CN103724408A - Effector protein derived from phytophthora capsici as well as coding gene and application thereof - Google Patents
Effector protein derived from phytophthora capsici as well as coding gene and application thereof Download PDFInfo
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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Abstract
The invention discloses an effector protein derived from phytophthora capsici as well as a coding gene and application thereof. The protein disclosed by the invention is (a) or (b) or (c): (a) protein consisting of amino acid sequences represented in the 19th-476th sites of a sequence 1 of a sequence list; (b) protein consisting of an amino acid sequence represented in the sequence 1 of the sequence list; and (c) protein obtained by substituting and/or deleting and/or adding the amino acid sequence of the protein defined by (a) or (b) through one or more amino acid residues and having an effector function. Experiments prove that the protein disclosed by the invention effectively participates in a process that phytophthora capsici infects a pepper host and has a function of inducing plant tissue cell death and even an obvious necrotic symptom on an infected part. The effector protein disclosed by the invention is significant for enriching molecular mechanism information of plant pathogenic oomycetes and host interaction and for establishing an oomycetes disease integrated control technological strategy.
Description
Technical field
The invention belongs to biology field, relate to a kind of effector albumen and encoding gene and application that derives from Phytophthora capsici.
Background technology
Effector albumen (effector) is pathogen invading the class producing in host's process and can overcome the molecule of host's system of defense.In infection processs, pathogen is captured host's system of defense with effector albumen, and the resistibility that can weaken on the one hand host is convenient to it and is successfully invaded, and can also utilize on the other hand host's nutrient for self-reproduction.Many pathogens can be secreted effector albumen, such as all having found this proteinoid in bacterium, fungi, oomycetes and nematode.In pathogen, there is multiclass effector, comprise NLP, Avr, CRN, PcF, ScR, carbohydrate lytic enzyme, polygalacturonase, chitinase and esterase etc.Wherein NLP protein gene ubiquity in oomycetes, fungi, bacterium.This genoid is comparatively small amt in bacterium fungal gene group, and in oomycetes genome One's name is legion, mainly with gene family form, exist.This proteinoid all has NLP gene dosage and the structural performance of distribution and congeneric species pathogenic bacteria also to have very big difference, reasoning NLP protein function tool diversity feature thus between the far species of evolutionary relationship according to one's analysis.The NLP effector Argine Monohydrochloride sequence high conservative of different plant species, middle portion tool GHRHDWE motif, sequence length is between 600-800bp, and coded amino acid length is generally more than 300 amino acid.It is reported that after various plants is processed by NLP albumen, showing as ethene produces, map kinase activation, plant protecting chemical is synthetic, the gene induced expression of PR, kytoplasm Ca
2+the blade anaphylaxis of release and multiple dicotyledons etc.But also have report to indicate the susceptible factor that NLP albumen is plant, can express by inducing plant adversity gene.The expression product of for example verticillium dahliae (Verticillium dahliae) effector gene can make cotton, tobacco, Arabidopsis leaf wilt, and illustrates that effector is inducible factor important in cotton and pathogen Interaction.But to the effector albumen research discovery of two kinds of bacterium Erwinia carotovora subsp.Carotovora and E.carotovora subsp.atroseptica, by after sub-thalline internal effect gene knockout, thalline declines greatly to the toxicity that infects of potato.Although studied at present the anaphylaxis of this proteinoid to plant, the function of this proteinoid and the binding mode in plant or the unknown, more do not have effector albumen in enough evidence explanation oomycetes whether the mould toxicity of epidemic disease to be played an important role.
Phytophthora capsici (Phytophthora capsici) belongs to Oomycete, is that class one zone is not in the eukaryote of fungi.Except causing that capsicum epidemic disease can also cause multiple Solanaceae and cucurbitaceous plant epidemic disease.Capsicum epidemic disease is that a kind of soil passes Plant diseases, worldwide has distribution, and rapid onset is popular wide, easily causes heavy losses.The a large amount of uses of traditional chemical agent not only make pathogen produce drug resistance, and the pesticide residue that cause especially serious threat the mankind's living environment.Therefore carry out the research of gene function analysis and the key gene thereof relevant to pathogenic variation, for enriching pathogenic oomycetes and the host molecular mechanism data information of work mutually, and it is all significant to set up the integrated control technique strategy of oomycetes disease.
Summary of the invention
The object of this invention is to provide a kind of effector albumen and encoding gene and application that derives from Phytophthora capsici.
Effector albumen provided by the present invention, specifically derives from pathogenic Phytophthora capsici (Phytophthora capsici) bacterial strain phyc12, and name is called Phcnlp1, is specially following (a) or (b) or (c):
(a) protein being formed by the aminoacid sequence shown in the 19-476 position of sequence in sequence table 1;
(b) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(c) aminoacid sequence of the protein by (a) or (b) limiting is through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue, and has the protein of effector function.
