CN117304285A - Fusarium pseudograminearum FpMCoL protein, and coding gene and application thereof - Google Patents

Fusarium pseudograminearum FpMCoL protein, and coding gene and application thereof Download PDF

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CN117304285A
CN117304285A CN202311221430.1A CN202311221430A CN117304285A CN 117304285 A CN117304285 A CN 117304285A CN 202311221430 A CN202311221430 A CN 202311221430A CN 117304285 A CN117304285 A CN 117304285A
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protein
fusarium pseudograminearum
fpmicrocol
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fpmcol
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吴玉星
韩森
王亚娇
孔令晓
栗秋生
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Plant Protection Institute hebei Academy Of Agricultural And Forestry Sciences
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Abstract

The invention belongs to the technical field of biology, and particularly relates to fusarium pseudograminearum FpMCoL protein, and a coding gene and application thereof. The FpMCoL protein provided by the invention is shown in a sequence 2; the coding gene is shown in sequence 1. Experiments prove that the protein provided by the invention plays an important role in the growth and development process of fusarium pseudograminearum per se, and is specifically characterized in that the growth rate of fusarium pseudograminearum hyphae is reduced, the generation amount of conidium is reduced, the pathogenicity is reduced and the like after the protein is deleted. Therefore, the invention provides a technical basis for exploring the growth and development of the fusarium graminearum and the pathogenic mechanism, and provides a potential molecular target for the research and development of novel bactericides in the future.

Description

Fusarium pseudograminearum FpMCoL protein, and coding gene and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to FpMCoL protein, and a coding gene and application thereof.
Background
Wheat stem rot (WCR) caused by fusarium pseudograminearum (Fusarium pseudograminearum) is an important soil-borne disease in Wheat production, and the main symptom is represented by browning and rot of the root base of Wheat, which can seriously cause seedling blight and white ears. Wheat stem rot occurs and is serious in australia, the united states, canada and other countries, and the average yield loss of wheat caused by the wheat stem rot is about 9.5% in normal years, and the pandemic year can reach 35%. In recent years, the disease commonly occurs in Huang-Huai-Hai winter wheat areas and has a trend of increasing year by year, and at present, the disease field rates of the disease in Henan province and Hebei province are respectively up to 65.1% and 81.4%, which seriously threatens the safe production of wheat.
Magnesium (magnesium) is one of the core nutrient elements necessary for the growth and development of organisms, and is also the divalent cation with the greatest content in cells. Magnesium has the largest hydration radius, the smallest ion radius and the largest charge density, and plays an important role in enzyme activation, genome stabilization, aging inhibition, aluminum harm alleviation, nitrogen metabolism regulation and other aspects, so that the special physicochemical property and biological function of magnesium ions make the transportation mode in organisms important. Cobalt-resistant protein family CorA belongs to a helical channel subclass transmembrane transporter among channel pore class transporters and is a major member of the superfamily of large divalent metal ion transporters. Family proteins are widely distributed in gram-positive bacteria, gram-negative bacteria, blue algae, archaea and yeast, and are regulating systems for absorption, transportation and excretion of divalent cations such as magnesium ions, nickel ions, drilling ions and the like; is the most important transport system for maintaining intracellular magnesium ion steady state of organisms, and the loss of the system can seriously affect the growth and development of organisms.
Disclosure of Invention
The invention develops the related research work of the function of the metal divalent cation transporter MIT_CorA-like (metal ion transporter CorA-like divalent cation transporter superfamily) homologous protein FpMCoL of the fusarium pseudograminearum, and provides reference and reference for the design of novel fungal control targets for the deeper understanding of the function of the metal divalent cation transporter system of the fusarium pseudograminearum in the processes of regulating and controlling the growth and development of bacteria and interaction with hosts.
