A gene FgTEM1 regulating pathogenicity of Fusarium graminearum and its application
The knockdown of the FgTEM1 gene in Fusarium gracilis has reduced the yield and pathogenicity of conidia, solved the problem of insufficient research on pathogenic genes of wheat gibberellia, and provided a new method for preventing and treating wheat gibberellia.
Patent Information
- Application Number
- CN202211183426.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-27
- Publication Date
- 2025-04-25
- Estimated Expiration
- 2042-09-27
AI Technical Summary
In the prior art, there are few studies on the pathogenic genes of wheat gibberellia, and it is difficult to effectively prevent and treat wheat gibberellia.
By knocking out the small and medium GTPase family genes of Fusarium granida, FgTEM1, a gene that regulates the pathogenicity of Fusarium granida and its knockout mutant ΔFgtem1 are identified and publicly regulated.
Knockout of FgTEM1 gene significantly reduced the conidia yield and pathogenicity of wheat ears in Fusarium grazing, providing a new target and research direction for the prevention and treatment of wheat gibberellia.
Smart Images
Figure 220927150402 
Figure 220927150406 
Figure 220927150410
Abstract
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 pathogenicity of Fusarium graminearum FgTEM1 and its applications. Background Art
[0002] Wheat is an important food crop in my country. During the cultivation and production process, it will be attacked by a variety of pathogens, resulting in a decrease in yield and quality. Among them, the most harmful is Fusarium graminearum, which is common in wheat-growing areas in my country. Wheat Fusarium graminearum is caused by infection with Fusarium graminearum. Fusarium graminearum is a type of plant pathogenic fungus that mainly infects wheat, barley, corn and other cereal crops, causing wheat Fusarium graminearum and corn stalk rot, reducing the yield and quality of food crops (Chen Y, Kistler HC, Ma Z (2019) Fusarium graminearum TrichotheceneMycotoxins: Biosynthesis, Regulation, and Management. Annu Rev Phytopathol57: 15-39.). Fusarium graminearum produces conidia through asexual reproduction and is the main primary infection source of Fusarium graminearum. Conidia are spread by wind, rain, etc., fall on wheat leaves and ears, and can infect wheat at all development stages. The most harmful thing is to infect wheat ears at the flowering stage, which can cause wheat crop failure in severe cases (Francl L, Shaner G, Bergstrom G, Gilbert J, Pedersen W, et al. (1999) Daily inoculum levels of Gibberella zeae on wheat spikes. Plant Disease 83: 662-666.).
[0003] Wheat fusarium rust is extremely harmful, and its prevention and control has important economic value. However, few key pathogenic genes of wheat fusarium rust (Fusarium graminearum) have been discovered so far. Therefore, the exploration and identification of key pathogenic genes of Fusarium graminearum infection can provide drug targets and research directions for the prevention and control of wheat fusarium rust, which has important scientific significance and application value, and can ultimately achieve green and ecological prevention and control of wheat fusarium rust.
[0004] In previous studies, we knocked out the small GTPase family genes in Fusarium graminearum and found a gene that had little effect on the growth rate of vegetative hyphae, but significantly reduced the conidia yield and pathogenicity of wheat ears. FgTEM1 The mutant, which we named Δ Fgtem1Therefore, the present invention discloses a gene that regulates the conidia production and pathogenicity of Fusarium graminearum FgTEM1 and its knockout mutants. Summary of the invention
[0005] The present invention aims to disclose a gene that regulates the pathogenicity of Fusarium graminearum FgTEM1 and its applications, FgTEM1 The gene only slightly affects the normal vegetative hyphae growth of Fusarium graminearum, does not affect the production of ascocarps and ascospores, but significantly reduces the yield of conidia and the pathogenicity to wheat ears, which indicates that FgTEM1 It is an important gene in the pathogenicity of Fusarium graminearum. FgTEM1 Genes are of great significance to the spread and green ecological control of Fusarium graminearum, and are also an important genetic resource for studying the pathogenic mechanism of Fusarium graminearum.
[0006] To achieve the above object, the technical solution adopted by the present invention is:
[0007] A gene that regulates pathogenicity of Fusarium graminearum FgTEM1 , as described FgTEM1 The gene has a nucleotide sequence shown in SEQ ID NO.1, and the protein encoded by the gene has an amino acid sequence shown in SEQ ID NO.2.
