FpMCoL protein of Pseudomonas graminearum and its encoding gene and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the FpMCoL protein of Pseudofusorium graminearum, its encoding gene and application. The FpMCoL protein provided by the present invention is shown in Sequence 2; its encoding gene is shown in Sequence 1. Experiments have shown that the protein provided by the present invention plays an important role in the growth and development of Pseudofusorium graminearum itself, which is specifically manifested in that after the protein is missing, the mycelial growth rate of Pseudofusorium graminearum slows down, the amount of conidia produced is reduced, and the pathogenicity is reduced. Therefore, the present invention provides a technical basis for exploring the growth and development and pathogenic mechanism of Pseudofusorium graminearum, and provides a potential molecular target for the research and development of new fungicides in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to FpMCoL protein and its encoding gene and application. Background Art

[0002] Wheatcrown rot (WCR), caused by Fusarium pseudograminearum, is an important soil-borne disease in wheat production. The main symptoms are browning and rotting of the base of the wheat rhizome, and severe cases can cause seedling blight and white spikes. Wheatcrown rot occurs in Australia, the United States, Canada and other countries and causes serious damage. In normal years, wheatcrown rot causes an average yield loss of about 9.5% and can reach 35% in epidemic years. In recent years, the disease has been prevalent in the winter wheat areas of the Huanghuaihai region and has shown a trend of increasing severity year by year. At present, the disease rate in Henan and Hebei provinces is as high as 65.1% and 81.4% respectively, which has seriously threatened the safe production of wheat.

[0003] Magnesium is one of the core nutrients necessary for biological growth and development, and is also the most abundant divalent cation in cells. Magnesium has the largest hydration radius, the smallest ion radius and the largest charge density. It plays an important role in enzyme activation, genome stability, inhibition of aging, reduction of aluminum damage and regulation of nitrogen metabolism. Therefore, the special physical and chemical properties and biological functions of magnesium ions make their transport mode in organisms particularly important. The cobalt resistance protein family CorA belongs to the transmembrane transporter of the helical channel subclass in the channel pore transporter, and is a major member of the large divalent metal ion transporter superfamily. Family proteins are widely distributed in Gram-positive and Gram-negative bacteria, cyanobacteria, archaea and yeast. They are the regulatory system for the absorption, transport and efflux of divalent cations such as magnesium ions, nickel ions and cobalt ions; they are the most important transport system for organisms to maintain intracellular magnesium ion homeostasis. The loss of this system will seriously affect the growth and development of organisms. Summary of the invention

[0004] The present invention carries out relevant research work on the function of the metal divalent cation transporter MIT_CorA-like (metalion transporter CorA-like divalent cation transporter superfamily) homologous protein FpMCoL of Pseudomonas graminearum, which will provide a deeper understanding of the role of the metal divalent cation transport system of Pseudomonas graminearum in regulating the growth and development of the pathogen and its interaction with the host, as well as provide reference and reference for the design of new fungal control targets.

[0005] The inventors have found that the FpMCoL protein in Pseudofusorium graminearum and its encoding gene are closely related to the mycelial growth rate, conidia production and pathogenicity of Pseudofusorium graminearum. These results show that the FpMCoL protein in Pseudofusorium graminearum has an important regulatory role in the growth and development of the pathogen and the pathogenic process. The gene development of FpMCoL protein as a molecular agent target for the pathogenic fungus Pseudofusorium graminearum has important application prospects.

[0006] Therefore, one of the purposes of the present invention is to provide a homologous protein of CorA of Pseudomonas graminearum, and to name it FpMCoL, which is derived from Pseudomonas graminearum strain 2035 and is a protein of the following A1) or A2) A3) or A4):

[0007] A1) The amino acid sequence is the protein shown in SEQ ID NO.2;

[0008] A2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2;

[0009] A3) a protein derived from the protein shown in SEQ ID NO. 2 having the same function as that obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2;

[0010] A4) An amino acid sequence that has a similarity of more than 75%, preferably more than 85%, more preferably more than 95% to the amino acid sequence shown in SEQ ID NO.2 and has the same function as the amino acid sequence shown in SEQ ID NO.2.