The inventor researches find that FpMCoL protein in the fusarium pseudograminearum and encoding genes thereof are closely related to the hypha growth rate, conidium generation and pathogenicity of the fusarium pseudograminearum. These results indicate that the FpMCoL protein in Fusarium pseudograminearum has important regulatory effects on the growth and development of pathogenic bacteria and the pathogenic process. The FpMCoL protein gene is used as a molecular medicament target of pathogenic fungi fusarium pseudograminearum, and has important application prospect.
Accordingly, one of the objects of the present invention is to provide a fusarium pseudograminearum CorA homologous protein, designated FpMCoL, derived from fusarium pseudograminearum strain 2035, which is a protein of A1) or A2) A3) or A4) as follows:
a1 Amino acid sequence is protein shown as SEQ ID NO. 2;
a2 A fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of a protein shown in SEQ ID NO. 2;
a3 A protein derived from a protein shown as SEQ ID NO.2, which has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown as SEQ ID NO. 2;
a4 An amino acid sequence having a similarity of 75% or more, preferably 85% or more, more preferably 95% or more with the amino acid sequence shown in SEQ ID NO.2, and having the same function as the amino acid sequence shown in SEQ ID NO. 2.
To facilitate purification of the protein of A1), the amino-terminal or carboxyl-terminal linkage of the protein consisting of the amino acid sequence shown in SEQ ID NO.2 may be provided with tags such as Poly Arg (RRRRR), poly His (HHHH), FLAG (DYKDDDDK), strep tag II (WSHPQFEK), c myc (EQKLISEEDL) and the like.
The protein in the A1) -A4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing. The coding gene of the protein in the A1) -A4) can be obtained by deleting one or more amino acid residues in the DNA sequence shown in SEQ ID NO.1 of the sequence list and/or carrying out missense mutation of one or more nucleotide pairs and/or connecting the coding sequences of the tags at the 5 'end and/or the 3' end of the coding sequence.
Wherein, in A1), SEQ ID NO.2 (FpMCoL protein) in the sequence Listing consists of 377 amino acid residues.
It is a further object of the present invention to provide nucleic acid molecules encoding the desired FpMCoL proteins. The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA, such as mRNA, hnRNA, tRNA or the like.
Wherein, the encoding gene of the FpMCoL protein is as follows B1) or B2) or B3):
b1 A DNA molecule shown in a nucleotide sequence shown in SEQ ID NO.1 of the sequence table;
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 FpMCoL protein described above;
b3 Under stringent conditions with a nucleotide sequence defined in B1) or B2), and a cDNA molecule or DNA molecule encoding the above-mentioned FpMCoL protein.
The coding gene is formed by 2044 nucleotides in SEQ ID NO.1 in a sequence table; the nucleotide numbers 1-102, 154-201, 265-275, 550-738, 793-980, 1303-1322, 1376-1807, 1863-1976 and 2015-2044 of the 5' end of SEQ ID NO.1 are the coding sequences, and the FpMCoL protein shown in SEQ ID NO.2 in the sequence table is coded.
The RNA molecule is obtained by transcription of the coding gene;
preferably, the sequence of the RNA molecule is C1) or C2) as follows:
c1 A RNA sequence having a similarity of 75% or more, more preferably 85% or more, still more preferably 95% or more, with the RNA sequence transcribed from the DNA sequence shown in SEQ ID NO. 1;
c2 RNA sequence transcribed from the DNA sequence shown in SEQ ID NO. 1.
