CN117721136A - Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt - Google Patents
Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt Download PDFInfo
- Publication number
- CN117721136A CN117721136A CN202311667882.2A CN202311667882A CN117721136A CN 117721136 A CN117721136 A CN 117721136A CN 202311667882 A CN202311667882 A CN 202311667882A CN 117721136 A CN117721136 A CN 117721136A
- Authority
- CN
- China
- Prior art keywords
- fobut
- protein
- banana
- fusarium wilt
- pathogenicity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 141
- 235000018290 Musa x paradisiaca Nutrition 0.000 title claims abstract description 113
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 241000223218 Fusarium Species 0.000 title claims abstract description 70
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 69
- 230000007918 pathogenicity Effects 0.000 title claims abstract description 45
- 230000001276 controlling effect Effects 0.000 title claims abstract description 14
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 8
- 240000005561 Musa balbisiana Species 0.000 title 1
- 241000234295 Musa Species 0.000 claims abstract description 112
- 230000002792 vascular Effects 0.000 claims description 30
- 238000002360 preparation method Methods 0.000 claims description 29
- 230000002401 inhibitory effect Effects 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 9
- 230000000903 blocking effect Effects 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 abstract description 17
- 238000004458 analytical method Methods 0.000 abstract description 16
- 210000001938 protoplast Anatomy 0.000 abstract description 13
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000012217 deletion Methods 0.000 abstract description 4
- 230000037430 deletion Effects 0.000 abstract description 4
- 235000018102 proteins Nutrition 0.000 description 38
- 239000012634 fragment Substances 0.000 description 32
- 239000013598 vector Substances 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 17
- 201000010099 disease Diseases 0.000 description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 16
- 239000000047 product Substances 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000012010 growth Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000007619 statistical method Methods 0.000 description 8
- 108010006654 Bleomycin Proteins 0.000 description 7
- 108020000949 Fungal DNA Proteins 0.000 description 7
- 229960001561 bleomycin Drugs 0.000 description 7
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 7
- 238000003209 gene knockout Methods 0.000 description 7
- 238000010200 validation analysis Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 5
- 240000000905 Nymphoides indica Species 0.000 description 5
- 235000017590 Nymphoides indica Nutrition 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000000600 sorbitol Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 230000035784 germination Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 238000004383 yellowing Methods 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 101150029559 hph gene Proteins 0.000 description 2
- 108700016226 indium-bleomycin Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 101710159648 Uncharacterized protein Proteins 0.000 description 1
- 101710100170 Unknown protein Proteins 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229940089206 anhydrous dextrose Drugs 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000007376 cm-medium Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000006081 fluorescent whitening agent Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010002685 hygromycin-B kinase Proteins 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000012966 insertion method Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt. The invention obtains the deletion mutant and the anaplerotic mutant of the Fobut by constructing knockout plasmid and anaplerotic plasmid of the gene (Fobut) encoding the Fobut of the banana fusarium wilt protein and respectively converting corresponding protoplast. Through observing the phenotypes of the deletion mutant and the anaplerotic mutant and carrying out pathogenicity analysis, the knock-out gene Fobut can obviously reduce the pathogenicity of banana fusarium wilt, and the pathogenicity of the anaplerotic gene Fobut is recovered after anaplerotic, which shows that the protein Fobut is related to the pathogenicity of the banana fusarium wilt and can be used as a target point for preventing and treating the banana fusarium wilt. The invention enriches protein databases related to pathogenicity of banana fusarium wilt and is beneficial to preventing and treating banana fusarium wilt.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering. More particularly relates to the application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt.
Background
Banana wilt (Banana Fusarium wilt) is a soil-borne vascular bundle fungal disease caused by banana wilt bacteria (Fusarium oxysporum f.sp.cube, foc) which can lead to banana yield reduction and even failure, severely restricting the development of banana industry. At present, no method for effectively preventing and treating banana vascular wilt exists.
Research on banana fusarium wilt pathogens and pathogenic mechanisms thereof is carried out, and searching for the banana fusarium wilt pathogens which can be used as a target point for preventing and controlling banana fusarium wilt is a key for effectively preventing and controlling banana fusarium wilt. Therefore, genes or proteins related to the pathogenicity of banana fusarium wilt bacteria are required to be continuously excavated, so that the banana fusarium wilt disease can be effectively prevented and treated.
