CN104212794B - The extracting method of Plasmodiophora brassicae Causing Cruciferae Clubroot DNA and application - Google Patents

Abstract

The invention provides a method and application for extracting the DNA of clubroot pathogen of cruciferous vegetables. The sample DNA of clubroot pathogen of cruciferous vegetables is extracted, and the diatomite method is used to isolate the host DNA of clubroot fungus. Molecular research lays the groundwork. It can be used for the isolation and detection of physiological races of clubroot of cruciferous vegetables. In the process of identification of plant disease resistance, specific molecular markers of clubroot are used at the same time, and the accuracy of identification of physiological race-specific disease resistance can reach 100%. . Using the diatomaceous earth method to extract the DNA of clubroot of cruciferous vegetables has good repeatability, high reliability, low detection cost, and saves time and effort.

Description

Extraction method and application of DNA of clubroot pathogen of cruciferous vegetables

technical field

The invention relates to a method for extracting DNA of plant pathogenic bacteria, in particular to a method for extracting DNA of pathogenic bacteria used for isolating host contamination.

Background technique

Clubroot of cruciferous vegetables is a worldwide disease caused by the infection of Brassica clubroot, and any susceptible variety of cruciferous vegetables will be infected by this pathogen. When the environmental conditions are favorable for the occurrence of the disease, the yield of cruciferous vegetables can be severely reduced, or even lost.

There are many physiological races of clubroot fungus, which makes it difficult to control clubroot. Clubroot of Brassicaceae in China has relatively large occurrence area and wide distribution. Shen Xiangqun et al. (2009) initially identified 16 clubroot bacteria from Chinese cabbage collected from Liaoning, Shandong, Yunnan, Sichuan, Jilin and other regions using the Williams identification host system. The dominant flora of a physiological race. However, the Williams identification host system has problems such as blurred boundaries in the identification of some physiological races, and further identification needs to be combined with the molecular differences of pathogenic bacteria.

The disease resistance of plants is not only related to its own genes, but also related to the genes of pathogens. A correct understanding of the gene interaction between host plants and pathogenic bacteria is extremely important for a comprehensive understanding of plant disease resistance. Therefore, whether it is plant protection research or disease resistance breeding, it is necessary to quickly and accurately separate and identify physiological races in order to carry out targeted control and disease resistance breeding. Clubroot invades the roots of cruciferous plants and survives in the host root mainly in the form of spores, zoospores and sporangia. The bacterium is an obligate parasite, and it has not yet been able to be cultured on a synthetic medium. Therefore, when the genomic DNA of pathogenic bacteria is extracted from plant clubroot tissue, the contamination of host DNA cannot be avoided, which affects the exploration of pathogenicity differences of different physiological races at the molecular level. Therefore, it is an urgent problem to study the molecular mechanism of clubroot action and remove the host DNA contamination. Wu Shaohui et al. reported using diatomite method to extract trace actinomycete DNA ^[3] ; Chen Qiang, Wu Shaohui et al. used diatomite method to directly extract rhizobia DNA from leguminous root nodules ^[4,5] . Because clubroot fungus invades the roots of Brassicaceae plants, it mainly lives in the host root in the form of spores, zoospores and sporangia, and the currently commonly used methods for isolating clubroot DNA are contaminated by DNA molecules of the host plant. See related reports on extraction of clubroot bacteria using diatomaceous earth method.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, and discloses a method for extracting clubroot DNA from hosts isolated with diatomite, which is beneficial to accurately identify the physiological races of clubroot from the molecular level, and to extract the DNA of cruciferous vegetables. Resistance identification of resistant source materials, thereby shortening the breeding cycle of new varieties of cruciferous clubroot resistant vegetables and speeding up the breeding process.

To achieve the above object, the present invention adopts the following technical solutions:

1. Diatomaceous earth method for extracting Plasmodium spore DNA, the specific steps include:

(1) Squeeze 1g of susceptible diseased root to release spores, mix with 50mL of sterilized distilled water, filter through 16 layers of gauze, filter the filtrate again through medium-speed filter paper, and distribute it into 50 mL centrifuge tubes, the filtrate is 4500 rpm , centrifuged for 10 minutes, discarded the supernatant solution, and continued to dissolve the precipitate with 50 mL of sterilized distilled water. The filtrate was centrifuged at 4500 rpm for 10 minutes, and the filtrate was centrifuged at 4500 rpm for 10 minutes. Add 5 Centrifuge at 3100 rpm with 25% (w/v) sucrose for 5 minutes, discard the supernatant, redissolve the precipitate in 30 mL distilled water, mix well, and centrifuge at 3100 rpm for 5 minutes; suspend the precipitate with 30 mL distilled water, That is to obtain clubroot spore suspension; centrifuge the prepared spore suspension (≥108) at 4000rpm for ⁵ minutes, remove the supernatant, suspend the precipitate in 1mL sterilized distilled water, store at 4°C, and use for DNA extraction;

