CN105176837A - Application of protein fogac in regulating and controlling toxicity of fusarium oxysporum f.sp.cubense on banana plants - Google Patents
Application of protein fogac in regulating and controlling toxicity of fusarium oxysporum f.sp.cubense on banana plants Download PDFInfo
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- CN105176837A CN105176837A CN201510555477.0A CN201510555477A CN105176837A CN 105176837 A CN105176837 A CN 105176837A CN 201510555477 A CN201510555477 A CN 201510555477A CN 105176837 A CN105176837 A CN 105176837A
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Abstract
The invention discloses application of fogac protein in regulating and controlling toxicity of fusarium oxysporum f.sp.cubense on banana plants. The amino terminal sequence of the fogac protein is as shown in sequence 3 in a sequence list. The invention also discloses a method for regulating and controlling the toxicity of fusarium oxysporum f.sp.cubense on banana plants, wherein the method comprises step (a1) or (a2) or (a3) as follows: (a1) regulating and controlling the level of fogac protein in the fusarium oxysporum f.sp.cubense as shown in sequence 3 in the sequence list; (a2) regulating and controlling the expression level of a coding gene of the fogac protein in the fusarium oxysporum f.sp.cubense as shown in the sequence 3 in the sequence list; and (a3) regulating and controlling the transcriptional level of the coding gene of the fogac protein in the fusarium oxysporum f.sp.cubense as shown in the sequence 3 in the sequence list. Upon experiments, the fogac protein can be used for regulating and controlling the toxicity of fusarium oxysporum f.sp.cubense on banana plants.
Description
Technical field
The present invention relates to biological technical field, particularly relate to protein fogac at regulation and control Fusarium oxysporum Cuba specialized form to the application in banana plant virulence.
Background technology
Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) belongs to Deuteromycotina fungi, is a kind of pathogenic bacteria causing banana blight.This fungi invades banana plant from root, causes vascular bundle to block, yellowing leaf, plant wither and banana total crop failure.Be a kind of destructive disease by this microbial banana blight, plant is once morbidity can only be rooted out, and this bacterium can at soil long-term survival, and this makes this disease control very difficult.This disease all has generation in banana plantation, the whole world, and just constantly spreads and expand.At present, prevent and treat the blight caused by Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense), adopt the fungicide effect such as derosal, prochloraz generally not ideal enough.Adopt new type bactericide and seed selection is disease-resistant or disease tolerant variety be from now on banana blight control main method.Can help to understand its pathogenesis to the research of banana blight bacteria Disease-causing gene, for the control of this disease provides theoretical foundation.
Protein fogac is one of Fusarium oxysporum Cuba specialized form G-protein α subunit, the full name of G-protein (Gprotein) is guanine nucleotide binding protein (guaninenucleotide-bindingprotein), be the different aggressiveness be made up of the subunit that α, β, γ etc. 3 are different, there is the ability in conjunction with GTP or GDP.Research finds, in fungi, G-protein α subunit participates in the biological procedureses such as growth, growth, cell mating, secondary metabolite synthesis.But in Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense), the function of G-protein α subunit there is no correlative study report.
Summary of the invention
Technical problem to be solved by this invention how to regulate and control Fusarium oxysporum Cuba specialized form to banana plant virulence.
For solving the problem, the present invention provide firstly a kind of fogac protein, and the aminoacid sequence of described fogac protein can as shown in sequence in sequence table 3.
Described fogac protein belongs to protection scope of the present invention at regulation and control Fusarium oxysporum Cuba specialized form to the application in banana plant virulence.
For solving the problem, the invention provides a kind of regulate and control Fusarium oxysporum Cuba specialized form to the method for banana plant virulence.
Regulation and control Fusarium oxysporum Cuba provided by the present invention specialized form, to the method for banana plant virulence, can comprise the steps (a1) or (a2) or (a3):
(a1) level of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled;
(a2) expression level of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled;
(a3) transcriptional level of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled.
For solving the problem, present invention also offers a kind of method of screening Fusarium oxysporum Cuba specialized form sterilant.
A kind of method of screening Fusarium oxysporum Cuba specialized form sterilant provided by the present invention, can comprise the steps (b1) or (b2) or (b3):
(b1) treat detection material with the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, by the material to be detected Fusarium oxysporum Cuba specialized form sterilant alternatively of suppression described fogac protein obtained;
(b2) treat detection material with the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, by the material to be detected Fusarium oxysporum Cuba specialized form sterilant alternatively that the described encoding gene of suppression obtained is expressed;
(b3) treat detection material with the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, the material to be detected that described for the suppression obtained encoding gene is transcribed Fusarium oxysporum Cuba specialized form sterilant alternatively.
For solving the problem, present invention also offers a kind of method of qualification or assistant identification Fusarium oxysporum Cuba specialized form candidate anti-microbial agents.