For the ease of the purifying of Phcnlp1 albumen, N-terminal that can the protein that the amino acid residue sequence of sequence 1 forms in by sequence table or C-terminal connect label as shown in the table.
Table: the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6(is generally 5) | RRRRR |
| Poly-His | 2-10(is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Protein in above-mentioned (c) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the protein in above-mentioned (c) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in the 55-1428 position of sequence in sequence table 2 or sequence 2, and/or carry out the missense mutation of one or several base pair.
The nucleic acid molecule of described Phcnlp1 albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially the gene (called after Phcnlp1) of the described Phcnlp1 albumen of coding; Described Phcnlp1 gene is following 1) to 4) in arbitrary described DNA molecular:
1) DNA molecular shown in 2 55-1428 position in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of the described Phcnlp1 albumen of encoding;
4) with 1) or 2) or 3) DNA molecular limiting has the DNA molecular of 90% above homology and the described Phcnlp1 albumen of encoding.
Above-mentioned stringent condition can be with 6 × SSC, the solution of 0.5%SDS, and at 65 ℃, hybridization, then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively washes film once.
Wherein, sequence 2 is comprised of 1431 Nucleotide, 1-1431 position is ORF, albumen shown in sequence 1 in code sequence list, sequence 1 is comprised of 476 amino acid altogether, wherein 1-18 amino acids is signal peptide sequence, and the sequence of finally processing ripe Phcnlp1 albumen is the 19-476 position of sequence 1, the 55-1428 position of corresponding sequence 2 on gene level.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can be used existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other promotor; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.
In an embodiment of the present invention, the promotor that starts described Phcnlp1 genetic transcription in described recombinant expression vector is specially 35S promoter.
More specifically, described recombinant expression vector is in the restriction enzyme site of pGR106 carrier, to insert the recombinant plasmid that described Phcnlp1 gene (the 55-1428 position of sequence 2) obtains.Described restriction enzyme site is specially Cla I and Sma I.
Described expression cassette is by the promotor that can start described Phcnlp1 genetic expression, described Phcnlp1 gene, and transcription termination sequence composition.
Described Phcnlp1 albumen, or described nucleic acid molecule, or the application in following arbitrary of described recombinant expression vector, expression cassette or recombinant bacterium also belongs to protection scope of the present invention:
(a1) inducing plant tissue necrocytosis;
(a2) for the preparation of the product of inducing plant tissue necrocytosis.
An also object of the present invention is to provide a kind of product for inducing plant tissue necrocytosis.
Product for inducing plant tissue necrocytosis provided by the present invention, its activeconstituents is described Phcnlp1 albumen, or described nucleic acid molecule, or described recombinant expression vector, expression cassette or recombinant bacterium.
In described application and described product, the described product for inducing plant tissue necrocytosis can be campelyco.
In described application and described product, described plant tissue is blade; Described plant can be dicotyledons, also can be monocotyledons.
In one embodiment of the invention, described plant is specially dicotyledon tobacco, and that more concrete is Ben Shi cigarette (Nicotiana Benthamiana).
In one embodiment of the invention, described Phcnlp1 albumen is expressed the albumen obtaining after being specially the DNA fragmentation shown in the 55-1428 position of sequence in sequence table 2 being inserted between the restriction enzyme site Cla I of pGR106 carrier and Sma I.
The present invention utilizes qPCR to analyze the expression level of Phcnlp1 gene at phytophthora blight of pepper infection processs; Simultaneously take Agrobacterium Gv3101 as host, utilize PVX virus expression carrier to carry out transient expression to this gene, adopt injection inoculation method inoculation Ben Shi cigarette (Nicotiana.Benthamiana), proved that effector albumen Phcnlp1 has participated in that Phytophthora capsici infects capsicum host process and inducing plant tissue necrocytosis thereof effectively and even the position that makes to be injured produces the function of obvious downright bad symptom.The present invention is for enriching pathogenic oomycetes and the host molecular mechanism data information of work mutually, and it is all significant to set up the integrated control technique strategy of oomycetes disease.
Accompanying drawing explanation
Fig. 1 is that fluorescence quantitative PCR detection Phcnlp1 gene infects expression level variation in host's process at Phytophthora capsici.Wherein, ordinate zou represents the relative expression quantity (take the expression amount of Phytophthora capsici 18S rRNA gene as 1) of Phcnlp1 gene, and X-coordinate represents after Phytophthora Capsici Zoospores inoculation Pepper Leaves respectively sampling in the 1st, 2,3,4,5,6,7 days.Phcnlp1 gene expression dose raises gradually in host's infection processs, reaches peak value at the 7th day.
Fig. 2 is the plasmid map of recombinant expression vector pGR106/Phcnlp1.