[0008] Fusarium graminearum FgTEM1 Knockout mutant Δ Fgtem1 , the mutant Δ Fgtem1 Hygromycin phosphotransferase HPH Knockout from the genome of Fusarium graminearum PH-1 by homologous recombination FgTEM1 Gene obtained; the mutant Δ Fgtem1 It has the following characteristics:
[0009] (1) Compared with the PH-1 strain of Fusarium graminearum, the Δ Fgtem1 The growth of vegetative hyphae in the mutant was slightly decreased;
[0010] (2) Compared with the PH-1 strain of Fusarium graminearum, the Δ Fgtem1 The conidia production of the mutant was significantly reduced;
[0011] (3) Compared with the PH-1 strain of Fusarium graminearum, the Δ Fgtem1 Pericarp and ascospore production were not affected in the mutant;
[0012] (4) Compared with the PH-1 strain of Fusarium graminearum, the Δ Fgtem1 The pathogenicity of the mutant to wheat ears was significantly reduced.
[0013] A gene that regulates pathogenicity of Fusarium graminearum FgTEM1 Application in the prevention and control of wheat fusarium wilt.
[0014] A gene that regulates pathogenicity of Fusarium graminearum FgTEM1 Application in studying the pathogenicity mechanism of Fusarium graminearum.
[0015] The invention is beneficial in that: FgTEM1 After the gene was successfully knocked out from the wild-type strain PH-1 of Fusarium graminearum, the FgTEM1 Knockout mutant Δ Fgtem1 The growth rate of vegetative hyphae was slightly reduced, the formation of sexual reproductive ascocarp and ascospores was not affected, the conidia production was significantly reduced, and the pathogenicity to wheat ears was greatly reduced. The above results show that FgTEM1 The gene mainly regulates the conidia production of Fusarium graminearum and its pathogenicity to wheat ears, and plays an important role in the spread of Fusarium graminearum and the prevention and control of wheat fusarium ergot. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1. Fusarium graminearum FgTEM1 Schematic diagram of the homologous recombination principle and restriction site map of gene knockout (1A), and Southern hybridization map (1B). Hin dⅢ digested genomic DNA, mutant Δ Fgtem1 A band of 2.48 kb was obtained, and a band of 2.12 kb was obtained for the wild-type PH-1 strain.
[0017] Figure 2. Wild-type PH-1, FgTEM1 Knockout mutant (Δ Fgtem1 ) and complement strain Δ Fgtem1-C Colony morphology (2A) and colony diameter (2B) in complete medium (CM) and minimal medium (MM). ** indicates Δ Fgtem1 There was a significant difference in the diameter of the mutant compared with the wild type, p<0.01.
[0018] Figure 3. FgTEM1 Conidia yield of knockout mutants. ** indicates Δ Fgtem1 There was a significant difference in the conidia production between the mutant and the wild type (p<0.01).
[0019] Figure 4. FgTEM1 Gene knockout did not affect the formation of ascocarps and ascospores during sexual reproduction.
[0020] Figure 5. FgTEM1 Gene knockout reduced the pathogenicity of Fusarium graminearum to wheat ears (5A) and pathogenicity index (5B). ** indicates ΔFgtem1 There was a significant difference in the pathogenicity index between the mutant and the wild type, p<0.01.
[0021] Figure 6. Subcellular localization of FgTem1 protein in Fusarium graminearum. DETAILED DESCRIPTION
[0022] The formula and preparation method of some culture media used in the examples are as follows:
[0023] Complete medium (CM) / L: 10 g sucrose, 6 g yeast extract, 6 g acid hydrolyzed casein, 20 g agar powder, and ddH2O to make up to 1 L.
[0024] Minimal medium (MM) / L: 10 g glucose, 6 g NaNO3, 0.52 g KCl, 1.52 g KH2PO4, 0.312 g MgSO4·7H2O, 0.01 g VB1, 20 g agar powder, 1 mL trace element stock solution, ddH2O to 1 L, pH 6.5. (Trace element stock solution formula / L: 400 mg CuSO4·5H2O, 8 g ZnSO4·7H2O, 800 mg MnSO4·2H2O, 1180 mgFeSO4·7H2O, 800 mg Na2MoO4, 40 mg Na2B4O7·10H2O).
[0025] Conidia induction medium (CMC) / L: 1 g yeast extract, 1 g NH4NO3, 1 g KH2PO4, 0.5 g MgSO4·7H2O, 15 g sodium carboxymethyl cellulose, and ddH2O to make up to 1 L.
[0026] The above culture media were all sterilized by high pressure at 121°C for 20 min.