The method of a kind of qualification provided by the present invention or assistant identification Fusarium oxysporum Cuba specialized form candidate anti-microbial agents, can comprise following (c1) or (c2) or (c3):
(c1) level that can test substance reduce the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is detected, as as described in determinand mass-energy reduce as described in fogac protein described in Fusarium oxysporum Cuba specialized form level, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not reduce as described in fogac protein described in Fusarium oxysporum Cuba specialized form level, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate;
(c2) expression that can test substance suppress the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is detected, as as described in determinand mass-energy suppress as described in encoding gene described in Fusarium oxysporum Cuba specialized form expression, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not suppress as described in encoding gene described in Fusarium oxysporum Cuba specialized form expression, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate;
(c3) detect test substance and can suppress transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form, as as described in determinand mass-energy suppress as described in the transcribing of encoding gene described in Fusarium oxysporum Cuba specialized form, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not suppress as described in the transcribing of encoding gene described in Fusarium oxysporum Cuba specialized form, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate.
There is d1) and/or d2) and/or d3) application of material in preparation Fusarium oxysporum Cuba specialized form sterilant of function also belong to protection scope of the present invention:
D1) the fogac protein level shown in sequence 3 in sequence table is reduced;
D2) expression of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced;
D3) transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced.
There is e1) and/or e2) and/or e3) application of material in the banana plant of preparation anti-Fusarium oxysporum Cuba specialized form of function also belong to protection scope of the present invention:
E1) the fogac protein level shown in sequence 3 in sequence table is reduced;
E2) expression of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced;
E3) transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced.
Above-mentioned arbitrary described encoding gene can be the DNA molecular shown in sequence 2 in sequence table.
Above-mentioned arbitrary described banana can be following f1)-f5) in any one: f1) Musaceae plant; F2) Brazilian any of several broadleaf plants; F3) Brazilian any of several broadleaf plants kind Musaspp.AAAgroupcv.Brazil; F4) dwarf banana; F5) dwarf banana kind Musaspp.AABgroupcv.Fenjiao.
Present invention also offers a kind of method preparing recombinant bacterium.
A kind of method preparing recombinant bacterium provided by the present invention, the encoding gene knocking out the fogac protein shown in sequence 3 in the sequence table in the genome of Fusarium oxysporum Cuba specialized form can be comprised the steps:, obtain the recombinant bacterium of virulence lower than described Fusarium oxysporum Cuba specialized form.
Above-mentionedly prepare in the method for recombinant bacterium, described in the encoding gene of protein fogac that knocks out in the genome of Fusarium oxysporum Cuba specialized form realize by homologous recombination.Described homologous recombination is realize by importing specific fragment.Described specific fragment can contain upstream homology arm and downstream homology arm, the nucleotide sequence of described upstream homology arm can be the sequence 1 of sequence table from the DNA molecular shown in 5' end the 233rd to the 776th, and the nucleotide sequence of described downstream homology arm can be the sequence 1 of sequence table from the DNA molecular shown in 5' end the 3213rd to the 4082nd.
The preparation method of described specific fragment specifically can be: the DNA small segment between the KpnI recognition sequence of plasmid pCT74 and XhoI recognition sequence is replaced with the sequence 1 of nucleotide sequence sequence table from the DNA molecular shown in 5' end the 233rd to the 776th by (1), DNA small segment between EcoRI recognition sequence and BamHI recognition sequence replace with nucleotide sequence be the sequence 1 of sequence table from the DNA molecular shown in 5' end the 3213rd to the 4082nd, obtain recombinant plasmid pCT-fogac; (2) recombinant plasmid pCT-fogac restriction enzyme KpnI and BamHI enzyme are cut, reclaim the DNA fragmentation of about 4.4kb, be specific fragment.
Above-mentioned arbitrary described specific fragment also belongs to protection scope of the present invention.
Experiment proves, fogac protein controllable Fusarium oxysporum Cuba specialized form is to banana plant virulence.Δ fogac is mutants which had, can reduce the expression of the encoding gene of fogac protein in Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2; Δ fogac+fogac is complement bacterial strain, can recover the expression of the encoding gene of fogac protein in Δ fogac.Result shows, the Brazilian any of several broadleaf plants disease index (3.0) of inoculation Δ fogac is significantly lower than the Brazilian any of several broadleaf plants disease index (55.0) of inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2, and the Brazilian any of several broadleaf plants disease index of the Brazilian any of several broadleaf plants disease index (50.3) and inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2 of inoculating Δ fogac+fogac is without significant difference (55.0); The disease index (3.2) of the dwarf banana of inoculation Δ fogac is significantly lower than the dwarf banana disease index (28.7) of inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2, and the dwarf banana disease index (28.7) of the disease index (26.5) and inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2 of inoculating Δ fogac+fogac is without significant difference.Visible, the virulence of Δ fogac to Brazilian any of several broadleaf plants or dwarf banana significantly declines, and Δ fogac+fogac can make the virulence of Δ fogac recover.