Disclosure of Invention
The invention provides the application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt, enriches a protein database related to the pathogenicity of banana fusarium wilt, and is beneficial to preventing and controlling banana fusarium wilt.
The first object of the invention is to provide an application of the protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt.
A second object of the present invention is to provide the use of an agent for inhibiting the expression of protein Fobut or an agent for knocking out the gene encoding protein Fobut for controlling banana vascular wilt.
A third object of the present invention is to provide the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for the preparation of a product for controlling banana vascular wilt.
A fourth object of the present invention is to provide the use of an agent for inhibiting the expression of protein Fobut or an agent for knocking out the gene encoding protein Fobut for reducing the pathogenicity of banana vascular wilt.
A fifth object of the present invention is to provide the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for the preparation of a product for reducing the pathogenicity of banana vascular wilt.
A sixth object of the invention is to provide a method for reducing the pathogenicity of banana vascular wilt.
The seventh object of the invention is to provide a method for preventing and treating banana vascular wilt.
The above object of the present invention is achieved by the following technical scheme:
in the course of research on Foc secretion proteomics, an unknown protein (uncharacterized protein) with high conservation in evolution is found and is named as protein FoBut. The phenotype of the obtained FoBut knockout mutant is observed and pathogenicity analysis is carried out by constructing a knockout plasmid of a gene (FoBut) for encoding the protein FoBut and transferring the FoBut into a banana fusarium wilt protoplast, and the obtained FoBut knockout gene FoBut obviously reduces the pathogenicity of the banana fusarium wilt, and after the FoBut is restored, the pathogenicity of the obtained restoring mutant is restored, so that the protein FoBut is a protein related to the pathogenicity of the banana fusarium wilt and regulates and controls the pathogenicity of the banana fusarium wilt. Accordingly, the present invention claims the following applications:
the invention discloses application of a protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt.
Specifically, the amino acid sequence of the protein Fobut is shown as SEQ ID NO. 2.
Specifically, the application is the application of the protein Fobut in reducing the pathogenicity of banana fusarium wilt.
More specifically, the use is achieved by inhibiting the expression of the protein Fobut or knocking out the gene encoding the protein Fobut.
As one of the choices, the nucleotide sequence of the gene encoding the banana fusarium wilt protein Fobut is shown in SEQ ID NO. 1.
In view of the fact that banana vascular wilt is a disease caused by banana vascular wilt, reducing pathogenicity of banana vascular wilt can prevent occurrence of banana vascular wilt and reduce harm of banana vascular wilt. The expression of the banana fusarium wilt gene Fobut or the inhibition gene Fobut can be knocked out to influence the expression of the protein Fobut, so that the pathogenicity of the banana fusarium wilt is reduced. Therefore, the invention also claims the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for controlling banana vascular wilt.
Optionally, the agent that inhibits protein Fobut expression is an siRNA that targets the gene Fobut.
The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in the preparation of the product for preventing and treating banana vascular wilt.
The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in reducing the pathogenicity of banana fusarium wilt.
The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in the preparation of products for reducing the pathogenicity of banana fusarium wilt.
Specifically, the amino acid sequence of the protein Fobut is shown as SEQ ID NO. 2.
Specifically, the preparation for knocking out the gene encoding the protein Fobut comprises a knocking-out plasmid thereof, wherein the knocking-out plasmid is constructed based on a filamentous fungus expression vector pCT 74.
The invention also provides a method for reducing the pathogenicity of banana fusarium wilt, which comprises the following steps: blocking or inhibiting the expression of banana fusarium wilt protein Fobut.
The invention also provides a method for preventing and treating banana vascular wilt, which comprises the following steps: blocking or inhibiting the expression of banana fusarium wilt protein Fobut.
Specifically, the banana fusarium wilt is caused by banana fusarium wilt, and the banana fusarium wilt is prevented and controlled by blocking or inhibiting the expression of banana fusarium wilt protein Fobut and reducing the pathogenicity of banana fusarium wilt.
More specifically, the banana fusarium wilt is banana fusarium wilt No. 4 race.
