CN105603105B - A kind of measurement soyabean phytophthora is to the molecular method of the toxicity of soybean line Chapman(Rps3a) - Google Patents
A kind of measurement soyabean phytophthora is to the molecular method of the toxicity of soybean line Chapman(Rps3a) Download PDFInfo
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Abstract
The present invention relates to a kind of measurement soyabean phytophthoras to disease-resistant geneRps3a(Chapman) molecular method of toxicity, comprising steps of extracting the genomic DNA of soyabean phytophthora to be detected, PCR amplification, which goes out, contains nontoxic geneAvr3aDNA fragmentation, use restriction enzymeBpu10I digestion carries out agarose gel electrophoresis to digestion products, obtains the agarose gel electrophoresis figure of digestion products；Digestion products agarose gel electrophoresis figure is observed, if there are 2 DNA bands within the scope of 250bp-500bp, corresponding soybean phytophthora bacteria strain to be detected is avirulent strains；If digestion rear electrophoresis figure has 1 DNA band in 750bp or less, corresponding soybean phytophthora bacteria strain to be detected is toxic strain.This method measure the period it is short, without intermediate form, accurate and reliable, can high throughput assay soyabean phytophthora to disease-resistant geneRps3a(Chapman) toxicity.
Description
Technical field
The present invention relates to a kind of molecular assay methods more particularly to a kind of measurement soyabean phytophthora to disease-resistant gene Rps3a
(Chapman) molecular method of toxicity.
Background technique
It is the Major Diseases that Soybean production is threatened in world wide that soyabean phytophthora, which infects root rot caused by soybean,
One of.Root rot was found for the first time in the Indiana State in the U.S. in 1948, then the major producing country of soybean all
The disease is had found in succession.China is separated to soybean phytophthora in northeast soybean main producing region for the first time by Shen Chong Yao and Su Yanchun in 1989
Bacterium, later Heilongjiang Province, Jilin Province and Beijing also in succession discovery have soyabean phytophthora, in recent years Shandong, Anhui, Henan,
15 provinces (urban district) such as Jiangsu, Zhejiang, the Inner Mongol, Hubei, Fujian, Xinjiang, Sichuan, Tianjin and Guizhou are also reported separation in succession
Identify soyabean phytophthora.It is wherein more serious in Heilungkiang and the generation of two province of Fujian, it is at present still the important external inspection in China
Epidemic disease object.
The evolutionary rate of soyabean phytophthora is very fast, and Physiological Race Differentiation is sufficiently complex, and the U.S. has identified 55 lifes at present
Reason microspecies and the pathological form for not naming biological strain formally more；China have found No. 1, No. 3, No. 8, No. 9, No. 11, No. 15,
10 biological strains such as No. 17, No. 21, No. 24 wherein No. 1 biological strain is the dominant races of Heilongjiang Province, while also identifying
The pathological form of biological strain is not named largely.
Prevention and treatment soybean phytophthora root rot most efficient method is plantation disease-resistant variety at present, and wants accurately to use Soybean Resistance
Sick kind just needs the toxicity of soyabean phytophthora (soyabean phytophthora of designated area separation) clearly to be detected to form, and (i.e. physiology is small
The composition of kind)；And the measurement of biological strain at present mainly using with 14 disease-resistant genes (Rps1a, Rps1b, Rps1c,
Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7 and Rps8) a set of near isogene
Be differential host carry out the measurement of hypocotyl wound inoculation, this method is although simple and easy, but measure period length, great work intensity,
It is easy to appear intermediate form, is not suitable for high-throughput Race Identification；Therefore seeking one kind can be quick, accurate, high-throughput
The method for measuring soybean phytophthora mushroom toxin composition is this field technical problem urgently to be resolved.