Accompanying drawing explanation
Fig. 1 is gene knockout plasmid pCT-fogac part-structure schematic diagram
Fig. 2 is the initial amplification position view of checking primer.
Fig. 3 is the PCR qualification result of fogac knock out mutants body and complement thereof.
Wherein, swimming lane 1 is with the genomic dna of fogac knock out mutants body for template, carries out pcr amplification with primer pair 1; Swimming lane 2 is with the genomic dna of the complement of fogac knock out mutants body for template, carries out pcr amplification with primer pair 1; Swimming lane 3 is with the genomic dna of B2 bacterial strain for template, carries out pcr amplification with primer pair 1; Swimming lane 4 is with the genomic dna of fogac knock out mutants body for template, carries out pcr amplification with primer pair 2; Swimming lane 5 is with the genomic dna of the complement of fogac knock out mutants body for template, carries out pcr amplification with primer pair 2; Swimming lane 6 is with the genomic dna of B2 bacterial strain for template, carries out pcr amplification with primer pair 2; Swimming lane 7 is with the genomic dna of fogac knock out mutants body for template, carries out pcr amplification with primer pair 3; Swimming lane 8 is with the genomic dna of the complement of fogac knock out mutants body for template, carries out pcr amplification with primer pair 3; Swimming lane 9 is with the genomic dna of B2 bacterial strain for template, carries out pcr amplification with primer pair 3.
Fig. 4 is the RT-PCR the result of fogac knock out mutants body and complement thereof.
Wherein, swimming lane 1 is with B2 bacterial strain cDNA for template, carries out pcr amplification with primer pair 4 or actin primer pair; Swimming lane 2 is with the cDNA of fogac knock out mutants body for template, carries out pcr amplification with primer pair 4 or actin primer pair; Swimming lane 3 is with the cDNA of the complement of fogac knock out mutants body for template, carries out pcr amplification with primer pair 4 or actin primer pair.
Fig. 5 is the colonial morphology of fogac knock out mutants body and complement thereof.
Fig. 6 is fogac knock out mutants body and the pathogenic comparison of complement in Brazilian any of several broadleaf plants thereof.
Wherein, " WT " is the Brazilian any of several broadleaf plants plant disease index of inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2, " Δ fogac " is the Brazilian any of several broadleaf plants plant disease index of inoculation fogac knock out mutants body, " Δ fogac+fogac " is the Brazilian any of several broadleaf plants plant disease index of the complement of the complementary fogac gene of inoculation, and CK is contrast.
Fig. 7 is fogac knock out mutants body and the pathogenic comparison of complement in dwarf banana thereof.
Wherein, " WT " is the dwarf banana plant disease index of inoculation Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2, " Δ fogac " is the dwarf banana plant disease index of inoculation fogac knock out mutants body, " Δ fogac+fogac " is the dwarf banana plant disease index of the complement of the complementary fogac gene of inoculation, and CK is contrast.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2 and plasmid pCT74 in following embodiment (Guo Lijia etc. Fusarium oxysporum Cuba specialized form fgb1 knocks out structure and the phenotype analytical [J] of mutant. tropical crops journal, 2014,35 (11): 2205-2210.), the public can obtain from Chinese Academy of Tropical Agricultural Sciences (i.e. applicant), to repeat the application's experiment.Fusarium oxysporum Cuba specialized form (Fusariumoxysporumf.sp.cubense) bacterial strain B2 is called for short B2 bacterial strain hereinafter.
Plasmid pSilent-Dual1 (Nguyen, Q.B., Kadotani, N., Kasahara, S., Tosa, Y., Mayama, S. & Nakayashiki, H. (2008) Systematicfunctionalanalysisofcalcium-signallingproteins inthegenomeoftherice-blastfungus, Magnaportheoryzae, usingahigh-throughputRNA-silencingsystem.Molecularmicrob iology, 68:1348-1365.) can obtain from FungalGeneticsStockCenter (USA), sequence accession number GenBank:AB512772.1.
In following embodiment
hDCloningPlus is Clontech Products.
Brazilian any of several broadleaf plants in following embodiment is specially Brazilian any of several broadleaf plants kind (Musaspp.AAAgroupcv.Brazil), dwarf banana is specially dwarf banana kind (Musaspp.AABgroupcv.Fenjiao), is all purchased from Chinese Academy of Tropical Agricultural Sciences Zu Pei center.
STC solution: solute and concentration thereof are: 1.2M sorbyl alcohol, 50mM calcium chloride, 10mMTris-Cl; Solvent is water; PH7.5.
PTC solution: solute and concentration thereof are: 40% (mass volume ratio) PEG3350,50mM calcium chloride, 10mMTris-Cl; Solvent is water; PH7.0.
