Gene FgTBC1 specifically regulating ascospore maturation of Fusarium graminearum and its application

Abstract

The present invention specifically relates to a gene that specifically regulates the maturation of ascospores of Fusarium graminearum FgTBC1 The invention and its application belong to the field of plant disease prevention and control. The invention finds a new gene that specifically regulates ascospore maturation in the sexual reproduction process from the genome of Fusarium graminearum. FgTBC1 The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. FgTBC1 The gene deletion mutant specifically regulates the maturation of ascospores in the sexual reproduction process of Fusarium graminearum, and ascospores are an important primary infection source of wheat fusarium head blight. FgTBC1 Genes play an important role in the spread and green ecological prevention and control of Fusarium graminearum, and are also an important resource for studying the sexual reproduction process of filamentous ascomycete fungi.

Description

Technical Field

[0001] The present invention belongs to the field of microbiology, and specifically relates to a gene that specifically regulates the maturation of ascospores of Fusarium graminearum FgTBC1 and its applications. Background Art

[0002] Fusarium graminearum is a plant pathogenic filamentous ascomycete fungus that mainly infects wheat, barley, corn and other cereal crops, causing wheat head blight and corn stalk rot, reducing the yield and quality of grain crops, and producing a large number of ascospores in wheat ears and straw through sexual reproduction (Stack R, Leonard K, Bushnell W. 2003History of Fusarium head blight with emphasis on North America. Fusarium headblight of wheat and barley: 1-34.). Ascospores are an important form of overwintering of Fusarium graminearum in the field. During the flowering stage of wheat, ascospores 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 head blight (Schmale DG, Arntsen QA, Bergstrom GC.2005. The forcible discharge distance of ascospores of Gibberelia zeae . Can JPlant Pathol 27: 376–382; Trail F, Gaffoor I, Vogel S (2005) Ejectionmechanics and trajectory of the ascospores of Gibberella zeae (anamorph Fuarium graminearum ). Fungal Genet Biol 42: 528-533.).

[0003] The above literature shows that ascospores play a vital role in the infection cycle of Fusarium graminearum. Therefore, identifying genes and mutants related to the specific regulation of ascospore development in sexual reproduction in Fusarium graminearum can not only reveal the formation mechanism of ascospores in 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 be sequenced with its entire genome, and is widely used in the genetic transformation and gene function research of Fusarium graminearum.

[0004] In previous studies, we knocked out 12 genes containing the TBC (Tre-2 / Bub2 / Cdc16) domain in the wild-type PH-1 strain of Fusarium graminearum and found a mutant that did not affect growth and perithecia production but specifically blocked the maturation and ejection of ascospores in perithecia. We named it Δ Fgtbc1 The corresponding gene is named FgTBC1 Therefore, the present invention discloses a gene that specifically regulates the maturation of ascospores of Fusarium graminearum FgTBC1 Genes and their knockout mutants and applications. Summary of the invention

[0005] The purpose of the present invention is to provide a gene that specifically regulates the maturation of ascospores of Fusarium graminearum FgTBC1 and its applications, FgTBC1 The gene does not affect the normal vegetative hyphal growth, conidia formation and ascocarp production of Fusarium graminearum, but specifically regulates the maturation and ejection of ascospores during the sexual reproduction of Fusarium graminearum. FgTBC1 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 mechanism of sexual reproduction in filamentous ascomycete fungi.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A gene that specifically regulates ascospore maturation in Fusarium graminearum FgTBC1 , the gene FgTBC1 The nucleotide sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the above FgTBC1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Fusarium graminearum FgTBC1 Knockout mutant Δ Fgtbc1 , the Fusarium graminearum FgTBC1 Knockout mutant Δ Fgtbc1 The preparation steps are as follows:

[0010] The target fragments A and B were amplified from the genomic DNA of wild-type Fusarium graminearum strain PH-1 using primers FgTBC1-AF / FgTBC1-AR and FgTBC1-BF / FgTBC1-BR, respectively; the target fragments H1 and H2 were amplified from plasmid pCB1003 using primers HYG-F / HY-R and YG-F / HYG-R, respectively; fragments A and H1 were connected, and fragments H2 and fragment B were connected to form long fragments AH and HB using the SOE-PCR method; AH and HB were added to the protoplasts of wild-type Fusarium graminearum strain PH-1 at the same time, and the target gene was amplified by homologous recombination. FgTBC1 Knock out the Fusarium graminearum FgTBC1 Knockout mutant Δ Fgtbc1 ;

[0011] The primer sequences used above are as follows:

[0012] FgTBC1-AF: 5'-GAAGTAGCGGAGCGGGTTC-3',

[0013] FgTBC1-AR: 5'-TTGACCTCCACTAGCTCCAGCCAAGCC-CGCCGAAGTGGCTGGTTAT-3',

[0014] FgTBC1-BF: 5'-GAATAGAGTAGATGCCGACCGCGGGTT-GGCATTCTCCTTTGTCTTC-3',

[0015] FgTBC1-BR: 5'-TCTTAGCTGGGCACTTGTAT-3',

[0016] YG-F: 5'-GATGTAGGAGGGCGTGGATATGTCCT-3',

[0017] HY-R: 5'-GTATTGACCGATTCCTTGCGGTCCGAA-3',

[0018] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3',

[0019] HYG-R: 5'-AACCCGCGGTCGGCATCTACTCTATTC-3'.