Common knowledge related to the present invention: common knowledge (1): pathogen carries nontoxic gene or virulent gene, mirror
Other host carries disease-resistant gene or susceptible gene, and wherein nontoxic gene and virulent gene are a pair of alleles, disease-resistant gene
It is a pair of alleles with susceptible gene；When the pathogen infection for carrying nontoxic gene carries the identification of corresponding disease-resistant gene
When host, it is disease-resistant that disease-resistant gene with nontoxic gene interaction shows as differential host；When the pathogen for carrying nontoxic gene is invaded
When dye carries the differential host of susceptible gene, differential host shows as susceptible；When the pathogen infection for carrying virulent gene
When differential host, regardless of whether differential host carries disease-resistant gene, show as susceptible.Common knowledge (2): for nontoxic base
It is in the world usually first to name nontoxic gene, and differential host will be made with nontoxic gene interaction because of the name with disease-resistant gene
Showing as disease-resistant unnamed gene is corresponding disease-resistant gene；May theoretically there are multiple disease-resistant genes and the same nothing
The case where virus gene interaction, and actually also really in this way, being for example initially considered that the nontoxic gene in soybean phytophthora in the world
Avr4 and Avr6 respectively with disease-resistant gene Rps4 and the Rps6 interaction in soybean, but further investigation revealed that Avr4 and Avr6
The actually same gene, is renamed as Avr4/6, and disease-resistant gene Rps4 and Rps6 can divide with nontoxic gene Avr4/6 interaction
Not Shi Bie the different parts of nontoxic gene Avr4/6 cause disease resistance response, analyzed by common knowledge (2), may theoretically be deposited
In multiple disease-resistant genes and nontoxic gene Avr3a interaction, i.e., the disease-resistant gene Rps3a from different disease-resistant materials may be
Complete different disease-resistant gene.
Summary of the invention
The purpose of the present invention is to provide a kind of measurement soyabean phytophthoras to disease-resistant gene Rps3a (Chapman) toxicity
Molecular method；It is to identify to post that the present invention, which measures the soyabean phytophthora disease-resistant gene Rps3a targeted to disease-resistant gene Rps3a toxicity,
Disease-resistant gene Rps3a in main Chapman, this method measure the period it is short, without intermediate form, accurate and reliable, can high throughput assay
Toxicity of the soyabean phytophthora to disease-resistant gene Rps3a (Chapman)；For quick, accurate, high throughput assay soybean phytophthora mushroom toxin
Composition is laid a good foundation.
In order to solve the above technical problems, the technical scheme adopted by the invention is that: a kind of measurement soyabean phytophthora is to disease-resistant
The molecular method of gene Rps3a (Chapman) toxicity, comprising the following steps:
Step 1: extracting the genomic DNA of soyabean phytophthora to be detected；
Step 2: design specific primer AVR3A1F/AVR3A1R carries out PCR using the genomic DNA of extraction as template
Amplification, amplifies the DNA fragmentation containing complete nontoxic gene Avr3a；The alkali of the specific primer AVR3A1F/AVR3A1R
Basic sequence are as follows:
AVR3A1F:5 ˊ-ATCGAAGCCATATATCTACAGG-3 ˊ (sequence 1 in such as sequence table)；
AVR3A1R:5 ˊ-CCTACTGTATGCAAAGTGATCG-3 ˊ (sequence 2 in such as sequence table)；
Step 3: the DNA fragmentation amplified with restriction enzyme Bpu10I digestion, obtains digestion products；
Step 4: carrying out agarose gel electrophoresis to digestion products, the agarose gel electrophoresis figure of digestion products is obtained；It sees
Digestion products agarose gel electrophoresis figure is examined, it is corresponding to be detected if there is 2 DNA bands within the scope of 250bp-500bp
Soybean phytophthora bacteria strain is avirulent strains；If digestion rear electrophoresis figure has 1 DNA band in 750bp or less, corresponding to be checked
Survey soybean phytophthora bacteria strain is toxic strain.
Further, the method for extraction soyabean phytophthora genomic DNA to be detected described in step 1 is CTAB method.
