CN106011137A - Specific molecular marker primer for identification of phytopthora sojae avirulence gene PsAvr1k and method - Google Patents
Specific molecular marker primer for identification of phytopthora sojae avirulence gene PsAvr1k and method Download PDFInfo
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Abstract
Belonging to the field of biotechnology, the invention discloses a specific molecular marker primer for identification of phytopthora sojae avirulence gene PsAvr1k and a method. The specific molecular marker primer pair has an upstream primer nucleotide sequence shown as SEQ ID No.3 and a downstream primer nucleotide sequence shown as SEQ ID No.4. Through the molecular marker PCR amplification by means of the molecular marker can rapidly and efficiently detect whether a field phytopthora sojae strain is a PsAvr1k virulent strain or avirulent strain. The research not only can be used for studying the distribution of PsAvr1k avirulence gene in the field, but also can reveal the pathotype differentiation variation characteristics of the phytopthora sojae PsAvr1k, and provides the basis for making of a resistant variety selection strategy.
Description
Technical field
The invention belongs to biological technical field, disclose a kind of specific molecular mark identifying soybean phytophthora nontoxic gene PsAvr1k
Note primer and method, the present invention quickly can effectively detect soybean phytophthora by the method for specific molecular marker in soybean phytophthora
Nontoxic gene PsAvr1k.
Background technology
The microbial root rot of soybean phytophthora is a kind of destructive disease in world wide on Soybean production, and soybean phytophthora is subordinate to
Belong to oomycetes, although such pathogen is morphologically similar to fungus, but relatively near with diatom and cyanophyceae relation on evolving, for
Flimmer biosphere, therefore general fungus antibacterial is often invalid to soybean phytophthora and other oomycetes.The application of plant disease-resistant kind,
Owing to overcoming the problem such as poisoning and environmental pollution, become and in agricultural production, realize disease-resistant important means.But in nature
The pathogen tachytelic evolution mediated by pathogen effector makes disease-resistant variety will soon be overcome by new pathogen, the most disease-resistant product
The application planted also is faced with certain challenge.Understand soybean phytophthora nontoxic gene distribution situation in different strains in nature,
Foundation is provided for formulating correct disease-resistant variety application strategy in agricultural production.
During the long-term coevolution of plant and pathogen, pathogen, in order to successfully infect plant, can secrete a large amount of effector and make
Different cell area for plant disturb the immunoreation of plant.Plant evolves disease-resistant egg to suppress infecting of pathogen
Identifying these effectors in vain, trigger the immunoreation of plant, this kind of effector is otherwise known as nontoxic gene.Soybean phytophthora is secreted
The identification of nontoxic gene and disease-resistant gene directly decide the generation of oomycetes disease.And nontoxic gene during evolution further through
Base mutation, aminoacid deletion, base is inserted and is caused the strategies such as frameshift mutation, escapes the identification of host's ill-resistant protein.Therefore
Understand the sequence characteristic of nontoxic gene in toxic strain and avirulent strains the virulence understanding nontoxic gene is changed, understands nontoxic base
Because the regularity of distribution in field, formulation disease-resistant variety use strategy to have great importance.
This patent strategy based on molecular marker, designs the specific primer labelling for avirulent strains, for detection field soybean epidemic disease
Trichoderma strain provides effective means for corresponding disease-resistant variety for toxic strain or avirulent strains.
Summary of the invention
In order to solve agricultural production is formulated a difficult problem for disease-resistant variety Breeding strategy, it is an object of the invention to provide a kind of detection big
Specific molecular marker primer, test kit and the application thereof of nontoxic gene PsAvr1k in bean Mtr mutant.
Another object of the present invention is to provide a kind of method identifying soybean phytophthora nontoxic gene PsAvr1k quick, easy.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of for expanding the primer pair of PsAvr1k full length sequence in soybean phytophthora different strains, forward primer nucleotide sequence is such as
Shown in SEQ ID No.1, downstream primer nucleotide sequence is as shown in SEQ ID No.2.
A kind of specific molecular marker primer pair detecting nontoxic gene PsAvr1k in soybean phytophthora bacterial strain, forward primer nucleotide
Sequence is as shown in SEQ ID No.3, and downstream primer nucleotide sequence is as shown in SEQ ID No.4.
A kind of for detecting the test kit of nontoxic gene PsAvr1k specific molecular marker in soybean phytophthora bacterial strain, comprise such as SEQ
Primer pair shown in ID No.3 and SEQ ID No.4.
A kind of for expanding the test kit of PsAvr1k full length sequence in soybean phytophthora different strains, comprise such as SEQ ID No.1 and
Primer pair shown in SEQ ID No.2.
Primer as shown in SEQ ID No.1 and SEQ ID No.2 to or comprise the test kit of this primer pair and expanding Semen sojae atricolor epidemic disease to be measured
Application in trichoderma strain PsAvr1k full length sequence.
Primer as shown in SEQ ID No.3 and SEQ ID No.4 to or comprise the test kit of this primer pair at detection soybean phytophthora
In bacterial strain nontoxic gene PsAvr1k specific molecular marker or detection soybean phytophthora bacterial strain toxicity in application.
A kind of method detecting soybean phytophthora bacterial strain toxicity, with soybean phytophthora strain gene group DNA as template, with SEQ ID No.1
It is that primer carries out PCR amplification acquisition PsAvr1k full length sequence with SEQ ID No.2, with described PsAvr1k full length sequence as template,
PCR amplification is carried out for primer, if it is possible to amplify the fragment of 504bp, then show with SEQ ID No.3 and SEQ ID No.4
This soybean phytophthora bacterial strain is PsAvr1k avirulent strains, if the fragment of 504bp can not be amplified, then shows this soybean phytophthora
Bacterial strain is PsAvr1k toxic strain.
Technical scheme particular by molecular markers for identification soybean phytophthora bacterial strain nontoxic gene PsAvr1k sequence and
Its polymorphism, its forward primer is FL-F (SEQ ID No.1), and downstream primer is FL-R (SEQ ID No.2).According to Semen sojae atricolor
The difference of the sequence of PsAvr1k in Mtr mutant avirulent strains and toxic strain, the primer mark that design avirulent strains is special
(STS), forward primer is SF-F (SEQ ID No.3), and downstream primer is SF-R (SEQ ID No.4).Draw if this is special
Thing can amplify the fragment of 504bp and be avirulent strains, if the fragment of 504bp can not be amplified, is then toxic strain.
The present invention utilizes the method that described primer identifies soybean phytophthora nontoxic gene PsAvr1k, specifically includes following steps:
(1) different strains phytophthora sojae gene group DNA is extracted；
(2) with phytophthora sojae gene group DNA as template, with described primers F L-F/R (SEQ ID No.1 and SEQ ID
No.2) PCR amplification is carried out；
(3) reclaim amplified fragments, carry out with described specific primer SF-F/R (SEQ ID No.3 and SEQ ID No.4)
PCR expands；If obtaining the fragment that size is 504bp, then show that described soybean phytophthora is avirulent strains, if 504bp cannot be obtained
Fragment, then show that described soybean phytophthora is toxic strain.
Advantage of the present invention and good effect show:
Utilize primer of the present invention and method can quickly, easy, accurate identification soybean phytophthora bacterial strain be toxic strain
Or avirulent strains.The research of the present inventor's early stage shows that PsAvr1k is the nontoxic base corresponding with Resistance genes of soybeans Rps1k
Cause, it is anti-that the soybean phytophthora avirulent strains containing PsAvr1k nontoxic gene can trigger that the soybean varieties containing Rps1k mediated
Property.The present invention is possible not only to the distribution situation studying this nontoxic gene in natural population, discloses soyabean phytophthora and occurs in field
The feature of pathological form dissociation, formulates disease-resistant by the distribution and change monitoring nontoxic gene for us in agricultural production
Strategy, control disease provide strong and instruct.
Accompanying drawing explanation
Fig. 1 soybean phytophthora difference microspecies expand PsAvr1k full-length gene
PsAvr1k full length sequence comparison in Fig. 2 soybean phytophthora avirulent strains and toxic strain
Fig. 3 soybean phytophthora bacterial strain expands the PsAvr1k fragment that PsAvr1k avirulent strains is special
Detailed description of the invention
PsAvr1k sequence polymorphism detection in embodiment 1 soybean phytophthora different strains
1. strains tested
Soybean phytophthora different virulence bacterial strain, should by Brett M.Tyler professor (Virginia bio information research department of the U.S.) present
Bacterial strain is known in those skilled in the art, Agricultural University Of Nanjing planting disease is that plant preserves with phytophthora interaction laboratory.Bacterial strain preserves
In 10%V8 solid medium, storage temperature is 10 DEG C.
2. for the preparation of examination soybean seedling
Differential host's soybean planting is inner in the plastic flowerpot (d=10cm) equipped with Vermiculitum (2-4mm), 15 kinds of every basin kind
Son, is put in the dark greenhouse of 14h illumination (strong illumination)/10h and grows, and after 7 days, two panels true leaf stays 10 after growing
Seedling is used for inoculating.Differential host used is Williams82.Susceptible variety is Williams.
3. soybean phytophthora bacterial strain pathological form measures
This research uses hypocotyl wound inoculation method to measure the soybean phytophthora bacterial strain toxicity to differential host.Soybean phytophthora bacterial strain exists
Grow after 3 days on V8 flat board, with scalpel at the mycelium of colony edge picking 1cm length；With scalpel embryo under Semen sojae atricolor
Axle (under cotyledon at about 1cm) draws downwards the wound of about 0.5cm, is then inoculated in this wound by mycelium.After inoculation
Semen sojae atricolor be put in the greenhouse that 14h illumination (strong illumination)/10h is dark and grow, add up incidence after 2d.With plant
Mortality rate, as anti-sense criteria for classification, plant mortality rate≤30%, is designated as disease-resistant (A)；Plant mortality rate >=70%, is designated as
Susceptible (V)；Plant mortality rate, between 30%-70%, is designated as intermediate form (M).Test is repeated 3 times.
4. phytophthora sojae gene group is extracted
This research uses CTAB method to extract phytophthora sojae gene group DNA, and concrete operations are as follows:
(1) take the cryodesiccated mycelium powder of about 50mg in 1.5ml EP pipe, add 900 μ L CTAB (hexadecanes
Base trimethylammonium bromide) extracting solution (2%CTAB (w/v, g/ml)；100mM Tris HCl, pH=8.0；20mM
EDTA；1.4M NaCl) and 90 μ L 10%SDS (dodecylbenzene sodium sulfonate) (w/v) whirlpools afterwards concussion instrument on fully
Concussion mixing；
(2) 55-60 DEG C of water-bath 1h, every 15min vibration mixing is once；
(3) 12000rpm/min is centrifuged 10min, draws supernatant, adds isopyknic phenol/chloroform/isoamyl alcohol (25:24:1)
(v/v) extracting 1 time；
(4) 12000rpm/min is centrifuged 10min, draws supernatant, adds equal-volume chloroform 1 time；
(5) 12000rpm/min is centrifuged 10min, draws supernatant, the 3M NaAc solution of 0.1 times of volume of addition and 2 times
The dehydrated alcohol overnight precipitation DNA of volume；
(6) 70% washing with alcohol of use pre-cooling twice, drying at room temperature；
(7) the dried DNA ddH of 40 μ L2O (containing 50 μ g/mL RNase) dissolves, 37 DEG C of degradation of rna
1h。
Take DNA sample electrophoresis on the agarose gel of 1% that 2 μ L extract, UV spectrophotometer measuring DNA concentration
And purity.DNA sample is stored in-20 DEG C of refrigerators after being finally diluted to about 25ng/ μ L.
5. the primer of design amplification nontoxic gene PsAvr1k
According to the primer of the sequential design amplification PsAvr1k of nontoxic gene PsAvr1k in avirulent strains Race 2 (P6497)
(FL-F/R), respectively from Race 1, Race 2, Race 11, Race 6, Race 7, Race19, Race 25 and Race 30
Bacterial strain expands the full length sequence of PsAvr1k.
6.PCR expands
PCR reaction system is: 2.5 μ L 10 × PCR reaction buffers；1.5μL 1.5mM MgCl2；0.5μL 2.5
mM dNTPs；0.25 μ L Taq archaeal dna polymerase (5.0U/ μ L)；0.5 μ L primer；0.5 μ L template；Sterilized water is mended
Sufficient to 25 μ L.
PCR response parameter: 95 DEG C of degeneration 5min；(72 DEG C extend 1min for 95 DEG C of degeneration 30S, 58 DEG C of annealing 30s)
Totally 33 circulations；72 DEG C extend 10min；4℃10min；PCR reacts in TaKaRa PCR instrument.
Electrophoresis: PCR primer 1% agarose gel electrophoresis, 120V electrophoresis 20-30min, with 0.5 μ g/mL ethidium bromide
Solution dyes, and observes (Fig. 1) with Bio-rad gel imaging system.
7.PCR product reclaims and order-checking
The PCR primer of PsAvr1k reclaims test kit by TaKaRa gel and reclaims, and send Jin Sirui company to check order.
The order-checking comparison of 8.PCR product
In avirulent strains and toxic strain, the sequence of PsAvr1k compares, and finds out toxic strain and avirulent strains
PsAvr1k sequence difference (Fig. 2).
Specific primer Marker Identification avirulent strains in embodiment 2 soybean phytophthora PsAvr1k avirulent strains
1. the labeled primer of PsAvr1k in design specific amplification avirulent strains
Specificity section design primer (table 1) (Fig. 2) according to avirulent strains and toxic strain.PsAvr1k gene is different bacterium
Sequence difference in strain sees SEQ ID No.5-12
Table 1PsAvr1k total length primer and the specificity labeled primers of avirulent strains
2.PCR amplification is verified with product
The PCR primer utilizing PsAvr1k total length is template, with for avirulent strains PsAvr1k specific primer (SEQ ID
No.3 and SEQ ID No.4) carry out PCR amplification.PCR primer is verified with the agarose gel of 1%.If able to expand
Increase the fragment 504bp, then show that this bacterial strain is PsAvr1k avirulent strains；If correspondingly sized sheet can not be amplified
Section, then show that this bacterial strain is PsAvr1k toxic strain (Fig. 3).
Claims (7)
1. one kind is used for expanding the primer pair of PsAvr1k full length sequence in soybean phytophthora different strains, it is characterised in that: upstream is drawn
Thing nucleotide sequence is as shown in SEQ ID No.1, and downstream primer nucleotide sequence is as shown in SEQ ID No.2.
2. the specific molecular marker primer pair being used for detecting nontoxic gene PsAvr1k in soybean phytophthora bacterial strain, its feature exists
In: forward primer nucleotide sequence is as shown in SEQ ID No.3, and downstream primer nucleotide sequence is as shown in SEQ ID No.4.
3. one kind is detected the test kit of nontoxic gene PsAvr1k specific molecular marker in soybean phytophthora bacterial strain, it is characterised in that:
Comprise the primer pair described in claim 2.
4. one kind is used for expanding the test kit of PsAvr1k full length sequence in soybean phytophthora different strains, it is characterised in that: comprise power
Profit requires the primer pair described in 1.
5. the primer described in claim 1 to or claim 4 described in test kit expand soybean phytophthora bacterial strain PsAvr1k to be measured
Application in full length sequence.
6. the primer described in claim 2 to or claim 3 described in test kit detection soybean phytophthora bacterial strain in nontoxic gene
Application in PsAvr1k specific molecular marker or detection soybean phytophthora bacterial strain toxicity.
7. the method detecting soybean phytophthora bacterial strain toxicity, it is characterised in that: with soybean phytophthora strain gene group as template,
Carry out PCR amplification with SEQ ID No.1 and SEQ ID No.2 for primer and obtain PsAvr1k full length sequence, complete with described PsAvr1k
Long sequence is template, carries out PCR amplification with SEQ ID No.3 and SEQ ID No.4 for primer, if it is possible to amplify 504bp
Fragment, then show that this soybean phytophthora bacterial strain is PsAvr1k avirulent strains, if the fragment of 504bp can not be amplified, then
Show that this soybean phytophthora bacterial strain is PsAvr1k toxic strain.
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| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
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| CN1526831A (en) * | 2003-09-19 | 2004-09-08 | 深圳出入境检验检疫局动植物与食品检 | Real-time fluorescence PCR detection probe and kit for soyabean phytophthora and the detection method thereof |
| CN101942520A (en) * | 2010-10-14 | 2011-01-12 | 南京农业大学 | Marker of avirulence gene PsAvr3b of phytophthora sojae |
| CN102220415A (en) * | 2011-04-15 | 2011-10-19 | 南京农业大学 | Markers of phytophthora sojae avirulence gene Avr5 |
| CN102676511A (en) * | 2012-05-16 | 2012-09-19 | 南京农业大学 | Detection target sequence A3apro of phytophthora sojae, and specific LAMP (loop-mediated isothermal amplification) primer composition and application thereof |
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| Title |
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| GENBANK: "KC312950.1", 《GENBANK》 * |
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