The DNA sequence according to the invention is capable of molecular hybridization under stringent conditions with the DNA sequence shown as SEQ ID NO.1 and encodes a protein shown as SEQ ID NO. 2. The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65℃followed by washing the membrane once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
It is a further object of the present invention 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 biological material, the vector may be a plasmid, cosmid, phage or viral vector; the microorganism may be a yeast, bacterium, algae or fungus, such as agrobacterium; the transgenic plant cell line does not include propagation material. Specifically, any one of the following D1) to D10) may be mentioned:
d1 An expression cassette comprising SEQ ID NO. 1;
d2 A recombinant vector comprising SEQ ID NO.1 or a recombinant vector comprising the expression cassette of D1);
d3 A recombinant microorganism comprising SEQ ID NO.1, or a recombinant microorganism comprising the expression cassette of D1), or a recombinant microorganism comprising the recombinant vector of D2);
d4 A transgenic plant cell line comprising SEQ ID NO.1, or a transgenic plant cell line comprising the expression cassette of D1);
d5 A transgenic plant tissue comprising SEQ ID NO.1, or a transgenic plant tissue comprising the expression cassette of D2);
d6 A transgenic plant organ comprising SEQ ID NO.1 or a transgenic plant organ comprising the expression cassette of D2);
d7 A nucleic acid molecule that inhibits expression of the gene shown in SEQ ID NO. 1;
d8 A expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line comprising the nucleic acid molecule of D7);
d9 A nucleic acid molecule that inhibits translation of the RNA molecule;
d10 Producing D9) an expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line of said nucleic acid molecule.
In the present invention, the nucleotide sequence encoding the protein FpMCoL may be inserted into an expression vector to form a recombinant expression vector. The term "expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements. The expression vector of the present invention is not limited to the vectors mentioned in the examples below, and 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 techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
The fifth object of the present invention is to provide the use of the FpMCoL protein of Fusarium pseudograminearum, a nucleic acid molecule encoding the FpMCoL protein, or a biological material comprising the nucleic acid molecule encoding the FpMCoL protein.
The application is any one or more of the following 1) -4):
1) The application in regulating and controlling (increasing or decreasing) the growth rate of fusarium pseudograminearum hyphae;
2) The application in regulating (increasing or decreasing) the yield of fusarium pseudograminearum conidium;
3) The application in regulating and controlling (improving or reducing) the pathogenicity of the fusarium pseudograminearum on a host;
4) The application in inhibiting and/or killing fusarium graminearum.
Preferably, among said uses, use is made of 1) to 4) by inhibiting transcription or inactivating the coding gene of SEQ ID NO.1, or inhibiting translation of said RNA molecule, or inhibiting and/or inactivating the activity of the FpMCoL protein of SEQ ID NO. 2.
In the application, the growth of fusarium pseudograminearum can be inhibited and/or killed by inhibiting the transcription of the coding genes, or the translation of the RNA sequences, or inhibiting and/or inactivating the activity of the FpMCoL protein to interfere with the growth rate of hyphae, influence the yield of conidia and regulate the capability of infecting a host.
The invention aims at providing an application of FpMCoL protein shown as SEQ ID NO.2 or a coding gene shown as SEQ ID NO.1 as a bacteriostatic or bactericidal agent target for screening a fusarium pseudograminearum bacteriostatic or bactericidal agent.
The seventh object of the invention is to provide a method for screening or assisting in screening fusarium pseudograminearum and/or bactericides.
The method comprises the step of applying a substance to be detected to fusarium pseudograminearum, wherein when the substance to be detected can inhibit transcription of a DNA sequence, inhibit translation of an RNA sequence, inhibit and/or inactivate FpMCoL protein as shown above, the substance to be detected is a candidate fusarium pseudograminearum antibacterial and/or bactericidal agent.
The eighth object of the present invention is to provide a method for reducing the activity of fusarium pseudograminearum, comprising the steps of: inhibiting transcription or deletion of the coding gene as described above, or inhibiting translation of the RNA molecule, or inhibiting and/or inactivating the activity of the fpmicrocol protein as described above.
Wherein, the reduction of the activity of the fusarium pseudograminearum is to reduce the infection capability of the fusarium pseudograminearum to a host and/or the pathogenicity of the fusarium pseudograminearum to the host, and/or reduce the growth speed of the bacterial body of the fusarium pseudograminearum and/or inhibit the conidium yield of the fusarium pseudograminearum.
In the above method, the inactivation of the protein is achieved by inhibiting or reducing the expression of the encoding gene of the protein to be inhibited or inactivated, specifically, by gene knockout or by gene silencing.
The gene knockout refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.
