CN103451204B - Chestnut epidemic disease bacterium virulence gene P5Cdh and application thereof - Google Patents

Abstract

The invention discloses a kind of the chestnut epidemic disease bacterium virulence gene P5Cdh and the application thereof that affect fungus virulence and produce spore.This gene contains 3 exons and 2 introns, DNA total length 2270bp, cDNA total length 1782bp, 594 amino acid of encoding, and the protein of expression is as shown in SEQ.ID.No.3, and this albumen can be used for regulating and controlling the pathogenesis of chestnut epidemic disease bacterium.Experiment proves, the deletion mutant that this gene obtains after being replaced by hygromycin gene hph forms the pathogenic spot that the spot that causes a disease extremely significantly is less than wild-type generation in vitro Chinese chestnut branch, and namely the disappearance of gene P5Cdh causes the virulence of chestnut epidemic disease bacterium to be lost.The gene P5Cdh that the present invention relates to and expression vector, host etc. control in research significant at control of plant disease medicine and chestnut epidemic disease.Hypovirus suppresses P5Cdh genetic transcription after infecting chestnut epidemic disease bacterium, the albumen of this genes encoding may be the target that hypovirus infects rear effect, and influencing each other of the hypovirus that the present invention relates to and this gene has great importance for studying the interactional molecular mechanism of virus-host.

Description

Chestnut epidemic disease bacterium virulence gene P5Cdh and application thereof

Technical field

The invention belongs to microbiological genetic engineering and plant protection art, particularly relate to a kind of the chestnut epidemic disease bacterium virulence gene P5Cdh and the application thereof that affect fungus virulence and produce spore.

Background technology

Chestnut epidemic disease bacterium (Cryphonectria parasitica) is the pathogenic bacteria causing chestnut epidemic disease, and classification belongs to the filamentous fungus in ascomycetes, has become now a pattern bacterium of research plant pathogenic fungi pathogenesis.

At present, about 70% ~ 80% Plant diseases is all caused by plant pathogenic fungi, and the infection processs of research pathogenic fungi is the basis of further investigation pathogenic fungi pathogenic molecular mechanism.The process infecting host according to pathogenic fungi recognizes that pathogenic fungi virulence factor mainly contains with Disease-causing gene: (1) adhere to host surfaces with plant pathogenic fungi relevant, and as esterase and at, fungi changes the characteristic on host surface by discharging these enzymes; (2) relevant with plant pathogenic fungi Infection structure, as appressorium and infect nail, fungi helps invade and set up family planning relation with host by producing the Infection structure formed by the mycelia of specialization; (3) relevant with intrusion, it is invaded by natural aperture that some pathogenic fungi penetrates, and as pore or wound, the invasion of some pathogenic fungi then directly can penetrate plant surface; (4) invade that the pathogenic fungi of host is corresponding will carry out detoxification to the defensive substance that host produces, also produce a series of degrading enzyme and virose meta-bolites destruction Sum decomposition host tissue simultaneously, and therefrom obtain moisture and nutritive ingredient, reach further in the effect of host's Colonization inside plants, as cell wall degrading enzyme (polygalacturonase, cellulase, hemicellulase, lignin-degrading enzymes) and cytolemma and cell content degrading enzyme (proteolytic enzyme, amylase, lipase).By analyzing virulence factor, being separated and cloning the pathogenic molecular mechanism that Disease-causing gene in depth studies pathogenic fungi, lasting for formulation, effective disease management strategy is significant.

At home, the main prevention and controls of existing endothia parasitica is integrated control, comprises breeding resistant variety, roots out pathogeny and scab medication.Medication mainly relies on chemical agent, but a large amount of uses of chemical pesticide can bring the negative impact such as environmental pollution, ecological damage, and biological control more and more comes into one's own.Qualification Disease-causing gene, further investigate its pathogenesis, be the target gene of biological Pesticide design candidate, especially the selected of environment-friendly high-efficiency sterilant design target has important directive significance.

Summary of the invention

The technical problem to be solved in the present invention is to provide a kind of chestnut epidemic disease bacterium virulence gene P5Cdh and application thereof, and this gene plays an important role in the mechanism of causing a disease of chestnut epidemic disease bacterium, significant to the target agent of screening control of plant disease.

For solving the problems of the technologies described above, the present invention is by the following technical solutions: chestnut epidemic disease bacterium virulence gene P5Cdh, there is the base sequence of sequence table SEQ .ID.No.1, by 2270 based compositions, first exon of what 3 exons and 2 introns laid respectively at SEQ ID No:1 from 5 ' the 1-180 bit base held is this genomic gene, from second exon that 5 ' the 254-361 bit base held is this genomic gene, from the 3rd exon that 5 ' the 438-1931 bit base held is this genomic gene, be the First Intron of this genomic gene from 5 ' the 181-253 bit base held, from second intron that 5 ' the 362-437 bit base held is this genomic gene, being this gene start codon ATG from 5 ' the 1-3 bit base held, is the terminator codon TGA of this genomic gene from 5 ' the 1929-1931 bit base held, from the 3 ' non-coding region that the 1932-2270 bit base of 3 ' end is this genomic gene, or there is the base sequence of polynucleotide SEQ.ID.No.3 aminoacid sequence.