The invention has the following beneficial effects:
the invention obtains the deletion mutant and the anaplerotic mutant of the Fobut by constructing knockout plasmid and anaplerotic plasmid of the gene (Fobut) encoding the Fobut of the banana fusarium wilt protein and respectively converting corresponding protoplast. Through observing the phenotypes of the deletion mutant and the anaplerotic mutant and carrying out pathogenicity analysis, the knock-out gene Fobut can obviously reduce the pathogenicity of banana fusarium wilt, and the pathogenicity of the anaplerotic gene Fobut is recovered after anaplerotic, which shows that the protein Fobut is related to the pathogenicity of the banana fusarium wilt and can be used as a target point for preventing and treating the banana fusarium wilt. The invention enriches protein databases related to pathogenicity of banana fusarium wilt and is beneficial to preventing and treating banana fusarium wilt.
Drawings
FIG. 1 is a schematic diagram of the principle of knocking out FoBut gene of banana vascular wilt.
FIG. 2 is a schematic diagram of the FoBut anaplerotic plasmid of the banana vascular wilt gene.
FIG. 3 shows the result of PCR-validated analysis of gene hph in a portion of hygromycin-positive transformants.
FIG. 4 shows the results of PCR-validated analysis of the FoBut gene in a portion of hygromycin resistant transformants.
FIG. 5 shows the results of PCR-validated analysis of the gene FoBut in bleomycin positive transformants.
FIG. 6 shows growth and statistical analysis results of FoBut knockout mutants under different stress conditions; a in the graph is the growth condition of FoBut knockout mutants under different stress conditions; b in the figure is the corresponding statistical analysis result.
FIG. 7 is a result of a pathogenicity analysis of FoBut knockout mutants on Brazilian banana; a in the figure is the brazil banana plant and bulb symptoms; b in the figure is the result of statistical analysis of the disease index.
The different letters in the graph represent significant differences, p < 0.05.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 construction of FoBut Gene knockout mutant and anaplerotic mutant of Banana fusarium wilt
1. Experimental materials
(1) Test strain and plant
The banana fusarium wilt bacterial strain used in the embodiment of the invention is banana fusarium wilt bacterial No. 4 race (Foc 4), and the plant to be tested is Brazil banana (Cavendsh, AAA).
(2) Host bacterium and plasmid vector
The cloning vector used in the embodiment of the invention is pMD18-T, the gene knockout vector used is pCT74, and the gene complement vector used is pCTZN. Wherein, the repayment vector pCTZN is obtained by replacing a fluorescent protein gene (SGFP) and a hygromycin phosphotransferase gene (hph) on pCT74 with bleomycin genes on the basis of a vector pCT 74.
2. Experimental method
The principle schematic diagram of the invention for knocking out FoBut gene of banana fusarium wilt is shown in figure 1. Namely, foBut genes in banana fusarium wilt are replaced by hph and SGFP through homologous recombination, so that the knockout of the FoBut genes is realized.
(1) Amplification of upstream and downstream homology arms of FoBut gene of banana fusarium wilt
The nucleotide sequence of the FoBut gene of banana fusarium wilt is shown as SEQ ID NO.1, and is a complete gene sequence, and the amino acid sequence of the encoded FoBut protein is shown as SEQ ID NO. 2. The invention finds the upstream and downstream homologous fragments of the FoBut gene by comparison in NCBI database, sequences with the length of about 1200bp are respectively selected as homology arms at the upstream and downstream of the FoBut gene and named as a homology arm A fragment and a homology arm B fragment respectively, corresponding amplification primers are designed, proper enzyme digestion sites are introduced, and the nucleotide sequences of the primers are designed as shown in the table 1.
TABLE 1 amplification primers for FoBut Gene homology arm A fragment and B fragment
Note that: the underlined position is the cleavage site.
Extracting Foc4 genomic DNA with a fungal DNA extraction kit (OMEGA Fungal DNA Kit); PCR amplification is carried out by using the extracted genome DNA as a template and using primers FoBut-AF and FoBut-AR to obtain a homologous arm A fragment (FoBut-A) of the FoBut gene; PCR amplification was performed using the primers FoBut-BF and FoBut-BR to obtain a homology arm B fragment (FoBut-B) of the FoBut gene.
The PCR reaction system used for amplifying the homology arm fragment is shown in Table 2.