The gene silencing refers to the phenomenon that the gene is not expressed or expressed under the condition that the original DNA is not damaged. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of a target RNA at the post-transcriptional level of the gene, including antisense RNA, co-inhibition, gene suppression, RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.
Preferably, the protein shown as SEQ ID NO.2 in the sequence table is inactivated by knocking out the gene shown as SEQ ID NO.1 in the sequence table in Fusarium pseudograminearum;
in one embodiment of the present invention, the method of gene knockout of the above-mentioned gene is a homologous recombination gene knockout method based on PEG transformation.
Specifically, the homologous recombination gene knockout method based on PEG transformation is a recombination vector of a sequence 800-1500bp upstream of a target gene to be knocked out and a sequence 800-1500bp downstream of the target gene to be knocked out, which are sequentially connected.
The application of the substance for inhibiting the expression and/or the activity of FpMCoL protein in preparing the fusarium pseudograminearum bactericide also belongs to the protection scope of the invention.
In the above application, the substance that inhibits the expression and/or activity of the fpmicrocol protein is a substance that inhibits the expression and/or transcription of the encoding gene of the fpmicrocol protein and/or translation of an RNA molecule obtained by transcription of the encoding gene of the fpmicrocol protein.
The invention has the beneficial effects that:
the invention discovers that FpMCoL protein and a coding gene thereof play a role in the growth and development process of fusarium pseudograminearum. The knocked-out transformant obtained by the homologous recombination gene knocked-out method of PEG transformation has obvious change in growth and development compared with a wild strain, and mainly comprises: fpMCoL gene knockout results in a reduced rate of hyphal growth, reduced conidium production, and a reduced ability to infect host plants. Therefore, fpMCoL protein and encoding genes thereof in the fusarium pseudograminearum can play an important role in a plurality of processes of the fusarium pseudograminearum, such as vegetative growth, asexual reproduction, infection of hosts and the like. The invention provides technical support for researching the pathogenic mechanism of fusarium pseudograminearum and provides a potential molecular action target for developing novel bactericides in the future.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the PCR detection results of F.pseudograminearum FpMCoL gene knockout mutants;
FIG. 2 shows the growth of Fusarium pseudograminearum 2035 strain (WT), fpMCoL gene knockout transformant series strain (11383-9, 11383-11) and gene revertant mutant strain 11383-C, wherein A is the growth of colonies (3 days of culture on PDA); b is a bar graph of hypha growth rate; c is the generation amount of conidium;
FIG. 3 shows the pathogenicity of Fusarium pseudograminearum 2035 strain (WT), fpMCoL gene knockout transformant series strain (11383-9, 11383-11) and gene revertant mutant strain 11383-C conidia at the basal part of wheat stem.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
The reagents that may be used in some embodiments of the invention:
fusarium pseudograminearum 2035 strain: is obtained from plant protection institute of academy of sciences of agriculture and forestry in Hebei province;
DNA gel recovery kit (Bio-Teke);
miniprep plasmid extraction kit (OMEGA);
fungal genomic DNA extraction kit (BioFlux);
digoxin-labeled DNA and detection kit (Roche);
yeast plasmid extraction kit (OMEGA);
PCR reagent: taq enzyme (Thermo), fastpfu enzyme (gold of full formula), restriction enzyme Fermentas;
conventional reagents: hygromycin B (Roche), ampicillin (AMP), geneticin G418, basic phenol: chloroform: isoamyl alcohol (25:24:1), chloroform, isopropanol, absolute ethanol, 70% ethanol, lyase (Sigma), crashing enzyme (Drisease) (Sigma).
PDA medium (1L): 200g of potato, 20g of glucose and 15g of agar.
TB3 Medium (1L): 3g of yeast extract, 3g of acid hydrolyzed casein and 200g of sucrose.
CMC liquid medium (1L): CMC (sigma Co.) 15g, NH 4 NO 3 1.0g，KH 2 PO 4 1.0g，MgSO 4 ·7H 2 O0.5 g, yeast extract 1.0g.