Further, the CTAB method extracts the specific steps of soyabean phytophthora genomic DNA to be detected are as follows:
(1) 10-20 block diameter 2 × 2mm mycelia block is cut from the soyabean phytophthora colony edge to be detected of culture 5-7 days,
It moves into the triangular flask equipped with the clear 10%V8 fluid nutrient medium of 100ml, is placed under 25 DEG C of dark conditions, 120r/min concussion
Culture 5-7 days, cultured mycelium is collected by filtration with sterilizing double gauze, distilled water flushing 1 time, abundant after freeze-dried
Smash into hypha powder to pieces；
(2) take the hypha powder of 50mg in 1.5ml EP pipe, be added 900 μ l mass concentrations be 2% CTAB extracting solution and
It is mixed well on eddy mixer after the SDS aqueous solution that 90 μ l mass concentrations are 10%；
(3) 55-60 DEG C of water-bath 1h, every 15min oscillation mix primary；
(4) 12000r/min is centrifuged 10min, takes supernatant A, and phenol, chloroform and the mixture of isoamyl alcohol three is added and takes out
It mentions 1 time, the weight ratio of phenol, chloroform and isoamyl alcohol is 25 in the mixture of the phenol being added, chloroform and isoamyl alcohol three:
The volume of 24:1, phenol, chloroform and isoamyl alcohol three's mixture are equal with the volume of supernatant A；
(5) 12000r/min be centrifuged 10min, Aspirate supernatant B, be added chloroform 1 time, the volume of chloroform be added and
The volume of supernatant B is equal；
(6) 12000r/min is centrifuged 10min, Aspirate supernatant C, and the NaAc aqueous solution and ice dehydrated alcohol of 3mol/L is added
The volume ratio of overnight precipitation genomic DNA, NaAc aqueous solution and ice dehydrated alcohol be added and supernatant C is 0.1:2:1；
(7) it is washed twice with the ethanol water that the volume ratio of pre-cooling is 70%, it is dry under the conditions of being subsequently placed in 37 DEG C；
(8) DNA after drying contains the ddH of 50 μ g/ml ribalgilases with 100 μ l2O dissolves, and after 37 DEG C of standing 1h, sets
It is spare in -20 DEG C of refrigerators.
Further, the amplification system and amplification condition of the PCR amplification are respectively as follows:
Amplification system: the PCR reaction buffer of 10 times of dilution, 2.5 μ l；The MgCl of 2.5mmol/L2, 1.5 μ l；
The dNTPs of 2.5mmol/L, 0.5 μ l；The Taq archaeal dna polymerase of 5U/ μ l, 0.2 μ l；The specific primer of 10 μm of ol/L
Each 0.5 μ l of AVR3A1F and AVR3A1R；Template DNA, 0.5 μ l；Add sterile ultrapure water to 25 μ l；
Amplification condition: 95 DEG C of denaturation 30S, 60 DEG C of annealing 30S, 72 DEG C of extension 30S, 30 recycle, 72 DEG C of extension 5min.
Further, the DNA fragmentation that PCR amplification described in step 2 goes out is before digestion with the recycling examination of AxyPrep DNA gel
The recycling of agent box；The digestion system and digestion condition of digestion described in step 3 are respectively as follows:
Digestion system: the enzyme cutting buffering liquid of 10 times of dilution, 2 μ l；The Bpu10I enzyme of 5U/ μ l, 0.5 μ l；With AxyPrep DNA
The DNA fragmentation of gel reclaims kit recycling, 6 μ l；Add ddH2O to 20 μ l；
Digestion condition: 37 DEG C of incubation 1.5h, then 80 DEG C of incubation 20min are inactivated.
Further, the condition of the agarose gel electrophoresis are as follows: 1% Ago-Gel, electrophoresis under 120V voltage
30min is dyed with 0.5 μ g/ml ethidium bromide solution, is taken a picture under ultraviolet lamp with Bio-rad gel imaging system.
The utility model has the advantages that the present invention provides one kind for measuring soyabean phytophthora to disease-resistant gene Rps3a (Chapman)
The molecular method of toxicity, the specific primer AVR3A1F/AVR3A1R that the present invention designs be capable of specificity with template DNA without
The upstream and downstream of virus gene Avr3a combines, so that the DNA fragmentation containing complete nontoxic gene Avr3a is amplified, and
The extra base quantity in the both ends nontoxic gene Avr3a is relatively reasonable in the DNA fragmentation amplified, has both saved needed for PCR amplification
Base, and subsequent electrophoretic effects can be made more obvious；The present invention is to be detected big by restriction enzyme Bpu10I digestion
The nontoxic gene Avr3a of beans phytophthora carrys out indirect determination soyabean phytophthora to be detected to disease-resistant gene Rps3a's (Chapman)
Toxicity, the measurement period is short, without intermediate form, accurate and reliable, can high throughput assay soyabean phytophthora to disease-resistant gene Rps3a
(Chapman) toxicity provides strong support for a large amount of, the quick biological strain with precise Identification soyabean phytophthora；The present invention
Judge whether nontoxic gene Avr3a is cut by restriction enzyme Bpu10I by carrying out agarose gel electrophoresis to digestion products
It opens, it is clearly to be digested into two sections, as avirulent strains that electrophoretogram, which is 2 DNA bands,.