[0011] In order to facilitate the purification of the protein in A1), tags such as Poly Arg (RRRRR), Poly His (HHHHHH), FLAG (DYKDDDDK), Strep tag II (WSHPQFEK), c myc (EQKLISEEDL) can be connected to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in SEQ ID NO.2.

[0012] The proteins in A1)-A4) above can be synthesized artificially, or their coding genes can be synthesized first and then expressed biologically. The coding genes of the proteins in A1)-A4) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO.1 in the sequence list, and / or performing missense mutations of one or several nucleotide pairs, and / or connecting the coding sequence of the above tag at its 5' end and / or 3' end.

[0013] Among them, in A1), SEQ ID NO.2 (FpMCoL protein) in the sequence list consists of 377 amino acid residues.

[0014] The second object of the present invention is to provide a nucleic acid molecule encoding the desired FpMCoL protein. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA.

[0015] Wherein, the coding gene of the above-mentioned FpMCoL protein is as follows B1) or B2) or B3):

[0016] B1) a DNA molecule represented by the nucleotide sequence of SEQ ID NO.1 in the sequence list;

[0017] B2) a cDNA molecule or a DNA molecule having 75% or more, 85% or more, or 95% or more identity with the nucleotide sequence shown in B1), and encoding the above-mentioned FpMCoL protein;

[0018] B3) a cDNA molecule or a DNA molecule which hybridizes with the nucleotide sequence defined in B1) or B2) under stringent conditions and encodes the above-mentioned FpMCoL protein.

[0019] The above-mentioned coding gene, SEQ ID NO.1 in the sequence table consists of 2044 nucleotides; the nucleotides 1-102, 154-201, 265-275, 550-738, 793-980, 1303-1322, 1376-1807, 1863-1976, and 2015-2044 from the 5' end of SEQ ID NO.1 are the coding sequence, encoding the protein FpMCoL shown in SEQ ID NO.2 in the sequence table.

[0020] The RNA molecule is an RNA molecule transcribed from the coding gene;

[0021] Preferably, the sequence of the RNA molecule is as follows C1) or C2):

[0022] C1) an RNA sequence having a similarity of 75% or more, more preferably 85% or more, and more preferably 95% or more to an RNA sequence transcribed from the DNA sequence as shown in SEQ ID NO.1 and having the same function as the RNA sequence transcribed from the DNA sequence as shown in SEQ ID NO.1;

[0023] C2) RNA sequence transcribed from the DNA sequence shown in SEQ ID NO.1.

[0024] For example, the DNA sequence of the present invention can be hybridized with the DNA sequence shown in SEQ ID NO.1 under stringent conditions and encodes the DNA sequence of the protein shown in SEQ ID NO.2. The stringent conditions may be hybridization at 65°C with a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0025] The third object of the present invention is to provide biological materials related to the above-mentioned nucleic acid molecules, including recombinant vectors, expression cassettes, recombinant microorganisms or transgenic plant cell lines. The recombinant vector can be a recombinant expression vector or a recombinant cloning vector. In the above-mentioned biological materials, the vector can be a plasmid, a cosmid, a phage or a viral vector; the microorganism can be a yeast, a bacterium, an algae or a fungus, such as Agrobacterium; the transgenic plant cell line does not include a propagation material. Specifically, it can be any one of the following D1) to D10):

[0026] D1) an expression cassette containing SEQ ID NO.1;

[0027] D2) a recombinant vector containing SEQ ID NO.1, or a recombinant vector containing the expression cassette described in D1);

[0028] D3) a recombinant microorganism containing SEQ ID NO.1, or a recombinant microorganism containing the expression cassette described in D1), or a recombinant microorganism containing the recombinant vector described in D2);

[0029] D4) a transgenic plant cell line containing SEQ ID NO.1, or a transgenic plant cell line containing the expression cassette described in D1);

[0030] D5) transgenic plant tissue containing SEQ ID NO.1, or transgenic plant tissue containing the expression cassette described in D2);

[0031] D6) a transgenic plant organ containing SEQ ID NO.1, or a transgenic plant organ containing the expression cassette described in D2);

[0032] D7) a nucleic acid molecule that inhibits the expression of the gene shown in SEQ ID NO.1;

[0033] D8) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in D7);

[0034] D9) a nucleic acid molecule that inhibits the translation of the above RNA molecule;

[0035] D10) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line producing the nucleic acid molecule described in D9).