TABLE 2 PCR reaction System for amplifying homology arm fragments
The PCR reaction conditions were: reacting at 94 ℃ for 5min; reacting at 98 ℃ for 10sec,58 ℃ for 30sec, and 72 ℃ for 1.2min for 30 cycles; the reaction was carried out at 72℃for 10min.
After the PCR reaction was completed, the PCR amplified product was purified and recovered by using OMEGA Cycle Pure Kit kit.
(2) Construction of FoBut Gene knockout vector
Referring to the specification of the pMD18-T Vector Cloning Kit (TakaRa) kit, foBut-A and FoBut-B were ligated to pMD18-T vectors, respectively, to obtain recombinant vectors pMD18-T-FoBut-A and pMD18-T-FoBut-B. The method comprises the following steps: taking 1 mu L of pMD18-T vector, adding 4 mu L of the PCR recovery product (homologous arm A fragment or homologous arm B fragment) and 5 mu L of solution I respectively, uniformly mixing, and connecting at 16 ℃ overnight; adding 10 μl of the ligation product into 100 μl of Escherichia coli DH5 α competent cells, standing on ice for 30min, performing water bath heat shock at 42deg.C for 90s, and cooling on ice for 5min; adding 800 mu L of LB liquid medium, and culturing at 37 ℃ for 1h by shaking at 150 rpm; centrifuging at 4000rpm for 5min, discarding supernatant, mixing 100 μl of bacterial liquid with the precipitate, and coating on LB solid medium (containing 50 μg/mL Amp); the culture was carried out in an incubator at 37℃overnight.
And selecting positive transformants with Amp resistance, extracting recombinant plasmid DNA, carrying out sequencing identification, and selecting recombinant plasmid DNA with correct sequencing results to construct a FoBut gene knockout vector. Specifically, the pMD18T-FoBut-A and pCT74 vectors are respectively subjected to enzyme digestion by Kpn I and Apa I, a homology arm A fragment and a linearized pCT74 vector are recovered, and the A fragment is connected with pCT74 by using T4 DNA ligase to transform escherichia coli DH5 alpha; the recombinant plasmid pCT74-FoBut-A was obtained. The pMD18-T-FoBut-B and the recombinant plasmid pCT74-FoBut-A are digested with EcoR I and SpeI in the same manner, the B fragment and the recombinant plasmid pCT74-FoBut-A are recovered, and the B fragment is ligated with pCT74-FoBut-A by using T4 DNA ligase to transform E.coli DH 5. Alpha; and obtaining the gene knockout vector pCT74-FoBut-KO through enzyme digestion identification.
(3) Amplification of the anaplerotic fragment of the FoBut Gene
The patch segment (FoBut-com) of the FoBut gene selected by the invention comprises the steps of selecting a promoter sequence with the length of 1932bp at the upstream of the FoBut gene and selecting a terminator sequence with the length of 475bp at the downstream of the FoBut gene, designing PCR amplification primers and introducing proper enzyme cutting sites. The amplification primers for the designed FoBut gene make-up fragments are shown in Table 3.
TABLE 3 amplification primers for FoBut Gene make-up fragments
Extracting Foc4 genomic DNA with a fungal DNA extraction kit (OMEGA Fungal DNA Kit); and (3) taking the extracted genome DNA as a template, and carrying out PCR amplification by using the primers FoBut-comF and FoBut-comR to obtain a patch fragment (FoBut-com) of the FoBut gene.
The PCR reaction system used for amplifying the FoBut gene anaplerotic fragment is shown in Table 4.
TABLE 4 PCR reaction System for amplifying FoBut Gene anaplerotic fragments
The PCR reaction conditions were: reacting at 94 ℃ for 5min;94℃for 1min,55℃for 1min and 72℃for 3.2min for 30 cycles; the reaction was carried out at 72℃for 10min.
After the PCR reaction, the PCR amplification product was purified and recovered using OMEGA Cycle Pure Kit kit.
(4) Construction of FoBut Gene repair vector
A schematic diagram of the banana fusarium wilt bacteria FoBut gene anaplerosis vector constructed by the invention is shown in figure 2. The construction process is as follows: digestion of the FoBut-com and pCTZN vectors with EcoR I and SpeI, respectively, to recover the FoBut-com fragment and linearized pCTZN vector; the FoBut-com fragment was ligated with pCTZN vector overnight at 16℃with T4 DNA ligase, the ligation product was transformed into E.coli DH 5. Alpha. And positive transformants were picked up, recombinant plasmids thereof were extracted and subjected to restriction enzyme identification. And successfully obtaining the gene compensation vector pCTZN-FoBut-com through enzyme digestion identification.