There is more than 50% homology and the base sequence of identical function protein of encoding with above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh.

Above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh or its disappearance, sudden change or the application of gene in switchboard Cryphonectria Parasitica causes a disease after modifying.

With the primer of base sequence design in arbitrary region in above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh.

Above-mentioned primer by pcr amplification for detecting the expression of P5Cdh gene under compound treatment situation.

The cDNA of above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh, has the base sequence of sequence table SEQ .ID.No.2, by 1782 based compositions; Or there is the base sequence of polynucleotide SEQ.ID.No.3 aminoacid sequence.

The protein that above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh encodes or its homologous protein, have sequence table SEQ .ID.No.3, be made up of 594 amino acid; Or SEQ.ID.No.4(rice blast fungus (Manapothia oryzae) homologous protein, consistence 73%), SEQ.ID.No.5(Fusarium oxysporum (Fusarium oxysporum) homologous protein, consistence 71%) aminoacid sequence.

The protein that above-mentioned chestnut epidemic disease bacterium virulence gene P5Cdh encodes or its homologous protein, aminoacid sequence is through being no more than the replacement of 10 amino-acid residues and or lacking and or add.

Above-mentioned protein or its homologous protein are the application of target in design and screening antifungal drug.

With above-mentioned protein or the polypeptide having arbitrary region amino acid sequence design in more than 40% conforming homologous protein with it.

Aforementioned polypeptides Dispersal risk is for detecting the expression of P5Cdh albumen under compound treatment situation.

The method for the treatment of chestnut epidemic disease, the expression of the P5Cdh of blocking-up or suppressing plate Cryphonectria Parasitica.

Chestnut epidemic disease bacterium coding 5-carboxyl-Δ provided by the present invention ¹-pyrroline dehydrogenase (Δ ¹-pyrroline-5-carboxylate dehydrogenase) albumen, name is called P5Cdh, derives from chestnut epidemic disease bacterium (Cryphonectria parasitica) EP155 bacterial strain (ATCC 38755).

The cluster analysis of homologous protein shows, P5Cdh and plant pathogenic fungi rice blast fungus sibship are recently (homology 70.4%), be Fusarium oxysporum (homology is 69.2%), sclerotinite (homology is 69.0%) afterwards, Aspergillus fumigatus and Penicllium chrysogenum (homology is respectively 62.8% and 62.0%) (Fig. 1), show that P5Cdh albumen may perform some identical basic functions in pathomycete.In order to identify P5Cdh protein function, P5Cdh albumen is carried out prokaryotic expression and Enzyme activity assay, be reaction substrate to recombinant protein with P5C, NAD is terminal electron acceptor, accepts the electronics that P5C transmits generate NADH to detect the kinetic constant of enzyme according to detecting a series of concentration gradient NAD.K is drawn according to two counting backward technique _mvalue is 1.48 ± 0.145mM and V _maxvalue is 1.69 ± 0.110 μm of ols ^-1, k _cat=0.77 ± 0.050s ^-1(as Fig. 2).In order to study the function of P5Cdh in chestnut epidemic disease bacterium, construct the deletion mutant Δ P5Cdh of P5Cdh with efficient homologous recombination system Δ ku80 for starting strain by methods of homologous recombination and carried out have complementary functions (Fig. 3), through Southern blot, the mutant of structure verifies that gene P5Cdh is replaced by hygromycin gene.Mutant Δ P5Cdh is inoculated into PDA slat chain conveyor and observes phenotype, this Strain phenotypes and EP155 and Δ ku80 are without significantly different, and pigment increases (Fig. 4); Mutant Δ P5Cdh is inoculated into Chinese chestnut branch and carries out virulence detection, what Δ P5Cdh produced causes the pathogenic spot (Fig. 4) that Lesion size is significantly less than EP155 and Δ ku80, and visible genetic P5Cdh is chestnut epidemic disease bacterium virulence genes involved.The sporulation quantity (2.51 × 10 of mutant Δ P5Cdh ⁷± 1.03 × 10 ⁶individual/mL) and EP155(2.08 × 10 ⁷± 3.38 × 10 ⁶individual/mL) and Δ ku80(1.79 × 10 ⁷± 1.63 × 10 ⁶individual/mL) without significant difference (Fig. 5).Hypovirus infects mutant Δ P5Cdh not only makes bacterial strain bleach and not produce spore, and colony growth is significantly suppressed; And the transcribing (Fig. 6) of hypovirus suppressor gene P5Cdh.Based on the above results, the disappearance demonstrating P5Cdh gene causes the virulence of chestnut epidemic disease bacterium significantly to reduce.Illustrate that P5Cdh gene is the necessary gene of chestnut epidemic disease bacterium mechanism of causing a disease.Therefore, the compound that screening can stop this genetic expression and its protein expression, modify and locate, can the virulence of effective switchboard Cryphonectria Parasitica, directly can utilize as the new drug candidates thing of new type bactericide.That is, an important use of P5Cdh provided by the present invention is, the expression of this gene and its protein expression, modify and locate can as important target site be used for antifungal medicine (particularly the medicament of anti-chestnut epidemic disease bacterium) screening and in designing.The mechanism of causing a disease that this gene of further parsing participates in, therefrom also can find that candidate's target site is in the screening of antifungal medicine (particularly anti-chestnut epidemic disease bacterium medicament) and design.In addition, also can be separated this sequence as probe or as the basis of PCR primer design again using a certain section of this gene nucleotide series in chestnut epidemic disease bacterium, also may be used for screening, be separated other fungi with this gene, there is the sequence of certain sequence homology.