(2) Add 200 μL guanidinium isothiocyanate buffer (50 mL buffer contains 0.2 M guanidinium isothiocyanate, 0.25 g EDTA, 40 mM Tris-HCl ) and mix well, place at room temperature for 10 minutes, centrifuge at 8000 rpm for 30 seconds;

(3) Transfer the supernatant to another centrifuge tube, add 20 mg/mL RNase, and bathe in water at 37°C for 30 minutes;

(4) Add 30 μL of diatomite adsorption solution (1g diatomaceous earth mixed with 1×TE buffer solution at a ratio of 1:1 (w/v), where 1×TE buffer solution contains 100mM Tris-Hcl and 10mM EDTA), After oscillating evenly, place at room temperature for 15 minutes, centrifuge at 13,000 rpm for 30 seconds, and discard the supernatant;

(5) Continue to add 200 μL of guanidine isothiocyanate buffer solution to the precipitation, mix well, place at room temperature for 10 minutes, centrifuge at 8000 rpm for 30 seconds, and discard the supernatant;

(6) Wash the precipitate with washing buffer (each 500 mL of washing buffer contains 60% (v/v) ethanol, 20 mM Tris-HCl, 0.25 g of EDTA, 400 mM NaCl) for 3 times, 300 μL each time, and then Wash the pellet with 200 μL of 70% (v/v) ethanol, centrifuge at 13000 rpm for 2 minutes, discard the supernatant, and place the pellet at room temperature for 1 hour;

(7) Add 30 μL of distilled water to the precipitate, mix thoroughly on a vortex shaker, bathe in a 55°C water bath for 10 minutes, and centrifuge at 13,000 rpm for 5 minutes. The supernatant is the DNA extraction solution.

2. PCR amplification

(1) Reaction system: 25 µL system, the content of each component is 25 ng template DNA; 5 pmol L ^-1 primer; 1.0 mmol L ^-1 dNTP; 1.0 μL 10×PCR buffer; 0.5 U Taq DNA polymerase; add 25 µL of ddH ₂ O and mix well;

(2) Amplification program: pre-denaturation at 94°C/5 min; 94°C/30 s; 60°C/45 s; 72°C/30 s; after 35 cycles; extension at 72°C for 10 min;

(3) Electrophoresis: TCR01, TC01 amplification products were mixed with an equal volume of loading buffer (30mM EDTA, 50% (v/v) glycerol, 0.25% (w/v) xylene cyanol FF, 0.25% (w/ v) bromophenol blue) were mixed, and then 1% (w/v) agarose gel electrophoresis was performed on an agarose gel electrophoresis apparatus, and electrophoresis was performed at 100V for 30 minutes. After electrophoresis, observe and image under ultraviolet light;

3. Judgment

(1) Whether the TCR01 marker can amplify a 195bp fragment, if the fragment can be amplified, it proves that there is host DNA contamination in the extracted DNA;

(2) TC01 marker detects the presence or absence of Plasmodium DNA, if the band can be amplified in the buffer without host DNA, it proves that the extracted DNA is the DNA of Plasmodium;

If there is no band in the amplification result in item (1), and a 552 bp electrophoresis band is amplified in item (2), then the DNA sample to be tested is the DNA of Clubroot, and there is no host contamination. It can be used for further molecular detection of physiological races of Plasmodium clubroot.

Beneficial effects of the present invention

1. This method can extract Plasmodium DNA easily, quickly and accurately without contamination of host DNA, which is the biggest feature of this technology.

2. This method can be used for the screening and identification of clubroot physiological races, and for the identification of resistance source materials of cruciferous vegetables, thereby shortening the breeding cycle of new varieties of cruciferous vegetables resistant to clubroot and accelerating the breeding process.