(5) Preparation of Foc protoplasts
Foc 4A culture medium (FeSO) 4 .7H 2 O 0.018g,KCl 0.5g,K 2 HPO 4 .3H 2 O1g,MgSO 4 .7H 2 O 0.5g,NaNO 3 3g, 30g of sucrose, and distilled water to 1L), and culturing at 28 ℃ for 3d at 150 rpm; filtering the culture solution with a cell sieve, centrifuging the filtrate at 5000rpm at 4deg.C for 10min, and discarding the supernatant; the precipitate was resuspended in CM broth (tryptone 2g, yeast extract 1g, casein hydrolysate 1g,20 Xnitrate 50mL,1000 Xvitamin 1mL,1000 Xtrace element 1mL, constant volume to 800mL, pH 6.5; glucose 10g, constant volume to 200 mL) and diluted to give a conidium suspension; inoculating the conidium suspension to a CM liquid culture medium, and shake culturing at 120rpm for 11-13 h at 28 ℃; filtering mycelium by using a cell sieve, and flushing 3-5 times by using 0.8mol/L NaCl solution to obtain fresh mycelium; mixing the enzymolysis liquid and mycelium in a volume-mass ratio of 10:1, and carrying out enzymolysis for 3 hours at 120rpm to obtain protoplast enzymolysis liquid; centrifuging at 2000rpm at 4deg.C for 10min, discarding supernatant, adding pre-chilled STC solution (containing 100mmol/L Tris-HCl pH7.5, 1.2mol/L sorbitol, 50mmol/L CaCl) 2 ) Re-suspending and precipitating; centrifuging, discarding the supernatant, adding pre-cooled STC, and re-suspending the precipitate to obtain Foc protoplast suspension.
Protoplast of the banana fusarium wilt knock-out mutant is prepared by referring to the preparation steps of Foc protoplast.
(6) Transformation of protoplasts
The transformation of protoplasts of the FoBut gene knockout plasmid of banana vascular wilt is exemplified. Cleavage of the knock-out vector pCT74-FoBut-KO with Kpn I and Spe IObtaining an A-hph-SGFP-B fragment; uniformly mixing the A-hph-SGFP-B fragment with the prepared protoplast of banana vascular wilt bacteria, and carrying out ice bath for 20min; fresh PTC conversion buffer (40% PEG4000,1.2mol/L sorbitol, 50 mmol/LCaCl) was added 2 10mmol/L Tris-HCl, pH 7.5), mixing, and standing on ice for 7min; adding the freshly prepared PTC solution again, mixing uniformly, standing on ice for 7min, adding pre-cooled STC, mixing uniformly, and standing at room temperature for 7min; centrifuging at 4000rpm at 4deg.C for 15min; adding 3mL of PDA liquid regeneration culture medium (200.0 g of potato, 273.6g of sucrose and distilled water to 1L) for re-suspending and precipitating, and performing shake culture at 28 ℃ and 100rpm for 13-16 h to regenerate cell walls; centrifuging the solution at 4000rpm for 15min, removing the supernatant, adding a PDA solid regeneration medium (1.5% agar powder and 150 mug/mL hygromycin are added into the PDA liquid regeneration medium), uniformly mixing, pouring into a plate, and culturing in darkness at 28 ℃ for 2-3 d; hygromycin resistant transformants were picked up and transferred to PDA medium (200.0 g potato, 20.0g anhydrous dextrose, 15.0g agar, distilled water to volume 1L) containing 150. Mu.g/mL hygromycin, and incubated in the dark at 28℃for 3-4 d, and single colonies were picked up for identification.
The transformation of the banana fusarium wilt FoBut gene anaplerotic plasmid is the same as that of the banana fusarium wilt knockout mutant, and the antibiotic used for screening positive transformants is bleomycin with the bleomycin concentration of 200 mug/mL.