YEPD medium (1L): 3.0g of yeast extract, 10g of peptone and 20g of glucose.
STC buffer (500 mL): sucrose 100g,0.5M Tris-Cl (pH 8.0) 50mL, caCl 2 ·2H 2 O3.6755g。
PTC buffer (100 mL): PEG8000 40g, STC solution was added to 100mL and stored at 4 ℃ after sterilization with bacterial filters.
1.2M KCl solution (1L): KCl 89.46g was made up to 1L.
Protoplast buffer (20 mL): 500mg of crashing enzyme and 100mg of lyase are added to make up to 20mL of KCl with the concentration of 1.2M, and the bacterial filter is used for filtration sterilization.
EXAMPLE 1 construction of homologous recombination fragments of the Gene of interest
1.1 Fusarium pseudograminearum FpMCoL protein and obtaining of coding gene thereof
The fusarium pseudograminearum FpMCoL protein machine coding gene (or cDNA) can be obtained by taking fusarium pseudograminearum 2035 strain DNA (or cDNA) as a template and amplifying through 11383-F/R primer pairs. Wherein the DNA or RNA extracted material can be fusarium pseudograminearum 2035 mycelium.
11383-F 5'-ATGGTGAAGCAGACTGTTAT-3'；
11383-R 5'-TTAACAACCGTAAATCTGAG-3'。
Wherein, the coding gene of the fusarium pseudograminearum FpMCoL is shown as SEQ ID NO.1 in a sequence table, and the SEQ ID NO.1 in the sequence table consists of 2044 nucleotides; the nucleotide numbers 1-102, 154-201, 265-275, 550-738, 793-980, 1303-1322, 1376-1807, 1863-1976 and 2015-2044 of the 5' end of SEQ ID NO.1 are the coding sequences, and the FpMCoL protein shown in SEQ ID NO.2 in the sequence table is coded.
The nucleotide sequence of SEQ ID NO.1 is as follows:
the FpMCoL protein or gene may also be synthesized artificially.
1.2 obtaining Fusarium pseudograminearum FpMCoL encoding Gene homologous recombination knockout fragment
Extracting fusarium pseudograminearum 2035 genome DNA by using a fungus genome DNA extraction kit, designing 11383-1F/2R primers and 11383-3F/4R primers according to FpMCoL coding genes (SEQ ID No. 1) by using the genome DNA as a template, and respectively amplifying upstream (L) and downstream (R) homologous sequence fragments of about 800-1500bp of a target fragment.
11383-1F：5'-TGACGCTGGACGAGACGA-3'；
11383-2R：
5'-TTGACCTCCACTAGCTCCAGCCAAGCCCTGCCGAGCATGGAAGAG-3'
11383-3F：
5'-GAATAGAGTAGATGCCGACCGCGGGTTGCCGTTCCTGACTTGTGA-3'
11383-4R：5'-CAAACCCGAAGAGTGCTG-3'。
The reaction system is thatFastPfu PCR SuperMix (2X) premix, 25. Mu.L, 1.5. Mu.L each of two primers, 2. Mu.L of DNA template, ddH 2 O20. Mu.L system.
The amplification procedure was 5min at 95℃for pre-denaturation followed by 35 cycles, each cycle comprising 10min at 95℃for 30s, 55℃for 30s, 72℃for 1min, and 72℃for 10min for 1 cycle.
Hygromycin resistance (hph) gene fragments are amplified by using HYG-F and HYG-R primers by taking pHIG2RHPH2-GFP-GUS plasmid as a template, and the amplification system conditions are the same.
HYG-F：5'-GGCTTGGCTGGAGCTAGTGGAGGTCAA-3'；
HYG-R：5'-AACCCGCGGTCGGCATCTACTCTATTC-3'。
The PCR product was separated by agarose gel electrophoresis and the band of interest was recovered (Bio-Kete gel recovery kit, methods see description). And (3) mixing and connecting the L, R and hph fragment glue recovery products into a knockout fragment by utilizing a Double-joint method for later use.