Description of drawings

Fig. 1 is a comparison of the DNA purity of clubroot bacteria extracted by the Chinese cabbage genome-specific SSR marker detection CTAB method and the diatomite method of the present invention. The amplified materials include No. 4 physiological race monospora and field dominant physiological races No. 1, No. 2, No. 4, No. 10, No. 14 physiological races and Non-susceptible host (CK) DNA. For the classification method of the physiological races of clubroot disease, please refer to the paper published by the inventor of the patent applicant, Shen Xiangqun et al. (Preliminary report on the identification of the population differentiation of the main physiological races of Chinese cabbage clubroot, Chinese Vegetables, 2009, (8):59-62).

Fig. 2 is the detection result of the TCO1 mark on the diatomaceous earth method of the present invention to extract the Plasmodium spore DNA. The amplified materials include diatomaceous earth method to extract the monosporus of physiological race 4 and the field dominant physiological race 1, 2, 3, 4, 7, 10, 11, 14, 16 physiological race and uninfected Disease host (CK) DNA. For the classification method of physiological races of clubroot disease, please refer to the paper published by the inventor of the patent applicant, Shen Xiangqun et al. (Preliminary report on identification of population differentiation of main physiological races of clubroot disease in Chinese cabbage, Chinese Vegetables, 2009, (8):59-62).

detailed description

The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings: this embodiment is implemented on the premise of the technical solution of the present invention, and provides detailed implementation methods and specific processes.

Embodiment 1: Chinese cabbage genome specific SSR marker detects CTAB method and diatomite method of the present invention extracts purity comparison

1. Collection of clubroot pathogenic bacteria: 5 sources of clubroot pathogenic bacteria were collected from the diseased roots of the diseased plants in the main producing areas of cruciferous vegetables such as Yunnan, Liaoning, Shandong, etc. Plasmodium. Hosts include major cruciferous vegetables such as Chinese cabbage, cabbage, hybrids of black mustard and cauliflower, and radishes. The clubroot tissue sections were examined under the microscope, and dormant sporangia and swimming dormant spores were observed under the microscope, which was determined to be Plasmodiophora brassicae Wor. The collected fresh mycorrhizae were washed, dried on the surface, and stored in a -20°C refrigerator.

Host identification: The identification of the physiological races of Plasmodium clubroot was carried out using the Williams differential host system (Williams, 1966). The 4 differential hosts included: 2 head cabbage varieties Jersey Queen and Badger Shipper, and 2 rutabaga varieties Laurentian and Wilhelmsburger. The set of hosts is introduced from SAKATA KOREA CO.,LTD (Korea Breeding Research Institute Jin Haidong E-mail: ssikeeper@hotmail.com or ch.cabbage@sakatakorea.co.kr), and the seeds are provided by the patent applicant.

The DNA extraction of clubroot pathogenic bacteria was carried out by diatomaceous earth method and CTAB method respectively:

A. Diatomaceous earth method:

Diatomaceous earth method extracts Plasmodium spore DNA, and the specific steps include:

(1) Squeeze 1g of the susceptible root, try to avoid damaging the root tissue, release the spores, mix with 50mL of sterilized distilled water, filter through 16 layers of gauze, filter the filtrate through medium-speed filter paper again, and divide it into a 50 mL centrifuge In the tube, centrifuge the filtrate at 4500 rpm for 10 minutes, discard the supernatant solution, continue to dissolve the precipitate with 50 mL of sterilized distilled water, centrifuge the filtrate at 4500 rpm for 10 minutes, and then centrifuge the filtrate at 4500 rpm for 10 minutes. Add 5 mL of 25% (w/v) sucrose at 3100 rpm to the precipitate obtained by the third centrifugation, centrifuge for 5 minutes, pour off the supernatant, redissolve the precipitate in 30 mL of distilled water, mix well, 3100 rpm, Centrifuge for 5 minutes. Suspend the precipitate with 30 mL of distilled water to obtain the clubroot spore suspension. Centrifuge the prepared spore suspension (≥108 spores) at 4000 rpm for ⁵ minutes, remove the supernatant, suspend the pellet with 1 mL of sterilized distilled water, store at 4°C, and use for DNA extraction.

(2) Add 200 μL guanidinium isothiocyanate buffer (50 mL buffer contains 0.2 M guanidinium isothiocyanate, 0.25 g EDTA, 40 mM Tris-HCl ) and mix well, place at room temperature for 10 minutes, centrifuge at 8000 rpm for 30 seconds.

(3) Transfer the supernatant to another centrifuge tube, add 20 mg/mL RNase, and incubate at 37°C for 30 minutes.

