CN114480468B - Method for inhibiting growth of fusarium by constructing Sch9 gene mutant strain - Google Patents

Method for inhibiting growth of fusarium by constructing Sch9 gene mutant strain Download PDF

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CN114480468B
CN114480468B CN202111622212.XA CN202111622212A CN114480468B CN 114480468 B CN114480468 B CN 114480468B CN 202111622212 A CN202111622212 A CN 202111622212A CN 114480468 B CN114480468 B CN 114480468B
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sch9
gene
fusarium oxysporum
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fosch9
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CN114480468A (en
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孙力军
邓义佳
邓旗
房志家
王雅玲
王润东
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Guangdong Ocean University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3535—Organic compounds containing sulfur
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi

Abstract

The invention belongs to the technical field of food preservation, and discloses a method for inhibiting the growth of fusarium oxysporum by constructing a Sch9 gene mutant strain.

Description

Method for inhibiting growth of fusarium by constructing Sch9 gene mutant strain
Technical Field
The invention relates to the technical field of food preservation, in particular to a method for inhibiting growth of fusarium by constructing a Sch9 gene mutant strain.
Background
In fungal cells, TORC1 senses signals such as nutrients, growth factors, pressure, etc. in the environment to participate in the regulation of signal pathways such as protein translation, ribosome synthesis, gene transcription, etc., thereby regulating the growth and metabolism of fungi. The upstream response elements Gtr and Gtr2 bind as heterodimers and mediate multiple upstream signal pathways that regulate the physiological processes of cells via downstream effectors, while in the downstream arms, sch9 and Tap42 act as metabolic response elements that respond to signal factors transmitted by the upstream pathways and regulate the growth metabolism of Fusarium via downstream. However, the regulation of hyphal growth and secondary metabolite production mediated by the TORC1 pathway by the Gtr, gtr, sch9 and Tap42 single elements has not been reported.
Amino acids are used as activators of the TORC1 signaling pathway and have the effect of significantly regulating cell functions. Amino acids can be classified into acidic amino acids, basic amino acids, branched amino acids, sulfur-containing amino acids, and the like, depending on their nature. Under different kinds of amino acid activation, different response signals can be transmitted through the fungal TORC1 element, respond to specific amino acids, and are utilized for growth metabolism through synergistic absorption by downstream branches. However, how to regulate the growth and metabolism of Fusarium with different kinds of amino acids has not been studied intensively.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention firstly provides the application of the amino acid in inhibiting the growth of fusarium oxysporum.
A second object of the present invention is to provide a method for inhibiting growth of Fusarium by constructing a Sch9 gene mutant strain.
A third object of the present invention is to provide the use of the above method for preserving food products.
The aim of the invention is achieved by the following technical scheme:
use of an amino acid selected from two or more of L-Arg, L-Asp, L-Glu, L-Cys, L-Met, L-Tyr, for inhibiting the growth of fusarium oxysporum.
The invention also provides a method for inhibiting growth of fusarium oxysporum by constructing a Sch9 gene mutant strain, which comprises the following steps:
s1, respectively constructing fusarium oxysporum gene knockout strain delta FoSch9 and fusarium oxysporum gene complementation strain delta FoSch9-C;
s2, adding amino acids to wild fusarium oxysporum strain, gene knockout strain delta FoSch9 and gene complement strain delta FoSch9-C, wherein the amino acids are selected from two or more of L-Arg, L-Asp, L-Glu, L-Cys, L-Met and L-Tyr.
Preferably, the construction method of the fusarium oxysporum gene knockout strain delta FoSch9 in S1 comprises the following steps:
(1) Designing primers for amplifying left and right homology arms of the Sch9 gene, and respectively amplifying the left and right homology arms of the Sch9 gene by using wild type bacteria DNA of Fusarium oxysporum as a template;
(2) The purified left and right homology arm PCR products are respectively connected to the front and rear multiple cloning sites of hygromycin resistance gene of a gene knockout vector pBluescript KS (+ -) (pBS) -HPH1 after enzyme digestion, so as to obtain a recombinant gene knockout vector pPBS-HPH-Sch9-5'3';
(3) Amplifying the recombinant vector pPBS-HPH-Sch9-5'3' serving as a template by using primers Sch9-A1 and Sch9-A4 to obtain a knockout large fragment HPH-Sch9-5'3' with the length of about 3200 bp;
(4) The knockout structure of Sch9 was transformed into Fusarium oxysporum protoplast cells by protoplast transformation. After transformation, protoplasts were screened by hygromycin HYG plates and cultured at 25℃until transformants developed.