The substratum related in following embodiment is as follows:
PDB liquid nutrient medium: peeled by 200g potato and be cut into small pieces, then adds 1L water, boils 30min, filtered through gauze, adds glucose 15g, is settled to 1L, pH7.0 ± 0.1 in filtrate, 121 DEG C of autoclaving 15min.
PDA solid medium: adding agar to its content to above-mentioned PDB liquid nutrient medium is the substratum that 20g/L obtains.
Regeneration culture medium 1: 200g potato is peeled and is cut into small pieces, then 1L water is added, boil 30min, filtered through gauze, in filtrate, add sucrose 174g, agar 9g, be settled to 1L, pH7.0 ± 0.1,121 DEG C of autoclaving 15min, add Totomycin when being cooled to 60 DEG C, make the concentration of Totomycin in system be 50 μ g/ml.
Regeneration culture medium 2: 200g potato is peeled and is cut into small pieces, then add 1L water, boil 30min, filtered through gauze, sucrose 174g is added in filtrate, agar 9g, is settled to 1L, pH7.0 ± 0.1,121 DEG C of autoclaving 15min, add G418 (also referred to as Geneticin, Geneticin) when being cooled to 60 DEG C, make the concentration of G418 in system be 50 μ g/ml.
The functional verification of embodiment 1, fogac gene
One, vector construction
1, the structure of gene knockout carrier
The construction step of gene knockout carrier is as follows:
(1) Design and synthesis of primer
Fogac gene order according to sequence in sequence table 1, designs and synthesizes primer LF, LR, RF and RR, and the sequence information of LF, LR, RF and RR is in table 1.Wherein LF (containing Restriction Enzyme KpnI restriction enzyme site) and LR (containing Restriction Enzyme XhoI restriction enzyme site) is the primer of amplification fogac gene left arm, and RF (containing Restriction Enzyme EcoRI restriction enzyme site) and RR (containing Restriction Enzyme BamHI restriction enzyme site) is the primer of amplification fogac gene right arm.
Table 1.PCR primer sequence information
| Primer | Sequence information 5'-3' |
| LF | 5'-gg ggtaccCAAGGTTTCGCTATTTGACA-3' |
| LR | 5'-ccg ctcgagGGGTAGACAAGAGAAAGAGAT-3' |
| RF | 5'-cg gaattcGCATTCCTATGGCTTCTGT-3' |
| RR | 5'-cg ggatccTGATACGGTCTGAGAGTGT-3' |
| P1 | 5'-GGATTGGATGTGGATGTGATGG-3' |
| P2 | 5'-CGTTGCAAGACCTGCCTGAA-3' |
| P3 | 5'-GACCACTACCAGCAGAACAC-3' |
| P4 | 5'-AGTTCAACGATACTCGGTCTC-3' |
| P5 | 5'-ATGGGCGCATGCATGAGCTCG-3' |
| P6 | 5'-AAGAATGCCCGAGTCCTTAAG-3' |
| fogac-RT-F | 5'-GTTCTGGCGAGAGTGGCAAGTC-3' |
| fogac-RT-R | 5'-TTCGGCGGAATCCATGAGGTAGA-3' |
| actin-F | 5'-CCAAGTCCAACCGTGAGAAGATGA-3' |
| actin-R | 5'-CCAGAGTCCAGAACGATACCAGTG-3' |
Note: underscore is labeled as restriction enzyme site.
(2) with B2 strain gene group DNA for template, adopt LF and LR composition primer pair carry out pcr amplification, obtain the pcr amplification product of about 540bp.
(3) with the pcr amplification product that restriction enzyme KpnI and XhoI double digestion step (2) obtain, digestion products is reclaimed.
(4) with restriction enzyme KpnI and XhoI double digestion plasmid pCT74, the carrier framework of about 6kb is reclaimed.
(5) carrier framework that digestion products step (3) obtained and step (4) obtain is connected, and obtains recombinant plasmid pCT74-L.
(6) with restriction enzyme EcoRI and BamHI double digestion recombinant plasmid pCT74-L, the carrier framework of about 6.5kb is reclaimed.
(7) with B2 strain gene group DNA for template, adopt RF and RR composition primer pair carry out pcr amplification, obtain the pcr amplification product of about 870bp.
(8) use the pcr amplification product of restriction enzyme EcoRI and BamHI double digestion step (7), reclaim digestion products.
(9) carrier framework that digestion products step (8) obtained and step (6) obtain is connected, and obtains the recombinant plasmid pCT-fogac of about 7.4kb.