Fig. 3 is the leaf tissue pathology result of Phcnlp1 gene transient expression in tobacco leaf.A-d:Phcnlp1 gene Agrobacterium-mediated Transformation inoculation tobacco leaf, the symptom of the 1st, 3,5,10 days, is inoculated blade and is produced yellow spot after 3 days, increase the weight of gradually until blade necrosis; E: control treatment, with empty carrier pGR106 inoculation tobacco leaf after 3 days without any symptom.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Pathogenic Phytophthora capsici (Phytophthora capsici) bacterial strain phyc12: be recorded in " B.Z.Feng; P.Q.Li.Cloning and expression of a novel laccase gene from phytophthora capsici.Journal of Plant Phathology (2013); 95 (2): 417-421. " literary composition, the public can obtain from Yuncheng Univercity.
PGR106 carrier: be recorded in " Sang-Keun Oh; Saet-Byul Kim; Seon-In Yeom; et al.Positive-Selection and Ligation-Independent Cloning Vectors for Large Scale in Planta Expression for Plant Functional Genomics.Mol.Cells; 2010 (30): 557-562. " literary composition, the public can obtain from Yuncheng Univercity.
Ben Shi cigarette (Nicotiana Benthamiana): be recorded in " Louise Jones; Andrew J.Hamilton; Olivier Voinnet; et al.RNA-DNA Interactions and DNA Methylation in Post-Transcriptional Gene Silencing.The Plant Cell; 1999 (11): 2291-2301. " literary composition, the public can obtain from Yuncheng Univercity.
The acquisition of the effector albumen Phcnlp1 encoding gene of embodiment 1, Phytophthora capsici
Choosing pathogenic Phytophthora capsici (Phytophthora capsici) bacterial strain phy12 is the material of participating in the experiment, according to the effector protein gene information analysis Phytophthora capsici genome sequence of having reported, identify effector protein gene in the full genome of Phytophthora capsici, design special primer p1 and p2, utilize PCR method amplification screening to obtain goal gene fragment, then according to the gene fragment design primer p3 and the p4 that obtain, carry out 3 '-RACE this full length gene that increases.
1, adopt CTAB method to extract high quality phytophthora blight of pepper genomic dna
Phytophthora capsici (Phytophthora capsici) bacterial strain phyc12 is in solid oat medium (formula: rolled oats 100g, agar 17g, water 1000ml boils 30 minutes, packing triangular flask, sterilizing, to be cooled standby) dull and stereotyped cultivation, in 28 ℃ of biochemical cultivation cases, cultivate after 3 days, getting the bacterium piece that 3 diameters are 4mm transplants in filling 100mL liquid oat medium (formula: rolled oats 100g, water 1000ml boils 30 minutes, filtered through gauze packing triangular flask, sterilizing, to be cooled standby) triangular flask in, in 28 ℃ of constant-temperature table shaking culture 5 days, filter mycelia, put and in mortar, add liquid nitrogen and be ground to Powdered.Then according to following steps, extract the strain gene group DNA of participating in the experiment:
(1) mycelia powder is proceeded to 1.5mL centrifuge tube, add 700 μ L DNA extraction damping fluids, mix gently, put 30-60min in 65 ℃ of water-baths.
(2) add equal-volume chloroform: different the eleventh of the twelve Earthly Branches alcohol (volume ratio 24:1), jog 10-20min, the centrifugal 10min of 10000r/min.
(3) get supernatant liquor in another centrifuge tube, add the freezing Virahol of equal-volume, standing 20min under room temperature, the centrifugal 10min of 10000r/min, removes supernatant, and dehydrated alcohol rinses 1-2 time, dries, and adds 600 μ L TE damping fluids, jog 2-3 time.
(4) add the saturated phenol of equal-volume: chloroform: primary isoamyl alcohol (volume ratio 25:24:1), jog 10-20min, the centrifugal 10min of 10000r/min.
(5) get supernatant liquor in another centrifuge tube, repeating step (2) and (3), but (3) step replaces freezing Virahol with dehydrated alcohol.
(6) outwell supernatant liquor, add 70%(volume fraction) alcohol flushing 2-3 time, dry at 37 ℃, add 300 μ LTE solution to dissolve.
Get 5 μ L DNA samples in 1% agarose gel electrophoresis, detect DNA segment length, then DNA is placed in to-20 ℃ of medium-term and long-term preservations of refrigerator-freezer, standby.
2, pcr amplification goal gene fragment
Primer is p1 and p2, and sequence is as follows:
p1：5’-tt(g/a/c)gga(c/t)cac(t)gactggga-3’；
p2：5’-ccacatg(a)atg(c)a(g)a(g)a(g)t-3’。
Note: p1 and p2 are degenerate primer, and any one base in round bracket can.
Reaction system is: ddH
2o(32.5 μ L), 10 × buffer(5 μ L), MgCL
2(4 μ L), dNTP(4 μ L), the each 1 μ L of upstream and downstream primer (p1 and p2), DNA(2 μ L) and, Taq enzyme (0.5 μ L).
PCR response procedures is: 95 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 40s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ are extended 10min.