Preferably, the construction method of the fusarium oxysporum gene anaplerotic strain delta FoSch9-C in S1 comprises the following steps:
(1) And amplifying the Sch9 gene by using a recombinant T vector connected with the Sch9 gene as a template and using a primer for constructing a compensation vector. After the PCR product is purified, in-HD Cloning Kit is connected to corresponding binary vector pCAMBIA1300-neo after double enzyme digestion, and sequencing is carried out for verification;
(2) Transformation of the Gene-complementing vector into Agrobacterium: adding 1 mug of plasmid DNA into agrobacterium, performing coating plate culture, picking up monoclonal colonies, extracting plasmids, and performing PCR amplification identification;
(3) Uniformly mixing the agrobacterium solution and the fungus spores in proportion, coating on a nitrocellulose membrane on an IM flat plate, and culturing for 3d at 25 ℃;
(4) Transferring the agrobacterium and fungal spore co-culture to a fungus screening culture medium, culturing at 30 ℃ for 3d in dark until colonies appear, transferring and performing monospore culture;
(5) The cultured colonies were subjected to extraction of genomic DNA of the strain to be verified using AxyPrep genomic DNA miniprep kit. PCR amplification was performed with primers NEO-F and NEO-R, and sequencing was performed.
Preferably, the inhibition of fusarium oxysporum growth refers to inhibition of at least one of colony growth, spore-forming ability and toxin-forming ability.
Preferably, the inhibition of fusarium oxysporum growth refers to simultaneous inhibition of colony growth, spore-forming ability and toxin-forming ability; the amino acid is L-Asp.
The invention also provides application of the method in food preservation. Those skilled in the art know that Fusarium oxysporum is widely existed in aquatic dry products such as dried fish and dried shrimp and the like, and causes a certain degree of harm to the aquatic dry products, so that the method can be used for preventing and controlling the outbreak of Fusarium oxysporum in the aquatic dry products.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, through a molecular biological method, a gene knockout strain delta FoSch9 and a gene complementing strain delta FoSch9-C are constructed, different types of amino acids are added into a wild strain and the constructed gene knockout strain delta FoSch9 and the constructed gene complementing strain delta FoSch9-C, and it is found that some specific types of amino acids can obviously inhibit the growth of fusarium oxysporum, so that the growth of fusarium oxysporum can be obviously inhibited by constructing the gene knockout strain delta FoSch9 and the gene complementing strain delta FoSch9-C in advance, and by adding some specific types of amino acids, the growth of fusarium oxysporum is obviously inhibited, and the method is used for preserving foods in the future.
Drawings
FIG. 1 is a schematic diagram of the structure of gene knockout constructed from the gene knockout vector pBluescript KS (+ -) (pBS) -HPH 1;
FIG. 2 is a PCR verification of amplified HYG gene in Sch9 knockout transformant (a) and Sch9 knockout transformant Sch9-9 (b); (a) M: DNA Markers;1. amplifying HYG gene by taking transformant Sch9-9 hypha DNA as a template; (b) M: labeling DNA molecules; 1-2, PCR amplification is carried out by using Sch9 knock-out transformant Sch9-9 hypha DNA as a template and primers A1 and A4; 3-4, PCR amplification is carried out by using Sch9 knock-out transformant Sch9-9 hypha DNA as a template and using primers A5 and A6; 5-6, PCR amplification is carried out by using Sch9-9 hypha DNA of Sch9 knockout transformant as template and primers A1 and HY; 7-8, PCR amplification is carried out by using Sch9 knock-out transformant Sch9-9 hypha DNA as a template and primers A4 and YG;
FIG. 3 PCR-validation M for Fusarium oxysporum Sch9 gene amplification: labeling DNA molecules; 3: PCR verification of Sch9 gene; all genes include a promoter region at the 5 'end and a terminator region at the 3' end;
FIG. 4 is a PCR validation of Fusarium oxysporum Sch9 gene knockout complement; m is DNA molecular marker; PCR amplification of NEO Gene (1-8) and HYG Gene (9-16) Using Fusarium oxysporum hypha DNA as templates [1: performing compensation verification on delta FoGtr 1; 2: performing compensation verification on the delta FoSch9 gene; 3: performing compensation verification of delta FoTap 42; (IM-AS, agrobacterium and Fusarium oxysporum co-culture without AS) ] [4: performing compensation verification on delta FoGtr 1; 5: performing compensation verification on delta FoSch 9; 6: performing compensation verification of delta FoTap 42; fusarium oxysporum for transformation of pCAMBIA 1300-NEO; (im+as, addition of AS during co-cultivation of agrobacterium and fusarium oxysporum) ]8: NEO gene PCR positive control [9: performing compensation verification on delta FoGtr 1; 10: performing compensation verification on delta FoSch 9; (IM-AS, agrobacterium and Fusarium oxysporum co-culture without AS) ] [11: performing compensation verification of delta FoTap 42; 12. performing compensation verification on ΔFoGtr 1; 13: performing compensation verification on delta FoSch 9; 14: performing compensation verification of delta FoTap 42; 15: fusarium oxysporum transformed into pCAMBIA 1300-NEO; (IM+AS, addition of AS during co-cultivation of Agrobacterium and Fusarium oxysporum) ];16: HYG PCR positive control;