[0027] In this patent, Fusarium graminearum FgTEM1 Genes are italicized FgTEM1 The protein is expressed as FgTem1. FgTEM1 Knockout mutants were denoted by Δ Fgtem1 Indicates that the complement strain is Δ Fgtem1-C Indicates that the wild-type Fusarium graminearum strain is PH-1. The present invention first knocks out the small GTPase family gene and identifies a mutant Δ Fgtem1 The corresponding gene is FgTEM1 , its nucleotide sequence is SEQ ID NO. 1, and the encoded protein amino acid sequence is SEQ ID NO. 2.
[0028] To further elucidate a gene that regulates pathogenicity of Fusarium graminearum FgTEM1 And its application, for example:
[0029] Example 1: Fusarium graminearum FgTEM1 Obtaining gene knockout and complementation strains
[0030] First, the target fragments A and B were amplified from the genomic DNA of Fusarium graminearum PH-1 strain using the following primers FgTEM1-AF / FgTEM1-AR and FgTEM1-BF / FgTEM1-BR, respectively, and the hygromycin phosphotransferase gene fragments H1 and H2 were amplified from the pCB1003 plasmid using primers HYG-F / HY-R and YG-F / HYG-R, respectively. H1 and H2 overlapped 315 bases. Then, the knockout fragment A fragment was connected to the H1 fragment, and the H2 fragment was connected to the B fragment using the overlap extension gene amplification technique (SOE-PCR) method to obtain AH and HB fragments, respectively. Then, the protoplasts of Fusarium graminearum PH-1 were prepared by enzymatic hydrolysis, and the protoplast transformation method mediated by polyethylene glycol PEG3350 was used to add fragments AH and HB (about 2 μg each) to the prepared protoplasts at the same time, mix, revive, and pour plates. After 3-5 days of cultivation, single colonies were picked and crude DNA was extracted. The candidate transformants were verified using the following primers: FgTEM1-OF / FgTEM1-OR, FgTEM1-UA / H853. The positive transformants were picked and further verified as single copy mutants using Southern hybridization. The principle is as follows: Figure 1 As shown in A, the experimental results showed that the four selected transformants Δ Fgtem1-1 / 3 / 33 / 34 are all correct single-copy insertion mutants ( Figure 1 B), select Δ Fgtem1-1 The / 3 mutant was used for subsequent studies.
[0031] Acquisition of complementation strains: The complementation fragment was amplified from the genomic DNA of Fusarium graminearum PH-1 strain using the following primers FgTEM1-CF / FgTEM1-CR, and then ligated into the pKNTG2 vector, transformed into E. coli, and the positive transformants were selected for sequencing. The vector with the correct sequence was transferred into Δ Fgtem1 The mutant protoplasts were used to obtain the complemented strain Δ Fgtem1-C .
[0032] The primer sequences used are:
[0033] FgTEM1-AF:5'-CATTTCACGCATAAGCAG-3'
[0034] FgTEM1-AR: 5'-TTGACCTCCACTAGCTCCAGCCAAGCCGGTTGAGTCGTCCAGTTT-3'
[0035] FgTEM1-BF: 5'-GAATAGAGTAGATGCCGACCGCGGGTTGACACGGCCATTTACGAG-3'
[0036] FgTEM1-BR:5'-TGCTCCCGCTGTCTACCTT-3'
[0037] FgTEM1-OF:5'-AGGCATACAACGACTACCG-3'
[0038] FgTEM1-OR:5'-GAACTGGCATACCTGATTT-3'
[0039] FgTEM1-UA:5'-GACAACACGCAAACGAAC-3'
[0040] H853:5'-GACAGACGTCGCGGTGAGTT-3'
[0041] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3'
[0042] HY-R: 5'-GTATTGACCGATTCCTTGCGGTCCGAA-3'
[0043] YG-F: 5'-GATGTAGGAGGGCGTGGATATGTCCT-3'
[0044] HYG-R: 5'-AACCCGCGGTCGGCATCTACTCTATTC-3'
[0045] FgTEM1-CF: 5'-AGGGAACAAAAGCTGGGTACCGAAGCTATTAAGACGCACGA-3'
[0046] FgTEM1-CR: 5'-GCCCTTGCTCACCATAAGCTTACAGACTGATATAACAGGA-3'
[0047] Example 2: Δ Fgtem1 Colony growth analysis of knockout mutants
[0048] The wild-type PH-1, FgTEM1 Knockout mutant (Δ Fgtem1) and complement strains (Δ Fgtem1-C ) were transferred to complete medium (CM) and minimal medium (MM), cultured inverted at 28°C for 3 days, and the colony morphology and colony diameter were observed. Figure 2 As shown in A, compared with PH-1 and Δ Fgtem1-C Compared with strain Δ Fgtem1 There was no significant difference in the colony morphology of the mutants. In MM medium, Δ Fgtem1 The colony diameter of the mutant did not change significantly, but decreased slightly in CM medium, indicating that FgTEM1 The knockout only slightly affected the colony growth of Fusarium graminearum.