(7) PCR validation analysis of FoBut knockout mutants
Genomic DNA of the hygromycin positive transformant was extracted and analyzed by PCR according to the instructions of the fungus DNA extraction kit (OMEGA Fungal DNA Kit). Firstly, carrying out PCR amplification on the hph gene fragment by using a primer hph-F/hph-R, and then carrying out PCR amplification analysis on the FoBut gene fragment by using a primer FoBut-F/FoBut-R; if the gene fragment hph can be amplified but the gene fragment FoBut cannot be amplified, the detected transformant is shown to be a FoBut knockout mutant.
hph-F:5′-TGCTGCTCCATACAAGCCAA-3′;
hph-R:5′-GACATTGGGGAGTTCAGCGA-3′。
FoBut-F:5′-CAGGTCATCCCAGTTGGTGAA-3′;
FoBut-R:5′-GTCTTCATTCCAGAGCCACCT-3′。
The PCR reaction system used for PCR validation analysis of FoBut knockout mutants is shown in Table 5.
TABLE 5 PCR reaction System for FoBut knockout mutant PCR validation analysis
The PCR reaction conditions were: reacting at 94 ℃ for 5min;94 ℃ for 1min,56 ℃ for 1min and 72 ℃ for 1min, 30 cycles; and (3) reacting for 10min at 72 ℃ to obtain an amplification product.
The invention utilizes a homologous recombination method to obtain 26 hygromycin positive transformants by transforming the gene knockout vector into banana fusarium wilt protoplast. Genomic DNA of 26 hygromycin positive transformants was extracted and amplified by using hph gene-specific primers, and it was found that gene hph was amplified in all of the 26 transformants, and the result of PCR verification analysis of gene hph of a part of hygromycin positive transformants is shown in FIG. 3. On this basis, the PCR verification analysis of the gene FoBut was performed on the 26 positive transformants amplified to the gene hph by the PCR using the FoBut gene-specific primers. The results of the PCR-validated analysis of the gene FoBut in a portion of hygromycin resistant transformants are shown in FIG. 4. As can be seen from the figure, of the 26 transformants, 8 transformants did not amplify the FoBut gene fragment, indicating that these 8 transformants were FoBut knockout mutants.
(9) PCR validation analysis of FoBut make-up mutants
Genomic DNA of the bleomycin positive transformant was extracted and analyzed by PCR as per the protocol of the fungal DNA extraction kit method (OMEGA Fungal DNA Kit). PCR amplification of the gene fragment FoBut was performed with the primer FoBut-F/FoBut-R.
The PCR reaction system used for the PCR validation analysis of the FoBut make-up mutant is shown in Table 6.
Table 6 PCR reaction System for FoBut make-up mutant PCR validation analysis
The PCR reaction conditions were: reacting at 94 ℃ for 5min;94 ℃ for 1min,56 ℃ for 1min and 72 ℃ for 1min, 30 cycles; and (3) reacting for 10min at 72 ℃ to obtain an amplification product.
The invention uses random insertion method to transform gene compensation plasmid pCTZN-FoBut-com into banana fusarium wilt delta FoBut protoplast, to obtain 4 bleomycin positive transformants. After extracting genomic DNA of bleomycin positive transformants, PCR validation analysis was performed on these bleomycin positive transformants using FoBut gene-specific primers. The results of PCR-validated analysis of the gene FoBut in bleomycin positive transformants are shown in FIG. 5. As can be seen from FIG. 5, 2 positive transformants were amplified to the target gene fragment, indicating that these 2 transformants contained the FoBut gene, i.e., these 2 transformants were FoBut-make-up mutants (. DELTA.FoBut-com).
Example 2 phenotypic observations and stress resistance analysis of FoBut knockout mutants
1. Phenotypic observations of FoBut knockout mutant (. DELTA.FoBut)
(1) Colony morphology observation and growth rate measurement
Respectively inoculating Foc wild type, knockout mutant delta FoBut and anaplerosis mutant delta FoBut-com on PDA culture medium, and culturing at 28deg.C in dark condition; colony diameter was measured at 5d and colony morphology was observed. The results show that there is no significant difference in colony morphology and growth rate of ΔFoBut compared to wild type banana fusarium wilt.