Present invention demonstrates that hypovirus infects causes the transcriptional level of P5Cdh gene to decline.Illustrate that P5Cdh gene is the potential target gene that low virus function chestnut epidemic disease bacterium makes its virulence decline.An important use of P5Cdh provided by the present invention is, this gene transcribe with its protein expression, modifying and locate can as important target site for the screening of antiviral agent (particularly the medicament of antimycotic virus) with in designing.The mechanism of causing a disease that this gene of further parsing participates in, therefrom also can find in the screening that mycovirus are used for antifungal medicine (particularly anti-chestnut epidemic disease bacterium medicament) and design.

The candidate anti-microbial agents applying qualification of the present invention or assistant identification chestnut epidemic disease bacterium can be adopted with the following method: be that target spot screens detection material with foregoing proteins, by the sterilant of the material to be detected chestnut epidemic disease bacterium alternatively of protein expression shown in the suppression sequence that obtains; Or be that target spot screens detection material with foregoing proteins, by the sterilant of the material to be detected chestnut epidemic disease bacterium alternatively of protein transcription shown in the suppression sequence that obtains; As do not suppressed, then test substance is the chestnut epidemic disease bacterium sterilant of non-candidate.

Accompanying drawing explanation

Fig. 1 is the cluster analysis figure of homologous protein in P5Cdh and other plant pathogenic fungi.

Fig. 2 is prokaryotic expression and the Enzyme activity assay lab diagram of P5Cdh.Wherein,

A is protein electrophoresis figure: the albumen P5Cdh Δ 45 obtained after GST sepherose resin purifying.

B is the movable mechanics graphic representation of enzyme: Lineweaver – Burk double-reciprocal plot method analyzes the kinetic constant V of recombinant protein GST-P5Cdh Δ 45 _maxand K _m; P5Cdh Activity determination 30 DEG C with 0-5mM NAD for substrate, measure the concentration of product NADH in 443nm, data presentation is the mean number repeating for three times to test.

Fig. 3 be P5Cdh gene knock out research experiment figure.Wherein,

A is that gene knockout box builds schematic diagram: left arm just, reverse primer is respectively P5Cdh-A (5 '-atttagccgtcgtgacatcttcctc-3 ') and P5Cdh-3 (5 '-tctttctagaggatccccgggtaccggattggatggattgagctgt-3 '), right arm just, reverse primer is respectively P5Cdh-2 (5 '-atatcatcttctgtcgacctgcaggctatccgagcaatgaggtctg-3 ') and P5Cdh-B (5 '-ggtgtgagagagaaggtggtcgttc-3 '), amplification template is EP155 STb gene, amplification object clip size is respectively 974bp and 1038bp.Hygromycin phosphotransferase gene (hph) forward and reverse primer is respectively hph-F (5 '-cggtacccggggatcctctag-3 ') and hph-R (5 '-gcctgcaggtcgacagaagatg-3 '), and amplification template is plasmid pCPXHY2.The pcr amplified fragment of left and right two-arm and Hph passes through the method for fusion PCR, left and right two-arm is fused to the both sides of hph respectively according to upstream and downstream order, form gene substitution and knock out box, the hph of 2145bp displacement be the functional domain of the 3rd this region of exon region 1454bp(of P5Cdh gene amino-acid residue of deducing containing complete ALDH_F4-17_P5CDH) respectively have Hind III restriction enzyme site being about 1.5kb upstream and 3 ' UTR place from first exon.

B is the structure electrophorogram of Southern blot witness plate Cryphonectria Parasitica P5Cdh deletion mutant body: use the left arm fragment that probe probe1 increase for primer P5Cdh-A/P5Cdh-3, the hph promoter region that use probe probe2 increases for primer hphF/hphR (5 '-agtagatggatccatccact-3 ').After STb gene Hind III enzyme of wild-type EP155, starting strain Δ ku80, mutant Δ P5Cdh and Δ P5Cdh-com is cut, electrophoresis order is as follows: M:GeneRuler1Kb Ladder; 1:EP155; 2: Δ ku80; 3: Δ P5Cdh; 4: Δ P5Cdh-com.As shown in A figure, after the 3rd the exon region 1454bp of gene P5Cdh is replaced by hph2145bp, probe1 is hybridized Δ P5Cdh band and is increased to 4157bp by the 3466bp of hybridizing EP155, Δ ku80 and Δ P5Cdh-com; Probe2 hybridizes EP155 and Δ ku80 without band, and hybridization Δ P5Cdh band is 4157bp.

Fig. 4 is gene-deleted strain bacterium colony phenotype and the ulceration lab diagram of P5Cdh gene.

In figure, by EP155, Δ ku80, EP713, Δ P5Cdh and Δ P5Cdh-com in the PDA illumination cultivation bacterium colony phenotype of 14 days; After EP155, Δ ku80, EP713, Δ P5Cdh and Δ P5Cdh-com being inoculated dormancy Chinese chestnut branch, place 30 days in 25 DEG C of moisturizings, branch ulceration is observed.