Figure of description
The DNA fragmentation that 5 plants of soyabean phytophthora genomic DNA amplifications to be detected shown in Fig. 1, embodiment go out is by restriction enzyme
Agarose gel electrophoresis figure before and after enzyme Bpu10I digestion, before a figure indicates digestion, after b figure indicates digestion；
Nontoxic gene in the DNA fragmentation that 5 plants of soyabean phytophthora genomic DNA amplifications to be detected shown in Fig. 2, embodiment go out
The gene order of Avr3a is to when restriction enzyme site figure；In figure: point indicates that base is identical as the Ith row base, and short-term indicates and the Ith
Row is compared and base deletion has occurred；
The DNA fragmentation that 16 plants of soyabean phytophthora genomic DNA amplifications go out in Fig. 3, test is by restriction enzyme Bpu10I
Agarose gel electrophoresis figure after digestion；
In Fig. 1-Fig. 3: A indicates avirulent strains, and V indicates toxic strain；I indicates bacterial strain Ps0702, and II indicates bacterial strain
Ps0301, III indicates bacterial strain Ps0705, and IV indicates bacterial strain Ps0903, and V indicates bacterial strain Ps0302.
Specific embodiment
The present invention will be further described in detail below with reference to specific embodiments.
Embodiment
5 plants of soyabean phytophthoras to be detected: bacterial strain Ps0702 (is separated) for 2007, and bacterial strain Ps0301 (is separated) for 2003, bacterium
Strain Ps0705 (is separated) for 2007, and bacterial strain Ps0903 (is separated) for 2009, and bacterial strain Ps0302 (is separated) for 2003, these bacterial strains
Be to be separated from Chinese soyabean field using Soybean Leaves dish mass trapping, then purified by single spore separation, switching in
It is saved at 12 DEG C on 10%V8 slant medium, the 10%V8 slant medium refers to, the V8 after 100mL filtered through gauze
In vegetable juice, 0.2g calcium carbonate and 15g agar powder is added, distilled water is added to complement to 1 000mL, heating melts agar completely,
Then it is divided in triangular flask, 121 DEG C of sterilizing 20min；
A kind of measurement soyabean phytophthora to the molecular method of disease-resistant gene Rps3a (Chapman) toxicity, respectively to 5 plants to
Detection soybean phytophthora bacteria strain be measured, specific measuring method the following steps are included:
Step 1: extracting the genomic DNA of soyabean phytophthora using CTAB method, the concrete operations of genomic DNA are extracted such as
Under:
(1) 10-20 block diameter 2 × 2mm mycelia block is cut from the soyabean phytophthora colony edge to be detected of culture 5-7 days,
Equipped with the clear 10%V8 fluid nutrient medium of 100ml, (10%V8 fluid nutrient medium refers to the V8 vegetable with 10 times of distilled water dilution for immigration
Vegetable juice, V8 vegetable juice be Campbell company of the U.S. production tinned drink, by tomato juice, carrot juice, Celery Juice, parsley juice,
8 kinds of beet juice, lettuce juice, Western vegetable juice and spinach juice vegetable juice compositions) triangular flask in, be placed under 25 DEG C of dark conditions,
120r/min shake culture 5-7 days, cultured mycelium are collected by filtration with sterilizing double gauze, and distilled water flushing 1 time, warp
Sufficiently smash into hypha powder after freeze-drying to pieces；
(2) it takes the hypha powder of 50mg in 1.5ml EP pipe, the CTAB extracting solution that 900 μ l mass concentrations are 2% is added
(CTAB, that is, cetyl trimethylammonium bromide, mass concentration be 2% CTAB extracting solution, that is, 100ml CTAB extracting solution in contain
Have the CTAB of 2g, surplus is water) and 90 μ l mass concentrations be 10% SDS aqueous solution (SDS i.e. neopelex；
10% SDS aqueous solution, that is, 10g SDS is soluble in water, is settled to 100ml) it mixes well on eddy mixer afterwards；
(3) 55-60 DEG C of water-bath 1h, every 15min oscillation mix primary；
(4) 12000r/min is centrifuged 10min, takes supernatant A, and phenol, chloroform and the mixture of isoamyl alcohol three is added and takes out