Fig. 1 is shown in by recombinant plasmid pCT-fogac part-structure schematic diagram.Structrual description carries out to recombinant plasmid pCT-fogac as follows: the DNA small segment between the KpnI recognition sequence of plasmid pCT74 and XhoI recognition sequence being replaced with nucleotide sequence is that the sequence 1 of sequence table is from the DNA molecular shown in 5' end the 233rd to the 776th, DNA small segment between EcoRI recognition sequence and BamHI recognition sequence replace with nucleotide sequence be the sequence 1 of sequence table from the DNA molecular shown in 5' end the 3213rd to the 4082nd, obtain recombinant plasmid pCT-fogac.
Cut recombinant plasmid pCT-fogac with restriction enzyme KpnI and BamHI enzyme, reclaim the DNA fragmentation of about 4.4kb.
2, the structure of gene complementation carrier
The construction step of gene complementation carrier is as follows:
(1) Design and synthesis of primer
Fogac gene order according to sequence 1, designs and synthesizes primers F and R (underscore is have 15 identical base sequences with plasmid pSilent-Dual1).The sequence information of F and R is as follows:
F：5'-ggttctcgaggtcgaCTTTAGAGTCAAACACGACAAGGT-3'
R：5'-
caagctggcagtcgaTACTCTTTTCGGAAGGGGGTTGC-3'。
(2) with B2 strain gene group DNA for template, carry out pcr amplification with F and R for primer, obtain double chain DNA molecule.
(3) with restriction enzyme SalI single endonuclease digestion plasmid pSilent-Dual1, large stretch of disconnected linearizing plasmid pSilent-Dual1 is reclaimed.
(4) utilize
hDCloningPlus, the double chain DNA molecule obtain step (2) and step (3) obtain linearizing plasmid pSilent-Dual1 and recombinate, and obtain recombinant plasmid pSD-fogac.
According to sequencing result, structrual description carries out to recombinant plasmid pSD-fogac as follows: insert in the SalI restriction enzyme site of plasmid pSilent-Dual1 nucleotide sequence be the sequence 1 of sequence table from the DNA molecular shown in 5' end the 215th to the 2941st, obtain recombinant plasmid pSD-fogac.
Two, fogac knock out mutants body is obtained
The step obtaining fogac knock out mutants body is as follows:
1, the preparation of B2 Strain Protoplast
(1) by B2 inoculation in PDB liquid nutrient medium, be placed in 28 DEG C, 180r/min shaking table concussion cultivation 5 days, then cross bacteriological filtration liquid removal mycelium with six layers of sterilizing lens wiping paper, obtain B2 spore liquid.
(2) B2 spore liquid 4ml (in practical application 3-5ml) prepared by step (1) is accessed to the triangular flask (250mL triangular flask liquid amount is 150mL) that PDB liquid nutrient medium is housed, 26 DEG C (in practical application 27 DEG C ± 1 DEG C), 110r/min cultivate 10h (in practical application 10h-15h), with three layers of sterilizing lens wiping paper collecting by filtration mycelium.
(3) with the mycelium that 0.8M aqueous NaCl wash step (2) is collected, then use three layers of sterilizing lens wiping paper collecting by filtration, and with filter paper, mycelium is pressed dry.
(4) in centrifuge tube, add mycelium and 4-6mL enzymolysis solution (add driselase in 0.8M sodium chloride aqueous solution, make the concentration of driselase in system be 20mg/mL) prepared by 0.1g step (3).26 DEG C (in practical application 27 DEG C ± 1 DEG C), 100r/min enzymolysis 3h (in practical application 3h-4h).
(5) after completing steps (4), in centrifuge tube, add 0.8M sodium chloride aqueous solution and cumulative volume is less than 45ml, filter with three layers of sterilizing lens wiping paper, then collect protoplastis in the centrifuge tube of 50mL.
(6) by centrifuge tube at normal temperature, the centrifugal 15min of 4000r/min, abandon supernatant, then use STC solution washing, centrifugal, abandon supernatant, it is 0.5 × 10 that the protoplastis STC solution of collection is fully suspended into concentration
8individual/mL is (in practical application 0.5 × 10
8-1 × 10
8individual/mL).
(7) protoplastis prepared by step (6) is sub-packed in the centrifuge tube of 2mL, often pipe 200 μ l.
2, DNA transforms
The DNA fragmentation that 2 μ g (in practical application 2 μ g-4 μ g) step one reclaims is added in the centrifuge tube of (7) in above-mentioned steps 1, room temperature places 20 minutes, then 1.2mLPTC solution is added, room temperature leaves standstill 20 minutes, and then added the regeneration culture medium 1 that 100mL (in practical application 100mL-150mL) is cooled to 45 DEG C, mixing, paves plate.Cultivate 4 days (in practical application 4 days-6 days) for 28 DEG C.To obtain after transformant carries out single spore separation purifying, carrying out next step pcr amplification qualification.
3, the PCR qualification of fogac knock out mutants body
Extract the genomic dna of transformant in above-mentioned steps 2 and as template, carry out pcr amplification with primer pair 1, primer pair 2 or primer pair 3.Primer pair 1 is made up of P1 and P2, and primer pair 2 is made up of P3 and P4, and primer pair 3 is made up of P5 and P6.Fig. 2 is seen in P1, P2, P3, P4, P5 and P6 initial amplification position, and the sequence information of primer is in table 1.