Get 10 μ L reaction product electrophoresis, determine containing target gene mono-clonal, order-checking.By the blast program in NCBI, the gene fragment obtaining is carried out to homologous sequence comparison, determine goal gene.
3, adopt Trizol method to extract phytophthora blight of pepper geneome RNA and reverse transcription
A. phytophthora blight of pepper geneome RNA extracts
(1) Phytophthora capsici (Phytophthora capsici) bacterial strain phy12 after 3 days, gets 0.1g sample through liquid nitrogen grinding in 28 ℃ of cultivations on liquid oat medium, moves into the 1ml Trizol gentle and quiet 5min that puts of solution chamber.
(2) add 0.2mL chloroform, fully mix, the centrifugal 15min of 12000r/min at 4 ℃, draws supernatant, repeats one to multiple time.
(3) draw supernatant, every 1ml Trizol adds 0.25 times of volume Virahol and 0.25 times of volume RNA precipitation agent, fully mixes, and room temperature is placed 10min, the centrifugal 10min of 12000r/min at 4 ℃.
(4) collect RNA precipitation, use 75%(volume fraction) washing with alcohol 2 times, repeated centrifugation.
(5) exhaust remaining ethanol, or make residual ethanol volatilization.
(6) add the water that 50-100 μ l DEPC processes, RNA solution is stored in to-70 ℃ and saves backup.
B. synthetic cDNA the first chain of reverse transcription
(1) get the total RNA of 1-2 μ g, add ddH
2o to 9.5 μ L, 75 ℃ of sex change 5 minutes, ice bath 5 minutes, centrifugal a little.
(2) add 10 × amplification buffer, 2 μ L.
(3) add the dNTP mixed solution 2 μ L that concentration is 10mmol/L.
(4) add the MgCl that concentration is 25mmol/L
2solution 4 μ L.
(5) add primer Oligo-dT1 μ L.
(6) add RNA enzyme inhibitors 0.5 μ L.
(7) add ThermoScript II M-MLV1 μ L.
(8) react total system 20 μ L.After reaction solution mixes, room temperature is placed 10 minutes, and 42 ℃ of temperature are bathed 60 minutes, and 85 ℃ of water-baths are placed 10 minutes.
(9) add 180 μ L ddH
2o mixes, centrifugal a little, preserves at-20 ℃.
(10) 3 of negative controls are set, respectively: 1. add the first chain cDNA to react required all reagent, do not add template ribonucleic acid; 2. add all reagent except ThermoScript II; 3. add all reagent except primer.
4,3 '-RACE amplifying target genes total length
According to the acquired gene fragment of step 2 design primer p3 and p4, carry out 3 '-RACE this full length gene that increases.
P3:5 '-cggactttggagacgccaatcccatagggacac-3 ' (the 629-661 position of sequence 2);
P4:5 '-gtgttcccagtcgtgacggtgaccgaagccc-3 ' (reverse complementary sequence of the 1071-1101 position of sequence 2).
Carry out respectively two-wheeled pcr amplification reaction.First round response procedures is: 95 ℃ of denaturation 1min; 95 ℃ of 30s, 68 ℃ of 1min, 72 ℃ of 2min, 5 circulations; 95 ℃ of 30s again, 65 ℃ of 1min, 72 ℃ of 2min, carry out 30 circulations; Last total extension 10min again.First round amplification reaction system is as follows: PCR-Grade Water(40 μ L), 10 × Advantage2PCR Buffer(5 μ L), the each 1 μ L of primer p3 and USP, 50 × Advantage2polymerasemix(1 μ L), template (step 3 gained cDNA the first chain) (1 μ L).Wherein, PCR-GradeWater, 10 × Advantage2PCR Buffer, primer USP, and 50 × Advantage2polymerasemix is the product in SMARTer RACE cDNA Amplification Kit test kit (Clontech, USA, CAT#:634923).
Take first round reaction product as template, carry out second and take turns reaction.Reaction system is: ddH
2o(32.5 μ L), 10 × buffer(5 μ L), MgCl
2(4 μ L), dNTP(4 μ L), the each 1 μ L of primer p4 and NUSP, template (2 μ L), Taq enzyme (0.5 μ L).Wherein, NUSP is product in SMARTer RACE cDNA Amplification Kit test kit.Reaction conditions is: 95 ℃ of 4min; 94 ℃ of 30S, 65 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ are extended 10min.
Through above two-wheeled pcr amplification, obtain goal gene total length, as shown in sequence in sequence table 2.
5, goal gene total length checking
In order to confirm the exactness of the goal gene full length sequence shown in sequence 2 that above step 4 obtains.The present inventor has designed primer p5 and the p6 for extension increasing sequence 2 total lengths according to sequence 2, and sequence is as follows:
P5:5 '-atgcaactacgtgccttcatctctg-3 ' (the 1-25 position of sequence 2);
P6:5 '-ctactggtaccaggcgttcgcgagcttcga-3 ' (reverse complementary sequence of the 1402-1431 position of sequence 2).