It mentions 1 time, the weight ratio of phenol, chloroform and isoamyl alcohol is 25 in the mixture of the phenol being added, chloroform and isoamyl alcohol three:
The volume of 24:1, phenol, chloroform and isoamyl alcohol three's mixture are equal with the volume of supernatant A；
(5) 12000r/min be centrifuged 10min, Aspirate supernatant B, be added chloroform 1 time, the volume of chloroform be added and
The volume of supernatant B is equal；
(6) 12000r/min is centrifuged 10min, Aspirate supernatant C, and the NaAc aqueous solution and ice dehydrated alcohol of 3mol/L is added
The volume ratio of overnight precipitation genomic DNA, NaAc aqueous solution and ice dehydrated alcohol be added and supernatant C is 0.1:2:1；
(7) it is washed twice with the ethanol water that the volume ratio of pre-cooling is 70%, it is dry under the conditions of being subsequently placed in 37 DEG C；
(8) DNA after drying contains the ddH of 50 μ g/ml ribalgilases with 100 μ l2O dissolves, and after 37 DEG C of standing 1h, sets
It is spare in -20 DEG C of refrigerators；
Step 2: design specific primer AVR3A1F/AVR3A1R carries out PCR using the genomic DNA of extraction as template
Amplification is recycled with AxyPrep DNA gel QIAquick Gel Extraction Kit, obtains the DNA fragmentation (amplification containing complete nontoxic gene Avr3a
DNA fragmentation out includes nontoxic gene Avr3a and its upstream and downstream base, the length of the DNA fragmentation about 700bp, wherein bacterium
The gene order of nontoxic gene Avr3a is corresponding in turn to sequence table in strain Ps0702, Ps0301, Ps0705, Ps0903 and Ps0302
In sequence 3, sequence 4, sequence 5, sequence 6 and sequence 7)；
The sequence of the specific primer AVR3A1F/AVR3A1R are as follows:
AVR3A1F:5 ˊ-ATCGAAGCCATATATCTACAGG-3 ˊ；
AVR3A1R:5 ˊ-CCTACTGTATGCAAAGTGATCG-3 ˊ；
The system and condition of the PCR amplification are respectively as follows:
Amplification system: the PCR reaction buffer of 10 times of dilution, 2.5 μ l；The MgCl2 of 2.5mmol/L, 1.5 μ l；
The dNTPs of 2.5mmol/L, 0.5 μ l；The Taq archaeal dna polymerase of 5U/ μ l, 0.2 μ l；The specific primer AVR3A1F of 10 μm of ol/L
With each 0.5 μ l of AVR3A1R；Template DNA, 0.5 μ l；Add sterile ultrapure water to 25 μ l；
Amplification condition: 95 DEG C of denaturation 30S, 60 DEG C of annealing 30S, 72 DEG C of extension 30S, 30 recycle, 72 DEG C of extension 5min；
Step 3: recycled with restriction enzyme Bpu10I digestion with AxyPrep DNA gel QIAquick Gel Extraction Kit
DNA fragmentation obtains digestion products；Digestion system and digestion condition are as follows:
Digestion system: 3, the 2 μ l of NEB buffer of 10 times of dilution；The Bpu10I enzyme of 5U/ μ l, 0.5 μ l；With AxyPrep DNA
The DNA fragmentation that gel reclaims kit recycles, 6 μ l；Add ddH2O to 20 μ l；
Digestion condition: 37 DEG C of incubation 1.5h, then 80 DEG C of incubation 20min are inactivated；
Step 4: carrying out agarose gel electrophoresis, deposition condition: 1% Ago-Gel (i.e. every 100ml to digestion products
TAE or tbe buffer liquid in 1g agar Icing Sugar is added), electrophoresis 30min under 120V voltage, with 0.5 μ g/ml ethidium bromide solution
It dyes, is taken a picture under ultraviolet lamp with Bio-rad gel imaging system；
Agarose gel electrophoresis after above step obtains the corresponding nontoxic gene Avr3a digestion of 5 plants of soyabean phytophthoras
Figure；As shown in Fig. 1, after the corresponding nontoxic gene Avr3a digestion of bacterial strain Ps0702 and Ps0301 within the scope of 250bp-500bp
There are 2 DNA bands, therefore bacterial strain Ps0702 and Ps0301 are that avirulent strains (can be such that the identification for carrying disease-resistant gene Rps3a posts
Main Chapman generates disease resistance response and shows disease-resistant)；The corresponding nontoxic gene of bacterial strain Ps0705, Ps0903 and Ps0302
There is 1 DNA band in 750bp or less after Avr3a digestion, therefore bacterial strain Ps0705, Ps0903 and Ps0302 are toxic strain
(the differential host Chapman for carrying disease-resistant gene Rps3a cannot be made to generate disease resistance response and show as susceptible).