Double-join PCR first step: the L and R fragments and the hph fragment were mixed for amplification. PCR system: 5 XBuffer: 5 μl, dNTPs:4 μl, fragment L:1 μl, fragment R:1 μL, hph: 3. Mu.L, fastpfu enzyme: 0.3. Mu.L ddH 2 O: make up to 25. Mu.L.
The PCR procedure was: 95 ℃ for 1min;95℃20s,55℃5min,72℃2min,15 cycles; and at 72℃for 10min.
Double-join PCR second step: the PCR product is taken as a template, a primer pair 1F/4R is added, and the PCR system is as follows: 5 XBuffer: 5 μL,11383-1F/4R: 0.4. Mu.L each, dNTPs: 2.5. Mu.L, fastpfu enzyme: 0.5 mu L ddH 2 O: make up to 25. Mu.L.
The PCR procedure was 95℃for 30s, 55℃for 30s, 72℃for 2min,35 cycles, 72℃for 10min,1 cycle.
After the PCR program is finished, 1.2% agarose gel electrophoresis is carried out, and after the target band is detected correctly, the PCR product is concentrated (> 150 mug), and the obtained concentrated product is the FpMCoL coding gene homologous recombination knockout fragment.
EXAMPLE 2 obtaining FpMCoL encoding Gene knockout mutant
2.1FpMCoL coding Gene knockout mutant
Inoculating wild Fusarium pseudograminearum 2035 strain into 250mL triangular flask containing 100mL CMC liquid medium, culturing at 25deg.C for 72 hr to induce spore production, transferring spores into 250mL triangular flask containing 100mL YEPD liquid medium, shaking at 25deg.C and 175rpm for 12 hr, collecting mycelium, lysing mycelium with 20mL protoplast Buffer per gram mycelium, shaking at 30deg.C and 90rpm for 2 hr, collecting protoplast, centrifuging with 10mL STC Buffer for 2 times, and re-suspending to 2-5×10 7 Each protoplast/mL.
Adding 5 mug of the gene homologous recombination knockout fragment constructed in the example 1, standing for 20min at room temperature, adding 1mL of 40% PTC into a tube, turning over and mixing uniformly, standing for 20min at room temperature, adding 5mL of TB3 (containing 50g/mL of ampicillin), shaking overnight at room temperature, adding 10mL of TB3 Agar (containing 250ug/mL of hygromycin) to a flat plate quickly, culturing for 10 hours at room temperature, pouring TB3 Agar containing 400 mug/mL of hygromycin, continuing culturing at room temperature, extracting transformant DNA by a fungus genome DNA extraction kit method until single colony grows, taking the transformant DNA as a template, carrying out PCR detection by using four pairs of primers,
11383-5F 5'-CGACAGCCATCCCACCTA-3'；
11383-6R 5'-AAGCAGCGCAGTTACGAC-3'；
11383-7F 5'-TCGTCCGCAGAAGTCCCA-3'；
11383-8R 5'-TCAAAGGCTGTGATAGTTC-3'；
H852 5'-AACTCACCGCGACGTCTGTC-3'；
H850 5'-TTGTCCGTCAGGACATTGTT-3'；
H855R 5'-GCTGATCTGACCAGTTGC-3'；
H856F 5'-GTCGATGCGACGCAATCGT-3'。
the detection fragment is shown in figure 1, 11383-5F/6R detects whether each transformant contains the target gene; H850/H852 detects whether the hph gene is transferred into each transformant; 11383-7F/H855R detects whether homologous recombination occurs upstream of the target gene; H856F/11183-8R detects whether homologous recombination occurs downstream of the target gene. Therefore, only when 11383-5F/6R could not detect the target band, but the other three pairs of primers could detect the correct band, the transformant could be determined to be a positive transformant, and a positive transformant could be obtained. Referring to the digoxin-labeled DNA and the detection kit instruction, southern blot hybridization is performed by using the hph gene fragment (750 bp) as a probe, and 11383-9 and 11383-11 are obtained by verification as knockout mutants.