Take step 3 reverse transcription gained cDNA as template, carry out pcr amplification with primer p5 and p6.Reaction finishes laggard row agarose gel electrophoresis, and result shows that PCR product size is about 1431bp, consistent with expected results.Further sequence verification, demonstration gained PCR product is just for shown in sequence 2 in sequence table.
By unnamed gene shown in sequence in sequence table 2, be Phcnlp1 gene, whole sequence 2 is its open reading frame (ORF), the protein shown in sequence 1 in code sequence list (called after Phcnlp1 albumen).Shown in sequence 1, the 1-18 amino acids of albumen is signal peptide sequence, in the processing ripening process of Phcnlp1 albumen, signal peptide sequence is cut, the aminoacid sequence of the ripe Phcnlp1 albumen finally obtaining is the 19-476 position of sequence 1 in sequence table, the 55-1428 position of sequence 2 in corresponding sequence table on gene level.
Embodiment 2, Phcnlp1 gene infect expression pattern analysis in capsicum host process at phytophthora blight of pepper
One, phytophthora blight of pepper zoospore inoculation Pepper Leaves
1, zoospore preparation
(1) will participate in the experiment Phytophthora capsici bacterial strain Phyc12 be transferred to V8 substratum (formula: V8 vegetables juice 200ml, CaCO
33.0g, agar 15g adding distil water is to 1000ml, boils the sterilizing of dress triangular flask standby; Wherein V8 vegetables juice market is on sale) cultivate 4d, from colony edge picking mycelia piece, move to fresh V8 substratum continuation cultivation 4d standby.
(2) picking colony edge mycelia piece is to V8 culture medium culturing, 25 ℃ of dark culturing 5-6d.
(3) add aqua sterilisa (being advisable with submergence mycelia), every 1d, change water to zoospore and produce, and regulate zoospore concentration to 1 × 10
5individual/ml.
2, zoospore inoculation Pepper Leaves
By zoospore inoculation capsicum (the 5-6 leaf phase) blade of induction, concrete steps are as follows:
To choose blade sterilization: 70%(volume fraction) Ethanol Treatment 30s, 0.1%(volume fraction) mercuric chloride processing 7min, in aqua sterilisa, rinse 3 times, dry standby.After drying, blade is placed in to the culture dish that is covered with 3 sterilizing filter paper, each plate is placed 3, and each blade inoculation 2 μ l steps 1 are adjusted the zoospore suspension of concentration.Inoculation blade is placed in 25 ℃ of illumination box dark culturing, and after inoculation, sampling 1-7 days every days, extracts for RNA.
Two, the total RNA of inoculation Pepper Leaves extracts and reverse transcription
1, the total RNA of inoculation Pepper Leaves extracts
(1) get 0.1g sample through liquid nitrogen grinding, move into the 1ml Trizol gentle and quiet 5min that puts of solution chamber.
(2) add 0.2mL chloroform, fully mix, the centrifugal 15min of 12000r/min at 4 ℃, draws supernatant, repeats one to multiple time.
(3) draw supernatant, every 1ml Trizol adds 0.25 times of volume Virahol and 0.25 times of volume RNA precipitation agent, fully mixes, and room temperature is placed 10min, the centrifugal 10min of 12000r/min at 4 ℃.
(4) collect RNA precipitation, use 75%(volume fraction) washing with alcohol 2 times, repeated centrifugation.
(5) exhaust remaining ethanol, or make residual ethanol volatilization.
(6) add the water that 50-100 μ l DEPC processes, RNA solution is stored in to-70 ℃ and saves backup.
2, synthetic cDNA the first chain of reverse transcription
(1) get the total RNA of 1-2 μ g, add ddH
2o to 9.5 μ L, 75 ℃ of sex change 5 minutes, ice bath 5 minutes, centrifugal a little.
(2) add 10 × amplification buffer, 2 μ L.
(3) add the dNTP mixed solution 2 μ L that concentration is 10mmol/L.
(4) add the MgCl that concentration is 25mmol/L
2solution 4 μ L.
(5) add primer Oligo-dT1 μ L.
(6) add RNA enzyme inhibitors 0.5 μ L.
(7) add ThermoScript II M-MLV1 μ L.
(8) react total system 20 μ L.After reaction solution mixes, room temperature is placed 10 minutes, and 42 ℃ of temperature are bathed 60 minutes, and 85 ℃ of water-baths are placed 10 minutes.
(9) add 180 μ L ddH
2o mixes, centrifugal a little, preserves at-20 ℃.
(10) 3 of negative controls are set, respectively: 1. add the first chain cDNA to react required all reagent, do not add template ribonucleic acid; 2. add all reagent except ThermoScript II; 3. add all reagent except primer.
Three, quantitative fluorescent PCR is analyzed Phcnlp1 gene expression pattern in phytophthora blight of pepper and host's Interaction
According to Phcnlp1 gene order design primer qPCR1 and qPCR2, sequence is as follows:
QPCR1:5 '-ccatagggacaccgatcaagct-3 ' (the 650-671 position of sequence 2);
QPCR2:5 '-tgtagccgctatgggccgacg-3 ' (reverse complementary sequence of the 1160-1180 position of sequence 2).
Take Phytophthora capsici 18S rRNA sequence as internal reference, design primer 18sRNAF and 18sRNAR, sequence is as follows:
18sRNAF：5’-tttcggtcctattacgttgg-3’；
18sRNAR：5’-ttcgcagttgttcgtctttc-3’。
Utilize Bio-Radi Cycler to react, condition is as follows:
95 ℃ of denaturation 2min, then carry out amplification cycles; 94 ℃ of sex change 15sec, 55 ℃ of annealing 15sec, 68 ℃ are extended 30sec, totally 45 circulations; Prepare solubility curve for last 65-95 ℃.Selection 18sRNA is reference gene, each sample in triplicate, with 2
-△ △ Ctmethod is carried out differential expression relative quantitative assay to sample gene, and method of calculation are △ △ Ct=(CT
phcnlp1-CT
18SrRNA) this – of Dai test sample (CT
phcnlp1-CT
18SrRNA) (wherein, Phcnlp1 gene is sample to be tested at the expression amount of the 2nd, 3,4,5,6,7 days to calibration sample; Calibration sample is Phcnlp1 gene at the expression amount of the 1st day).
As shown in Figure 1, as can be seen from the figure, Phcnlp1 gene expression dose infects in capsicum host process and raises gradually at phytophthora blight of pepper result, within the 7th day after inoculation, reaches peak value.
Embodiment 3, Phcnlp1 gene transient expression in tobacco body
One, Phcnlp1 gene mature peptide sequence separates
According to acquired Phcnlp1 full length gene sequence (sequence 2), design following primer pvxF and pvxR:
PvxF:5 '-cc
gctgttatcgaccacgaccaggtcgt-3 ' (underscore part is the recognition sequence of Cla I, and sequence is thereafter the 55-80 position of sequence 2);
PvxR:5 '-tcc
ctggtaccaggcgttcgcgagct-3 ' (underscore part is the recognition sequence of Sma I, and sequence is thereafter the reverse complementary sequence of the 1406-1428 position of sequence 2).
Take the cDNA of the Phytophthora capsici bacterial strain Phyc12 that participates in the experiment as template, carry out pcr amplification with primer pvxF and pvxR.Reaction system is 50 μ L:cDNA templates (2 μ L), 10 × buffer(5 μ L), Mg
2+(4 μ L), dNTP(4 μ L), the each 1 μ L of upstream and downstream primer, Taq enzyme (0.5 μ L), ddH
2o(32.5 μ L).PCR response procedures is: 94 ℃ of 4min; 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ are extended 10min.
After reaction finishes, get 10 μ L reaction product and carry out 1% agarose gel electrophoresis, then goal gene is reclaimed and connects pGEM-T Easy carrier (Promega, USA, Cat.#A1360), transform bacillus coli DH 5 alpha, blue hickie screening, plasmid DNA enzyme are cut evaluation.By identify correct recombinant plasmid (obtain size and be about 2650bp and two object bands of 1374bp) through Cla I and Sma I double digestion, serve the order-checking of the biological company limited of Hai Boya.To show that external source Insert Fragment is " cc through order-checking
the 55-1428 position of+sequence 2+
gga " recombinant plasmid called after pGEM-T/Phcnlp1.Wherein, the 55-1428 position of sequence 2 is Phcnlp1 gene mature peptide sequence, the Phcnlp1 maturation protein shown in the 19-476 amino acids of sequence 1 in code sequence list.
Two, PVX recombinant expression vector builds
(1) Cla I and Sma I double digestion step 1 build the pGEM-T/Phcnlp1 plasmid obtaining, and reclaim and insert segment, and size is about 1374bp.
(2) connect with the pGR106 carrier of same double digestion, transform bacillus coli DH 5 alpha.
(3) the DH5 α after conversion, through Kan resistance screening, obtains bacterium colony and extract plasmid after 37 ℃ of shaking tables spends the night.
(4) by restriction enzyme Cla I and Sma I, recombinant plasmid is carried out to double digestion evaluation.Enzyme is cut to the recombinant plasmid that preliminary evaluation is correct (obtain size and be about 11164bp and two object bands of 1374bp) and serve the order-checking of the biological company limited of Hai Boya.By the recombinant plasmid called after pGR106/Phcnlp1(plasmid map that shows DNA fragmentation shown in the 55-1428 position of insertion sequence 2 between the restriction enzyme site Cla I of pGR106 carrier and Sma I through order-checking as shown in Figure 2).In recombinant expression vector pGR106/Phcnlp1, the promotor that shown in the 55-1428 position of initiating sequence 2, DNA fragmentation is transcribed is 35S promoter.