(2) Observation of yield and germination of conidia
Foc4 wild type, knockout mutant ΔFoBut and anaplerotic mutant ΔFoBut-com were inoculated into Charles medium respectively, and cultured with shaking at 28℃and 120rpm for 3d, and then the spore yield was counted. In addition, a suspension of the conidia of Foc wild type, knockout mutant ΔFoBut and anaplerotic mutant ΔFoBut-com was inoculated in CM medium, shake-cultured at 28℃and 120rpm, sampled at 7 hours, and the germination of the conidia was observed and recorded. The result shows that the spore yield of the mutant delta FoBut is not obviously different from that of the wild type, and the germination of the conidium is also not different from that of the wild type, so that the generation and the germination of the conidium of the banana fusarium wilt are not influenced after the FoBut is knocked out.
2. Stress resistance analysis of FoBut knockout mutants
Foc4 wild type, knockout mutant ΔFoBut and make-up mutant ΔFoBut-com were inoculated with 1mol/L NaCl, 1mol/L Sorbitol (Sorbitol), 300mmol/L H, respectively 2 O 2 Growth was observed and statistically analyzed on PDA medium, 0.05% SDS, 100. Mu.g/mL Congo Red (CR), 50. Mu.g/mL fluorescent whitening agent (CFW), after 5d incubation in an incubator at 28℃in an inverted position.
The growth conditions and statistical analysis results of the FoBut knockout mutants under different stress conditions are shown in FIG. 6; a in fig. 6 is the growth of the FoBut knockout mutant under different stress conditions; b in fig. 6 is the corresponding statistical analysis result. As can be seen from fig. 6: (1) in PDA medium containing NaCl and sorbitol, there is no obvious difference between ΔFoBut and wild type, indicating that FoBut has no effect on Foc ability of resisting high osmotic pressure; (2) at H 2 O 2 Under the stress condition, compared with the wild type, the bacterial colony of the delta FoBut mutant has no obvious difference with the wild type, which proves that the FoBut has no influence on the oxidation stress resistance of Foc 4; (3) the growth of mutant ΔFoBut was significantly different from wild type in PDA medium containing 100 μg/mL CR (different letters in the figure indicate significant differences, p < 0.05), indicating that knockout of FoBut affects the cell wall integrity of Foc.
Example 3 pathogenic analysis of knockout mutant ΔFoBut
Brazil banana with consistent growth vigor and 4 leaf stage is taken, and conidia (1×10) of Foc wild type, knockout mutant ΔFoBut and complementation mutant ΔFoBut-com are respectively used 5 individual/mL) suspension was root-dipped, and the fresh water treatment group was used as a control; transplanting the banana seedlings into sterile nutrient soil after 40min of treatment, placing the banana seedlings into a plant culture room at 28 ℃ for culture, alternately culturing the banana seedlings in 12h/12h of light and dark, observing the disease condition of the banana seedlings and the bulbs after 28d, calculating the disease condition index, and carrying out statistical analysis on the disease condition index. Disease grading criteria reference Huang Yonghui (2016) method (Table 7).
TABLE 7 Banana wilt disease grading Standard (Huang Yonghui, 2016)
The disease index calculating method comprises the following steps: disease index = Σ (disease grade x number of plants with disease grade)/(highest grade x total number of plants investigated) ×100
The pathogenicity analysis result of the FoBut knockout mutant on Brazilian banana is shown in FIG. 7; a in fig. 7 is the brazil banana plant and bulb symptoms; b in FIG. 7 is the result of statistical analysis of the disease index. After 28d of culture, the observation shows that no leaf yellowing phenomenon occurs in the Brazilian banana seedlings treated by clear water, and the bulb does not change color. After Foc wild type inoculation, the whole banana plant is obviously yellowing from the lower part to the upper part, and more than 50% of bulb areas are browned; after the delta FoBut inoculation, only the lower leaves of banana plants are yellowing, and the bulb discoloration area is not more than 20%; after inoculation of the anaplerotic mutant ΔFoBut-com, the upper and lower leaves of the banana plants also showed large area yellowing, and more than 50% of the bulb area was browned (A in FIG. 7). Statistical analysis of the disease index shows that ΔFoBut-com is similar to Foc wild type disease index, indicating that the pathogenicity of ΔFoBut-com is restored to wild type level. Meanwhile, the disease index of delta FoBut is obviously lower than that of wild type and anaplerotic mutants, which indicates that after FoBut gene is knocked out, the pathogenicity of banana fusarium wilt is obviously reduced. The result shows that the FoBut is knocked out to reduce the pathogenicity of banana fusarium wilt.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
2. The use according to claim 1, characterized in that the use is of the protein Fobut for reducing the pathogenicity of banana vascular wilt.
