Application of FgGCV1 gene and its knockout mutant in the prevention and control of Fusarium graminearum

Abstract

The present invention discloses FgGCV1 The invention discloses the application of a gene and its knockout mutant in preventing and controlling Fusarium graminearum, belonging to the field of microbial technology. FgGCV1 The gene can inhibit the production of ascospores during the sexual reproduction of Fusarium graminearum, thereby preventing and controlling Fusarium graminearum and wheat fusarium head blight. FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. FgGCV1 The gene deletion mutant specifically regulates the maturation of ascospores in the sexual reproduction process of Fusarium graminearum. Ascospores are an important primary infection source of wheat fusarium head blight, so the knockout FgGCV1 The gene can block the transmission pathway of wheat fusarium wilt, so the invention plays an important role in the prevention and control of Fusarium graminearum and green ecological prevention and control, and is also an important resource for studying the sexual reproduction process of filamentous ascomycete fungi.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to the field of FgGCV1 Application of genes and their knockout mutants in the prevention and control of Fusarium graminearum. Background Art

[0002] Fusarium graminearum Fusarium graminearum ) is a plant pathogenic filamentous ascomycete fungus of the same ancestry. It mainly infects wheat, barley, corn and other cereal crops, causing wheat fusarium head blight and corn stalk rot, reducing the yield and quality of grain crops, and producing a large number of ascocarps in wheat ears and straw through sexual reproduction. Ascocarps are an important form of overwintering of Fusarium graminearum in the field. During the flowering stage of wheat, ascocarps are ejected to produce mature ascospores, which are spread to wheat by wind, rain or insects, and infect wheat ears as a primary infection source, causing wheat fusarium head blight.

[0003] Ascospores play a vital role in the infection cycle of Fusarium graminearum. Therefore, identifying genes and mutants that specifically regulate ascospore development during sexual reproduction in Fusarium graminearum can not only reveal the formation mechanism of ascospores during sexual reproduction, but also provide scientific strategies for the green ecological prevention and spread of wheat fusarium head blight. The PH-1 strain is the first strain of Fusarium graminearum to have its genome sequenced, and is widely used in genetic transformation and gene function research of Fusarium graminearum.

[0004] However, although some progress has been made in the functional research of genes related to sexual reproduction and ascospore development of Fusarium graminearum, the specific functions and mechanisms of action of many genes are still unclear. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a knockout FgGCV1 Application of genes and their knockout mutants in the prevention and control of Fusarium graminearum.

[0006] The present invention knocks out an aminomethyltransferase gene in the wild-type PH-1 strain of Fusarium graminearum and finds a mutant that does not affect growth and perithecia production but specifically blocks the production of ascospores in perithecia, and names it Δ FgGCV1 The corresponding gene is named FgGCV1 , and study the applications of the above genes and their knockout mutants.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides FgGCV1 Use of a gene in any of the following (1)-(3):

[0009] (1) Prevention and control of Fusarium graminearum;

[0010] (2) inhibiting ascospore formation of Fusarium graminearum;

[0011] (3) Prevent and control wheat fusarium rust;

[0012] Among them, the FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0013] Furthermore, the FgGCV1 The gene inhibits the production of ascospores during the sexual reproduction of Fusarium graminearum, thereby preventing and controlling Fusarium graminearum.