FIG. 5 shows colony morphology of Fusarium oxysporum wild type strains Fo17, ΔFoSch9 and ΔFoSch9-C on PDA medium supplemented with 18 amino acids;
FIG. 6 shows the sporulation amounts of the Fo17 wild strain and the Sch9 gene-deleted and anaplerotic strain in CDA liquid medium (note: based on the sporulation amount of Fo17 in CDA medium, single-factor analysis of variance was performed, and the result was marked with p <0.05 #, and p <0.01 #);
FIG. 7 is a heat map of the sporogenesis rules of the Fo17 wild strain, the Sch9 knockout and anaplerotic strain in CDA liquid medium to which specific amino acids are added respectively;
FIG. 8 shows the amount of T-2 toxin produced by wild-type strain Fo17 after 14d culture in GYM medium supplemented with 18 amino acids, respectively;
FIG. 9 shows the amounts of T-2 toxin produced after culturing DeltaFoSch 9 and DeltaFoSch 9-C in GYM medium supplemented with 18 amino acids respectively for 14d (note: significant difference analysis was performed on the basis of the amounts of T-2 toxin produced in GYM medium by DeltaFoSch 9 and DeltaFoSch 9-C, p <0.05 is marked #);
FIG. 10 is a heat map of T-2 toxin production rules of Fo17 wild strain, sch9 gene knockout and anaplerotic strain in CDA liquid medium supplemented with specific amino acids;
FIG. 11 shows the regulation of amino acids on growth, spore production and T-2 toxin production by Fusarium oxysporum, which is deleted from the TORC1-Sch9 gene.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Test strain: fusarium oxysporum wild strain, gene knockout strain delta FoSch9 and gene complement strain delta Foch9-C, and the construction of the gene knockout strain and the gene complement strain belong to conventional genetic engineering and molecular biology technologies.
The information on the preservation of wild Fusarium oxysporum strain shows that: deposit No. GDMCC No:60824, taxonomic name Fusarium oxysporum Fo, date of deposit: 10.23 days 2019, deposit unit: the collection address of the microorganism strain collection in Guangdong province: building 5 of No. 59 of Qinghui No. 100 university in Guangzhou City of Guangdong.
Experimental data are expressed as Mean ± standard deviation (Mean ± SD), and colony growth diameter, spore yield, and T-2 production were analyzed by SPSS 19.0 as having significant levels of variance defined by p < 0.05.
Example 1 construction of Gene knockout Strain DeltaFoSch 9 and Gene anaplerotic Strain DeltaFoSch 9-C
1. Construction of Fusarium oxysporum Sch9 Gene knockout Strain
1. Small amount of genomic DNA extraction by steel ball method: about 0.1g of fresh mycelium is picked up on a flat plate, placed in a 2mL centrifuge tube, 600 mu L of cell lysate is added into the centrifuge tube filled with mycelium, 2 grinding balls are added, and the 2mL centrifuge tube is placed in a sample grinder and oscillated for 1min at a frequency of 60 Hz. After the ground sample was allowed to stand for 10min, it was centrifuged at 13,200rpm at 4℃for 10min. The supernatant was pipetted into a new centrifuge tube, 2.5 volumes of absolute ethanol were added, mixed slowly upside down, and centrifuged at 12,000rpm for 15min at 4 ℃. The supernatant was discarded and the precipitate was washed with 70% ethanol. The precipitate was dried and then treated with 100. Mu.L of ddH 2 O is dissolved.
2. Extraction of Fusarium oxysporum DNA by CTAB method
Freshly cultured Fusarium oxysporum pieces were inoculated into 200mL of PDB medium and cultured at 25℃at 180rpm/min for 24h. Filtering mycelium with gauze after culturing, washing mycelium with sterilized distilled water for 3 times, wiping off mycelium water with absorbent paper, adding liquid nitrogen, and grinding into fine powder. Then adding 10mL of CTAB extract preheated at 65 ℃, slightly shaking, heating in a water bath at 65 ℃ for 1h, centrifuging at 9,000rpm for 10min at 4 ℃ after the completion, taking supernatant, adding an equal volume of phenol/chloroform/isoamyl alcohol (v: v=25:24:1) into a centrifuge tube, centrifuging at 12,000rpm for 10min after shaking and mixing, taking supernatant, adding 2 times of pre-cooled absolute ethyl alcohol at-20 ℃, standing for 2h after mixing, centrifuging at 1,000rpm for 10min, removing supernatant, washing 5 times by using 10mL of 70% ethyl alcohol, centrifuging at 1,000rpm for 5min after washing, retaining precipitate and drying. After drying, 400. Mu.L of double distilled aqueous DNA was added and 1. Mu.L of 10mg/mL RNase was added to the culture at 37℃for 2 hours.