2.2 functional recovery verification of FpMCoL encoding Gene
The method comprises the steps of using wild type fusarium pseudograminearum strain 2035 genome DNA as a template, amplifying a revertant gene fragment by using a primer 11383-CF/CR containing 1-1.5kb upstream of a start codon of a target gene and a full-length gene fragment without a stop codon, co-transferring the revertant fragment obtained and a pFL2 vector subjected to Xho I digestion to an XK1-25 yeast strain, performing plating culture on an SD-Trp culture medium, detecting single colonies by using the primer 11383-5F/6R, detecting correct shaking, extracting yeast plasmids by using a yeast plasmid extraction kit, amplifying the yeast recombinant plasmids into escherichia coli, transferring the recombinant plasmids into FpMCoL gene knockout mutants according to the step 1.2, and screening transformants by using G418. Transformants were subjected to PCR detection with primers 11383-5F/6R at a final G418 concentration of 300ug/mL in Bottom Agar and 500 ug/mL in Top Agar, PCR detected as positive clones and phenotypically reverted to wild-type trait transformants on PDA plates for phenotypic verification.
11383-CF：5'-TCTCATCACCATCACCATCACATCGCAATAGGAAAGATGGG-3'；
11383-CR：5'-TCGCCCTTGCTCACCCTCGAACAACCGTAAATCTGAGGCT-3'。
EXAMPLE 3 analysis of biological Properties of mutants of the Gene encoding FpMCoL
3.1 hypha growth Rate determination
The wild type fusarium pseudograminearum strain 2035, the knockout mutant and the revertant mutant were activated with PDA plates and cultured at room temperature for 3 to 4 days.
The patties were punched with a punch of 5mm diameter at the same distance from the center of the colony, and the wild type and mutant were each punched three times. The bacterial cakes are respectively connected to the center position of a 9cm culture dish plate containing 20mLPDA culture medium, the front surface of the bacterial cakes faces downwards, the bacterial cakes are ensured to be uniformly contacted with the plate, and three repetitions are respectively carried out. The plates were inverted and incubated at room temperature for 72 hours, and the diameters were measured.
The results showed that the hyphal growth rate of the FpMCoL knockout mutant (11383-9, 11383-11) was significantly reduced compared to the wild-type fusarium pseudograminearum strain 2035 (WT) (fig. 2), and the growth rate of the revertant mutant was restored. Experimental results show that FpMCoL protein participates in regulating and controlling hypha growth of fusarium pseudograminearum.
3.2 conidium production assay
The patties were removed from the activated 3-4d wild type F.pseudograminea strain and mutant plates using a punch with a diameter of 5 mm. 5 cakes were transferred to a triangular flask containing 100mL CMC medium, 3 replicates per strain, and after shaking at 25℃and 150rpm for 5d, the spore concentration in suspension was measured with a hemocytometer.
As shown in FIG. 2C, the FpMCoL knockout mutant (11383-9, 11383-11) had significantly reduced production of conidia under induction conditions compared to wild-type F.pseudograminearum strain 2035 (WT), indicating that the FpMCoL protein affects the production of Fusarium pseudograminearum conidia.
3.3 determination of pathogenicity
Culturing mutant strain and wild strain on PDA plate for 7d, taking bacterial disc with hole puncher with diameter of 6mm, placing into 100mL CMC liquid culture medium, shake culturing at 25deg.C and 170r/min for 5-7d, and concentrating spore suspension of each strain to 1×10 5 Is used for wheat inoculation. Soaking wheat seeds in spore suspension for 15min, pouring out the bacterial liquid, sowing in a culture dish containing sterile filter paper and sterilized water, repeating for 3 times, and culturing 10 seeds in a light-dark alternate culture chamber at 22 ℃ for 12 h; investigation and calculation of disease index when wild type strain is heavily developedA number.