Fig. 5 is the gene-deleted strain sporulation quantity comparison diagram of P5Cdh gene.

In figure, the illumination cultivation bacterium colony conidium of 14 days is collected, counts with blood counting chamber.。

Fig. 6 is that hypovirus infects the gene-deleted strain phenotype of P5Cdh gene and the transcriptional level lab diagram of hypovirus suppression P5Cdh gene.Wherein,

A is bacterium colony phenotype comparison diagram: after EP713 and Δ P5Cdh Hyphal anastomosis, the Δ P5Cdh away from EP713 is connected to the PDA illumination cultivation bacterium colony phenotype of 7 days.

B is transcriptional level comparison diagram: the total serum IgE extracting EP155 and EP713, reverse transcription synthesis cDNA first chain, the cDNA of primer P5Cdh-qf (5 '-ttctacatcaactgcaagag-3 ') and P5Cdh-qr (5 '-actcttccttcatggtcctc-3 ') amplification P5Cdh gene, after being corrected by CP18S rDNA, compare the relative transcript levels of the P5Cdh gene of EP155 and EP713.

Embodiment

Further describe the present invention below by way of embodiments and drawings, wherein, experimental technique if no special instructions, is ordinary method; Experiment material and reagent if no special instructions, all obtain by commercial sources; Percentage composition if no special instructions, is mass percentage.

The cluster analysis of homologous protein in embodiment 1, P5Cdh and other fungi

By the aminoacid sequence of P5Cdh input GenBank(http: //www.ncbi.nlm.nih.gov) carry out BlastP retrieval, obtain the aminoacid sequence of multiple species homologous protein.JGI344979 is chestnut epidemic disease bacterium P5Cdh albumen, the homologous protein of P5Cdh albumen in other fungi is: XP_003718931 is from rice blast fungus (Magnaporthe oryzae), EGU80836 is from Fusarium oxysporum (Fusarium oxysporum), XP_750764 is from Aspergillus fumigatus (Aspergillusfumigatus), CAP80517 is from Penicllium chrysogenum (Penicillium chrysogenum), and XP_001596907 is from sclerotinite (Sclerotinia sclerotiorum).Use the adjacent method (Neighbor-joining) of MEGA4.0 software to build evolutionary tree, achievement process choosing bootstrap checks 1000 times, shows with TREEVIEW.As shown in Figure 1, the conservative property that this albumen of cluster analysis of homologous protein is evolved in P5Cdh and other plant pathogenic fungi and sibship (Fig. 1), nearest with Fusarium oxysporum sibship, homology is 69.2%, nearer with sclerotinite sibship, homology is 69.0%, comparatively far away with rice blast fungus sibship, homology is 70.4%, and Aspergillus fumigatus and Penicllium chrysogenum are polymerized to cluster, with the sibship of chestnut epidemic disease bacterium farthest, homology is respectively 62.8% and 62.0%.

The prokaryotic expression of embodiment 2, P5Cdh and Enzyme activity assay

1) prokaryotic expression of P5Cdh: predict that P5Cdh signal peptide is 1-45aa with software MitoPro II, as 1 of embodiment 4) and 3) prepare the total serum IgE of EP155, synthesis cDNA first chain, be cloned in the cloning site of the Hind III/Sal I of the prokaryotic expression carrier pGEX-4T-1 containing GST label by the cDNA sequence of primer P5Cdh-f-Hind III (5 '-gcgaagcttatggcttctcgcagggtcagtctcc-3 ') and P5Cdh-r-Xba I (5 '-gcctctagagacctcattgctcggatactcgac-3 ') amplification P5Cdh deleted signal peptide (1-45aa), obtain recombinant expression plasmid pGEX-4T-P5Cdh Δ 45.Recombinant plasmid is imported to the competent cell of escherichia coli expression host BL21 by chemical transformation, coat on the LA flat board containing 100 μ g/mL ampicillin and carry out recombinant screen.The transformant obtained is after qualification is cut in electrophoretic analysis and enzyme, abduction delivering is carried out to the BL21 carrying pGEX-4T-P5Cdh Δ 45: by activation overnight culture by 1% inoculum size enlarged culturing in contain 50 μ g/mL ampicillin LB substratum in, 37 DEG C of 200rpm are cultured to OD ₆₀₀reach 0.4; Adding IPTG to final concentration is 0.3mM, culture is gone to 20 DEG C of 200rpm and continues cultivation after 6 hours, can collected by centrifugation thalline.Add the resuspended thalline of 10ml binding buffer by 1g thalline, adding lysozyme to final concentration is that 0.2mg/ml carries out 4 DEG C of slowly vibration 30min enzyme digestion reactions, within centrifugal 20 minutes, removes cell debris in 4 DEG C of 10000rpm.Slowly joined by supernatant liquor in Glutathione Sepharose resin prepacked column, flow rate control, at 1ml/min, after again adding 10ml binding buffer rinsing foreign protein, adds 1-2 times of column volume elution buffer and carries out wash-out.Target protein predicted molecular weight is about 64KD, and GST label protein is about 26KD, then fusion rotein size is expected to be 90KD.Eluted protein is carried out SDS-PAGE electrophoretic analysis display object band slightly larger than 90KD, with expection (accompanying drawing 2A) in the same size.Do not need excision GST label, directly carry out Enzyme activity assay.

2) Enzyme activity assay of P5Cdh: add 10 times of volume enzyme liquid preservation buffer to target protein and dilute, after concentrated with Amicon10KD super filter tube, be about 198 μ g/ μ l by Bradford method Quantitative Western concentration, namely 2.2 μMs.Enzyme reaction alive is reaction substrate with P5C, and NAD is terminal electron acceptor, and the electronics accepting P5C transmission according to a series of concentration gradient NAD of detection generates NADH, survey OD ₃₃₀value.Specific absorbance is recorded for 6.22cm with standard substance NADH preparation standard curve ^-1mM ^-1.Getting speed of reaction V 1/V reciprocal is ordinate zou, with substrate proline concentration [S] 1/ [S] reciprocal for X-coordinate mapping, draws K according to two counting backward technique _mvalue is 1.48 ± 0.145mM and V _maxvalue is 1.69 ± 0.110 μm of ols ^-1, k _cat=0.77 ± 0.050s ^-1(as accompanying drawing 2B).

Embodiment 3, the functional study of P5Cdh gene in chestnut epidemic disease bacterium

1) structure of box is knocked out

Gene knockout adopts the method for homologous recombination, and replace the coding region of P5Cdh gene in the efficient homologous recombination system of chestnut epidemic disease bacterium with hygromycin phosphotransferase gene, specific strategy is shown in accompanying drawing 2A.The forward and reverse primer of left arm is respectively P5Cdh-A (5 '-atttagccgtcgtgacatcttcctc-3 ') and P5Cdh-3 (5 '-tctttctagaggatccccgggtaccggattggatggattgagctgt-3 '), the forward and reverse primer of right arm is respectively P5Cdh-2 (5 '-atatcatcttctgtcgacctgcaggctatccgagcaatgaggtctg-3 ') and P5Cdh-B (5 '-ggtgtgagagagaaggtggtcgttc-3 '), amplification template is EP155 STb gene, and amplification object clip size is respectively 929bp and 1058bp.Hygromycin phosphotransferase gene (hph) forward and reverse primer is respectively hph-F (5 '-cggtacccggggatcctctag-3 ') and hph-R (5 '-gcctgcaggtcgacagaagatg-3 '), and amplification template is plasmid pCPXHY2.By the method for fusion PCR, left and right two-arm is fused to respectively the both sides of hph according to upstream and downstream order, form gene substitution and knock out box, the hph of 2145bp displacement be the functional domain of the 3rd this region of exon region 1454bp(of P5Cdh gene amino-acid residue of deducing containing complete ALDH_F4-17_P5CDH).

The Fusion PCR method adopted in the present invention is as follows:

Reclaim upstream and downstream fragment and resistant gene respectively and the ratio of 1 ︰ 3 ︰ 1 carries out fusion DNA vaccine reaction in molar ratio.Fusion DNA vaccine reaction conditions is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30sec, 58 DEG C of annealing 10min, 72 DEG C extend 4min, carry out 15 circulations altogether; Last 72 DEG C of reaction 10min.To dilute the fusion DNA vaccine product 1 μ l after 10 times for template, with primer P5Cdh-A/P5Cdh-B conveniently PCR method increase in a large number.The PCR primer obtained is 4132bp, and being concentrated into concentration through alcohol settling is 1-2 μ g/ μ L, can be directly used in fungal transformation.

2) the structure primer P5Cdh-F (5 '-tgctcaccttgctgctcttggaagt-3 ') of complementary recombinant plasmid and P5Cdh-B (5 '-ggtgtgagagagaaggtggtcgttc-3 ') amplification comprises the full length gene 4343bp of promoter region 1.5kb and terminator region 1kb, and is cloned into Sma I place, flush end site of the complementing vector containing G418 resistant gene.

3) chestnut epidemic disease bacterium conversion

In the present invention, chestnut epidemic disease bacterium conversion adopt CaCl ₂the method of the conversion fungal protoplasts of/PEG mediation, preparation and the method for transformation of protoplastis are specific as follows:

A. the preparation of protoplastis

Be equipped with solution 0.6M MgSO ₄, 1M Sorbitol, OM soluiton(1.2M MgSO ₄, 10mM NaH ₂pO ₄, pH5.8), Trapping Buffer(0.4M Sorbitol, 0.1M Tris-HCl, pH7.0), STC(1M Sorbitol, 0.1M Tris-HCl pH8.0,0.1M CaCl ₂), PTC(40%PEG3350,0.1M Tris-HCl, 0.1M CaCl ₂, pH8.0) and be stored in 4 DEG C after autoclaving.