[0020] A gene that specifically regulates ascospore maturation in Fusarium graminearum FgTBC1 Application in the prevention and control of Fusarium graminearum.

[0021] A gene that specifically regulates ascospore maturation in Fusarium graminearum FgTBC1 Application in studying the pathogenicity mechanism of Fusarium graminearum.

[0022] Fusarium graminearum FgTBC1 Knockout mutant Δ Fgtbc1 Application in studying the pathogenicity mechanism of Fusarium graminearum.

[0023] Compared with the wild-type Fusarium graminearum PH-1 strain, the above Δ Fgtbc1 The mutants have the following characteristics:

[0024] (1) Compared with the wild-type Fusarium graminearum PH-1 strain, Δ Fgtbc1 The vegetative hyphal growth of the mutant was not affected.

[0025] (2) Compared with the wild-type Fusarium graminearum PH-1 strain, Δ Fgtbc1 Conidia production was not affected in the mutant.

[0026] (3) Compared with the wild-type Fusarium graminearum PH-1 strain, Δ Fgtbc1 Pericary production was not affected in the mutant.

[0027] (4) Compared with the wild-type Fusarium graminearum PH-1 strain, Δ Fgtbc1 The ascospore maturation and ejection process of the mutant were affected.

[0028] The advantages of the present invention are:

[0029] The present invention has been tested in large quantities. FgTBC1 After the gene was successfully knocked out from Fusarium graminearum, the resulting Fusarium graminearum knockout mutant Δ Fgtbc1 The vegetative hyphae growth, conidia formation and yield were not affected. The results of sexual reproduction experiments showed that the knockout mutant Δ Fgtbc1 The mutant Δ Fgtbc1 The maturation of ascospores was blocked and normal ascospores could not be produced. Further ascospore ejection experiments also showed that the mutant Δ Fgtbc1 Ascospores cannot be ejected. The above results show FgTBC1 Gene-specific regulation of ascospore formation and development during sexual reproduction of Fusarium graminearum. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . Knockout in Fusarium graminearum FgTBC1 Schematic diagram of the homologous recombination strategy and restriction site map of the gene (1A), and Southern hybridization map (1B). BamHⅠ (B) digestion of genomic DNA, mutant Δ Fgtbc1 The obtained band was 2.79 kb, the wild-type PH-1 band was 4.98 kb, and the complemented strain Δ Fgtbc1 -C The above two bands appear at the same time.

[0031] Figure 2 . Wild-type PH-1, FgTBC1 Knockout mutant (Δ Fgtbc1 ) and complement strain Δ Fgtbc1-C Colony morphology (2A) and colony diameter (2B).

[0032] Figure 3 . FgTBC1 Knockout of the gene did not affect conidia production.

[0033] Figure 4 . FgTBC1 Gene knockout did not affect the formation of ascocarp during sexual reproduction.

[0034] Figure 5 . FgTBC1 Genes regulating ascospore maturation during sexual reproduction in Fusarium graminearum.

[0035] Figure 6 . FgTBC1 Genes regulating ascospore ejection during sexual reproduction in Fusarium graminearum.

[0036] Figure 7 . FgTBC1 The gene is highly expressed during the sexual development stage of Fusarium graminearum. DETAILED DESCRIPTION

[0037] The following description is only a preferred implementation case of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The test materials used in the following embodiments, unless otherwise specified, are conventional biochemical reagents.

[0038] The composition and preparation method of some culture media used in the following examples:

[0039] CM complete medium: sucrose 10 g, yeast extract 6 g, acid hydrolyzed casein 6 g, agar powder 20 g, dilute to 1 L with ddH2O, autoclave at 121°C for 20 min for later use.

[0040] CMC conidia production medium: yeast extract 1 g, NH4NO3 1 g / , KH2PO4 1 g, MgSO47H2O0.5 g, sodium carboxymethyl cellulose 15 g, ddH2O to 1 L, 121℃, high pressure sterilization for 20 min for use.

[0041] Carrot induction medium: 200 g peeled carrots, add appropriate amount of water, blend with a juicer, add 20 g agar powder, dilute to 1 L with ddH2O, sterilize at 121℃ for 20 min and set aside.