(4) Add 30 μL of diatomite adsorption solution (1g diatomaceous earth mixed with 1×TE buffer solution at a ratio of 1:1 (w/v), where 1×TE buffer solution contains 100mM Tris-Hcl and 10mM EDTA), After oscillating evenly, place at room temperature for 15 minutes, centrifuge at 13000 rpm for 30 seconds, and discard the supernatant.

(5) Continue to add 200 μL of guanidine isothiocyanate buffer solution to the precipitation, mix well, place at room temperature for 10 minutes, centrifuge at 8000 rpm for 30 seconds, and discard the supernatant.

(6) The precipitate was washed 3 times with washing buffer (500 mL washing buffer containing 60% (v/v) ethanol, 20 mM Tris-HCl, 0.25 g EDTA, 400 mM NaCl), 300 μL each time, and then washed with Wash the pellet with 200 μL of 70% (v/v) ethanol, centrifuge at 13000 rpm for 2 minutes, discard the supernatant, and place the pellet at room temperature for 1 hour.

(7) Add 30 μL of distilled water to the precipitate, mix thoroughly on a vortex shaker, bathe in a 55°C water bath for 10 minutes, and centrifuge at 13,000 rpm for 5 minutes. The supernatant is the DNA extraction solution.

B. CTAB method:

(1) Add 600 µL of extraction buffer to the test tube containing the precipitate, vortex to mix, then place in a water bath at 60°C for 2 hours, and mix intermittently;

(2) Carefully draw the upper layer solution with a pipette, transfer to a new centrifuge tube, add 600 µL of chloroform:isoamyl alcohol (24:1, v/v), mix vigorously, and centrifuge at 12,000 rpm for 5 minutes;

(3) Aspirate the upper layer (be careful not to absorb the lower layer of chloroform and isoamyl alcohol, otherwise it will have a great impact on the subsequent test of the extracted DNA), transfer to a new centrifuge tube and add 0.8 times the volume of isoamyl alcohol, 4°C Leave it for 40 min;

(4) Transfer the supernatant to another centrifuge tube, add 20 µg/mL RNase, and bathe in water at 37°C for 30 minutes;

(5) Repeat steps (3) to (4) once, centrifuge at 12,000 rpm for 15 minutes, discard the supernatant, add 600 µL of 75% (v/v) ethanol to wash, centrifuge at 2,000 rpm for 2 minutes, and discard the supernatant;

(6) Place the centrifuge tube at room temperature for 1 hour, add 50-100 μL of sterilized distilled water, and mix well to obtain the DNA solution.

PCR amplification: 25 µL reaction system, the content of each component is 25 ng template DNA; 5 pmol L ^-1 primer; 1.0 mmol L ^-1 dNTP; 1.0 μL 10×PCR buffer; 0.5 U Taq DNA polymerase; make up to 25 µL with sterilized distilled water and mix well. The primer used is a molecular marker closely linked to the Chinese cabbage clubroot resistance CRb gene: TCR01 (195bp), which is characterized in that this marker has the following nucleotide sequence:

5’-ttgagacgagttgacgaagtaacagaacttggtattgtcaagttgcaccttcgacacattcgacgagaaggcttcttttcact caccaa cttatccatttctctcgagatctaattgttgttgttgttgttgtgggggtgtcttcttatatagggtatt gatattgaaggagagataccg’acttcct

Its specific amplification primers are:

Forward primer: 5'-GGCACGTGAGAAAAACGTAT-3'

Reverse primer: 5'-GAGACGAGTTGACGAAGTAA-3'

Amplification program: pre-denaturation at 94°C/5min; 94°C/30s; 60°C/45s; 72°C/30s; after 35 cycles; extension at 72°C for 10min; final storage at 4°C.

Electrophoresis: Mix the TCR01 amplification product with an equal volume of loading buffer (30mM EDTA, 50% (v/v) glycerol, 0.25% (w/v) xylene cyanol FF, 0.25% (w/v) bromophenol blue ) to mix, and then perform 1% (w/v) agarose gel electrophoresis on an agarose gel electrophoresis apparatus, and electrophoresis at 100V for 30 minutes. After electrophoresis, observe and image under ultraviolet light.