[0014] In a second aspect, the present invention provides a kind of Fusarium graminearum FgGCV1 Knockout mutant Δ FgGCV1 , the mutant Δ FgGCV1 The target fragment was amplified by primers, the homologous recombination fragment was constructed by double-joint PCR method, and it was introduced into the protoplasts of Fusarium graminearum strain PH-1 to knock out the FgGCV1 Gene obtained, the FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] In a third aspect, the present invention provides a medicament for resisting wheat scab, wherein the medicament contains an inhibitory FgGCV1 Gene expression substance, the FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] In a fourth aspect, the present invention provides a method for preventing and controlling wheat head blight caused by Fusarium graminearum, comprising the following steps: during the growth of wheat, spraying an exogenous FgGCV1 Gene expression substance, the FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discovered a new gene that specifically regulates the sexual reproduction process from the genome of Fusarium graminearum FgGCV1 ,Will FgGCV1 After the gene was successfully knocked out from Fusarium graminearum, the resulting Fusarium graminearum knockout mutant Δ FgGCV1 The growth of vegetative hyphae, conidia formation, perithecia production and yield of Fusarium graminearum were not affected, but the ascospore formation during the sexual reproduction of Fusarium graminearum was significantly affected. The results of sexual reproduction experiments showed that the knockout mutant ΔFgGCV1 The mutant Δ FgGCV1 The maturation of ascospores was impaired, and further ascospore ejection experiments also showed that the mutant Δ FgGCV1 Ascospores cannot be ejected. The above results show FgGCV1 The gene specifically regulates the formation and development of ascospores during sexual reproduction of Fusarium graminearum. FgGCV1 The gene, its encoded protein and knockout mutant can all be used as important candidate targets, which are of great significance to the spread and green ecological control of Fusarium graminearum. The anti-wheat fusarium ergot agent developed with this gene or protein as the target can effectively prevent and control fungal diseases. FgGCV1 The gene is also an important genetic resource for studying the mechanism of sexual reproduction in filamentous ascomycete fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The present invention knocks out FgGCV1 Schematic diagram of the homologous recombination strategy of the gene and the PCR verification result diagram; wherein, A: schematic diagram of the homologous recombination strategy; B: PCR verification result diagram.

[0021] Figure 2 The wild type PH-1 of the present invention, FgGCV1 Knockout mutant (Δ FgGCV1 ) and complement strain Δ FgGCV1 - C1 / C2 Colony morphology and growth rate; A: colony morphology; B: growth rate; ns: nosignificance, no significant difference.

[0022] Figure 3 For the present invention FgGCV1 Effects of gene knockout on conidia production; ns: no significance, no significant difference.

[0023] Figure 4 For the present invention FgGCV1 The effect of gene knockout on sexual reproduction; A: FgGCV1 Gene knockout does not affect the formation of ascocarp during sexual reproduction; B: Compared with the wild type, FgGCV1Gene knockout can form yellow whiskers at the top of the ascus opening; C: Compared with the wild type, FgGCV1 Gene knockout cannot produce asci; D: Compared with the wild type, FgGCV1 The knockout gene did not form ascospores.

[0024] Figure 5 For the present invention FgGCV1 The gene does not affect the pathogenicity of Fusarium graminearum. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are conventional biochemical reagents unless otherwise specified.

[0027] Complete medium (CM): sucrose 10 g, yeast extract 6 g, acid hydrolyzed casein 6 g, agar powder 20 g, add water to 1 L, autoclave at 121 °C for 20 min for later use.

[0028] Minimal medium (MM): 0.5 g potassium chloride, 2 g sodium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.01 g ferrous sulfate heptahydrate, 30 g sucrose, 15 g agar, add water to 1 L, adjust pH to 6.9, and sterilize by autoclave for 20 min; before use, add 200 µL of 1×Trace elements mixed solution per 1 L. Among them, 1×Trace elements mixed solution is obtained by diluting 1000×Trace elements mixed solution 1000 times, and 1000×Trace elements mixed solution (80 mL) is prepared as follows: 0.5 g ferrous sulfate heptahydrate, 2.2 g zinc sulfate heptahydrate, 0.16 g copper sulfate pentahydrate, 25 mg manganese sulfate tetrahydrate, 0.17 g cobalt chloride hexahydrate, 1.1 g boric acid, 0.15 g sodium molybdate, 5 g tetrasodium ethylenediaminetetraacetic acid, add water to 80 mL.

[0029] CMC (carboxylmethyl cellulose) conidia production medium: 1 g yeast extract, 1 g ammonium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 15 g sodium carboxymethyl cellulose, add water to 1 L, 121°C, autoclave for 20 min and set aside.

[0030] Carrot induction medium: 200 g peeled carrots, add appropriate amount of water, and crush with a juicer, 20 g agar powder, add water to 1 L, and sterilize at 121 °C under high pressure for 20 min.

[0031] YEPD medium (Yeast Extract Peptone Dextrose Medium): 3 g yeast extract, 10 g peptone, 20 g glucose, add water to make up to 1 L, and sterilize by high pressure for 15 min after aliquoting.

[0032] RM regeneration medium: 1 g acid hydrolyzed casein, 1 g yeast extract, 274 g sucrose, 15 g agar, add water to make up to 1 L, and sterilize by high pressure for 20 min.

[0033] Water agar medium: 1.5% agar (w / v) was dissolved in water and sterilized by autoclaving for 20 min.