According to the method described above, the genomic dna of transformant in step 2 is replaced with the genomic dna of B2 bacterial strain, and other step is all identical, in contrast.
Result judges as follows: with the genomic dna of transformant as template, if with primer pair 1 carry out pcr amplification display 1.6kb band, to carry out pcr amplification with primer pair 2 and show the band of 1.7kb and carry out with primer pair 3 band that pcr amplification do not show 1.3kb, then transformant is fogac gene knockout transformant, otherwise is ectopic integration transformant or B2 bacterial strain.
PCR qualification result is as shown in Fig. 3 (in Fig. 3 swimming lane 1, swimming lane 3, swimming lane 4, swimming lane 6, swimming lane 7 and swimming lane 9): carry out pcr amplification with primer pair 1, transformant Δ fogac can detect the object band of 1.6kb, and B2 bacterial strain then can not detect the object band of 1.6kb; Carry out pcr amplification with primer pair 2, transformant Δ fogac can detect the object band of 1.7kb, and B2 bacterial strain then can not detect the object band of 1.7kb; Carry out pcr amplification with primer pair 3, transformant Δ fogac can not detect the object band of 1.3kb; And B2 bacterial strain can detect the object band of 1.3kb.Result shows, transformant Δ fogac is that fogac gene masculine knocks out mutant.
4, the RT-PCR checking of fogac knock out mutants body
Extract the total serum IgE of transformant Δ fogac in above-mentioned steps 3, then reverse transcription is cDNA and as template, carries out RT-PCR amplification with primer pair 4 or actin primer pair.Primer pair 4 is made up of fogac-RT-F and fogac-RT-R, and actin primer pair is made up of actin-F and actin-R.The sequence information of fogac-RT-F, fogac-RT-R, actin-F and actin-R is in table 1.The target gene of primer pair 4 is for the sequence 2 in sequence table is from the DNA molecular shown in 5' end the 125th to the 458th.
According to the method described above, the total serum IgE of transformant Δ fogac in step 3 is replaced with the total serum IgE of B2 bacterial strain, and other step is all identical, in contrast.
Result judges as follows: with the cDNA of transformant Δ fogac as template, if carry out RT-PCR amplification with primer pair 4 not show the band of 344bp, carry out the band of RT-PCR amplification display 130bp with actin primer pair, then transformant Δ fogac is fogac gene knockout transformant, otherwise is ectopic integration transformant or B2 bacterial strain.
RT-PCR qualification result is as shown in Fig. 4 (in Fig. 4 swimming lane 1 and swimming lane 2): carry out RT-PCR amplification with actin primer pair, transformant Δ fogac can detect the object band of 130bp, and B2 bacterial strain also can detect the object band of 130bp; Carry out RT-PCR amplification with primer pair 4, transformant Δ fogac can not detect the object band of 344bp, and B2 bacterial strain can detect the object band of 344bp.Result shows, transformant Δ fogac is fogac knock out mutants body, hereinafter referred to as Δ fogac.
The fogac knock out mutants body identified through step 3 and step 4 is inoculated in PDB liquid nutrient medium to cultivate, obtains spore suspension, spore suspension and 20% (volume percent) aqueous glycerin solution equal-volume is mixed, is placed in-70 DEG C of preservations.
Three, the acquisition of the complement of fogac knock out mutants body
(1) acquisition of the complement of fogac knock out mutants body
DNA fragmentation 1 in step 2 is replaced with the recombinant plasmid pSD-fogac in step one, B2 bacterial strain is replaced with fogac knock out mutants body, regeneration culture medium 1 replaces with regeneration culture medium 2, in repeating step two 1 to 4, obtains complementary transformant.
(2) the PCR qualification of complementary transformant
Extract complementary transformant in above-mentioned steps (1) genomic dna and as template, with in step 23 primer pair 1, primer pair 2 or primer pair 3 carry out pcr amplification.
Result judges as follows: with the genomic dna of complementary transformant as template, if with primer pair 1 carry out pcr amplification display 1.6kb band, to carry out with primer pair 2 pcr amplification display 1.7kb band and carry out with primer pair 3 band that pcr amplification shows 1.3kb, then complementary transformant is positive complementary transformant.
The PCR qualification result of complementary transformant is as shown in Fig. 3 (in Fig. 3 swimming lane 2, swimming lane 5 and swimming lane 8): carry out pcr amplification with primer pair 1, complementary transformant Δ fogac+fogac can detect the object band of 1.6kb, and B2 bacterial strain then can not detect the object band of 1.6kb; Carry out pcr amplification with primer pair 2, complementary transformant Δ fogac+fogac can detect the object band of 1.7kb, and B2 bacterial strain then can not detect the object band of 1.7kb; Carry out pcr amplification with primer pair 3, complementary transformant Δ fogac+fogac can detect the object band of 1.3kb; And B2 bacterial strain can detect the object band of 1.3kb.Result shows, complementary transformant Δ fogac+fogac is complement.