To preserve strain inoculation dull and stereotyped to PDA, room temperature illumination cultivation a couple of days, to colony radius 2cm, scrapes the mycelia that takes a morsel in 100ml EP perfect medium, can be used for preparing protoplastis after 25 DEG C of quiescent culture 3d.Now be equipped with enzymolysis solution, add cellulase 0.5g, helicase 0.3g and N,O-Diacetylmuramidase 0.2g in 50mLOM soluiton, stand-by after filtration sterilization.Collected by centrifugation thalline, adds 30mL0.6M MgSO ₄rinsing thalline twice, add 50mL enzymolysis solution digestion somatic cells wall, after 28 DEG C of 200rpm reaction 3-4h observe mycelium formation protoplastis to microscopy, be dispensed in 50ml corning pipe by every pipe 12.5ml enzyme digestion reaction liquid, liquid level slowly adds the Trapping Buffer of 2 times of volumes, after centrifugal 30 minutes, draws protoplastis from two-layer liquid level intersection carefully with pasteur pipet for 3500g4 DEG C, and be transferred in new corning pipe, put on ice; Add 2 times of volume 1M Sorbitol rinsing protoplastiss; Repeat once; With 15ml STC rinsing protoplastis once; Count protoplastis with blood counting chamber, with STC:PTC in the resuspended protoplastis of the ratio of 4:1, add DMSO to final concentration is 1% simultaneously, makes protoplast concentration be 8 × 10 ⁷-1 × 10 ⁸individual/more than ml; Divide by every pipe 100 μ l and be filled to EP pipe, be placed in-80 DEG C and save backup.

B. the conversion of chestnut epidemic disease bacterium

Be equipped with regeneration culture medium: containing Casein enzymatic hydrolysate0.2g, yeast extracts0.2g, surcose68.4g, Bacto in 200ml regeneration culture medium ^tMagar3.2g, autoclaving after heating for dissolving, 3h heating for dissolving before conversion, is put in 46 DEG C of incubations stand-by after being cooled to 50 DEG C.

Get DNA and the 1 μ l spermidine(100mM of 2-5 μ g purifying) mix after, add in 100 μ l protoplastiss, mix gently, ice bath 30min; Add 1ml PTC again to mix, room temperature places 25 minutes; 7000g4 DEG C of centrifugal 5min; Abandon supernatant, add 500 μ l STC and to suspend gently precipitation, 5000g4 DEG C of centrifugal 1min; Abandon supernatant, again add 500 μ l STC and to suspend gently precipitation, evenly put in the culture dish of cleaning sterile by 170 μ l/ wares, get 12ml regeneration culture medium and cover, and shake up gently; After cultivating 18-24 hour in 28 DEG C, add 12ml and cover upper strata containing antibiotic regeneration culture medium, within incubated at room temperature 3-4 days, grow to bacterium colony.

C. the purifying of resistant transformants

From regeneration culture medium, first the transformant of picking through the resistance screening of 2-3 wheel, will be made it have stable antibiotics resistance, is then carried out the purifying of bacterial strain by single spore separation or protoplast regeneration.The primary process of single spore separation is: be first inoculated in by transformant on the PDA flat board of antibiotic-free, within about illumination cultivation 14-21 days, it is made to produce spore, lower spore is washed with sterilized water or 0.02%Tween80, be applied to containing on antibiotic PDA substratum after diluting different concentration, incubated at room temperature 3 days, picking list bacterium colony detects.

D. the extraction of STb gene

Take 1g thalline and put into mortar, add enough liquid nitrogen fully to cool, by thalline grind into powder, add 5ml STb gene extraction buffer(0.1M Tris-Cl [pH8.0] successively, 0.2M NaCl, 4mM EDTA [pH8.0], 2%SDS) and the saturated phenol of 5ml Tris, constantly stir with grinding pestle in the process of melting and make it fully act on.The mixture of thawing is proceeded in 30mL Corning pipe, the centrifugal 40min of 3000g.Sucking-off supernatant, adds isopyknic phenol/chloroform, each extracting of isopyknic chloroform once.Gone to by supernatant liquor in EP pipe, add the Virahol of 0.7 times of volume, after mixing, centrifugal 8 minutes of room temperature 10000g, outwells supernatant liquor, washes precipitation 2-3 time, naturally dry, add 100 μ lddH with precooling 75% ethanol ₂o or TE dissolves.

e.Southern blot

The enzyme of chestnut epidemic disease bacterium STb gene is cut, electrophoresis and transferring film

Get the genomic dna of 30-50 μ g, add Hind III and RNaseA, reaction cumulative volume 400 μ l, after 37 DEG C of enzymes cut 8-16h to electrophoresis detection complete degestion, alcohol settling, is dissolved in 50 μ l ddH ₂in O; The sepharose being splined on 1.0% is separated digestion products in the buffer system of 0.5 × TBE.Pour depurination solution (0.25N HCl) into not having gel, slowly concussion 15min turns yellow to bromjophenol blue, ddH ₂o rinsing 2 times; Pour sex change liquid (0.5M NaOH, 1.5M NaCl) into not having gel, slowly concussion 15min becomes blue to bromjophenol blue, repeats once, ddH after removing sex change liquid ₂o rinsing 2 times; Pour neutralizer (1M Tris-Cl, 1.5M NaCl, pH8.0) into not having gel, the neutralizer renewed after slow concussion 15min slowly shakes 15min again, removes neutralizer, adds 20 × SSC(0.3M Trisodium Citrate, 3M NaCl) to not having gel, slowly concussion more than 10min.