3. The use according to claim 2, wherein the use is effected by inhibiting the expression of the protein Fobut or by knocking out the gene encoding the protein Fobut.
4. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in preventing and treating banana vascular wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
5. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in the preparation of a product for preventing and treating banana vascular wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
6. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in reducing the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
7. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in the preparation of a product for reducing the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
8. A method for reducing pathogenicity of banana fusarium wilt is characterized by blocking or inhibiting expression of banana fusarium wilt protein Fobut; the amino acid sequence of the protein is shown as SEQ ID NO. 2.
9. A method for preventing and treating banana vascular wilt is characterized by blocking or inhibiting the expression of banana vascular wilt protein Fobut.
10. The method according to claim 9, wherein the banana vascular wilt is caused by banana vascular wilt and the banana vascular wilt is controlled by blocking or inhibiting expression of banana vascular wilt protein Fobut and reducing pathogenicity of banana vascular wilt.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311667882.2A CN117721136A (en) | 2023-12-06 | 2023-12-06 | Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311667882.2A CN117721136A (en) | 2023-12-06 | 2023-12-06 | Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117721136A true CN117721136A (en) | 2024-03-19 |
Family
ID=90202715
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311667882.2A Pending CN117721136A (en) | 2023-12-06 | 2023-12-06 | Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117721136A (en) |
-
2023
- 2023-12-06 CN CN202311667882.2A patent/CN117721136A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111560384B (en) | Application of gene FoRnt in regulation and control of pathogenicity of banana fusarium wilt | |
CN110669773B (en) | Application of gene FoPDCD5 in regulation and control of pathogenicity of banana fusarium oxysporum | |
CN110183521B (en) | Application of magnaporthe grisea gene MoRMD1 in regulation and control of pathogenicity of magnaporthe grisea | |
CN113201054B (en) | Application of protein FoUPE1 in regulation and control of pathogenicity of banana vascular wilt | |
Ushimaru et al. | Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer | |
CN110656116B (en) | Application of gene FoCWM in regulation and control of pathogenicity of banana vascular wilt | |
CN112094852B (en) | Application of MODIP gene in regulation of growth and development of rice blast fungi and sporulation | |
CN112646011A (en) | Protein PHD-Finger17 related to plant stress resistance and coding gene and application thereof | |
CN113956337B (en) | Application of gene FoUPE3 in preventing and treating banana vascular wilt | |
Xin et al. | MrMid2, encoding a cell wall stress sensor protein, is required for conidium production, stress tolerance, microsclerotium formation and virulence in the entomopathogenic fungus Metarhizium rileyi | |
CN114426995B (en) | Method for improving yield of extracellular polysaccharide of cordyceps militaris by utilizing over-expressed hexokinase gene HK | |
CN117721136A (en) | Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt | |
CN116284284A (en) | Myosin containing FYVE structural domain, encoding gene and application thereof | |
CN114807187A (en) | Ural chart wheat receptor protein kinase gene TuRLK1 and application thereof | |
AU785214B2 (en) | Mutant bank | |
CN109456985B (en) | Pathogenicity-related botrytis cinerea gene BcMBF1 and application thereof | |
CN118085044A (en) | Application of protein FoUPE7 in reducing pathogenicity of banana fusarium wilt | |
CN114773440B (en) | Application of protein FoUpe2 in regulating and controlling pathogenicity of banana fusarium wilt | |
CN114807208B (en) | Application of protein FoAtg27 in regulating and controlling pathogenicity of banana fusarium wilt | |
CN114196681B (en) | Application of FoCupin1 gene in regulation and control of pathogenicity of banana fusarium wilt | |
CN116606877A (en) | Application of gene MoUPE5 in regulation and control of pathogenic force of rice blast fungi | |
CN118324881A (en) | Verticillium dahliae pathogenic protein VdPGL1 and application thereof | |
CN103451203B (en) | Sporulation gene Pro2 of cryphonectria parasitica and application thereof | |
CN103451214B (en) | Chestnut epidemic disease bacterium produces spore gene Pro1 and application thereof | |
CN117304285A (en) | Fusarium pseudograminearum FpMCoL protein, and coding gene and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |