CN102634585B

CN102634585B - Primer for detecting pytophthora capsici developed on basis of plygalacturonase Pcipg8 gene and method therefor

Info

Publication number: CN102634585B
Application number: CN 201210123020
Authority: CN
Inventors: 张修国
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2012-04-25
Filing date: 2012-04-25
Publication date: 2013-08-14
Anticipated expiration: 2032-04-25
Also published as: CN102634585A

Abstract

The invention discloses a primer for detecting pytophthora capsici developed on the basis of plygalacturonase Pcipg8 gene and a method for detecting the pytophthora capsici. The primer for detecting the pytophthora capsici developed on the basis of the plygalacturonase Pcipg8 gene consists of single-strained DNA (deoxyribonucleic acid) shown by a sequence 1 in a sequence table and single-strained DNA shown by a sequence 2 in the sequence table. The primer can be used for carrying out qualitative and quantitative molecule detection on the phytophthora capsici leonian and different phytophthoracapsici leonian materials at different periods, and detecting and easily warning the primary infection source of the phytophthora capsici leonian and the dynamic status of the growth and decline of the germs at different periods in the process of infection, so that the optimum disease preventing and controlling period can be conveniently and timely confirmed, particularly the comprehensive prevention, control and treatment can be timely carried out in the early activity period of the germs, according to the existence and the quantity for detecting the oospore in the soil, the soil can be timely treated, the breeding and spreading of source germs can be cut off, and the disease preventing and controlling cost can be reduced.

Description

Primer and method based on the detection phytophthora blight of pepper of polygalacturonase Pcipg8 gene exploitation

Technical field

The present invention relates to a kind of primer and method of the detection phytophthora blight of pepper based on polygalacturonase Pcipg8 gene exploitation.

Background technology

The pathogenic oomycetes is the important phytopathogen of a class, can infect the harm various plants, cause the destructive disease of various plants, give birth to epidemic disease mould (P.hibemalis) as phytophthora infestans (Phytophthora infestans), Phytophthora capsici (P.capsici), soybean phytophthora (P.sojae), phytophthora parasitica (P.parasitic), camphor tree epidemic disease mould (P.cinnamomi) and winter and cause the important eqpidemic disease of potato, capsicum, soybean, tobacco, camphor tree and oranges and tangerines respectively, serious time and even total crop failure.Oomycetes is mainly spent poor environment with mycelium or oospore and as primary source of infection in invalid body or soil, under optimum conditions, mycelium or oospore all can be sprouted generation sporocyst-release zoospore, propagate, infect and cause Plant diseases by rainwater or air-flow.Therefore, such germ disease is carried out in good time prosecution in early days, be beneficial to the generation of control disease.Traditional disease prevention and control strategy mainly relies on prophylactico-therapeutic measuress such as kind, cultivation, chemoprevention and ecological regulation and control, these prevention and control measures are mainly implemented when disease outburst and even the obvious harm of generation, ignored primary source of infection or infect the host and take comprehensive prevention and control and efficient control measures in early days in good time, closely get twice the result with half the effort, preventive effect is very little, the generation of final very difficult control disease and popular.

In recent years, round pcr and associated molecule detection technique thereof have been widely used in the dynamic Molecular Detection of phytopathogen growth and decline and early warning, wherein utilize rrna rDNA/ITS sequences Design special primer, carried out targetedly that soybean phytophthora, phytophthora infestans, winter give birth to that epidemic disease is mould, phytophthora parasitica, the research of Phytophthora capsici molecular detection technology, but these technical research and development achievements only can dynamically detect and early warning the germ activity, but can not to the germ pathogenic property and occurrence tendency detects and early warning.Studies show that phytophthora-host makes the multiple important cell wall degrading enzyme of normal secretion in the process mutually, mainly comprise polygalacturonase (Pg), pectin methyl esterase (Pme), pectin lyase (Pel), these important virulence factors mainly pass through main component pectin, mesogloea and the pectin polymer of degraded or softening plant cell wall, and impel intrusion and the field planting of germ, the host is produced parasitic and pathogenic.Such zymin is encoded by polygene, and (be respectively for GenBank number: DQ415987, DQ415988), Pcipg5 (is for GenBank number: EF558847) as disclosed Phytophthora capsici polygalactunonic acid enzyme gene at present Pcipg 2-3.Wherein the minority key gene is important pathogenic target gene, so target gene of these important zymins of isolation identification, utilize gene information to design and synthesize the special primer of target gene, carry out Molecular Detection and early warning at germ different times pathogenic property, in order to take comprehensive prevention and control measure in the germ phase of just infecting in good time, reduce medicament access times and usage quantity, the expansion of blocking-up germ with spread, the generation of control disease with spread.Therefore, carry out important phytophthora and cause a disease enzyme target gene clone and special primer design thereof with synthetic, development germ Molecular Detection, early warning technology and test kit thereof have important theory and practice significance to the comprehensive prevention and control of phytophthora root rot.

Summary of the invention

A technical problem to be solved by this invention provides a kind of PCR primer that can special detection Phytophthora capsici to (reagent).

The PCR primer of special detection Phytophthora capsici provided by the present invention is right, the special primer that is polygalacturonase Pcipg8 is right, its name is called ipg8F-R, is made up of the single stranded DNA (ipg8R) shown in the sequence 2 in the single stranded DNA (ipg8F) shown in the sequence in the sequence table 1 and the sequence table.

Wherein, the sequence 1 in the sequence table is made up of 21 Nucleotide, and sequence 2 is made up of 19 Nucleotide.

Contain the detection of ipg8F-R or the PCR test kit of auxiliary detection Phytophthora capsici and also belong to protection scope of the present invention, this test kit also can contain other PCR reagent such as archaeal dna polymerase, dATP, dTTP, dCTP and dGTP.

Another technical problem to be solved by this invention provides the method for a kind of detection or auxiliary detection Phytophthora capsici.

The method of detection provided by the present invention or auxiliary detection Phytophthora capsici comprises the steps: to carry out PCR with the testing sample of ipg8F_R, determines whether to contain in the testing sample content of phytophthora blight of pepper in Phytophthora capsici or the definite testing sample.

Described testing sample can be soil or is tissue and/or the organ of the host plant of described Phytophthora capsici, as the blade of capsicum, stem, fruit etc.

In the aforesaid method, can determine whether to contain in the testing sample Phytophthora capsici as follows: the cDNA that obtains with the genomic dna of testing sample or total RNA reverse transcription is template, carry out pcr amplification with ipg8F-R, the pcr amplification product that detection obtains, if described pcr amplification product satisfies following 1) or 2) condition, then described testing sample contains Phytophthora capsici or the candidate is contained Phytophthora capsici; If described pcr amplification product do not satisfy described 1) or 2) condition, then described testing sample does not contain Phytophthora capsici or the candidate is not contained Phytophthora capsici;

1) described pcr amplification product is the fragment of 176bp;

2) described pcr amplification product is shown as the band of a 100bp-200bp at agarose gel electrophoresis.

Wherein, determine that the condition that whether contains the pcr amplification of Phytophthora capsici in the testing sample can be wherein, the condition of described pcr amplification can be: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations.

In the aforesaid method, can adopt real-time fluorescence quantitative PCR to determine the content of phytophthora blight of pepper in the testing sample.Described real-time fluorescence quantitative PCR condition can be: 95 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 20s, 68 ℃ are extended 55s, 40 circulations.

Experimental result shows, the special primer of polygalactunonic acid enzyme gene Pcipg8 of the present invention is to ipg8F-R, can be to Phytophthora capsici and different capsicum epidemic disease material (the sick field soil of different times, sick stem, sick fruit, sick leaf) carry out qualitative, quantitative molecular detects, can dynamically detect and early warning Phytophthora capsici primary source of infection and each germ growth and decline in period of infection processs thereof, be convenient in time determine the best prevention and control of disease periods, particularly carry out comprehensive prevention and control and improvement period at the germ early ambulant in good time, as it's too late according to having that oospore in the soil is detected what, handle soil in good time, the breeding of blocking-up source germ with spread, reduced disease prevention and control cost.

Description of drawings

Fig. 1 is 14 Pcipg genes rna transcription expression level qRT-PCR detected result synoptic diagram in the inoculation pepper fruit.

Among the figure, X-coordinate 1-14 represent respectively 14 Pcipg genes (1:Pcipg2,2:Pcipg 3,3:Pcipg5,4:Pcipg 8,5:Pcipg9, and 6:Pcipg 10,7:Pcipg11,8:Pcipg 12, and 9:Pcipg 13,10:Pcipg 14,11:Pcipg15,12:Pcipg16,13:Pcipg 17, and 14:Pcipg 18), a, b, c, d represent phytophthora blight of pepper inoculation pepper fruit fate 1d, 3d, 5d, 7d respectively.Standard error is from three repeated experiments.

Fig. 2 for Pcipg 8 gene specific primers to the participate in the experiment PCR detected result synoptic diagram of kind of bacterium DNA of difference.

Among the figure, A is that a pair of special primer of Pcipg 8 genes is to the PCR detected result of ipg8F-R to bacterium DNA not of the same race; B is that a pair of special primer of Pcipg5 gene is to the PCR detected result of ipg5F-R to bacterium DNA not of the same race; 1: standard DNA; 2: negative control water; 3-7: Phytophthora capsici; 8: soybean phytophthora; 9: phytophthora parasitica; 10: phytophthora infestans; 11: the camphor tree epidemic disease is mould; 12: the Jue Shi epidemic disease is mould; 13: palm mould; 14: tropical epidemic disease is mould; 15: ramie mould; 16: Phytophthora cactorum; 17: the clover epidemic disease is mould; 18: the taro epidemic disease is mould; 19: standard DNA; 20: latent ground epidemic disease is mould; 21: the pea epidemic disease is mould; 22: the strawberry epidemic disease is mould; 23: big male epidemic disease is mould; 24: the Kidney bean epidemic disease is mould; 25: the green onion epidemic disease is mould; 26: it is mould that the winter is given birth to epidemic disease; 27: the melon and fruit corruption is mould; 28: water mold; 29: the sweet potato head mold; 30: miliary damping-off; 31: fusarium moniliforme; 32: fusarium solani; 33: alternaria solani sorauer; 34: Penicillium notatum; 35: aspergillus cremeus.