[0042] To further elucidate a gene that specifically regulates ascospore maturation in Fusarium graminearum FgTBC1 The following are examples of its applications.

[0043] As described in this patent, Fusarium graminearum FgTBC1 Genes are italicized FgTBC1 The protein is expressed as FgTbc1. FgTBC1 Knockout mutants were denoted by Δ Fgtbc1 Indicates that the complement strain is Δ Fgtbc1-C The wild-type strain of Fusarium graminearum is represented by PH-1.

[0044] After a long period of extensive screening, the present invention identified a knockout mutant Δ Fgtbc1 The corresponding gene is FgTBC1 , its nucleotide sequence is SEQ ID NO. 1, and the encoded protein amino acid sequence is SEQ ID NO. 2.

[0045] Example 1: Fusarium graminearum FgTBC1 Gene knockout

[0046] Primers FgTBC1-AF / FgTBC1-AR, FgTBC1-BF / FgTBC1-BR were used to amplify target fragments A and B from the genomic DNA of wild-type Fusarium graminearum strain PH-1, respectively; primers HYG-F / HY-R, YG-F / HYG-R were used to amplify target fragments H1 and H2 from plasmid pCB1003, respectively. Then, using the SOE-PCR method, fragments A and H1, H2 and fragment B were connected to form long fragments AH and HB, and AH and HB were added to the protoplasts of wild-type Fusarium graminearum strain PH-1 at the same time, and the target gene was amplified by homologous recombination. FgTBC1 Knockout (basic principle as Figure 1 A), so as to obtain FgTBC1 Knockout mutant Δ Fgtbc1 The results of Southern hybridization verification are as follows Figure 1 As shown in B, throughBam HI digestion of genomic DNA, mutant Δ Fgtbc1 The obtained band was 2.79 kb, the wild-type PH-1 band was 4.98 kb, and the complemented strain Δ Fgtbc1-C The simultaneous appearance of the above two bands is consistent with the predicted results.

[0047] The primer sequences used are:

[0048] FgTBC1-AF: 5'-GAAGTAGCGGAGCGGGTTC-3',

[0049] FgTBC1-AR: 5'-TTGACCTCCACTAGCTCCAGCCAAGCC-CGCCGAAGTGGCTGGTTAT-3',

[0050] FgTBC1-BF: 5'-GAATAGAGTAGATGCCGACCGCGGGTT-GGCATTCTCCTTTGTCTTC-3',

[0051] FgTBC1-BR: 5'-TCTTAGCTGGGCACTTGTAT-3',

[0052] YG-F: 5'-GATGTAGGAGGGCGTGGATATGTCCT-3',

[0053] HY-R: 5'-GTATTGACCGATTCCTTGCGGTCCGAA-3',

[0054] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3',

[0055] HYG-R: 5'-AACCCGCGGTCGGCATCTACTCTATTC-3'.

[0056] Example 2: Δ Fgtbc1 Vegetative growth and conidia assay of mutants

[0057] The wild-type PH-1, FgTBC1 Knockout mutant (Δ Fgtbc1 ) and complement strains (Δ Fgtbc1-C ) were transferred to CM solid medium and inverted for 3 days at 28°C. The colony morphology was observed and the colony diameter was measured. Figure 2 A and Figure 2 As shown in B, PH-1, Δ Fgtbc1 and Δ Fgtbc1-C There was no significant change in the colony morphology and diameter, indicating FgTBC1The deletion did not affect the vegetative growth of Fusarium graminearum.

[0058] To analyze the effect of FgTbc1 on the conidia production of Fusarium graminearum, we isolated wild-type PH-1, Δ Fgtbc1 and Δ Fgtbc1-C Inoculate into CMC liquid conidia medium and culture at 28℃, 180 rpm for 3 days. Take out the conidia solution, dilute it by the same multiple, and count the number of conidia with a hemocytometer. Figure 3 As shown, the mutant Δ Fgtbc1 The conidia production of wild-type PH-1 and complemented strain Δ Fgtbc1-C There was no significant difference compared with FgTBC1 The deletion of the gene did not affect the conidia production of Fusarium graminearum.