[0034] STC solution: 1.2 M sorbitol 21.86 g, 1 M Tri-HCl (pH=8) 1 mL, 1 M calcium chloride 5 mL, add water to make up to 100 mL.

[0035] PTC solution: 50% PEG 3350 (w / v), fixed to volume with STC solution, and filtered through a bacterial filter.

[0036] Enzyme hydrolysate: 200 mg of lytic enzyme, 150 mg of wall-lytic enzyme, 1 mg of chitinase, dilute to 15 ml with 1 M ammonium chloride, filter with a bacterial filter and set aside.

[0037] Wild-type Fusarium graminearum strain PH-1: The PH-1 strain is the first strain of Fusarium graminearum to have its entire genome sequenced. It is widely used in genetic transformation and gene function research of Fusarium graminearum and can be obtained through conventional channels.

[0038] pUCH18 plasmid: constructed by ligating a fragment containing the hygromycin resistance gene from pUCATPH into the SalI–XbaI sites of pUC18; References: Yang, G, Tang, L, Gong, Y, Xie, J, Fu, Y, Jiang, D, Li, G, Collinge, DB, Chen, W. Cheng, J. A cerato-platanin protein SsCP1targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum . (The cerato-platanin protein SsCP1 targets plant PR1 and enhances the pathogenicity of Sclerotinia sclerotiorum) New Phytol. 2018,217, 739-755.

[0039] pKNT vector: from reference Zheng H, Miao P, Lin X, Li L, Wu C, Chen X, Abubakar YS, Norvienyeku J, Li G, Zhou J, Wang Z, Zheng W. Small GTPase Rab7-mediated FgAtg9 trafficking is essential for autophagy-dependent developmentand pathogenicity in Fusarium graminearum . (Small GTPase Rab7-mediated trafficking of FgAtg9 is essential for autophagy-dependent development and pathogenicity of Fusarium graminearum) PLoS Genet. 2018 Jul 25; 14(7):e1007546.

[0040] In this patent, Fusarium graminearum FgGCV1 Genes are italicized FgGCV1 The protein is represented by FgGCV1. FgGCV1 Knockout mutants were denoted by Δ FgGCV1 Indicates that the two complement strains are represented by Δ FgGCV1 - C1 , Δ FgGCV1 - C2 The wild-type strain of Fusarium graminearum is represented by PH-1.

[0041] After long-term and extensive screening, the present invention identified a knockout mutant Δ FgGCV1 The corresponding gene is FgGCV1The nucleotide sequence is SEQ ID NO.1, and the amino acid sequence of the encoded protein is SEQ ID NO.2.

[0042] SEQ ID NO.1:

[0043]

[0044] SEQ ID NO.2:

[0045] MKSLSLAARAGPRVLSSTTARPTSLAALRVSQQSVRCASGGSSSDLKKTPLYDFHIANGGKLVPFAGYSLPVQYSGLSLAQSHHFTREHASLFDVSHMVQHIFKGKDAAA FLEKVTPSWTNQGNMQSKLTTFLWPNTGGIVDDSVVTRIGEDTYYVVTNGACLDKDTKYIDEQLGKFGGDVQWTRLDNSGLVALQGPQSAEVLNEVLASDVDLTKLYFG NAVWAELKLSDGSKTHPVLISRGGYTGEDGFEISFNGKDYPAFETTTPAIQSLMSKAGPERLQLAGLGARDSLRLEAGMCLYGHDLDDTTTPVEAGLSWIIPPARRESGG FHGAETIIPQLTPKSKGGSGVERRRIGLYVDGAPAREGAEEIHKDGEKIGVITSGVPSPTLGKNIAMGYIKSGNQKAGTEVDVVVRGKARKGTVTKMPFIQTKYWKGTAPA.

[0046] The present invention is further described below with reference to the embodiments.

[0047] Example 1 Fusarium graminearum FgGCV1 Gene knockout

[0048] Primers FgGCV1-1F / FgGCV1-1R and FgGCV1-2F / FgGCV1-2R were used to amplify the target fragments U (856 bp) and D (861 bp) from the genomic DNA of the wild-type Fusarium graminearum strain PH-1, respectively; primers HYG-F / HY-R and YG-F / HYG-R were used to amplify the target fragments H1 (1049 bp) and H2 (1316 bp) from the plasmid pUCH18, respectively.

[0049] The PCR reaction system was as follows: 50 µL of the reaction system, including 25 µL of 2×Phanta Max Master Mix, 1 µL of upstream and downstream primers, 1 µL of template DNA (20 ng / µL), and ddH2O was added to adjust the total volume to 50 µL; the PCR reaction conditions were as follows: 95 °C pre-denaturation for 2 min; 95 °C denaturation for 20 s, 56 °C annealing for 20 s, 72 °C extension for 45 s, a total of 34 cycles; 72 °C final extension for 5 min; 16 °C cooling for 2 min. The PCR amplification product was recovered by agarose gel electrophoresis (Vazyme gel recovery kit, see the instructions for detailed methods).

[0050] Then, using the double-joint PCR method, fragments U and H1, H2 and fragment D were connected to obtain fusion fragments UH (1905 bp, sequence shown in SEQ ID NO.3) and HD (2177 bp, sequence shown in SEQ ID NO.4).

[0051] The PCR reaction is divided into two steps. The first step is amplification:

[0052] The PCR reaction system was 50 µL, including 25 µL of 2×Phanta Max Master Mix, 2 µL of template DNA U+H1 (or H2+D) (20 ng / µL), and ddH2O was added to adjust the total volume to 50 µL. The PCR reaction conditions were: 95°C pre-denaturation for 2 min; 95°C denaturation for 20 s, 60°C annealing for 20 s, and 72°C extension for 1 min 30 s, for a total of 15 cycles; 72°C final extension for 5 min; 16°C cooling for 2 min. The amplified product UH1 (or H2D) was obtained.

[0053] Second step amplification:

[0054] Add 1 µL of FgGCV1-1F+HY-R (or YG-F+FgGCV1-2R) and 2 µL of 2×Phanta Max Master Mix to the first step amplification product UH1 (or H2D). The PCR reaction conditions are as follows: 95°C pre-denaturation for 2 min; 95°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 1 min 30 s, for a total of 34 cycles; 72°C final extension for 5 min; 16°C cooling for 2 min.

[0055] The PCR amplification products were recovered by agarose gel electrophoresis.

[0056] Then, the conidia of Fusarium graminearum PH-1 were shaken in CMC conidia production medium. After 3 days of culture, they were added to 50 mL of YEPD medium and cultured at 28 ℃ and 200 rpm for 12 h. The medium was filtered with 3 layers of sterile lens paper, and the medium and other impurities on the mycelium were washed with 1 M NH4Cl solution. 0.5 g of wild-type PH-1 mycelium was collected in a 50 mL sterile centrifuge tube, 15 mL of protoplast hydrolysate was added and mixed, and lysed at 30 ℃ and 100 rpm for 4 h; the protoplasts were filtered with 3 layers of sterile lens and dispensed into 1.5 mL EP tubes, centrifuged at 6000 rpm for 7 min, the supernatant was removed, and 1 mL STC was added to the centrifuge tube to resuspend the protoplasts. 10 μL of protoplasts were aspirated onto a hemocytometer to count the protoplast concentration, and the protoplast concentration was adjusted to 3×10 with STC. 7 protoplasts / mL, dispense 100 μL into 1.5 mL sterile centrifuge tubes; add fragments UH and HD (about 2 μg each) to the above-dispensed protoplasts at the same time, mix thoroughly and place on ice for 10 min; add 1 mL of PTC solution to the above-mentioned transformation mixture after standing, vortex mix evenly, add 1 mL of STC, let stand at room temperature for 20 min, and spread on 5 RM regeneration medium plates, which are placed in a constant temperature incubator at 25°C for culture. After about 20 hours of cultivation, a layer of about 10 mL of water agar medium (containing hygromycin at a final concentration of 225 µg / mL) was covered on the culture of the RM regeneration medium. After culturing at 25°C for 1-3 days, single colonies screened by hygromycin will grow on the plate. These single colonies were picked and cultured on PDA (potato dextrose agar) medium containing hygromycin at a final concentration of 150 μg / mL for re-screening. After about 48 hours of cultivation on PDA medium, a small amount of DNA was extracted from young mycelium and obtained by PCR verification. FgGCV1 Knockout mutant Δ FgGCV1 (Basic principles such as Figure 1 The PCR verification results are shown in A. Figure 1 As shown in B, three pairs of primers P1 (1F1 / HY-R), P2 (2R1 / YG-F), and P3 (F / R) were used to verify the transformants: P1 was the upstream fragment of the amplified gene and part of the hygromycin gene; P2 was the downstream fragment of the amplified gene and part of the hygromycin gene; P3 was amplified by the specific primers of the knocked-out gene, and the PH-1 strain was used as a control (primer P3) ( Figure 1 B), experimental results show that Δ FgGCV1 It is a homozygous knockout transformant.

[0057] The sequence of UH is shown in SEQ ID NO.3:

[0058]

[0059] The sequence of HD is shown in SEQ ID NO.4:

[0060]

[0061] The primer sequences used are as follows:

[0062] FgGCV1-1F1 (1F1): 5'-GTCACCTTCGATGGTCGCCCCCTC-3'; SEQ ID NO.5;

[0063] FgGCV1-1F: 5'-CAAGCAGGTGCTCGCCATTGCTTGC-3'; SEQ ID NO.6;

[0064] FgGCV1-1R: 5'-ACCTCCACTAGCTCCAGCCAAGGATGTATGCAATTGACGGAATTAAG-3'; SEQ ID NO.7;

[0065] FgGCV1-2F: 5'-TGAGTCGTTTACCCAGAATGCACAGAAGGGGAGACGATGGGTTGGTGTATC-3'; SEQ ID NO.8;

[0066] FgGCV1-2R: 5'-CATCATTGAAGGGGAGGCTGTTGC-3'; SEQ ID NO.9;

[0067] FgGCV1-2R1 (2R1): 5'-CTAACTTTAGTACATAAGGTATGC-3'; SEQ ID NO.10;

[0068] FgGCV1-F (F): 5'-GCAAGGACGCAGCTGCTTTCCTGG-3'; SEQ ID NO.11;

[0069] FgGCV1-R (R): 5'-GCCTCGACTGGTGTTGTGGTGTCG-3'; SEQ ID NO.12;

[0070] HYG-F: 5'-CTTGGCTGGAGCTAGTGGAGGT-3'; SEQ ID NO.13;

[0071] HY-R: 5'-AACCACGGCCTCCAGAAGAAGAT-3'; SEQ ID NO.14;

[0072] HYG-R: 5'-CTGTGCATTCTGGGTAAACGACTCA-3'; SEQ ID NO.15;

[0073] YG-F: 5'-GAATCGGTCAATACACTACATGGCG-3'; SEQ ID NO. 16.

[0074] Construction and acquisition of complementation strains:

[0075] The following primers FgGCV1-CF / FgGCV1-CR were used to amplify the FgGCV1 open reading frame ORF (without the stop codon TAA) and the upstream 1.5 kb complement fragment (sequence as SEQ ID NO.19) from the genomic DNA of Fusarium graminearum PH-1 strain, and then ligated into the pKNT vector (after Kpn Ⅰ and Hind Ⅲ double enzyme digestion linearization), transformed into E. coli, selected positive transformants for sequencing, and the vector with the correct sequence was transferred into Δ FgGCV1 The mutant protoplasts were transformed by PEG-mediated protoplast transformation. FgGCV1 The complement strain Δ FgGCV1-C1 / C2 .

[0076] The primer sequences used are as follows:

[0077] FgGCV1-CF:

[0078] 5'-AGGGAACAAAAGCTGGGTACCGCCCCTCTTATCAAGATCCTCCAGAC-3'; SEQ ID NO.17;

[0079] FgGCV1-CR:

[0080] 5'-GAACAGCTCCTCGCCCTTGCTCACAGCAGGCGCAGTGCCCTTCCAGTAC-3'; SEQ ID NO. 18.

[0081] The sequence of the patched fragments is as follows:

[0082]

[0083] Example 2 Fusarium graminearum FgGCV1 Knockout mutant (Δ FgGCV1 )

[0084] The wild-type PH-1, FgGCV1 Knockout mutant (Δ FgGCV1 ) and complement strains (Δ FgGCV1 - C1 / C2 ) were transferred to PDA, MM, or CM medium and inverted at 28°C for 3 days. The colony morphology was observed and the colony diameter was measured. Figure 2 A and Figure 2 As shown in B, PH-1, Δ FgGCV1 and Δ FgGCV1 - C1 / C2 There was no significant change in colony morphology and growth rate, indicating FgGCV1 The deletion did not affect the vegetative growth of Fusarium graminearum.

[0085] For analysis FgGCV1 Effects of wild-type PH-1, Δ FgGCV1 and Δ FgGCV1 - C1 / C2 Inoculate into CMC conidia production medium and culture at 28℃, 180 rpm for 5 days. Take out the spore liquid and count the number of conidia with a hemocytometer. Figure 3 As shown, the mutant Δ FgGCV1 The conidia production of wild-type PH-1 and complemented strain Δ FgGCV1 - C1 / C2 There was no significant difference compared with FgGCV1 The deletion of the gene did not affect the conidia production of Fusarium graminearum.

[0086] Example 3 Fusarium graminearum FgGCV1 Knockout mutant (Δ FgGCV1 Analysis of sexual reproduction process

[0087] The wild-type PH-1, FgGCV1 Knockout mutant (Δ FgGCV1 ) and complement strain Δ FgGCV1 - C1 / C2 Inoculate into carrot induction medium and place in a 25 ℃ incubator for about 7 days. When the mycelium is fully grown, add 2.5% Tween 60 in a clean bench and compact the mycelium with a sterile coating stick. Then place in a 25 ℃ incubator under black light. After 7 days, black ascus ( Figure 4 As the culture time increases, the wild-type PH-1FgGCV1 Knockout mutant (Δ FgGCV1 ) The number of ascocarps produced gradually increases, and yellow whiskers appear on the surface of the ascocarps ( Figure 4 B). The ascospores of 7-day-old ascocarp were pressed into slices to further observe the ascospore morphology. The wild type PH-1 formed typical ascospores, while FgGCV1 Knockout mutant (Δ FgGCV1 ) do not produce asci, and the perithecia fail to eject ascospores ( Figure 4 C and Figure 4 The above results show that FgGCV1 It plays an important role in the sexual reproduction of Fusarium graminearum. FgGCV1 Knockout does not affect the production of asci, but specifically regulates the formation of asci and ascospores.

[0088] Example 4 Fusarium graminearum FgGCV1 Knockout mutant (Δ FgGCV1 )

[0089] To explore FgGCV1 Whether it is involved in the pathogenicity of Fusarium graminearum, wild-type PH-1, FgGCV1 Knockout mutant (Δ FgGCV1 ) and complement strain Δ FgGCV1-C1 The conidia solution was adjusted to a concentration of 2 × 10 5 Conidia / mL. During the flowering period of wheat, 10 μL of the conidia suspension of each strain was inoculated into the floret of the spikelet in the middle of the wheat ear (generally the 5th or 6th spikelet from the bottom to the top), and the inoculation point was marked with a black marker ( Figure 5 The black dots in the middle are marked by arrows. The CK group was not inoculated with any strains and was bagged for moisture retention. After 14 days, the incidence of different strains was counted and photographed. Figure 5 As shown, compared with wild-type PH-1, knockout FgGCV1 There was no significant difference in the pathogenicity of Fusarium graminearum to wheat ears after the gene was isolated. FgGCV1 It does not affect the pathogenic process of Fusarium graminearum.

[0090] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. FgGCV1 Genes in the prevention and control of Fusarium graminearum ( Fusarium graminearum ), characterized in that Said FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; knocking out the FgGCV1 The gene inhibits the production of ascospores during the sexual reproduction of Fusarium graminearum, thereby preventing and controlling Fusarium graminearum.

2. Knockout FgGCV1 The use of a gene in inhibiting ascospore formation of Fusarium graminearum is characterized in that: Said FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. FgGCV1 The application of the gene in preventing and controlling wheat scab is characterized in that: Said FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; knocking out the FgGCV1 The gene inhibits the production of ascospores during the sexual reproduction of Fusarium graminearum, thereby preventing and controlling Fusarium graminearum.

4. Fusarium graminearum FgGCV1 Knockout mutant Δ FgGCV1 The application in preventing and controlling wheat scab is characterized in that: The mutant Δ FgGCV1 The target fragment was amplified by primers, the homologous recombination fragment was constructed by double joint PCR method, and it was introduced into the protoplasts of Fusarium graminearum strain PH-1, and then knocked out by homologous recombination. FgGCV1 Gene obtained, the FgGCV1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the mutant Δ FgGCV1 Inhibit the production of ascospores during the sexual reproduction of Fusarium graminearum, thereby preventing and controlling ergot disease.

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