3. PCR amplification
PCR reaction system:
PCR reaction conditions: denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 5s/kb,35 cycles; extending at 72deg.C for 5min, and storing at 4deg.C.
4. DNA electrophoresis, PCR product purification and DNA enzyme digestion: conventional means are employed.
5. Ligation of DNA fragments with vectors
The DNA fragment was ligated with the vector using T4 DNA Ligase.
The connection system is as follows:
10×ligation buffe 2μL；
vector DNA 50ng;
insert DNA fragment (molar ratio to vector DNA about 3);
T4 DNA Ligase(350U/μL) 1μL；
ddH 2 O add to 50μL；
connection conditions: 16 ℃ for 2h.
6. The plasmid DNA is transformed into competent cells of escherichia coli (E.coil DH 5 alpha), and the plasmid DNA is extracted by adopting a conventional means; the preparation of conidium and protoplast are all conventional methods;
7. fusarium oxysporum transformation: 200. Mu.L of protoplast cells to be transformed are added to a 1.5mL centrifuge tube pre-cooled on ice, 100. Mu.L of DNA sample, 10. Mu.L of 5mg/mL heparin sodium and 250. Mu.L of SPTC solution are added, and the mixture is thoroughly sucked and stirred uniformly, and the mixture is placed on ice in a dark place for 30min. Then 400. Mu.L of SPTC solution was added dropwise to the mixture and mixed well and left at room temperature for 20min in the absence of light. The mixture was added to a triangle flask containing 25ml of TB3 liquid medium, mixed slowly and placed in the dark for 30min. The flasks were then incubated overnight at 100rpm in a 25℃shaker. The next day the mycelium was added to PDA medium and 100. Mu.g/mL hygromycin was added and incubated at 25℃until transformants developed.
8. Determination of the nucleotide sequence of the coding region of the Sch9 Gene: the nucleotide sequence of the Sch9 (P11792) gene is downloaded from the website https:// blast.ncbi.nih.gov/blast.cgi, and is compared, and primers Sch9_5F and Sch9_3R for amplifying the gene are designed according to the comparison result. The genomic DNA extracted from Fusarium oxysporum is used as a template, and the coding frame region of the gene is amplified by PCR. The PCR product is subjected to electrophoresis, gel cutting and purification, and is connected with a T carrier for sequencing. The sequencing results were aligned with the relevant sequences and used as reference sequences in the design of subsequent use in gene knockout primers.
9. Construction method of Sch9 gene knockout structure
Primers Sch9-A1 (SacII restriction enzyme site is added at the 5 'end) and Sch9-A2 (BamHI restriction enzyme site is added at the 5' end), sch9-A3 (SalI restriction enzyme site is added at the 5 'end) and Sch9-A4 (KpnI restriction enzyme site is added at the 5' end) for amplifying the left and right homology arms of the Sch9 gene are respectively designed, fusarium oxysporum wild type bacteria DNA is used as a template, the left and right homology arms of the Sch9 gene are respectively amplified by PCR, and the purified PCR products of the left and right homology arms are respectively connected to the polyclonal sites before and after the hygromycin resistance gene of the gene knockout vector pBluescript KS (+ -) (pBS) -HPH1 to obtain a recombinant gene knockout vector pPBS-HPH-Sch9-5'3'. The recombinant vector pPBS-HPH-Sch9-5'3' is used as a template, and primers Sch9-A1 and Sch9-A4 are used for amplification to obtain a knockout large fragment HPH-Sch9-5'3' with the length of about 3200 bp. The schematic of the gene knockout structure constructed using the gene knockout vector pBluescript KS (+ -) (pBS) -HPH1 is shown in FIG. 1.
10. Fusarium oxysporum transformation of Sch9 gene knockout structure
Referring to the transformation method of Fusarium oxysporum protoplast, the knockout large fragment HPH-Sch9-5'3' of the Sch9 gene was transformed into Fusarium oxysporum protoplast cells. The transformed protoplast is spread on a plate containing hygromycin HYG for screening, and the transformant is cultured at 25 ℃ until the transformant grows out.