(3) the RT-PCR checking of complementary transformant
Extract the total serum IgE of complementary transformant Δ fogac+fogac in above-mentioned steps (2), then reverse transcription is cDNA and as template, with in step 24 primer pair 4 or actin primer pair carry out RT-PCR amplification.
If with primer pair 4 for primer, show the band of about 344bp, and carry out pcr amplification with actin primer pair, show the target stripe of about 130bp, then this complementary transformant positive complement of being fogac gene complementary, otherwise be the complementary transformant of false positive.The RT-PCR identification experiment of complement the results are shown in Figure 3.
Result judges as follows: with the cDNA of complementary transformant Δ fogac+fogac as template, if carry out the band of RT-PCR amplification display 344bp with primer pair 4, carry out the band of RT-PCR amplification display 130bp with actin primer pair, then complementary transformant Δ fogac+fogac is the positive complement of complementary fogac gene, otherwise is the complementary transformant of false positive.
RT-PCR qualification result is as shown in Fig. 4 (in Fig. 4 swimming lane 3): carry out RT-PCR amplification with actin primer pair, and complementary transformant Δ fogac+fogac can detect the object band of 130bp, and B2 bacterial strain can detect the object band of 130bp; Carry out RT-PCR amplification with primer pair 4, complementary transformant Δ fogac+fogac can detect the object band of 344bp, and B2 bacterial strain can detect the object band of 344bp.Result shows, complementary transformant Δ fogac+fogac is the complement of complementary fogac gene, hereinafter referred to as Δ fogac+fogac.
Four, colonial morphology is observed
By test strains (B2 bacterial strain, Δ fogac and Δ fogac+fogac) streak culture 36h on PDA solid medium, then Stereo microscope (SZX7 is used, Olympus) observe single colonial morphology and take pictures, (the left figure of Fig. 5 is B2 bacterial strain to the results are shown in Figure 5, middle figure is Δ fogac, right figure is Δ fogac+fogac), result shows that Δ fogac, Δ fogac+fogac and B2 bacterial strain do not have considerable change on colonial morphology.
Five, virulence compares
Detect test strains (B2 bacterial strain, Δ fogac and Δ fogac+fogac) respectively to the virulence of banana plant (Brazilian any of several broadleaf plants seedling or dwarf banana seedling), experiment in triplicate, is averaged.
By test strains list colony inoculation in 300mLPDB liquid nutrient medium, 28 DEG C, 180rpm cultivates 7 days.Collected by centrifugation thalline, obtains test strains spore liquid with sterilized water is resuspended, with blood counting chamber, test strains spore liquid concentration is adjusted to 1 × 10
7individual/mL.Random selecting 30 strain banana plant (plant height is about 30cm), inoculates above-mentioned test strains spore liquid (every strain plant root inoculation 50mL spore liquid) at its root, cultivates after process according to daily cultivation management method; Test strains spore liquid is replaced with sterilized water, and other step is all identical, as blank.Timing from after plant root inoculation test strains spore liquid to be measured or sterilized water, cultivates after 30 days, longitudinally cuts in the middle of banana plant bulb, observe the browning degree of bulb, evaluate the virulence of test strains, statistics disease index.
Evaluate the virulence of test strains, carry out according to following grade scale: 0 grade, bulb is without brown stain; 1 grade, bulb brown stain, bulb browned area accounts for less than 20% of the bulb total area; 2 grades, bulb brown stain, bulb browned area accounts for 20% to 40% (not containing 20%, containing 40%) of the bulb total area; 3 grades, bulb brown stain, bulb browned area accounts for 40% to 60% (not containing 40%, containing 60%) of the bulb total area; 4 grades, bulb brown stain or plant to be measured death, wherein bulb browned area accounts for more than 60% (not containing 60%) of the bulb total area.
The calculation formula of disease index is: disease index=100 × ∑ (banana plant morbidity progression × corresponding strain number)/(banana plant fall ill highest number × total strain number).
Statistics shows (Fig. 6), the Brazilian any of several broadleaf plants disease index (3.0) of inoculation Δ fogac is significantly lower than the Brazilian any of several broadleaf plants disease index (55.0) of inoculation B2 bacterial strain, and the Brazilian any of several broadleaf plants disease index of the Brazilian any of several broadleaf plants disease index (50.3) and inoculation B2 bacterial strain of inoculating Δ fogac+fogac is without significant difference (55.0).Statistics also shows (Fig. 7), the disease index (3.2) of the dwarf banana of inoculation Δ fogac is significantly lower than the dwarf banana disease index (28.7) of inoculation B2 bacterial strain, and the dwarf banana disease index (28.7) of the disease index (26.5) and inoculation B2 bacterial strain of inoculating Δ fogac+fogac is without significant difference.Visible, due to the disappearance of fogac gene, cause the virulence of Δ fogac to Brazilian any of several broadleaf plants or dwarf banana significantly to decline, Δ fogac+fogac can make the virulence of Δ fogac recover, and obtains the toxicity equal with B2 bacterial strain.