Prepare the nylon membrane 1 onesize with gel and filter paper 3, carry out mark with pencil in the front of film.

Get the container being greater than gel and load 20 × SSC, a sheet glass is put in top, getting a long filter paper puts on a glass, thieving paper two ends are also completely moistening from sheet glass hangs down immersion 20 × SSC, be salt bridge, lay gel, nylon membrane, filter paper, thieving paper tower, about 1kg weight successively on salt bridge, gel DNA is upwards transferred to the nylon membrane used time by capillary action and is about 8h, take off nylon membrane, DNA faces up and puts into UV-crosslinked instrument, with 1200J, 3min uv irradiating fixed dna, nylon membrane dries stand-by.Can wait and dry as carried out hybridization immediately and can directly use.

The preparation of probe

Get in 1kb ladder marker to the 1.5mLEP pipe of 15 μ l template DNA to be marked (0.3-1 μ g) and 1 μ l50ng/ μ l, boiling water bath 10min makes DNA sex change, rapidly in cooled on ice; Add 4 μ l DIG-High prime in denatured DNA, fully mix, 37 DEG C of temperature bath 1h, the time, longer labeling effciency was higher, but did not exceed 20h.65 DEG C of incubation 10min stopped reaction.

Prehybridization: nylon membrane DNA is faced up and puts into hybridization cylinder, by every 100cm ²nylon membrane adds the 10mL of 42 DEG C of preheatings, and 42 DEG C are rotated at least 30min.

Hybridization: the 1 μ l DNA probe (about 25ng/mL DIG Easy Hyb) that DIG marks is added to 49 μ l ddH ₂in O, be put in ice-water bath rapidly after boiling water bath 5min sex change and cool; The probe of cooling is added the DIG Easy Hyb(3.5mL/100cm of 42 DEG C of preheatings ²nylon membrane) in and mix; Outwell prehybridization solution and add probe/hybridization solution mixed solution, rotating gently in hybrid heater, 42 DEG C of hybridization 6-20h.

Wash film: under 25 DEG C of continuous rotation conditions (hybrid heater), wash 2 times with 2 enough × SSC, 0.1%SDS, each 5min.Under 68 DEG C of continuous rotation conditions (hybrid heater), wash 2 times with 0.5 × SSC, the 0.1%SDS being preheating to 68 DEG C, each 15min.

Immunity colour developing: be 100cm below ²the immunoassay procedures of Hybond membrane, all processes are carried out at 25 DEG C, rotate gently.Add l00mLwashing buffer, shake 1-5min; Remove washing buffer, add l00mL Blockingsolution, shake 30min; Remove Blocking solution, add 20mL Antibody solution, shake 30min; Discard Antibody solution, add l00mL Washing buffer and shake 15min, repeat once; Add 20mL Detection buffer and shake 2-5min; Film is put into hybridization bag, adds lmL CPSD, room temperature reaction 5min, suck unnecessary CPSD, sealing, put into 37 DEG C of 10min activator development reactions, in darkroom, carry out X film compressing tablet, developing liquid developing after 30min, stop bath is fixing.Scanned picture is preserved.

4) what wild type strain grew with the gene-deleted strain of P5Cdh gene compares

Receive on PDA plate by EP155, Δ ku80, EP713, Δ P5Cdh and Δ P5Cdh-com, each inoculation 3-5 part, observe phenotype (Fig. 4) in 25 DEG C of illumination cultivation after 14 days, Δ P5Cdh phenotype and EP155 and Δ ku80 are without significant difference, and pigment increases.

5) the gene-deleted strain virulence test experience of wild type strain and P5Cdh gene

In the Chinese chestnut trunk that annual 1-2 month cut-off footpath is about 5cm, to be gone out by all the other withes and with otch of sealing with wax, be that 5mm punch tool and branch punch with internal diameter diameter, hole depth is about 1-2mm, and every span is about 10-12cm; Chestnut epidemic disease bacterium illumination cultivation 2-3d on PDA flat board is about 2-3cm to colony diameter, taking out bacterium block with punch tool transfers in Chinese chestnut branch new bore place, each inoculation five parts, seal with sealed membrane, keep humidity, after trunk room temperature is placed 30 days, carefully the bark around punching place is pruned, namely can be observed the ulceration that bacterium block produces, measure the length diameter of ulceration and reference area, lesion area=3.14 × scab major axis radius × scab minor axis radius.The Lesion size that causes of Δ P5Cdh is significantly less than the pathogenic spot (Fig. 4) that EP155 and Δ ku80 produce.

6) the comparing of gene-deleted strain sporulation quantity of wild type strain and P5Cdh gene

Receive on PDA plate by EP155, Δ ku80, EP713, Δ P5Cdh and Δ P5Cdh-com, each inoculation 3-5 part, in 25 DEG C of illumination cultivation after 14 days, conidium is rinsed, in counted under microscope by every ware 10ml0.02%Tween80.Each bacterial strain analyzes numerical value at least three repetition.The departure spore quantity obtained is the unit surface sporulation quantity of this bacterium colony divided by area of colony.The sporulation quantity (2.51 × 10 of mutant Δ P5Cdh ⁷± 1.03 × 10 ⁶individual/mL) and EP155(2.08 × 10 ⁷± 3.38 × 10 ⁶individual/mL) and Δ ku80(1.79 × 10 ⁷± 1.63 × 10 ⁶individual/mL) without significant difference (Fig. 5).