11. Verification of Sch9 knockout transformants
Mycelium is picked from colonies growing on a screening plate of the Sch9 gene knockout transformant, mycelium genome DNA is extracted, and the mycelium DNA is used as a template for PCR amplification of HYG genes. If HYG can be successfully amplified, PCR amplification is continued with primers Sch9-A1 and Sch9-A4, sch9-A5 and Sch9-A6, sch9-A1 and HY, sch9-A4 and YG to amplify Sch9-5'-HY and Sch9-3' -YG, respectively, with four pairs of primers each amplifying DNA bands of the desired size. However, sch9-A1 and Sch9-A4, sch9-A1 and HY have weaker band intensities. The PCR products amplified with Sch9-A5 and Sch9-A6, and Sch9-A4 and YG were excised and sequenced. Sequencing results are shown in appendix 5: sch9-A5A6 and Sch9-A4YG. Sequencing results showed that the Sch9 gene was successfully knocked out in the verified strain (FIG. 2).
2. Anaplerosis of fusarium oxysporum Sch9 gene knockout strain
1. Amplification of the complement Gene sequence
The full-length sequence of the gene to be complemented (comprising a promoter region at the 5 'end and a terminator region at the 3' end of the gene) is amplified, and the reference sequence for gene amplification is obtained by homologous searching of the gene of Fusarium oxysporum Fusarium oxysporium (http:// www.broadinstitute.org/animation/genome/fusarium_group/multisome. HtmL). The primer sequences required for gene amplification are shown in Table 1. The full-length sequence of the Sch9 gene was amplified using the corresponding primers with wild Fusarium oxysporum genomic DNA as a template. The PCR products were purified and ligated into T vector for sequencing.
TABLE 1
As shown in FIG. 3, the result of PCR amplification revealed that the full-length sequence of the Sch9 gene was about 7000 bp.
2. Construction of a gene complement vector:
and respectively amplifying the genes by using the recombinant T vectors connected with the full-length genes as templates and using the primers for constructing the make-up vectors. After the PCR product is purified, the PCR product is utilizedThe HD Cloning Kit was ligated to the corresponding double digested binary vector pCAMBIA1300-neo to give the complementing vector pCA-FoSch9-C.
The construction of the primers for the make-up vector was done using on-line software (http:// www.takarabio.com/US/Products/cloning_and_components_cells/selection_guides/online_in-fusion_tools). The correctness of the connection is verified by sequencing.
3. The transformation of the gene anaplerotic vector into agrobacterium comprises the following steps:
(1) One tube of Agrobacterium competent cells (200. Mu.L) was taken and placed on ice for thawing, 1. Mu.g of plasmid DNA was added, gently mixed and placed on ice for 30min.
(2) Then quick-freezing in liquid nitrogen for l min, and water-bathing at 37 ℃ for 5min.
(3) 400. Mu.L of LB liquid medium (without antibiotics) was added and the culture was continued at 28℃for 2 days with shaking until single colonies were grown. Monoclonal colonies were picked, plasmids were extracted and identified by PCR amplification.
4. Agrobacterium-mediated transformation of Fusarium oxysporum
(1) Preparation of fungal spores: picking up newAdding fresh cultured Fusarium graminearum limbic mass into 150mLCMC spore culture solution, culturing at 180rpm in 25 deg.C shaking table for 6d, filtering the cultured bacterial solution with 2 layers of gauze, washing the gauze with a small amount of sterile water, collecting filtrate, centrifuging at 4 deg.C for 10min at 2,500×g, and collecting spores. Suspending spores in IM liquid medium to reach spore concentration of 1×10 7 CFU/mL。
(2) Preparation of agrobacterium:
a. streaking the agrobacterium with binary vector on LB plate (containing Kan 50 μg/mL, rif 50 μg/mL), culturing at 28 ℃ for 3 days to obtain agrobacterium monoclonal; b. one Agrobacterium was picked up and inoculated into 10mL of liquid LB medium (containing Kan 50. Mu.g/mL, rif 50. Mu.g/mL), and cultured at 28℃and 250rpm for 24 hours; c. 1.5mL of the Agrobacterium solution was taken and centrifuged at 2,400g for 10min at room temperature. Discarding the supernatant, slowly suspending and precipitating with 250 μl of liquid IM culture medium, centrifuging at room temperature for 10min with 2,400g, and discarding the supernatant; d. the pellet was suspended in 5mL of liquid IM medium (containing 200. Mu.M AS), and the bacterial liquid was transferred to a 100mL Erlenmeyer flask, and cultured at 28℃and 100rpm for 5 hours, so that the OD 600nm of the bacterial liquid was adjusted to 0.8.
5. Co-cultivation of Agrobacterium-fungi
100. Mu.L of the above Agrobacterium solution and 100. Mu.L of fungal spores (concentration about 1X 10) 7 CFU/mL) was mixed at a ratio of 1:1. Using sterilized forceps, 0.45. Mu.M nitrocellulose membrane was placed on a 15cm diameter IM plate (containing 200. Mu.M AS), 200. Mu.L of the mixture of Agrobacterium and fungal spores was aspirated, and the mixture was applied to the nitrocellulose membrane on the plate and spread with a glass coater. The plates were incubated at 25℃for 3d.
6. Screening of transformants
Nitrocellulose membranes with co-cultures of Agrobacterium and fungal spores were transferred to fungal screening medium (PDA medium containing Kan 50. Mu.M, cef 200. Mu.M, geneticin 100. Mu.M) using sterilized forceps. Culturing for 3d at 30 ℃ under dark condition until colonies appear. The transformant was transferred to a selection medium again, and subjected to monospore culture.
7. Verification of anaplerotic Strain
The genomic DNA of the strain to be verified was extracted using the AxyPrep genomic DNA miniprep kit. PCR amplification was performed with primers NEO-F and NEO-R.
The PCR verification system is as follows:
the repayment vector pCA-FoSch9-C is transferred into competent cells of agrobacterium Agrobacterium tumefaciens C C1, and then transferred into Sch9 gene knockout fusarium oxysporum by an agrobacterium-mediated transformation method. The strain is subjected to geneticin resistance culture medium screening and PCR verification, and the No. 2 band PCR verification is successful, so that the Sch9 gene strain is finally obtained (figure 4).
EXAMPLE 2 Effect of amino acid addition on Fusarium colony growth
PDA medium containing 5mg/mL of specific amino acid (sAA) was prepared, and 10. Mu.g/mL of hygromycin was added to the fed-back strain medium. PDA culture medium was used as control group, and 0.5mmol/L rapamycin was added to PDA culture medium to set as positive control group. After the culture medium is sterilized and cooled at 121 ℃, 5mm fungus cakes are respectively made on a Sch9 gene knockout strain and a anaplerotic strain plate of the Fo17 wild strain, the culture medium is inoculated to an sAA-PDA culture medium, and the culture medium is cultured for 7d at 28 ℃, and colonies are observed through photographing.
1. Effect of different amino acids on the growth of DeltaFoSch 9 and DeltaFoSch 9-C colonies
As can be seen from FIG. 5, in PDA medium, ΔFoSch9 grows normally with red pigment in the middle, sch9 gene complements and then grows normally, similar to the wild strain. After L-Arg, L-Iso, L-Leu, L-Lys, L-Phe, L-Pro, L-Thr and L-Tyr are added, the delta FoSch9 is obviously inhibited from growing compared with a control group, mycelia are smaller and the edge development is incomplete, aerial mycelia are reduced, and after gene anaplerosis, the growth of the mycelia is recovered to be complete under the addition of L-Pro and L-Iso. In comparison with the wild strain, delta FoSch9 and delta FoSch9-C growth was significantly inhibited after addition of L-Cys, L-Met, L-Asp and L-Glu, aerial hyphae were reduced and mycelia were smaller.
2. Analysis of Fusarium colony growth Rate by amino acids
Based on the Fo17 wild strain, the TORC1 gene-deleted strain and the complemented strain (100%) in PDA medium, the colony growth rate in PDA medium with different amino acids was calculated, and the results showed that: the growth rates of Fo17 in the medium added with L-Arg, L-Cys, L-Glu and L-Iso are 80.0%, 44.3%, 49.3% and 72.8%, respectively, which shows that the growth of the Fo17 is significantly inhibited. The growth rate of delta FoSch9 in the L-Iso medium is only 59.4%, and the growth rate of delta FoSch9-C is 90.3%, which indicates that the absorption of L-Iso by Fusarium is regulated by the Sch9 gene.
The growth rate of the Fo17 and Sch9 gene mutants and the anaplerotic strain in the L-Met culture medium is lower than 80%, which shows that the L-Met obviously inhibits the growth of Fusarium oxysporum and is not regulated by a target gene. The growth rate of FoSch9 in the medium added with L-Phe and L-Pro is only 63.0% and 60.9%, and the growth rate is obviously increased after gene complementation, which indicates that the Sch9 gene can regulate the absorption effect of Fusarium on the two amino acids.
Example 3 influence of the critical gene-mediated amino acids of TORC1 on fusarium spores
1. Influence on spore production and spore morphology in CDA liquid Medium
Preparing sAA-CDA culture media, wherein each culture medium contains 10mg/mL of specific amino acid, and inoculating the gene deletion strain and the anaplerotic strain into the specific 10mL of sAA-CDA culture medium respectively, and performing shake culture at 28 ℃ for 7d. After the culture is finished, the spores are uniformly mixed and smashed by a magnetic stirrer at 500rpm/min, and three layers of gauze are used for filtration. 50 μl of the filtrate was used to determine the spore count under a microscope using a hemocytometer.
As can be seen from FIG. 6, the yield of Fo17 wild strain in CDA liquid medium was 12.3X10 5 CFU/mL, ΔFoSch9 sporulation was significantly reduced compared to the wild strain (p<0.05 And the normal state is recovered after gene anaplerosis.
The sporulation amount of the Fo17 wild strain on CDA liquid culture medium is 12.3X10 5 CFU/mL, after L-Asp, L-Cys, gly, L-His and L-Leu are added into the culture medium, the spore yield of Fo17 is obviously reduced to 2.3-7.6X10 5 CFU/mL, manifests as negative amino acid sporulation inhibition effects. L-His significantly activated the sporulation of ΔFoSch9 in CDA medium and decreased to normal levels after gene complementation.
2. Law of Fusarium spore production in CDA liquid culture medium
As can be seen from the cluster analysis heat map of FIG. 7, the Fo17 wild strain and the Sch9 gene knockout and anaplerotic strain have significantly lower spore yield in CDA medium added with L-Cys, L-Leu, L-Val, L-Try, L-Ala and Gly, indicating that the 6 amino acids have an inhibiting effect on the spore-producing ability of Fusarium. And with the addition of L-Glu, L-Phe, L-Tyr, L-Met, L-Arg and L-Lys, the spore yield of the strain is higher.
Example 4 influence of the critical gene-mediated amino acids of TORC1 on fusarium production of T-2 toxin
Preparing GYM culture medium containing 5mg/mL of specific amino acid, adding 1mL of sAA-GYM culture medium solution into a 2mL centrifuge tube, respectively beating 25 mm fungus cakes of different types of fusarium, inoculating into a liquid culture medium, performing shake culture at 28 ℃ for 15d at 120r/min, centrifuging the culture solution at 5,000rpm for 15min after the culture is finished, adding equal amount of ethyl acetate into the supernatant, performing shake for 3min, collecting the supernatant, repeating the operation for 3 times, combining the supernatants, adding 1mL of 30% methanol solution for re-dissolution after nitrogen blow-drying at 60 ℃, performing shake until the bottom sediment is dissolved, filtering by a 0.22 mu m filter membrane, and determining the T-2 content by an LC-MS/MS method.
1. Effect of amino acid addition on T-2 production by wild strain Fo17
As can be seen from FIG. 8, the wild-type strain Fo17 produced 22.8ng/mL of T-2 in GYM medium, and the toxicity was significantly reduced by adding L-Asp and L-Glu, which were only 14.8ng/mL and 12.6ng/mL (p < 0.05). The amount of T-2 produced after the addition of L-Cys, L-Phe and L-Pro was significantly increased to 33.6, 30.5 and 31.0ng/mL, respectively. Fusarium T-2 production in GYM medium with other amino acid addition was not significantly different from control (p > 0.05).
2. Effect of amino acid addition on T-2 toxin production by DeltaFoSch 9 and DeltaFoSch 9-C
As can be seen from FIG. 9, the amount of T-2 produced in GYM medium by DeltaFoSch 9 was 18.1ng/mL, and the amount of toxin produced increased to 25.1ng/mL after the gene of Sch9 was complemented. The delta FoSch9 produced significantly reduced (p < 0.05) in the GYM medium supplemented with L-Asp, L-Glu, and slightly increased after gene supplementation, but still below the control level. Delta FoSch9 significantly increased the T-2 production (p < 0.05) in GYM medium supplemented with L-His, L-Iso, L-Met, L-Phe, L-Pro and L-Try to 22.4-30.5 ng/mL, and restored the control levels after gene supplementation. Delta FoSch9-C produced significantly reduced amounts of T-2 in L-Tyr medium (p < 0.05).
3. Amino acid addition to regulate and control Fusarium toxicity production law
As can be seen from FIG. 10, the Fo17 wild strain, the Sch9 knockout and anaplerotic strain showed a significant decrease in the toxicity production in the L-Asp and L-Glu added GYM medium, indicating that these two amino acids significantly inhibited the toxicity production capacity of Fusarium.
EXAMPLE 5 growth and metabolism of Fusarium with amino acids mediated by the critical gene for TORC1
As can be seen from FIG. 11, only L-Arg and L-Tyr showed negative regulation of Fusarium growth after Sch9 gene knockout, except L-Asp, L-Glu, L-Met and L-Cys which had a general negative regulation effect. In contrast, most of the amino acids have remarkable positive regulation effect on spore production and toxigenic capacity of delta FoSch9, and the amino acids with coactivation of the two mainly comprise L-Met, L-Asp, L-Iso, L-Pro, L-His, L-Tyr, L-Arg, L-Thr and L-Lys.