Fig. 3 is that Pcipg 8 gene specific primer PCR detect phytophthora blight of pepper zoospore DNA result schematic diagram.

Among the figure, A is that a pair of special primer of Pcipg8 is to the detected result of the different concns zoospore of ipg8F-R DNA; B is that a pair of special primer of reference gene Pcipg5 is to the detected result of the different concns zoospore of ipg5F-R DNA; 1: standard DNA; 2: negative control water; 3: positive control Phytophthora capsici DNA; 4-19: different volumes zoospore DNA (6.0,5.5,5.0,4.5,4.0,3.5,3.0,2.5,2.0,1.5,1.0,0.5,0.4,0.3,0.2,0.1 μ L) is detected effect (7 zoospore/μ L).

Fig. 4 is that Pcipg 8 gene specific primer PCR detect phytophthora blight of pepper oospore DNA synoptic diagram in the soil.

Among the figure, A is that Pcipg 8 a pair of special primers are to the phytophthora blight of pepper oospore of ipg8F-R DNA detection result; B is that Pcipg 5 a pair of special primers are to the phytophthora blight of pepper oospore of ipg 5F-R DNA detection result; 1: standard DNA; 2: negative control water; 3: positive control Phytophthora capsici DNA; 4-15: to oospore DNA (4.0,3.5,3.0,2.5,2.0,1.5,1.0,0.5,0.4,0.3,0.2,0.1 μ L) the DNA detection effect (7 oospore/μ L) of different volumes.

Fig. 5 be Pcipg 8 gene specific primers to ipg8F-R to different phytophthora blight of pepper material pcr amplification result schematic diagrams.

Among the figure, 1: standard DNA; 2-3: sterilized water and healthy capsicum tissue DNA; 4: Phytophthora capsici mycelium DNA; 5: sick stem tissue DNA; 6: sick fruit tissue DNA; 7: the sick DNA of leaf texture; 8: the soil thallus DNA of artificial inoculation oospore; 9: the sick field soil thallus DNA that contains oospore.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment, if no special instructions, be ordinary method, as Sambrook equimolecular cloning experimentation handbook (New York:Gold Spring Harbor Laboratory Press, 1989), or Draper, J etc. (1988) operative technique rules or the experiment condition of advising according to manufacturer.Used test reagent among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.

The primer of embodiment 1, special detection Phytophthora capsici is to acquisition and specificity and the sensitivity of ipg8F-R

Present embodiment is the material of participating in the experiment with Phytophthora capsici, there is Pcipg 2-3 (to be respectively for GenBank number: DQ415987 according to qRT-PCR technology for detection Phytophthora capsici polygalactunonic acid enzyme gene, DQ415988), Pcipg5 (is for GenBank number: EF558847), the difference of these 14 gene members of Pcipg 8-18 (its nucleotide sequence is respectively the sequence 3-13 in the sequence table) rna transcription expression level in the sick fruit of capsicum, compare related data information, Pcipg 8 is defined as the target Disease-causing gene, design and synthesize the many to special primer of Pcipg 8 genes (sequence 3), tested these primers to the specificity of bacterial classification that all are participated in the experiment and the susceptibility that phytophthora blight of pepper is detected.All higher primer is right as the primer of special detection Phytophthora capsici to ipg8F-R therefrom to have selected a pair of specificity and sensitivity, and upstream primer is ipg8F (21bp), and sequence is 5`-CAAGAAGCTGAGACAACGGCC-3`-3`; Downstream primer is ipg8R (19bp), and sequence is 5`-GTAAATTCTATTGTAGCGC-3`, and amplifying target genes length is 176bp.Concrete steps are as follows:

1,14 Pcipg gene rna transcription expression level qRT-PCR in the inoculation pepper fruit detect step:

1.1, phytophthora blight of pepper SD33 (Shandong Agricultural University) (Yong Jian Jia, Bao Zhen Feng, Wen Xiu Sun and Xiu Guo Zhang, J.Phytopathol157:585-591,2009) zoospore inoculation pepper fruit, concrete grammar is as follows:

1.1.1, with Phytophthora capsici bacterial strain SD33 dislocation after the 10%V8 solid medium is cultivated 4 days, picking colony edge mycelia piece was cultivated 4 days to the 10%V8 liquid nutrient medium, 25 ℃ dark culturing 5-6 days.

1.1.2, add aqua sterilisa, just the submergence mycelia is advisable, and changes water every 1 day to producing zoospore, collects zoospore and regulates concentration to 1 * 10 ⁵Individual/ml, configuration zoospore suspension is used for the pepper fruit inoculation.

1.1.3, phytophthora blight of pepper zoospore suspension inoculation pepper fruit

1.1.4, get the tender pepper fruit of fresh children, disinfect through 75% dehydrated alcohol, put the aseptic technique platform and dry.

1.1.5, scalpel organizes fruit surface gently and scratches, and causes the about 1cm of the degree of depth, the about 0.25cm of area ²Wicresoft hinders.

1.1.6, the aseptic rayon balls that will soak 30min in the zoospore suspension of suitable concentration is covered in the wound, then with preservative film wrapping sealing.

1.1.7, the inoculation fruit places and cultivates 7d in 28 ℃ of constant incubators, according to 1,3,5,7d takes a sample at interval, cut the strong intersection of disease preserve put-80 ℃ under standby extraction RNA.

1.2, extract inoculation pepper fruit RNA, measure RNA concentration with trace of albumin nucleic acid determination instrument (IMPLEN), cDNA first chain synthesize in reverse transcription, real-time fluorescence quantitative PCR (qRT-PCR) detect 14 genes in the inoculation capsicum sick really in the rna transcription expression level.Concrete grammar is as follows:

1.2.1, the sick fruit of inoculation phytophthora blight of pepper capsicum RNA extracts

1) gets the sick fruit of capsicum 0.1g and in liquid nitrogen, fully grind, move among the 1ml Trizol, leave standstill 5min under the room temperature behind the mixing, make nucleoprotein complex dissociate fully (sample volume be no more than used Trizol volume 10%).

2) add the 0.2mL chloroform, acutely shake or with vortex oscillator mixing, 4 ℃ of centrifugal 15min of following 12000rpm/min draw supernatant, this step of repetition one or many.

3) draw supernatant, every 1ml Trizol adds 0.25 times of volume Virahol and 0.25 times of RNA precipitation agent, abundant mixing, and room temperature is placed 10min, in 4 ℃ of centrifugal 10min of following 12000rpm/min.

4) collect the RNA precipitation, with 75% washing with alcohol 2 times, repeat above-mentioned centrifugal.

5) disposable tip exhausts remaining ethanol, opens the centrifuge tube of pipe lid and places the experiment table several minutes, and residual ethanol is vapored away.

6) add the water that 50-100 μ l DEPC handles, RNA solution is stored in-70 ℃.

1.2.2, RNA detects

Identify the RNA integrity by RNA denaturing formaldehyde electrophoresis, rRNA accounts for total RNA80-85%, and sepharose can clearly be seen 28S and 18SrRNA.The 28SrRNA amount is the twice of 18SrRNA approximately, illustrates that the RNA integrity is better.Get 2.5 μ l RNA samples, measure RNA with trace of albumin nucleic acid determination instrument (IMPLEN), the result shows that A260/A280 ratio is 1.9-2.1, obtains the higher RNA of purity.

1.2.3, reverse transcription cDNA first chain is synthetic

1) reaction system is for finally being 20 μ L, 10 * RT mix, 2 μ L, 2.5mmol/LdNTP mixed solution 2 μ L, primer Oligo-dT 2 μ L, Quant Reverse Transcriptase 1 μ L, RNase Free ddH ₂O 13-x μ L, the 4th step of template ribonucleic acid adds x μ L.

2) template ribonucleic acid is thawed primer, 10 * RT mix, dNTP mixed solution, RNase Free ddH on ice ₂O thaws in room temperature, places on ice rapidly then.With various solution vortex vibration mixings, brief centrifugal collection residues in the liquid on the tube wall before using.

3) according to 1) reverse transcription system preparation mixed solution, thorough mixing, the vortex vibration was no more than for 5 seconds, and is briefly centrifugal, places on ice.

4) as a plurality of reverse transcription reactions of need, the mixed solution for preparing can be divided in the single reaction pipe, place on ice.

5) template ribonucleic acid is added in the mixed solution thorough mixing, residual liquid on the brief centrifugal collection tube wall.

6) hatched 60 minutes for 37 ℃, back reverse transcription product carries out follow-up PCR reaction.

1.2.4, real-time fluorescence quantitative PCR (qRT-PCR)

The qRT-PCR primer sequence that adopts is as follows:

The upstream primer of Pcipg2: 5`-ACCTGTCTTCGTGAAGCTGCAA-3`, the downstream primer 5`-GCCGTCCGTGTTTTGAGCAA-3` of Pcipg2.

The upstream primer of Pcipg3: 5`-ACTGTGTCGGGCTTCACCCTC-3`, downstream primer: 5`-AGACGCCGTTGTTGCCGCTC-3`.

The upstream primer of Pcipg5: upstream primer is 5`-CGTTCCGTACCTTCAGCATTGT-3`, and downstream primer is 5`-GGTGTTCGTACTGGACTGCAT-3`.

The upstream primer of Pcipg8: 5`-CAAGAAGCTGAGACAACGGCC-3`, downstream primer: 5`-GTAAATTCTATTGTAGCGC-3`p.

The upstream primer of Pcipg9: 5`-TTATTTTCTACTGTTACCGCTGCCTT-3`, the downstream primer of Pcipg9: 5`-GGTCCAAAGTCACACCTGCG-3`.

The upstream primer of Pcipg10: 5`-GTCTGCTGCCTTTGCAGCGT-3`, the downstream primer of Pcipg10: 5`-TAAATTCTATCGTCGCCCCGGAC-3`.

The upstream primer of Pcipg11: 5`-CAAGCAGAAGAGGCGTCGACAT-3`, downstream primer: 5`-ACTTATTTTGATGTTTGCGCC--3`.

The upstream primer of Pcipg12: 5`-ATGAAATTTTTATCTGCTACTA-3`, the downstream primer of Pcipg12: 5`-TCTGTTTGGCCGGTCTGAGAAGC-3`.

The upstream primer of Pcipg13: 5`-AGCAACAGTCGAGTACAAGC-3`, downstream primer: 5`-GGTAAAAGTTATCGTGGCACC--3`

The upstream primer of Pcipg14: 5`-TTGCATTGATCGCGTCTATAAC-3`, the downstream primer of Pcipg14: 5`-GTCGGCTACATCAGACAGGTCT-3`;

The upstream primer 5`-CCCTGTGCAGCAACAGCAAC-3` of Pcipg15, downstream primer: 5`-AAGAAATTCAATTATAGCACC-3`.

The upstream primer of Pcipg16: 5`-CAAGCCGAAGAGGGCTCATCG-3`, downstream primer: 5`-GGTAAATTCTATAGTAGCTCC-3`.

The upstream primer of Pcipg17: 5`-TTGGCTGCCGCAGTTGCGCTTTTC-3`, the downstream primer of Pcipg17: 5`-CTTTAGACAAGTCTAAAGTCAC-3`.

The upstream primer of Pcipg18: 5`-AGCAGAGGGCGGAAACCAGGT-3`, downstream primer: 5`-TGTGAACTGAATGGTTGCCCCC-3`.

Be internal control gene with capsicum host 18S rRNA, its upstream primer: 5`-TTTCGGTCCTATTACGTTGG-3`; Amplification downstream primer: 5`-TTCGCAGTTGTTCGTCTTTC-3`.

The cDNA synthetic with the total RNA reverse transcription of different vaccination time pepper fruit is template, utilize above-mentioned primer to carry out qRT-PCR, the pcr amplification system is 20 μ L, 2.5 * realMasterMix:8 μ L, 20 * SYBR solution, 1 μ L, forward primer 0.5 μ L, reverse primer 0.5 μ L, cDNA template 2 μ L add dd H ₂O to 20 μ L.PCR parameter: 95 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 20s, 68 ℃ are extended 55s, 40 circulations, 65-95 ℃ of preparation solubility curve.

Selecting capsicum host 18S rRNA is internal control gene, and each sample is done three repetitions, with 2 ^{-Δ Δ Ct}Method is carried out the differential expression relative quantitative assay to the sample gene.Δ Δ Ct=(C _{T target}-C _{T 18S rRNA}) _{Sample to be tested}-(C _{T target}-C _{T 18S rRNA}) _{Calibration sample}, select each gene in the calibration sample that is expressed as of inoculating first day, the 3rd, 5,7 day is sample to be tested, for example:

The sample title	JUN Mean(Ct)	18SrRNA Mean(Ct)	ΔCt	ΔΔCt		2 ^-ΔΔCt
							1	27.35	16.98	10.37	0	1
2	27.52	16.86	10.66	0.29	0.82

Be calibration sample with No. 1 sample, No. 2 is sample to be tested, uses Δ Δ Ct method and analyzes:

The first step, use reference gene calibration sample and sample to be tested are proofreaied and correct:

Δ Ct _{(calibration sample)}=JUN (Mean Ct) ₁-reference gene 18S rRNA (Mean Ct) ₁

＝27.35-16.98＝10.37

Δ Ct _{(sample to be tested)}=JUN (Mean Ct) ₂-reference gene 18S rRNA (Mean Ct) ₂

＝27.52-16.86＝10.66

In second step, the Δ Ct of calibration sample and sample to be tested carries out normalization method:

Δ Δ Ct=Δ Ct _{(sample to be tested)}-Δ Ct _{(calibration sample)}=10.66-10.37=0.29

In the 3rd step, differential expression calculates:

2 ^-ΔΔCt＝2 ^-0.29＝0.82

Therefore the JUN expression of gene level of No. 2 samples is hanged down 0.82 times than No. 1.

The experiment establish three repetitions, the result shows, Pcipg 8 gene RNA transcriptional expression levels higher relatively (Fig. 1) among 14 gene members, and inoculate the 5th day the highest, thus it is defined as the target Disease-causing gene.

2, Pcipg 8 gene gene specific primer ipg8F-R

Design and synthesize the some to special primer of Pcipg 8 genes, tested these primers to the specificity of bacterial classification that all are participated in the experiment and the susceptibility that phytophthora blight of pepper is detected.All higher primer is right as the primer of special detection Phytophthora capsici to ipg8F-R therefrom to have selected a pair of specificity and sensitivity, and upstream primer is ipg8F (21bp), and sequence is 5`-CAAGAAGCTGAGACAACGGCC-3` (sequence 1); Downstream primer is ipg8R (24bp), and sequence is 5`-GTAAATTCTATTGTAGCGC-3` (sequence 2), and amplifying target genes length is 176bp.Simultaneously with Pcipg 5 (be for GenBank number: EF558847) be the reference gene, Pcipg 5 a pair of Auele Specific Primers, name is called ipg5F-R, upstream primer is ipg5F, sequence is 5`-acgggcgctaacatcaagatc-3`, downstream primer is ipg5R, and sequence is 5`-gatatggttgttcttagtcaggtc-3`, and amplifying target genes length is 357bp.

2.1, the specificity of a pair of special primer of Pcipg 8 and Pcipg5

2.1.1. for planting experimentally bacterium and cultivating and make

Strains tested: Phytophthora capsici (P.capsici) SD33, Jul02101, Jul02104 (J.Phytopathol157:585-591,2009), ATCC7858, ATCC7859, soybean phytophthora (P.sojae) SCRP555 (Molecular Plant Pathology, 2006,7 (5) 365-379), phytophthora parasitica (P.parasitic) SD-1 (J.Phytopathol157:585-591,2009), phytophthora infestans (P.infestans) Aug0206-7 (J.Phytopathol157:585-591,2009), camphor tree epidemic disease mould (P.cinnamomi) (CBS341.72), Jue Shi epidemic disease mould (P.drechsleri) (CBS 111342), palm mould (P.palmivora) (CBS 111346), torrid zone epidemic disease mould (P.tropicalis) (CBS121658), ramie mould (P.boehmeriae) (CBS 111328), Phytophthora cactorum (P.cactorum) (CBS128432), clover epidemic disease mould (P.medicaginis) (CBS119902), taro epidemic disease mould (P.colocasiae) (CBS 192.91), latent ground epidemic disease mould (P.cryptogea) (CBS 130886), pea epidemic disease mould (P.erythroseptica) (CBS 111343), strawberry epidemic disease mould (P.fragariae) (CBS309.62), big male epidemic disease mould (P.megasperma) (CBS 118733), Kidney bean epidemic disease mould (P.phaseoli) (CBS120373), green onion epidemic disease mould (P.porri) (CBS127101), winter is given birth to epidemic disease mould (P.hibemalis) (CBS 119904), melon and fruit corruption mould (Pythium aphanidermatum) (CBS 127509), water mold (Saprolegniasp.) (CBS 342.62) is used the V8 substratum respectively, and (substratum is made with reference to planting the disease research method, 1998,37-52).Sweet potato head mold (Rhizopus batatas) (CBS 387.34), dry thread Pyrenomycetes (Rhizoctonia solani) (CBS124594), fusarium moniliforme (Fusarium moniliforme) (CBS 130180), fusarium solani (F.solani) (CBS 130328), alternaria solani sorauer (Alternaria solani) (CBS 347.79), Penicillium notatum (Penicillium sp.) (CBS339.61) and aspergillus cremeus (Aspergillus cremeus) (CBS 477.65) all use potato culture (PDA) to cultivate that (substratum is made with reference to planting the disease research method, 1998,37-52).The preparation of Phytophthora capsici zoospore suspension and soil oospore and numeration method be with reference to the mould investigative technique of epidemic disease, and 1997,78-83).

2.1.2. yeast culture and collection

Move on the oat plate culture medium for the various phytophthoras of examination, after in 25 ℃ of biochemical incubators, cultivating 2-3d, cut the bacterium piece from colony edge, go to the V8 substratum, and other fungi of participating in the experiment moves on the PDA substratum, and 5-7d is cultivated in the equal 25 ℃ of concussions of all kind of bacterium of participating in the experiment, and sterile gauze filters, be developed into powder through freezing draining ,-20 ℃ of preservations are standby.

The phytophthora blight of pepper bacterium material not of the same race DNA extraction 2.1.3. participate in the experiment

Adopt the CTAB method, the DNA of extraction is standby in-20 ℃ of preservations, and gets 2 μ L (50ng/ μ L) respectively and carry out pcr amplification as template.

2.1.4.Pcipg 8 and the Pcipg5 gene specific primer to kind of the pcr amplification of bacterium DNA of participating in the experiment

The PCR reaction system of Pcipg5 and Pcipg 8 is: 10 * PCR damping fluid, 5 μ L, template DNA (50ng) 2 μ L, each 1 μ L (Pcipg 5 is all identical with Pcipg 8 gene upstream and downstream primer concentrations, is 10 μ M) of upstream and downstream primer, dNTP (2.5mmol/L) 4 μ L, MgCl ₂(25mmol/L) 4 μ L, Taq archaeal dna polymerase 0.5 μ L (5 units/μ L), ddH ₂O (32.5 μ L).

The PCR trace routine of Pcipg 8 and Pcipg 5 is: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations; Last 72 ℃ are extended 10min.Get 10 μ L pcr amplification products and detect with 1.5% TBE glue, compare with DL2000DNA Marker.After electrophoresis finishes, with Bi o Imag ing Sys tem Gene CeniusLP-400 full automatic gel imaging system observations and scan in computer software and preserve.

Three repetitions are established in experiment, and the result shows that a pair of special primer ipg8F-R of a pair of special primer ipg8F-R of Pcipg 8 genes and reference Pcipg 5 genes is all to 5 special detection performances of phytophthora blight of pepper sample tool.Ipg8F-R has all obtained the fragment of 176bp to the amplification system of 5 phytophthora blight of pepper samples, and the sequencing result of PCR product shows that the sequence of the pcr amplification product of this 5 strain Phytophthora capsici is the 76-251 position of sequence 3 in the sequence table; Ipg5F-R has all obtained the fragment (Fig. 2) of 357bp to the amplification system of 5 phytophthora blight of pepper samples.

2.2Pcipg 8 and the sensitivity of Pcipg5 gene specific primer

This step embodies the sensitivity of Pcipg 8 and Pcipg5 gene specific primer by the experiment of detection by quantitative phytophthora blight of pepper zoospore and oospore.

2.2.1. the PCR to Phytophthora capsici zoospore DNA

Get adding 350 phytophthora blight of pepper SD33 zoospores (microscopically is is directly recorded and narrated) in the 50 μ L sterilized waters, add 0.05g quartz sand, concuss 3min on vortice is centrifugal slightly, supernatant liquor is the DNA crude extract of zoospore, gets 6.0,5.5,5.0 respectively, 4.5,4.0,3.5,3.0,2.5,2.0,1.5,1.0,0.5,0.4,0.3 0.2,0.1 μ L supernatant liquor is directly as the pcr amplification template.

In the pcr amplification of special primer to ipg8F-R and ipg5F-R, the PCR reaction system that adopts is 50 μ L.

The PCR reaction system of Pcipg 5 and Pcipg 8 is: 10 * PCR damping fluid, 5 μ L, template DNA (50ng) 2 μ L, each 1 μ L (Pcipg 5 is all identical with Pcipg 8 gene upstream and downstream primer concentrations, is 10 μ M) of upstream and downstream primer, dNTP (2.5mmol/L) 4 μ L, MgCl ₂(25mmol/L) 4 μ L, Taq archaeal dna polymerase 0.5 μ L (5 units/μ L), ddH ₂O (32.5 μ L).

The PCR trace routine of Pcipg 5 and Pcipg 8 is: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations; Last 72 ℃ are extended 10min.Get 10 μ L pcr amplification products and detect with 1.5% TBE glue, compare with DL2000DNA Marker.After electrophoresis finishes, with Bio Imaging System Gene Cenius LP-400 full automatic gel imaging system observations and scan in computer software and preserve.

Three repetitions are established in experiment, the result shows, the a pair of specific primer I pg8F-R of Pcipg 8 genes all significantly detects effect to the zoospore tool of different concns, especially be 0.4 to zoospore DNA sample, 0.3,0.2,0.1 the equal tool of μ L significantly detects effect, has all obtained the fragment of 176bp, detects minimum 0.7/50 μ L that reach of zoospore precision, and the lowest detection volume of the zoospore DNA of a pair of specific primer I pg5F-R of Pcipg 5 genes sample is 0.5 μ L, to 0.4,0.3,0.2,0.1 μ L sample not tool detects effect, detects minimum 3.5/50 μ L (Fig. 3) that reach of zoospore precision.Therefore, Pcipg 8 gene specific primer Ipg8F-R are higher than the detection sensitivity of the Phytophthora capsici zoospore of reference gene Pcipg 5 gene specific primer Ipg5F-R.

2.2.2. the PCR to Phytophthora capsici oospore DNA

Get adding 350 phytophthora blight of pepper SD33 oospore (microscopically is is directly recorded and narrated) in the 50 μ L sterilized waters, add 0.05g quartz sand, concuss 3min on vortice, centrifugal slightly, supernatant liquor is the DNA crude extract of zoospore, gets 4.0 respectively, 3.5,3.0,2.5,2.0,1.5,1.0,0.5,0.4,0.3,0.2 0.1 μ L supernatant liquor is directly as the pcr amplification template.

The PCR trace routine of Pcipg 5 and Pcipg 8 is: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations; Last 72 ℃ are extended 10min.Get 10 μ L pcr amplification products and detect with 1.5% TBE glue, compare with DL2000DNA Marker.After electrophoresis finishes, with Bio Imaging System GeneCenius LP-400 full automatic gel imaging system observations and scan in computer software and preserve.

Three repetitions are established in experiment, the result shows, the a pair of special primer ipg8F-R of Pcipg 8 genes all significantly detects effect to the oospore tool of different concns, especially be 0.4 to oospore DNA sample, 0.3,0.2,0.1 the equal tool of μ L significantly detects effect, has all obtained the fragment of 176bp, detects minimum 0.7/50 μ L that reach of oospore precision, and the lowest detection volume of the oospore DNA of a pair of specific primer I pg5F-R of Pcipg5 gene sample is 0.5 μ L, to 0.4,0.3,0.2,0.1 μ L sample not tool detects effect, detects minimum 3.5/50 μ L (Fig. 4) that reach of oospore precision.Therefore, Pcipg 8 gene specific primer Ipg8F-R are higher than the detection sensitivity of the Phytophthora capsici oospore of reference gene Pcipg 5 gene specific primer Ipg5F-R.

Embodiment 2, Pcipg 8 gene specific primers detect different diseased tissuess and sick field soil DNA PCR Ipg8F-R

Present embodiment has been tested the detection effect of I pg8F-R to Phytophthora capsici in sick field soil, sick stem, sick fruit, the sick leaf, and concrete grammar is as follows:

1.DNA extract

Phytophthora blight of pepper SD33 is gone in the V8 solid plate, behind 25 ℃ of dark culturing 2-3d, gas is got bacterium colony piece (2mm * 2mm) from colony edge with fanning the air, wound is inoculated in capsicum (middle green pepper No. 6) seedling (7-9 sheet leaf) basal part of stem, fruit, onesize bacterium piece microtrauma mouth is inoculated in the capsicum blade, cut the different onset tissue then respectively, with reference to (Nucleic Acids Research such as Wang, 1993, NaOH method 21:4153-4154) is extracted the DNA of diseased tissues: get one section sick stem respectively, sick fruit, sick leaf, the 1mg diseased tissues adds 10 μ L0.5mo l/L NaOH, go in the eppendorf centrifuge tube of 1.5mL after fully grinding, the centrifugal 5min of 10000rpm gets 5 μ L supernatant liquors and adds 495 μ L0.1mmo l/L Tris (pH 8.0), gets 1 μ L after being mixed and is directly used in pcr amplification as template.The hybrid dna of healthy capsicum (the middle green pepper No. 6) stem that extracts with present method, fruit, blade is blank.With the DNA of phytophthora blight of pepper SD33 as positive control.

The sick field soil DNA that phytophthora blight of pepper SD33 is infected and the sick field soil DNA extracting method of artificial inoculation phytophthora blight of pepper SD33 oospore are got 1 μ LDNA as template with embodiment 1.

2.Pcipg 8 gene specific primers are to the pcr amplification of different capsicum epidemic disease tissue DNAs

The PCR reaction system is: 10 * PCR damping fluid, 5 μ L, template DNA (50ng) 2 μ L, each 1 μ L (Pcipg 5 is all identical with Pcipg 8 gene upstream and downstream primer concentrations, is 10 μ M) of upstream and downstream primer, dNTP (2.5mmol/L) 4 μ L, MgCl ₂(25mmol/L) 4 μ L, Taq archaeal dna polymerase 0.5 μ L (5 units/μ L), ddH ₂O (32.5 μ L).

The PCR trace routine is: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations; Last 72 ℃ are extended 10min.Get 10 μ LPCR amplified productions and detect with 1.5% TBE glue, compare with DL2000DNA Marker.After electrophoresis finishes, with Bio Imaging System Gene Cenius LP-400 full automatic gel imaging system observations and scan in computer software and preserve.

The result as shown in Figure 5, the soil thallus DNA of sick stem tissue DNA, sick fruit tissue DNA, the sick DNA of leaf texture, artificial inoculation oospore and the sick field soil thallus DNA that contains oospore are through the pcr amplification of Auele Specific Primer to Ipg8F-R, all obtained the fragment of 176bp, illustrated that Ipg8F-R significantly detects effect to the phytophthora blight of pepper tool in different Phytophthora capsici diseased tissuess and the sick field soil.The a pair of specific primer I pg8F-R of Pcipg 8 genes provided by the present invention can carry out qualitative and quantitative analysis and early warning to the phytophthora blight of pepper of different times.

Claims

1. the special primer of detection or auxiliary detection Phytophthora capsici is right, and it is characterized in that: described primer is to being made up of the single stranded DNA shown in the sequence 2 in the single stranded DNA shown in the sequence in the sequence table 1 and the sequence table.

2. detect or the PCR reagent of auxiliary detection Phytophthora capsici, it is characterized in that: it is right that described reagent contains the described special primer of claim 1.

3. detect or the PCR test kit of auxiliary detection Phytophthora capsici, it is characterized in that: it is right that described test kit contains the described special primer of claim 1.

4. the described special primer of claim 1 is to detecting or the reagent of auxiliary detection Phytophthora capsici or the application in the test kit in preparation.

5. the described special primer of claim 1 is to the application in detection or auxiliary detection Phytophthora capsici.

6. detect or the method for auxiliary detection Phytophthora capsici, it is characterized in that: described method comprises the steps: with the described special primer of claim 1 to determine whether to contain in the testing sample content of Phytophthora capsici in Phytophthora capsici or the definite testing sample to testing sample is carried out PCR.

7. method according to claim 6, it is characterized in that: determine whether contain Phytophthora capsici in the testing sample as follows: the cDNA that obtains with genomic dna or total RNA reverse transcription of described testing sample is template, with the described special primer of claim 1 to carrying out pcr amplification, the pcr amplification product that detection obtains, if described pcr amplification product satisfies following 1) condition, then described testing sample contains Phytophthora capsici or the candidate is contained Phytophthora capsici; If described pcr amplification product do not satisfy described 1) condition, then described testing sample does not contain Phytophthora capsici or the candidate is not contained Phytophthora capsici;

1) described pcr amplification product is the fragment of 176bp.

8. method according to claim 7 is characterized in that: adopt the pcr amplification of following condition to determine whether contain Phytophthora capsici in the testing sample in the described method: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 2min, 50 ℃ of 30s, 72 ℃ of 1min, 40 circulations.

9. method according to claim 6 is characterized in that: adopt real-time fluorescence quantitative PCR to determine the content of Phytophthora capsici in the testing sample in the described method.

10. method according to claim 9, it is characterized in that: the cycling condition of described real-time fluorescence quantitative PCR is as follows: 95 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 20s, 68 ℃ are extended 55s, 40 circulations.