The result shows that the disease index of the wild strain 2035 reaches 83%, the disease index of the mutant is reduced by 42% compared with that of the wild strain, and the pathogenicity of the FpMCoL protein is obviously reduced after knocking out the FpMCoL gene, which indicates that the FpMCoL protein participates in pathogenic bacteria.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (10)

  1. FpMCoL protein, which is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.
  2. 2. A coding gene encoding the fpmicrocol protein of claim 1.
  3. 3. The coding gene according to claim 2, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.
  4. 4. A biomaterial associated with the fpmicrocol protein of claim 1, comprising any one of the following:
    d1 An expression cassette containing the gene encoding the fpmicrocol protein;
    d2 A recombinant vector containing the gene encoding the FpMCoL protein, or a recombinant vector containing the expression cassette of D1);
    d3 A recombinant microorganism containing the gene encoding the FpMCoL protein, or a recombinant microorganism containing the expression cassette of D1), or a recombinant microorganism containing the recombinant vector of D2);
    d4 A transgenic plant cell line containing the gene encoding the fpmicrocol protein, or a transgenic plant cell line containing the expression cassette of D1);
    d5 A transgenic plant tissue containing the gene encoding the fpmicrocol protein, or a transgenic plant tissue containing D2) the expression cassette;
    d6 A transgenic plant organ containing the gene encoding the fpmicrocol protein, or a transgenic plant organ containing D2) the expression cassette;
    d7 A nucleic acid molecule that inhibits expression of a gene encoding the fpmicrocol protein;
    d8 A expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line comprising the nucleic acid molecule of D7);
    d9 A nucleic acid molecule that inhibits translation of an RNA molecule transcribed from a gene encoding the fpmicrocol protein;
    d10 Producing D9) an expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line of said nucleic acid molecule.
  5. 5. The use of the fpmicrocol protein of claim 1, the coding gene of claim 2 or the biomaterial of claim 4, characterized in that: the application is any one or more of the following 1) -4):
    1) Application in regulating and controlling growth rate of fusarium pseudograminearum hyphae
    2) Application in regulating and controlling the yield of fusarium pseudograminearum conidium;
    3) The application in regulating and controlling the pathogenicity of the fusarium pseudograminearum on a host;
    4) The application in inhibiting and/or killing fusarium graminearum.
  6. 6. The use of the fpmicrocol protein of claim 1, the coding gene of claim 2 or the biomaterial of claim 4, characterized in that: the application is any one or more of the following:
    1) Application in plant breeding;
    2) The application in cultivating disease-resistant plants.
  7. 7. Use of the fpmicrocol protein of claim 1, the coding gene of claim 2 or the biomaterial of claim 4 as a bacteriostatic or bacteriocidal target in the screening of products regulating the growth of fusarium pseudograminearum.
  8. 8. The use according to claim 7, wherein the product is a fusarium pseudograminearum bactericide or a fusarium pseudograminearum bacteriostatic agent.
  9. 9. A method of screening for a product that modulates the growth of fusarium pseudograminearum, the method comprising the step of applying an assay to said fusarium pseudograminearum, wherein said assay is determined to be a product that modulates the growth of fusarium pseudograminearum when said assay is capable of inhibiting transcription of the coding gene of claim 2, or inhibiting or inactivating the activity of the fpmicrocol protein of claim 1.
  10. 10. A method of reducing the activity of fusarium pseudograminearum comprising the steps of: inhibiting or deleting the transcription of the encoding gene of claim 2, or inhibiting or inactivating the activity of the fpmicrocol protein of claim 1;
    wherein, the reduction of the activity of the fusarium pseudograminearum is to reduce the pathogenicity of the fusarium pseudograminearum to a host, and/or reduce the hypha growth speed of the fusarium pseudograminearum, and/or reduce the yield of conidia of the fusarium pseudograminearum.
CN202311221430.1A 2023-09-21 2023-09-21 Fusarium pseudograminearum FpMCoL protein, and coding gene and application thereof Pending CN117304285A (en)

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