Three, Agrobacterium-mediated Transformation
1, recombinant plasmid pGR106/Phcnlp1 extracts
(1) intestinal bacteria that contain recombinant plasmid pGR106/Phcnlp1 are inoculated in containing in appropriate antibiotic LB substratum, 37 ℃, 220-250rpm concussion is cultured to logarithmic phase.
(2) get 1mL bacterium liquid to the centrifuge tube of 1.5mL, the centrifugal 1min of 8000rpm.
(3) remove supernatant, collect thalline.
(4) add the solution I (formula: 50mM glucose, 25mM Tris-HCl, 10mMEDTA, pH8.0) of 200 μ L precoolings, concussion suspension thalline.
(5) add the freshly prepared solution II of 400 μ L (formula: 0.2M NaCl, 1%SDS), put upside down centrifuge tube and mix for several times, the centrifugal 5min of 12000rpm.
(6) add the solution III of 300 μ L precoolings (to fill a prescription: 3M K
+, 5M Ac
-), put upside down and mix liquid, put ice sheet 5min, the centrifugal 5min of 12000rpm, supernatant proceeds in another centrifuge tube.
(7) add equal-volume phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1), concussion mixes, the centrifugal 5min of 12000rpm.
(8) upper strata water proceeds in another centrifuge tube, adds equal-volume Virahol, mixes rear room temperature and places 10min, and the centrifugal 10min of 12000rpm, removes supernatant.
(9) precipitation use 70%(volume fraction) ethanol washes 2 times, is inverted and is dried.
(10) back dissolving in 30 μ L TE(containing the RNA enzyme of 20 μ g) in, get 5 μ L electrophoresis detection, preserve at all the other-20 ℃.
2, the preparation of Agrobacterium competence and conversion
(1) choose nine divisions of China in remote antiquity, Agrobacterium GV3101(Beijing Tianrui Science and Technology Ltd.) single bacterium colony (containing Rifampin 50mg/ml) in 3ml LB liquid nutrient medium, at 28 ℃, 24h is cultivated in 200rpm concussion.
(2) in 1:100(volume ratio) in ratio switching 100ml LB liquid nutrient medium (containing Rifampin 50mg/ml), continue under the same conditions to cultivate 6-7h to OD600=0.6 left and right, for the preparation of competence.
(3) get the centrifugal 30s of 1.5ml bacterium liquid 13000rpm, abandon supernatant, with 800 μ l CaCl
2resuspended thalline, places 30min in ice sheet, and then the centrifugal 30s of 13000rpm abandons supernatant.
(4) use again 100 μ l CaCl
2resuspended thalline, ice sheet is placed standby.
(5) get 10 μ l recombinant plasmid pGR106/Phcnlp1 and add in 200 μ l competent cells, ice sheet is placed 30min, then in liquid nitrogen, places 1min, then goes at once thermal shock 5min in 37 ℃ of water-baths.
(6) add rapidly 800 μ l LB liquid culture based at 28 ℃, 2h is cultivated in 200rpm concussion; .
(7) then slightly try centrifugal collection thalline, coated LB liquid nutrient medium (containing kan and the each 50mg/ml of Rifampin), at 28 ℃, be inverted and cultivate 48h.
(8) Agrobacterium-mediated Transformation screening, the single spot transformant on picking flat board shakes cultivation in LB liquid nutrient medium, then extracts plasmid and carries out double digestion and PCR checking.
Cla I and Sma I double digestion and pcr amplification all can obtain size to be about the restructuring Agrobacterium of 1374bp object band positive, by its called after GV3101/pGR106/Phcnlp1.
Gained restructuring Agrobacterium called after GV3101/pGR106 is set to the contrast that proceeds to pGR106 empty carrier in Agrobacterium GV3101 simultaneously.
Four, restructuring Agrobacterium positive transformant injection Ben Shi cigarette seedling leaves
Take Ben Shi cigarette (Nicotiana Benthamiana) as test plant.
Screen and identify that the positive restructuring Agrobacterium-mediated Transformation single bacterium colony GV3101/pGR106/Phcnlp1 of son and GV3101/pGR106 are inoculated in respectively LB liquid nutrient medium (containing kan and the each 50mg/ml of Rifampin) by toothpick picking step 3.At 28 ℃, 200rpm concussion is cultivated after 24h, and centrifugal collection thalline is filled a prescription in equal-volume MMA(: 10mmol/L MgCl
2, 10mmol/L MES and 100mmol/L AS) in continue inducing culture 3 hours, the thalline after induction is inoculated in the Ben Shi cigarette seedling of 5-6 leaf phase, agrobacterium suspension is pressed in tobacco leaf vein with the needle-less asepsis injector of 5mL.Each processing repeats 3 times, and postvaccinal tobacco leaf dark culturing in 22 ℃ of incubators of atmospheric moisture 75%, after 2 days, proceeds to phytotron and cultivates, and the symptom variation of observed and recorded inoculation every day part after inoculation, till the 10th day.