[0059] Example 3: Δ Fgtbc1 Analysis of sexual reproduction process

[0060] The wild-type PH-1, mutant Δ Fgtbc1 and complement strain Δ Fgtbc1-C Inoculate into carrot culture medium and place in a 28℃ incubator for about 5 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 22℃ incubator under black light. After 5 days, black perithecia ( Figure 4 ). As the culture time increases, the wild-type PH-1 and FgTBC1 The knockout mutant produced an increasing number of perithecia, and a yellow substance called Cirrhi appeared on the surface of the perithecia. Figure 5 A, arrowhead), while the mutant Δ Fgtbc1 No yellow Cirrhi was observed ( Figure 5 A). The ascospores cultured for 9 days were selected for tablet pressing and further observation of ascospore morphology. Figure 5 As shown in A, the wild type PH-1 forms typical ascospores, Δ Fgtbc1-1 The mutant released a large number of short, club-shaped, deformed asci, and no mature ascospores were observed ( Figure 5 B). In order to observe the release of asci and ascospores more objectively, we compared the wild-type PH-1 with FgTBC1 Knockout mutants were used to observe ascospore ejection and the results showed that FgTBC1 Knockout mutant perithecia fail to eject ascospores ( Figure 6 The above results indicate that FgTbc1 plays an important role in the sexual reproduction of Fusarium graminearum. FgTBC1 The knockout did not affect the production of asci, but specifically regulated the maturation and ejection of asci and ascospores.

[0061] Example 4: Fusarium graminearum FgTBC1 Expression levels during the formation of vegetative hyphae and perithecia

[0062] To explore FgTBC1 In order to investigate the expression changes of Fusarium graminearum at different growth and development stages, we extracted total RNA from wild-type PH-1 mycelium (36 h) and perithecia (5 days, 9 days, and 13 days) and reverse transcribed them into cDNA. FgTBC1 The transcription levels at the above stages were taken as the reference for the hyphae stage.

[0063] The primer sequences used are:

[0064] FgTBC1-QF: 5'-AGATCCGAGAACAAGCCTACTGC-3';

[0065] FgTBC1-QR:5'-TTCCGCCTTGACCACCTTCATC-3';

[0066] FgActin-QF: 5'-ACGGAAACATTGTCATGTCTGGTG-3';

[0067] FgActin-QR: 5'-CTCTCGTCGTACTCCTGCTTGG-3'.

[0068] The results are as follows Figure 7 As shown, FgTBC1 At each stage of perithecia growth and development, there was an upregulation to varying degrees, indicating that FgTBC1 It is highly expressed during the sexual reproduction stage of Fusarium graminearum and plays an important function.

Claims

1. Genes that specifically regulate ascospore maturation in Fusarium graminearum FgTBC1 The application in preventing and controlling Fusarium graminearum is characterized by: The gene FgTBC1 The nucleotide sequence is shown in SEQ ID NO.1; the control of Fusarium graminearum refers to knocking out the gene from Fusarium graminearum FgTBC1 It specifically blocks the maturation and ejection of asci and ascospores in the sexual reproduction stage of Fusarium graminearum.

2. A strain of Fusarium graminearum FgTBC1 Knockout mutants ΔFgtbc1 The application in preventing and controlling Fusarium graminearum is characterized by: Said FgTBC1 The nucleotide sequence of the gene is as shown in SEQ ID NO.1; the control of Fusarium graminearum refers to knocking out the gene from Fusarium graminearum FgTBC1 The method specifically blocks the maturation and ejection of asci and ascospores in the sexual reproduction stage of Fusarium graminearum; the Fusarium graminearum FgTBC1 Knockout mutants ΔFgtbc1 The preparation steps are as follows: The target fragments A and B were amplified from the genomic DNA of wild-type Fusarium graminearum strain PH-1 using primers FgTBC1-AF / FgTBC1-AR and FgTBC1-BF / FgTBC1-BR, respectively; the target fragments H1 and H2 were amplified from plasmid pCB1003 using primers HYG-F / HY-R and YG-F / HYG-R, respectively; fragments A and H1 were connected, and fragments H2 and fragment B were connected to form long fragments AH and HB using the SOE-PCR method; AH and HB were added to the protoplasts of wild-type Fusarium graminearum strain PH-1 at the same time, and the target gene was amplified by homologous recombination. FgTBC1 Fusarium graminearum FgTBC1 Knockout mutants ΔFgtbc1 ; The primer sequences used above are as follows: FgTBC1-AF: 5’-GAAGTAGCGGAGCGGGTTC-3’ FgTBC1-AR: 5’-TTGACCTCCACTAGCTCCAGCCAAGCC-CGCCGAAGTGGCTGGTTAT-3’ FgTBC1-BF: 5’-GAATAGAGTAGATGCCGACCGCGGGTT-GGCATTCTCCTTTGTCTTC-3’ FgTBC1-BR: 5’-TCTTAGCTGGGCACTTGTAT-3’ YG-F: 5’-GATGTAGGAGGGCGTGGATATGTCCT-3’ HY-R: 5’-GTATTGACCGATTCCTTGCGGTCCGAA-3’’ HYG-F: 5’-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3’ HYG-R: 5’-AACCCGCGGTCGGCATCTACTCTATTC-3’

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