[0036] In the present invention, the nucleotide sequence encoding the protein FpMCoL can be inserted into an expression vector to form a recombinant expression vector. The term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element. The expression vector in the present invention is not limited to the vectors mentioned in the following embodiments. Methods well known to those skilled in the art can be used to construct an expression vector containing a nucleotide sequence encoding the protein FpMCoL and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0037] The fifth object of the present invention is to provide the application of the FpMCoL protein of Pseudomonas graminearum and the nucleic acid molecules encoding the FpMCoL protein or the biological materials containing the nucleic acid molecules encoding the FpMCoL protein.

[0038] The application is any one or more of the following 1)-4):

[0039] 1) Application in regulating (increasing or decreasing) the mycelial growth rate of Fusarium graminearum;

[0040] 2) Application in regulating (increasing or decreasing) the conidia yield of Fusarium graminearum;

[0041] 3) Application in regulating (increasing or decreasing) the pathogenicity of Fusarium graminearum to the host;

[0042] 4) Application in inhibiting and / or killing Fusarium graminearum.

[0043] Preferably, the application includes achieving the application described in 1)-4) by inhibiting the transcription of the coding gene described in SEQ ID NO.1 or inactivating it, or inhibiting the translation of the RNA molecule, or inhibiting and / or inactivating the activity of the FpMCoL protein described in SEQ ID NO.2.

[0044] In the application, by inhibiting the transcription of the above-mentioned coding gene, or inhibiting the translation of the above-mentioned RNA sequence, or inhibiting and / or inactivating the activity of the above-mentioned FpMCoL protein, the growth rate of hyphae is interfered with, the conidia yield is affected, and the ability to infect the host is regulated, thereby inhibiting and / or killing the growth of Pseudomonas graminearum.

[0045] The sixth object of the present invention is to provide the use of the FpMCoL protein shown in SEQ ID NO.2 or the encoding gene shown in SEQ ID NO.1 as an antibacterial or fungicide target in screening antibacterial or fungicide agents for Fusarium graminearum.

[0046] The seventh object of the present invention is to provide a method for screening or auxiliary screening of Pseudomonas graminearum and / or fungicides.

[0047] The method comprises applying the substance to be detected to F. graminearum. When the substance to be detected can inhibit the transcription of the above DNA sequence, or inhibit the translation of the above RNA sequence, or inhibit and / or inactivate the FpMCoL protein shown above, the substance to be detected is a candidate antibacterial and / or fungicide for F. graminearum.

[0048] An eighth object of the present invention is to provide a method for reducing the activity of Fusarium graminearum, comprising the following steps: inhibiting the transcription of the encoding gene as described above or making it deleted, or inhibiting the translation of the RNA molecule, or inhibiting and / or inactivating the activity of the FpMCoL protein as described above.

[0049] The reducing the activity of Pseudofusorium graminearum is to reduce the infection ability and / or pathogenicity of Pseudofusorium graminearum to the host, and / or reduce the growth rate of Pseudofusorium graminearum, and / or inhibit the conidia yield of Pseudofusorium graminearum.

[0050] In the above method, the inactivation of the protein is achieved by inhibiting or reducing the expression of the gene encoding the protein whose activity is to be inhibited or inactivated. Specifically, it can be achieved by gene knockout or gene silencing.

[0051] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0052] Gene silencing refers to the phenomenon of making a gene non-expressed or low-expressed without damaging the original DNA. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, gene inactivation by specific inhibition of target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition.

[0053] Preferably, the gene shown in SEQ ID NO.1 in the sequence list of Pseudomonas graminearum is knocked out to inactivate the protein shown in SEQ ID NO.2 in the sequence list;

[0054] In one embodiment of the present invention, the method for knocking out the above-mentioned gene is a homologous recombination gene knockout method based on PEG transformation.

[0055] Specifically, the homologous recombination gene knockout method based on PEG transformation is a recombinant vector that sequentially connects the upstream 800-1500bp sequence of the target gene to be knocked out, the hygromycin resistance gene sequence (hph gene sequence) and the downstream 800-1500bp sequence of the target gene to be knocked out.