Test
16 plants of soybean phytophthora bacterial strains are measured to differential host Chapman (only with disease-resistant using hypocotyl wound inoculation method
Gene Rps3a) pathogenic (i.e. to the toxicity of disease-resistant gene Rps3a), 16 plants of soybean phytophthora bacterial strains are using Soybean Leaves dish
Mass trapping is separated from Chinese soyabean field, is then purified by single spore separation, is transferred in 10%V8 slant medium
It is saved at upper 12 DEG C, 16 plants of soybean phytophthora bacterial strains and its separation time are shown in Table 1:
1, table is tested 16 plants of soybean phytophthora bacterial strains used
The soybean phytophthora bacterial strain cultivated 7 days on V8 culture medium flat plate is cut into the fritter of 2mm × 3mm as inoculum；
The wound for drawing about 0.5cm long at 1cm downwards under the soybean seedling cotyledon of plantation 7 days with scalpel, then by inoculum mycelia
Wound is attached in facing.Moisturizing 12h after inoculation is then placed in 24-28 DEG C, in 14h illumination/10h dark greenhouse, after 5 days
Investigation result.It is each 10 plants of differential host's inoculation, dead with plant with Williams (without any disease-resistant gene) for susceptible control
Classification standard of the rate as anti-sense is died, the plant death rate≤30% is denoted as disease-resistant；The plant death rate >=70% is denoted as susceptible；It plants
The strain death rate is denoted as intermediate form, test is repeated 2 times, and the disease resistance response of each bacterial strain is shown in Table 1 between 30%-70%；
Above-mentioned 16 plants of soybean phytophthora bacterial strains are carried out such as the step in embodiment respectively, extract genomic DNA, PCR amplification,
Digestion with restriction enzyme, agarose gel electrophoresis obtain agarose gel electrophoresis figure, as shown in Figure 3.
Knot of the more above two measurement soyabean phytophthora to the method measurement of disease-resistant gene Rps3a (Chapman) toxicity
Fruit, the results showed that the result of above two measuring method measurement is identical, illustrates that molecular assay method provided by the invention is
True and reliable.