[0049] Example 3: Δ Fgtem1 Analysis of conidia production in knockout mutants
[0050] The wild-type PH-1, FgTEM1 Knockout mutant (Δ Fgtem1 ) and complement strains (Δ Fgtem1-C ) were transferred to CMC conidia medium and cultured in a shaking incubator at 28°C and 180 rpm for 3 days. 600 μL of conidia solution was taken and diluted 3 times with sterilized ddH2O, filtered through a single layer of lens paper, and the filtrate was thoroughly mixed. The conidia yield was counted using a hemocytometer. Figure 3 As shown, wild-type PH-1 and Δ Fgtem1-C Compared with the conidia yield of strain Δ Fgtem1 The conidia production of the mutant decreased significantly, by about one third, indicating that FgTEM1 Genes play an important regulatory role in the conidia production of Fusarium graminearum.
[0051] Example 4: Δ Fgtem1 Analysis of sexual reproduction process
[0052] For research FgTEM1 To determine whether gene knockout affects the sexual reproduction of Fusarium graminearum, we firstly isolated PH-1, Δ Fgtem1 Mutant and complement strain Δ Fgtem1-C Transfer to carrot culture medium, invert and culture at 28℃ for 5 days until the mycelium fills the entire culture dish. Then take it out and place it in a clean bench, add 2.5% Tween 60, compact the mycelium, and then culture it at 22℃ under black light for 7 days to observe the formation of ascocarp. Figure 4 As shown in A, Δ Fgtem1 The mutant produced ascocarps of the same size as the wild type and complemented strain, indicating that FgTem1 does not affect the formation of ascocarps of Fusarium graminearum. We further picked ascocarps on the surface of the culture medium, placed them on the water droplet in the center of the slide, pressed them into pieces, and observed the morphology of asci and ascospores under a microscope. We found that Δ Fgtem1Mutants and PH-1 and complemented strain Δ Fgtem1-C There is no significant difference in the morphology of asci and ascospores ( Figure 4 B). In summary, our results indicate that FgTem1 does not affect the sexual reproduction process of F. graminearum.
[0053] Example 5: Δ Fgtem1 Pathogenicity analysis of knockout mutants
[0054] To investigate whether FgTem1 is involved in the pathogenicity of Fusarium graminearum, wild type, Δ Fgtem1 Mutant and complement strain Δ Fgtem1-C Inoculate the middle of wheat ears at the flowering stage and mark the inoculation point with a black marker ( Figure 5 The black dots in A were placed in bags to keep the moisture. After 14 days, photos were taken and the incidence of different strains was counted. Figure 5 A and Figure 5 As shown in B, knockout FgTEM1 The pathogenicity of Fusarium graminearum to wheat ears decreased significantly after the gene was added, which was about 80% lower than that of wild-type PH-1. The above results show that FgTem1 plays a vital role in the pathogenicity of Fusarium graminearum.
[0055] Example 6: Subcellular localization of FgTem1 protein in Fusarium graminearum
[0056] In order to observe where the FgTem1 protein is localized in Fusarium graminearum, we transformed the complemented strain Δ Fgtem1-C Inoculated CMC and CM culture media, it was found that FgTem1-GFP was mainly localized in the cytoplasm in the form of dots in different developmental stages such as vegetative hyphae, conidiophores and conidia ( Figure 6 In order to clarify its specific subcellular localization, the spindle marker protein FgAlp6-mCherry was further co-transfected. The results showed that FgTem1-GFP and FgAlp6-mCherry were co-localized in vegetative hyphae, conidiophores and conidia, indicating that FgTem1-GFP is mainly localized in the spindle and may be involved in cell division-related processes, thereby regulating the infection and pathogenicity of Fusarium graminearum.
Claims
1. Fusarium graminearum FgTEM1 Knockout mutants ΔFgtem1 The application in the process of preventing and controlling wheat scab is characterized by: The mutant ΔFgtem1 Hygromycin phosphotransferase HPH Knockout from the genome of Fusarium graminearum by homologous recombination FgTEM1 Gene acquisition; FgTEM1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the pathogen of wheat fusarium rust is Fusarium graminearum.