[0056] The use of substances that inhibit the expression and / or activity of FpMCoL protein in the preparation of plant Fusarium graminearum fungicides also falls within the protection scope of the present invention.

[0057] In the above application, the substance that inhibits the expression and / or activity of FpMCoL protein is a substance that inhibits the expression of FpMCoL protein and / or inhibits the transcription of the gene encoding FpMCoL protein and / or inhibits the translation of RNA molecules transcribed from the gene encoding FpMCoL protein.

[0058] Beneficial effects of the present invention:

[0059] The present invention finds that the FpMCoL protein and its encoding gene play a role in the growth and development of Pseudofusorium graminearum itself. The knockout transformants obtained by homologous recombination gene knockout method using PEG transformation have obvious changes in growth and development compared with wild-type strains, mainly including: FpMCoL gene knockout leads to slower mycelial growth rate, reduced conidia production, and weakened ability to infect host plants. Therefore, the FpMCoL protein and its encoding gene in Pseudofusorium graminearum can play an important role in multiple processes such as vegetative growth, asexual reproduction and host infection of Pseudofusorium graminearum. The present invention provides technical support for the study of the pathogenic mechanism of Pseudofusorium graminearum, and provides potential molecular action targets for the research and development of new fungicides in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] 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 some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 This is the PCR test result of the FpMCoL gene knockout mutant of Pseudomonas graminearum;

[0062] Figure 2The growth of the pseudo-Fusarium graminearum 2035 strain (WT), the FpMCoL gene knockout transformant series strains (11383-9, 11383-11) and the gene revertant mutant strain 11383-C, where A is the growth of the colony (cultured on PDA for 3 days); B is a bar graph of hyphae growth rate; C is the amount of conidia produced;

[0063] Figure 3 The pathogenicity of conidia of F. graminearum 2035 strain (WT), FpMCoL gene knockout transformant series strains (11383-9, 11383-11) and gene revertant strain 11383-C at the base of wheat stems is shown. DETAILED DESCRIPTION

[0064] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0065] Optional reagents for some embodiments of the present invention:

[0066] Pseudoceminalis Fusarium graminearum 2035 strain: obtained from the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences;

[0067] DNA gel recovery kit (Bio-Teke);

[0068] Small-volume plasmid extraction kit (OMEGA);

[0069] Fungal genomic DNA extraction kit (BioFlux);

[0070] digoxigenin-labeled DNA and detection kit (Roche);

[0071] Yeast plasmid extraction kit (OMEGA);

[0072] PCR reagents: Taq enzyme (Thermo), FastPfu enzyme (Full Gold), restriction endonuclease Fermentas;

[0073] Conventional reagents: Hygromycin B (Roche), ampicillin (AMP), Geneticin G418, alkaline phenol:chloroform:isoamyl alcohol (25:24:1), chloroform, isopropanol, anhydrous ethanol, 70% ethanol, Lysing enzyme (Sigma), Driselase (Sigma).

[0074] PDA medium (1 L): 200 g potato, 20 g glucose, 15 g agar.

[0075] TB3 medium (1 L): 3 g yeast extract, 3 g acid hydrolyzed casein, 200 g sucrose.

[0076] CMC liquid culture medium (1 L): CMC (Sigma) 15 g, NH4NO3 1.0 g, KH2PO4 1.0 g, MgSO4·7H2O 0.5 g, yeast extract 1.0 g.

[0077] YEPD medium (1L): 3.0 g yeast extract, 10 g peptone, and 20 g glucose.

[0078] STC buffer (500 mL): sucrose 100 g, 0.5 M Tris-Cl (PH 8.0) 50 mL, CaCl2·2H2O 3.6755 g.

[0079] PTC buffer (100 mL): PEG8000 40 g, add STC solution to 100 mL, sterilize with a bacterial filter and store at 4°C.

[0080] 1.2M KCl solution (1L): KCl 89.46g, add water to 1L.

[0081] Protoplast buffer (20 mL): 500 mg of lyase, 100 mg of lyase, add 1.2 M KCl to make up to 20 mL, and filter through a bacterial filter for sterilization.

