 3 Fgphm1 , characterized in that: The mutant Δ Fgphm1 Compared with the PH-1 strain, the synthesis of toxin DON was significantly decreased, the growth of vegetative hyphae was significantly decreased, the conidia production was significantly decreased, and the pathogenicity to wheat ears was basically lost.

5. According to claim 3 FgPHM1 Knockout mutant Δ Fgphm1 , characterized in that: The mutant Δ Fgphm1 DON toxin synthesis gene FgTRI4 , FgTRI6 , FgTRI10 and FgTRI12 The expression level was significantly downregulated.

6. The gene for regulating the synthesis of DON toxin of Fusarium graminearum according to claim 1 FgPHM1 Application in preventing and controlling DON contamination in wheat agricultural products.

7. The gene for regulating pathogenicity of Fusarium graminearum according to claim 1 FgPHM1 Application in the pathogenic mechanism of Fusarium graminearum infection of wheat ears.

8. A method for reducing DON synthesis by Fusarium graminearum, characterized in that: Including making the FgPHM1 The gene or the gene according to claim 2 FgPHM1 The encoded protein expression is reduced or not expressed.

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