Embodiment 4, hypovirus suppress transcribing of P5Cdh gene

1) extraction of total serum IgE: the thalline being inoculated in PDA illumination cultivation 7d is scraped from substratum, sucks excess moisture with thieving paper.Take 0.5g thalline and add enough liquid nitrogen grinding powdereds, add the equal-volume mixture of ready 5ml STb gene extraction buffer and the saturated phenol of 5ml Tris immediately, constantly stir with grinding pestle in the process of dissolving and make it fully act on.The mixture of thawing is proceeded in Corning pipe, the centrifugal 40min of 3500g.Abandon supernatant, add isopyknic phenol/chloroform, each extracting of isopyknic chloroform once.Gone to by supernatant liquor in EP pipe, add the Virahol of 0.7 times of volume, after mixing, centrifugal 8 minutes of room temperature 10000g, outwells supernatant liquor, washes precipitation 2-3 time, naturally dry, add the ddH of 100 μ l DEPC process with precooling 75% ethanol ₂o dissolves.The quality of electrophoresis detection gained total serum IgE and concentration.

2) purifying of dsRNA: remove small residue STb gene with RNase-Free DNase I (Roche) at 25-37 DEG C of digestion 15-20min, the consumption of DNase I is 1u/ μ g DNA, after phenol/chloroform and alcohol settling, is dissolved in appropriate DEPC-H ₂in O, the concentration of spectrophotometric determination RNA.Sepharose with 1.0% is at 1 × TAE(40mM Tris-HAc, 1mM EDTA, pH8.0) condition under the quality of RNA after electrophoresis detection digestion.

3) synthesis of cDNA first chain: reaction system: 0.2-2 μ g total serum IgE, 1 μ l10mM primer, add DEPC-process water and be settled to 12 μ l; Of short duration centrifugal after gentle mixing, after being placed in 70 DEG C of water-bath 5min, return ice bath cooling immediately, of short duration centrifugal after continue to add 4 μ l5 × reaction buffer, 2 μ l10mM dNTP, 1 μ l RiboLock ^tMrNaseInhibitor (20U), of short duration centrifugal after gentle mixing, after being placed in 37 DEG C of water-bath 5min, add 1 μ l RiboLock ^tMh Minus Reverse Transcriptase (200U) also mixes gently, 42 DEG C of incubation 60min, 70 DEG C of water-bath 5min termination reactions.Reaction solution is placed in-20 DEG C of preservations.

4) quantitative fluorescent PCR reaction system is as follows: dilute the first chain cDNA2 μ l, 10 × Ex Taq Buffer2.5 μ l of suitable multiple, 2.5mM dNTP1 μ l, 25mM MgCl ₂0.8 μ l, Primer1(5uM) 1 μ l, Primer2(5uM) 1 μ l, 20 × SYBG I1 μ l, Ex Taq0.1 μ l, add ddH ₂o to cumulative volume 25 μ l.Reaction system is placed in quantitative real time PCR Instrument (MJ Opticon II) and carries out PCR reaction, reaction conditions is set to: 94 DEG C of denaturations, 5min; 40-50 PCR circulation is carried out: 94 DEG C of 20sec according to following parameter; Annealing 20sec; 72 DEG C of 20sec; Read fluorescent signal (Plate read).72 DEG C, 5min, rises to 95 DEG C from 65 DEG C, often heats up 0.2 DEG C and reads first order fluorescence signal, thus carry out melting curve analysis (Melting curve analysis).Institute responds in triplicate, with C (t) mean value calculation result, is within the scope of 80-105% at amplification efficiency, uses 2 ^{-Δ Δ C (t)}method validation Relative gene differential expression.Repeat experiment through three times, result shows that in EP713, P5Cdh gene expression amount is only 8.5% of EP155, has lowered (Fig. 6) more than 10 times.

Claims (6)

1. a chestnut epidemic disease bacterium virulence gene P5Cdh, it is characterized in that the base sequence of sequence table SEQ .ID.No.1, or the base sequence of polynucleotide SEQ.ID.No.3 aminoacid sequence.

2. the application of chestnut epidemic disease bacterium virulence gene P5Cdh in switchboard Cryphonectria Parasitica causes a disease described in claim 1.

3. the cDNA of chestnut epidemic disease bacterium virulence gene P5Cdh described in claim 1, it is characterized in that the base sequence of sequence table SEQ .ID.No.2, or the base sequence of polynucleotide SEQ.ID.No.3 aminoacid sequence.

4. described in claim 1 chestnut epidemic disease bacterium virulence gene P5Cdh encode protein or its homologous protein, it is characterized in that the aminoacid sequence of sequence table SEQ .ID.No.3.

5. the protein that chestnut epidemic disease bacterium virulence gene P5Cdh described in claim 4 encodes is the application of target in design and screening antifungal drug.

6. treat a method for chestnut epidemic disease, it is characterized in that the expression of the P5Cdh of blocking-up or suppressing plate Cryphonectria Parasitica, described P5Cdh is the base sequence of sequence table SEQ .ID.No.1.