[0082] Example 1 Construction of homologous recombination fragments of target genes

[0083] 1.1 Obtaining the FpMCoL protein and its encoding gene from F. graminearum

[0084] The gene encoding the FpMCoL protein machinery of F. graminearum (or cDNA) can be obtained by amplification with the 11383-F / R primer pair using the DNA (or cDNA) of F. graminearum 2035 strain as a template. The material for DNA or RNA extraction can be the mycelium of F. graminearum 2035.

[0085] 11383-F 5'-ATGGTGAAGCAGACTGTTAT-3';

[0086] 11383-R 5'-TTAACAACCGTAAATCTGAG-3'.

[0087] Among them, the coding gene of FpMCoL of Pseudomonas graminearum is shown in SEQ ID NO.1 in the sequence list, and SEQ ID NO.1 in the sequence list consists of 2044 nucleotides; from the 5' end of SEQ ID NO.1, the nucleotides at positions 1-102, 154-201, 265-275, 550-738, 793-980, 1303-1322, 1376-1807, 1863-1976, and 2015-2044 are the coding sequence, encoding the protein FpMCoL shown in SEQ ID NO.2 in the sequence list.

[0088] The nucleotide sequence of SEQ ID NO.1 is as follows:

[0089]

[0090]

[0091] The above-mentioned FpMCoL protein or gene can also be artificially synthesized.

[0092] 1.2 Obtaining the homologous recombination knockout fragment of the FpMCoL encoding gene of F. graminearum

[0093] The genomic DNA of Fusarium graminearum 2035 was extracted using a fungal genomic DNA extraction kit. Using the genomic DNA as a template, 11383-1F / 2R primers and 11383-3F / 4R primers were designed based on the FpMCoL coding gene (SEQ ID No. 1) to amplify the upstream (L) and downstream (R) homologous sequence fragments of about 800-1500 bp of the target fragment, respectively.

[0094] 11383-1F: 5'-TGACGCTGGACGAGACGA-3';

[0095] 11383-2R:

[0096] 5'-TTGACCTCCACTAGCTCCAGCCAAGCCCTGCCGAGCATGGAAGAG-3'

[0097] 11383-3F:

[0098] 5'-GAATAGAGTAGATGCCGACCGCGGGTTGCCGTTCCTGACTTGTGA-3'

[0099] 11383-4R: 5'-CAAACCCGAAGAGTGCTG-3'.

[0100] The reaction system is FastPfu PCR SuperMix (2X) premix, 25 μL, two primers 1.5 μL each, DNA template 2 μL, ddH2O 20 μL system.

[0101] The amplification program was 1 cycle of pre-denaturation at 95°C for 5 min, followed by 35 cycles, each cycle consisting of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and extension at 72°C for 10 min.

[0102] The pHIG2RHPH2-GFP-GUS plasmid was used as a template and the hygromycin resistance (hph) gene fragment was amplified using primers HYG-F and HYG-R. The amplification system conditions were the same as above.

[0103] HYG-F: 5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3';

[0104] HYG-R: 5'-AACCCGCGGTCGGCATCTACTCTATTC-3'.

[0105] The PCR products were separated by agarose gel electrophoresis, and the target bands were recovered (Bio-Kete gel recovery kit, see the instructions for the method). The gel recovery products of L, R and hph fragments were mixed and connected into a knockout fragment using the double-joint method for later use.

[0106] Double-joint PCR step 1: Mix L and R fragments and hph fragment for amplification. PCR system: 5×Buffer: 5μL, dNTPs: 4μL, fragment L: 1μL, fragment R: 1μL, hph: 3μL, Fastpfu enzyme: 0.3μL, ddH2O: make up to 25μL.

[0107] The PCR program was: 95°C for 1 min; 95°C for 20 s, 55°C for 5 min, 72°C for 2 min, 15 cycles; 72°C for 10 min.

[0108] Double-joint PCR step 2: Using the PCR product from the previous step as a template, add primer pair 1F / 4R, and the PCR system is as follows: 5×Buffer: 5μL, 11383-1F / 4R: 0.4μL each, dNTPs: 2.5μL, Fastpfu enzyme: 0.5μL, ddH2O: make up to 25μL.

[0109] The PCR program was 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 2 min, and 1 cycle of extension at 72°C for 10 min.