Sequence listing
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Claims (2)

1. A method for inhibiting the growth of fusarium oxysporum by constructing a Sch9 gene mutant strain, comprising the steps of:
s1, respectively constructing fusarium oxysporum gene knockout strain delta FoSch9 and fusarium oxysporum gene complementation strain delta FoSch9-C;
s2, adding amino acid to wild fusarium oxysporum strain, fusarium oxysporum gene knockout strain delta FoSch9 and fusarium oxysporum gene complementation strain delta FoSch9-C, wherein the amino acid is one of L-Arg, L-Asp, L-Glu, L-Cys, L-Met or L-Tyr;
the construction method of the fusarium oxysporum gene knockout strain delta FoSch9 comprises the following steps:
(1) Designing primers Sch9-A1, sch9-A2, sch9-A3 and Sch9-A4 for amplifying left and right homology arms of the Sch9 gene, and respectively amplifying the left and right homology arms of the Sch9 gene by using the wild strain DNA of Fusarium oxysporum as a template;
(2) The purified left and right homology arm PCR products are respectively connected to the front and rear multiple cloning sites of hygromycin resistance gene of a gene knockout vector pBluescript KS (+ -) (pBS) -HPH1 after enzyme digestion, so as to obtain a recombinant gene knockout vector pPBS-HPH-Sch9-5'3';
(3) Amplifying the recombinant vector pPBS-HPH-Sch9-5'3' serving as a template by using primers Sch9-A1 and Sch9-A4 to obtain a knocked-out large fragment HPH-Sch9-5'3';
(4) Transforming protoplast cells of a wild strain of Fusarium oxysporum by using a knock-out structure of Sch9 through protoplast transformation, screening the transformed protoplasts by using a hygromycin HYG plate, and culturing at 25 ℃ until transformants grow out;
the fusarium oxysporum gene anaplerotic strain delta FoSch9-C is obtained by transferring anaplerotic vector pCA-FoSch9-C into agrobacterium Agrobacterium tumefaciens C C1 competent cells and transferring the agrobacterium-mediated transformation method into a fusarium oxysporum gene knockout strain delta FoSch9 with a Sch9 gene knockout, and the construction method of the fusarium oxysporum gene anaplerotic strain delta FoSch9-C comprises the following steps:
(1) The recombinant T vector connected with the Sch9 gene is used as a template, the Sch9 gene is amplified by utilizing primers Sch9-infu-5F and Sch9-infu-3R constructing a repair vector, and after the PCR product is purified, the PCR product is utilizedHD Cloning Kit was ligated to the corresponding double digested fragmentsOn the binary vector pCAMBIA1300-neo, a complementing vector pCA-FoSch9-C is obtained and sequenced for verification, and the sequences of the sch9-infu-5F and the sch9-infu-3R are respectively shown as SEQ ID NO:3 and SEQ ID NO:4 is shown in the figure;
(2) Transformation of the Gene-complementing vector into Agrobacterium: adding 1 mug of plasmid DNA into agrobacterium, performing coating plate culture, picking up monoclonal colonies, extracting plasmids, and performing PCR amplification identification;
(3) Uniformly mixing the agrobacterium solution and the fungus spores in proportion, coating on a nitrocellulose membrane on an IM flat plate, and culturing for 3d at 25 ℃;
(4) Transferring the agrobacterium and fungal spore co-culture to a fungus screening culture medium, culturing for 3d at 30 ℃ in dark until colonies appear, transferring and performing monospore culture;
(5) Extracting genome DNA of the strain to be verified from the cultured colony, carrying out PCR amplification by using primers NEO-F and NEO-R, and carrying out sequencing verification; the sequences of the primers NEO-F and NEO-R are shown in SEQ ID NO:5 and SEQ ID NO:6 is shown in the figure;
the inhibition of fusarium oxysporum growth refers to inhibition of colony growth; the preservation number of the fusarium oxysporum wild strain is GDMCCNo:60824, taxonomic name Fusarium oxysporum Fo, date of deposit 2019, 10 month 23, and collection unit, collection of microorganisms and strains of Guangdong province.
2. Use of the method of claim 1 for preserving food products.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2018213464A2 (en) * 2017-05-16 2018-11-22 Children's Medical Center Corporation Inhibiting the fungal cell-surface phosphate transporter pho84
CN110004184A (en) * 2019-04-16 2019-07-12 中国热带农业科学院环境与植物保护研究所 A kind of construction method of banana blight bacteria myosin-1 knock out mutants body

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Publication number Priority date Publication date Assignee Title
WO2018213464A2 (en) * 2017-05-16 2018-11-22 Children's Medical Center Corporation Inhibiting the fungal cell-surface phosphate transporter pho84
CN110004184A (en) * 2019-04-16 2019-07-12 中国热带农业科学院环境与植物保护研究所 A kind of construction method of banana blight bacteria myosin-1 knock out mutants body

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红鱼干中游离氨基酸介导TORC1信号通路对镰孢菌生长及T-2毒素合成的调控机制;邓义佳;《中国博士学位论文全文数据库 工程科技Ⅰ辑》(第02期);第B024-50页 *

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