Claims (10)
1. the fogac protein in sequence table shown in sequence 3 is regulating and controlling Fusarium oxysporum Cuba specialized form to the application in banana plant virulence.
2. regulate and control Fusarium oxysporum Cuba specialized form to the method for banana plant virulence, comprise the steps (a1) or (a2) or (a3):
(a1) level of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled;
(a2) expression level of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled;
(a3) transcriptional level of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is regulated and controled.
3. screen a method for Fusarium oxysporum Cuba specialized form sterilant, comprise the steps (b1) or (b2) or (b3):
(b1) treat detection material with the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, by the material to be detected Fusarium oxysporum Cuba specialized form sterilant alternatively of suppression described fogac protein obtained;
(b2) treat detection material with the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, by the material to be detected Fusarium oxysporum Cuba specialized form sterilant alternatively that the described encoding gene of suppression obtained is expressed;
(b3) treat detection material with the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form for target spot to screen, the material to be detected that described for the suppression obtained encoding gene is transcribed Fusarium oxysporum Cuba specialized form sterilant alternatively.
4. a method for qualification or assistant identification Fusarium oxysporum Cuba specialized form candidate anti-microbial agents, comprises following (c1) or (c2) or (c3):
(c1) level that can test substance reduce the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is detected, as as described in determinand mass-energy reduce as described in fogac protein described in Fusarium oxysporum Cuba specialized form level, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not reduce as described in fogac protein described in Fusarium oxysporum Cuba specialized form level, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate;
(c2) expression that can test substance suppress the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form is detected, as as described in determinand mass-energy suppress as described in encoding gene described in Fusarium oxysporum Cuba specialized form expression, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not suppress as described in encoding gene described in Fusarium oxysporum Cuba specialized form expression, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate;
(c3) detect test substance and can suppress transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table in Fusarium oxysporum Cuba specialized form, as as described in determinand mass-energy suppress as described in the transcribing of encoding gene described in Fusarium oxysporum Cuba specialized form, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in candidate, as described in test substance can not suppress as described in the transcribing of encoding gene described in Fusarium oxysporum Cuba specialized form, then as described in test substance be Fusarium oxysporum Cuba specialized form sterilant as described in non-candidate.
5. there is d1) and/or d2) and/or d3) application of material in preparation Fusarium oxysporum Cuba specialized form sterilant of function:
D1) the fogac protein level shown in sequence 3 in sequence table is reduced;
D2) expression of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced;
D3) transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced.
6. there is e1) and/or e2) and/or e3) application of material in the banana plant of preparation anti-Fusarium oxysporum Cuba specialized form of function:
E1) the fogac protein level shown in sequence 3 in sequence table is reduced;
E2) expression of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced;
E3) transcribing of the encoding gene of the fogac protein shown in sequence 3 in sequence table is reduced.
7. as the method as described in arbitrary in claim 2 to 4, or the application described in claim 5 or 6, is characterized in that: described encoding gene is the DNA molecular shown in sequence in sequence table 2.
8. apply as claimed in claim 1, the arbitrary described method of claim 2-4, or in claim 6-7, arbitrary described application, is characterized in that: described banana is following f1)-f5) in any one: f1) Musaceae plant; F2) Brazilian any of several broadleaf plants; F3) Brazilian any of several broadleaf plants kind Musaspp.AAAgroupcv.Brazil; F4) dwarf banana; F5) dwarf banana kind Musaspp.AABgroupcv.Fenjiao.
9. prepare the method for recombinant bacterium for one kind, comprise the steps: the encoding gene knocking out the fogac protein shown in sequence 3 in the sequence table in the genome of Fusarium oxysporum Cuba specialized form, obtain the recombinant bacterium of virulence lower than described Fusarium oxysporum Cuba specialized form.
10. a specific fragment, containing upstream homology arm and downstream homology arm; The nucleotides sequence of described upstream homology arm is classified as the sequence 1 of sequence table from the DNA molecular shown in 5' end the 233rd to the 776th; The nucleotides sequence of described downstream homology arm is classified as the sequence 1 of sequence table from the DNA molecular shown in 5' end the 3213rd to the 4082nd.
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| CN113584077A (en) * | 2021-08-31 | 2021-11-02 | 西南大学 | Silencing vector for silencing cupula mori key pathogenic gene CsGPA1 and application and method thereof |
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