[0110] After the PCR program is completed, 1.2% agarose gel electrophoresis is performed. After the target band is detected correctly, the PCR product is concentrated (>150 μg). The concentrated product obtained is the homologous recombination knockout fragment of the FpMCoL encoding gene.

[0111] Example 2 Obtaining FpMCoL encoding gene knockout mutant

[0112] 2.1 Obtaining FpMCoL encoding gene knockout mutants

[0113] The wild-type Fusarium graminearum 2035 strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL CMC liquid medium and cultured at 25°C and 150 rpm for 72 h to induce spore production. The spores were transferred to a 250 mL Erlenmeyer flask containing 100 mL YEPD liquid medium and cultured at 25°C and 175 rpm for 12 h. Mycelia were collected and lysed at a ratio of 20 mL protoplast buffer per gram of mycelia. The mycelia were cultured at 30°C and 90 rpm for 2 h. The protoplasts were collected and washed twice by centrifugation with 10 mL STC Buffer and resuspended to 2-5 × 10 7 protoplasts / mL.

[0114] Add 5 μg of the gene homologous recombination knockout fragment constructed in Example 1, let stand at room temperature for 20 minutes, add 1 mL of 40% PTC to the tube, turn over and mix evenly, let stand at room temperature for 20 minutes, add 5 mL of TB3 (containing 50 g / mL ampicillin), shake at room temperature overnight, add 10 mL of TB3 Agar (containing 250 ug / mL hygromycin) quickly to the plate, incubate at room temperature for 10 hours, then pour TB3 Agar containing 400 μg / mL hygromycin, continue to incubate at room temperature, wait for transformants with single colonies to grow, extract transformant DNA using the fungal genomic DNA extraction kit method, use the transformant DNA as a template, and perform PCR detection with four pairs of primers.

[0115] 11383-5F 5'-CGACAGCCATCCCACCTA-3';

[0116] 11383-6R 5'-AAGCAGCGCAGTTACGAC-3';

[0117] 11383-7F 5'-TCGTCCGCAGAAAGTCCCA-3';

[0118] 11383-8R 5'-TCAAAGGCTGTGATAGTTC-3';

[0119] H852 5'-AACTCACCGCGACGTCTGTC-3';

[0120] H850 5'-TTGTCCGTCAGGACATTGTT-3';

[0121] H855R 5'-GCTGATCTGACCAGTTGC-3';

[0122] H856F 5'-GTCGATGCGACGCAATCGT-3'.

[0123] Detection fragments as attached Figure 1 As shown, 11383-5F / 6R detects whether each transformant contains the target gene; H850 / H852 detects whether each transformant has the hph gene transferred; 11383-7F / H855R detects whether homologous recombination occurs upstream of the target gene; H856F / 11383-8R detects whether homologous recombination occurs downstream of the target gene. Therefore, only when 11383-5F / 6R cannot detect the target band, and the other three pairs of primers can detect the correct band, can the transformant be determined as a positive transformant and a positive transformant strain be obtained. Referring to the instructions for digoxigenin-labeled DNA and the detection kit, Southern blot hybridization was performed using the hph gene fragment (750bp) as a probe, and it was verified that 11383-9 and 11383-11 were knockout mutants.

[0124] 2.2 Verification of functional restoration of the gene encoding FpMCoL

[0125] Using the genomic DNA of wild-type Fusarium graminearum strain 2035 as a template, primers 11383-CF / CR containing 1-1.5 kb upstream of the start codon of the target gene and the full-length gene fragment without the stop codon were used to amplify the restored gene fragment. The restored fragment and the pFL2 vector digested with Xho I were co-transfected into the XK1-25 yeast strain, plated and cultured on SD-Trp medium, and single colonies were detected with primers 11383-5F / 6R. After the correct colony was detected, the yeast plasmid was extracted using a yeast plasmid extraction kit, the yeast recombinant plasmid was transformed into Escherichia coli for propagation, and the recombinant plasmid was transferred into the FpMCoL gene knockout mutant according to step 1.2, and the transformants were selected with G418. The final concentration of G418 in Bottom Agar was 300ug / mL, and the final concentration of G418 in Top Agar was 500μg / mL. The transformants were detected by PCR using primers 11383-5F / 6R, and the transformants that were positive clones by PCR and whose phenotypes on PDA plates reverted to wild-type traits were used for phenotypic verification.