SEQUENCE LISTING
<110>University Of Science and Technology Of He'nan
<120>molecular method of a kind of measurement soyabean phytophthora to disease-resistant gene Rps3a(Chapman) toxicity
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<170> PatentIn version 3.3
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<213>soyabean phytophthora
<400> 6
atgcgcctcg ctcaagttgt ggtcgtcatc gctgcttcct tcctggttgc cactgacgct 60
ctttcgacca cgaacgcaaa ccaggccaag atcatcaaag gaacgtcgcc cggtggccac 120
agcccacgac ttctgagggc ctaccaacct gatgatgaag gcgattcccc cgaagatagg 180
acgctgtcgc cttctcaggt gacaaaaatc cttaagaaac taggcaaagg cgtcacctgg 240
gacgacgtca tgcgggaccc ggctttgttc caaagttacc agaagaaggc taacaagatc 300
attgagaagc aaaaggcagc agcgaaaaac gcgtag 336
<210> 7
<211> 330
<212> DNA
<213>soyabean phytophthora
<400> 7
atgcgcctcg ttcaagttgt ggtcgtcacc gctgcttcct tcctggttgc cactgacgct 60
ctttcgacca cgaacgcaaa ccaggccaag atcatcaaag gaacgtcgcc cggtggccac 120
agcccacgac ttctgagggc ctaccaacct gatgatgaag gcgattcccc cgaagaaagg 180
acgctgccga attctcaggt ggcaaaaatc cttaacaaac taggcgtcac ctgggacgac 240
gtcctgcggg actcggcttt gttcgaaagg taccaggaga aggctaacaa gatcattgag 300
aagcaaaagg cagcagcgaa caacgcgaag 330
Claims (6)
1. a kind of measurement soyabean phytophthora is to soybean line Chapman(Rps3a) toxicity molecular method, it is characterised in that:
The following steps are included:
Step 1: extracting the genomic DNA of soyabean phytophthora to be detected；
Step 2: design specific primer AVR3A1F/AVR3A1R carries out PCR amplification using the genomic DNA of extraction as template,
It amplifies containing complete nontoxic geneAvr3aDNA fragmentation；The base sequence of the specific primer AVR3A1F/AVR3A1R
Are as follows:
AVR3A1F:5 ˊ-ATCGAAGCCATATATCTACAGG-3 ˊ；
AVR3A1R:5 ˊ-CCTACTGTATGCAAAGTGATCG-3 ˊ；
Step 3: using restriction enzymeBpuThe DNA fragmentation that 10I digestion amplifies, obtains digestion products；
Step 4: carrying out agarose gel electrophoresis to digestion products, the agarose gel electrophoresis figure of digestion products is obtained；Observe enzyme
Product agarose gel electrophoresis figure is cut, if there is 2 DNA bands within the scope of 250bp-500bp, corresponding soybean to be detected
Phytophthora bacteria strain is relative to soybean line Chapman(Rps3a) it is avirulent strains；If there was only 1 in 750bp or less after digestion
DNA band, then corresponding soybean phytophthora bacteria strain to be detected is relative to soybean line Chapman(Rps3a) it is toxic strain.
2. a kind of measurement soyabean phytophthora according to claim 1 is to soybean line Chapman(Rps3a) toxicity point
Submethod, it is characterised in that: the method for extraction soyabean phytophthora genomic DNA to be detected described in step 1 is CTAB method.
3. a kind of measurement soyabean phytophthora according to claim 2 is to soybean line Chapman(Rps3a) toxicity point
Submethod, it is characterised in that: the CTAB method extracts the specific steps of soyabean phytophthora genomic DNA to be detected are as follows:
(1) 10-20 block diameter 2 × 2mm mycelia block is cut from the soyabean phytophthora colony edge to be detected of culture 5-7 days, moved into
It in triangular flask equipped with the clear 10% V8 fluid nutrient medium of 100mL, is placed under 25 DEG C of dark conditions, 120 r/min concussion training
It supports 5-7 days, cultured mycelium is collected by filtration with sterilizing double gauze, distilled water flushing 1 time, is sufficiently smash after freeze-dried
It is broken into hypha powder；The 10% V8 fluid nutrient medium refers to the V8 that 10 times of Campbell company of U.S. production is diluted with distilled water
Vegetable juice；
(2) it takes the hypha powder of 50mg in 1.5mL EP pipe, CTAB extracting solution and 90 μ L matter that 900 μ L mass concentrations are 2% is added
It is mixed well on eddy mixer after the SDS aqueous solution that amount concentration is 10%；
(3) 55-60 DEG C of water-bath 1h, every 15min oscillation mix primary；
(4) 12000 r/min are centrifuged 10min, take supernatant A, and phenol, chloroform and the mixture of isoamyl alcohol three extracting 1 is added
Secondary, the weight ratio of phenol, chloroform and isoamyl alcohol is 25:24:1 in the mixture of the phenol, chloroform and the isoamyl alcohol three that are added,
The volume of phenol, chloroform and isoamyl alcohol three's mixture is equal with the volume of supernatant A；
(5) 12000 r/min be centrifuged 10min, Aspirate supernatant B, be added chloroform 1 time, the volume of chloroform be added with it is upper
The volume of clear liquid B is equal；
(6) 12000 r/min are centrifuged 10min, Aspirate supernatant C, and the NaAc aqueous solution and ice dehydrated alcohol mistake of 3mol/L is added
The volume ratio of night precipitating genomic DNA, NaAc aqueous solution and ice dehydrated alcohol be added and supernatant C is 0.1:2:1；
(7) it is washed twice with the ethanol water that the volume ratio of pre-cooling is 70%, it is dry under the conditions of being subsequently placed in 37 DEG C；
(8) DNA after drying contains the ddH of 50 μ g/mL ribalgilases with 100 μ L2O dissolves, and after 37 DEG C of standing 1h, is placed in -20 DEG C
It is spare in refrigerator.
4. a kind of measurement soyabean phytophthora according to claim 1 is to soybean line Chapman(Rps3a) toxicity point
Submethod, it is characterised in that: the amplification system and amplification condition of the PCR amplification are respectively as follows:
Amplification system: the PCR reaction buffer of 10 times of dilution, 2.5 μ L；The MgCl of 2.5mmol/L2, 1.5 μ L；2.5mmol/L
DNTPs, 0.5 μ l；The Taq archaeal dna polymerase of 5U/ μ L, 0.2 μ L；The specific primer AVR3A1F and AVR3A1R of 10 μm of ol/L is each
0.5μL；Template DNA, 0.5 μ L；Add sterile ultrapure water to 25 μ L；
Amplification condition: 95 DEG C of denaturation 30S, 60 DEG C of annealing 30S, 72 DEG C of extension 30S, 30 recycle, 72 DEG C of extension 5min.
5. a kind of measurement soyabean phytophthora according to claim 1 is to soybean line Chapman(Rps3a) toxicity point
Submethod, it is characterised in that: the DNA fragmentation that PCR amplification described in step 2 goes out is before digestion with the recycling examination of AxyPrep DNA gel
The recycling of agent box；The digestion system and digestion condition of digestion described in step 3 are respectively as follows:
Digestion system: the enzyme cutting buffering liquid of 10 times of dilution, 2 μ L；5U/ μ L'sBpu10I enzyme, 0.5 μ L；With AxyPrep DNA gel
The DNA fragmentation of QIAquick Gel Extraction Kit recycling, 6 μ L；Add ddH2O to 20 μ L；
Digestion condition: 37 DEG C of incubation 1.5h, then 80 DEG C of incubation 20min are inactivated.
6. a kind of measurement soyabean phytophthora according to claim 1 is to soybean line Chapman(Rps3a) toxicity point
Submethod, it is characterised in that: the condition of the agarose gel electrophoresis are as follows: 1% Ago-Gel, electrophoresis under 120V voltage
30min is dyed with 0.5 μ g/mL ethidium bromide solution, is taken a picture under ultraviolet lamp with Bio-rad gel imaging system.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220415A (en) * | 2011-04-15 | 2011-10-19 | 南京农业大学 | Markers of phytophthora sojae avirulence gene Avr5 |
| WO2013009935A2 (en) * | 2011-07-12 | 2013-01-17 | Two Blades Foundation | Late blight resistance genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220415A (en) * | 2011-04-15 | 2011-10-19 | 南京农业大学 | Markers of phytophthora sojae avirulence gene Avr5 |
| WO2013009935A2 (en) * | 2011-07-12 | 2013-01-17 | Two Blades Foundation | Late blight resistance genes |
Non-Patent Citations (4)
| Title |
|---|
| Copy Number Variation and Transcriptional Polymorphisms of Phytophthora sojae RXLR Effector Genes Avr1a and Avr3a;Dinah Qutob等;《PLoS ONE》;20090403;第4卷(第4期);第7页图5 * |
| Sequence Variants of the Phytophthora sojae RXLR Effector Avr3a/5 Are Differentially Recognized by Rps3a and Rps5 in Soybean;Suomeng Dong等;《PLoS ONE》;20110714;第6卷(第7期);第3页表2，第4页图2 * |
| 大豆疫霉无毒基因Avr3a识别位点及亚细胞定位分析;翟路翀;《中国优秀硕士学位论文全文数据库农业科技辑》;20140815(第8期);第21页最后一段，第34页图1-2 * |
| 大豆疫霉菌(Phytophthora sojae)无毒基因 Avr1a、Avr1k 及 Avr3a 的分子鉴定;孙龙 等;《微生物学通报》;20121020;第39卷(第10期);1533-1539 * |
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