As shown in Figure 3, as can be seen from the figure, tobacco leaf blade in the time of the 3rd day of having inoculated restructuring Agrobacterium GV3101/pGR106/Phcnlp1 produces yellow spot to result, and along with the carrying out of time, scab increases the weight of gradually until blade necrosis.And the tobacco leaf of control group inoculation restructuring Agrobacterium GV3101/pGR106 is to beginning to eventually without any symptom.
Claims (10)
1. protein is following (a) or (b) or (c):
(a) protein being formed by the aminoacid sequence shown in the 19-476 position of sequence in sequence table 1;
(b) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;
(c) aminoacid sequence of the protein by (a) or (b) limiting is through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue, and has the protein of effector function.
2. the nucleic acid molecule of protein described in coding claim 1.
3. nucleic acid molecule according to claim 2, is characterized in that: described nucleic acid molecule is the gene of protein described in coding claim 1; Described gene is following 1) to 4) in arbitrary described DNA molecular:
1) DNA molecular shown in the 55-1428 position of sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) protein DNA molecule described in the DNA molecule hybridize that limits and coding claim 1;
4) with 1) or 2) or 3) DNA molecular that limits there is 90% above homology and the claim 1 of encoding described in protein DNA molecule.
4. contain recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium of nucleic acid molecule described in claim 2 or 3.
5. recombinant vectors according to claim 4, is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5, is characterized in that: in described recombinant expression vector, the promotor of transcribing that starts described gene is 35S promoter.
7. protein claimed in claim 1, or the nucleic acid molecule described in claim 2 or 3, or arbitrary described recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium application in following arbitrary in claim 4-6:
(a1) inducing plant tissue necrocytosis;
(a2) for the preparation of the product of inducing plant tissue necrocytosis.
8. the product for inducing plant tissue necrocytosis, its activeconstituents is protein claimed in claim 1, or the nucleic acid molecule described in claim 2 or 3, or recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium described in arbitrary in claim 4-6.
9. application according to claim 7, or product claimed in claim 8, is characterized in that: described plant is dicotyledons or monocotyledons.
10. application according to claim 9 or method, is characterized in that: described dicotyledons is tobacco.
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Cited By (6)
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| CN104447966A (en) * | 2014-12-01 | 2015-03-25 | 山东农业大学 | Phytophthora capsici cell division protein as well as encoding gene and application thereof |
| CN109912699A (en) * | 2019-05-05 | 2019-06-21 | 南京林业大学 | Camphor tree phytophthora effector albumin A vh87 and its encoding gene and application |
| CN110734918A (en) * | 2019-11-04 | 2020-01-31 | 山东农业大学 | Phytophthora capsici effector RxLR19781 gene and application thereof |
| CN110804615A (en) * | 2019-11-04 | 2020-02-18 | 山东农业大学 | Phytophthora capsici effector RxLR553394 gene and application thereof |
| CN112251450A (en) * | 2020-09-16 | 2021-01-22 | 中国农业科学院烟草研究所 | Phytophthora parasitica avirulence gene PnAvr1 and encoding protein and application thereof |
| CN113121659A (en) * | 2021-04-23 | 2021-07-16 | 南京林业大学 | Phytophthora camphora effector protein Avh57 and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104447966A (en) * | 2014-12-01 | 2015-03-25 | 山东农业大学 | Phytophthora capsici cell division protein as well as encoding gene and application thereof |
| CN104447966B (en) * | 2014-12-01 | 2017-05-10 | 山东农业大学 | Phytophthora capsici cell division protein as well as encoding gene and application thereof |
| CN109912699A (en) * | 2019-05-05 | 2019-06-21 | 南京林业大学 | Camphor tree phytophthora effector albumin A vh87 and its encoding gene and application |
| CN109912699B (en) * | 2019-05-05 | 2020-09-29 | 南京林业大学 | Phytophthora camphora effector protein Avh87 and encoding gene and application thereof |
| CN110734918A (en) * | 2019-11-04 | 2020-01-31 | 山东农业大学 | Phytophthora capsici effector RxLR19781 gene and application thereof |
| CN110804615A (en) * | 2019-11-04 | 2020-02-18 | 山东农业大学 | Phytophthora capsici effector RxLR553394 gene and application thereof |
| CN110804615B (en) * | 2019-11-04 | 2021-06-29 | 山东农业大学 | Phytophthora capsici effector RxLR553394 gene and application thereof |
| CN112251450A (en) * | 2020-09-16 | 2021-01-22 | 中国农业科学院烟草研究所 | Phytophthora parasitica avirulence gene PnAvr1 and encoding protein and application thereof |
| CN113121659A (en) * | 2021-04-23 | 2021-07-16 | 南京林业大学 | Phytophthora camphora effector protein Avh57 and application thereof |
| CN113121659B (en) * | 2021-04-23 | 2021-10-22 | 南京林业大学 | Phytophthora camphora effector protein Avh57 and application thereof |
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