[0126] 11383-CF: 5'-TCTCATCACCATCACCATCACATCGCAATAGGAAAGATGGG-3';

[0127] 11383-CR: 5'-TCGCCCTTGCTCACCCTCGAACAACCGTAAATCTGAGGCT-3'.

[0128] Example 3 Analysis of biological characteristics of FpMCoL encoding gene mutants

[0129] 3.1 Determination of mycelial growth rate

[0130] Wild-type F. graminearum strain 2035, knockout mutants and revertant mutants were activated on PDA plates and cultured at room temperature for 3-4 days.

[0131] Use a 5mm diameter puncher to punch the bacterial cakes at the same distance from the center of the colony, three for each of the wild type and mutant. Place the bacterial cakes in the center of a 9cm culture plate containing 20mL PDA medium, with the front of the bacterial cake facing down and ensuring that the bacterial cake is in uniform contact with the plate, and repeat three times. Invert the plate and culture at room temperature for 72 hours, and measure the diameter.

[0132] The results showed that the mycelial growth rate of the FpMCoL knockout mutants (11383-9, 11383-11) was significantly decreased compared with the wild-type F. graminearum strain 2035 (WT). Figure 2 ), the growth rate of the revertant mutant was restored. The experimental results show that FpMCoL protein is involved in regulating the hyphal growth of F. graminearum.

[0133] 3.2 Conidia production assay

[0134] Use a 5 mm diameter puncher to punch out bacterial cakes on the plates of wild-type F. graminearum strains and mutants activated for 3-4 days. Transfer 5 bacterial cakes to a triangular flask containing 100 mL CMC medium, with 3 replicates for each strain, shake at 25°C and 150 rpm for 5 days, and measure the spore concentration in the suspension using a hemocytometer.

[0135] like Figure 2 As shown in C, compared with the wild-type F. graminearum strain 2035 (WT), the amount of conidia produced by the FpMCoL knockout mutants (11383-9, 11383-11) under induction conditions was significantly reduced, indicating that the FpMCoL protein affects the production of conidia of F. graminearum.

[0136] 3.3 Pathogenicity determination

[0137] The mutants and wild-type strains were cultured on PDA plates for 7 days, and then the plates were punched with a 6-mm-diameter cork puncher and placed in 100 mL CMC liquid medium. The plates were shaken and cultured at 25°C and 170 rpm for 5-7 days. The spore suspension concentration of each strain was 1 × 10 5 For wheat inoculation. Soak wheat seeds in spore suspension for 15 minutes, then pour out the bacterial solution and sow them in a culture dish with sterile filter paper and sterilized water. Repeat 3 times for each treatment, with 10 seeds per replicate, and culture them in a culture room at 22°C with 12h alternating light and dark. When the disease of wild-type strain becomes more serious, start the investigation and calculate the disease index.

[0138] The results showed that the disease index of the wild-type strain 2035 reached 83%, and the disease index of the mutant was 42% lower than that of the wild-type strain. The pathogenicity was significantly reduced after the FpMCoL gene was knocked out, which indicated that the FpMCoL protein was involved in the pathogenicity of the bacteria.

[0139] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. The use of knocking out the FpMCoL encoding gene, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.1, and the application is any one or more of the following 1) - 3): 1) Application in reducing the mycelial growth rate of Fusarium graminearum; 2) Application in reducing the conidia yield of Pseudomonas graminearum; 3) Application in reducing the pathogenicity of Pseudomonas graminearum to the host.

2. The use according to claim 1, characterized in that: The host is wheat seeds.

3. A method for reducing the activity of Pseudomonas graminearum, characterized in that: The method comprises the steps of knocking out the FpMCoL encoding gene of Pseudomonas graminearum, wherein the nucleotide sequence of the encoding gene is shown in SE Q ID NO.1; Wherein, the activity of reducing Pseudomonas graminearum is any one or more of the following 1) - 3): 1) Reduce the mycelial growth rate of Fusarium graminearum; 2) reducing the conidia yield of Pseudomonas graminearum; 3) Reduce the pathogenicity of Pseudomonas graminearum to the host.

4. The method according to claim 3, characterized in that: The host is wheat seeds.

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