Result analysis: Accompanying drawing 1 utilizes TCR01 molecular marker to compare the detection result of CTAB method and diatomite method of the present invention to extract Plasmodium host DNA. The amplification results of the 6 lanes on the left in Figure 1 are all Plasmodium extracted by the diatomaceous earth method; the amplification results of the 6 lanes on the right are the Plasmodium extracted by the improved CTAB method, and the amplification of the host DNA as a template appears in all The band was 195 bp; CK2 was the diatomaceous earth method to extract the host sample DNA, and there was no amplified band; CK1 was the improved CTAB method to extract the host sample DNA, and there were 195 bp amplified bands.

By comparing the Plasmodium DNA extracted by the CTAB method and the diatomaceous earth method, the results show that the whole process of extracting Plasmodium by using the CTAB method needs at least the steps of water bath, extraction, precipitation, washing, drying and dissolution, which takes a long time and the DNA Purity not guaranteed. The diatomite method only needs three steps of water bath, precipitation and dissolution in the whole extraction process, which requires less time, is simple to operate, and has high DNA purity of clubroot. The absence of host DNA contamination was detected using the Chinese cabbage genome-specific SSR marker TCR01. It can be seen that the diatomite method is superior to the CTAB method in the reagents, steps and time spent in the process of extracting the DNA of Plasmodium.

Example 2

Comparison of the amplification results of the Genome Specific SSR Marker TCO1 of Plasmodium Plasmodium in the CTAB method and the diatomite method of the present invention.

1. TC01 is based on the 18S rRNA (GenBank accession no. AF231027, 552bp) of the clubroot pathogen genome, which has the following nucleotide sequence:

5’-gtggtcgaacttcattaaatttgggctcttagaagaaggagaagtcgtaacaaggtttccgtaggtgaacctgcagaagg atcattaaca cagtgggcgg ccctagcgctgcatcccaca

tcccaacccccaaccccatgtgaaccggtgacgtgcggcgtaagctgcgtgtttcattttcgaaccatgctagccgaaacac aactgaagtt ccatacatac acatgttacaactcttagca

atggatatcttggttctcacaacgatgaagaacgcagcgaactgcgatacgtagtgcgaattgcagaattcagtgaatca tcaaatcttt gaacgcaagt tgcgcttccgagataccctt

ggaagcatgcctctttgagtgtcggttcctgtttgtttgggcgccccgcgcgcgcaagacaatgagctttgctgcggcat ggcttgaactagcgagacccgggaccgtgcgtgcattgttgagaacaccatcgagagaatgccggagcgg tccccgccga tctgcgcgca atcgcagaca-3acacac’

Its specific amplification primers are:

Forward primer: 5'-GTGGTCGAACTTCATTAAATTTGGGCTCTT-3'

Reverse primer: 5'-TTCACCTACGGAACGTATATGTGCATGTGA-3'.

The total length of the amplified fragments involved by the two primers was 552 bp.

Except that the annealing temperature is different, the specific operation methods such as DNA extraction of clubroot are the same as in Example 1.

references:

[1]Piao ZY, Park YJ, Choi SR et al. Conversion of AFLP markerlinked to clubroot resistance gene into SCAR marker. Journal of the Korean Society Horticultural Science, 2002, 43:653-767.

[2]Cao T, Tewari J, Strelkov S E. Molecular detection of Plasmodiophora brassicae , causal agent of clubroot ofcrucifers, in plant and soil. Plant Disease, 2007, 91(3): 80-87.

[3] Fu Shaobin, Wang Haiying, Meng Qingfeng, Sun Di'an. High-efficiency extraction method of trace DNA. Anhui Agricultural Sciences, 2011, 39(15): 8868-8869.

[4] Chen Qiang, Zhang Xiaoping, Li Dengyu, etc. A method for directly extracting rhizobia DNA from root nodules of legumes. Microbiology Bulletin, 2002, 29(6): 63-66.

[5] Wu Shaohui, Liu Zhong, Zhang Chenggang. Extraction and identification of trace Frankia DNA in plant root nodules. Journal of Applied and Environmental Biology, 2001(01):76-78.

sequence listing

SEQUENCE LISTING

<110> Shenyang Agricultural University

<120> Extraction method and application of DNA of clubroot pathogen of cruciferous vegetables

<130>

<160>6

<210>1

<211>195

<212> DNA

<213> Chinese cabbage (Brassica rapa spp. pekinnesis)

<400>1

ttgagacgag ttgacgaagt aacagaactt ggtattgtca gagttgcacc ttcgacacat 1

tcgacgagaa ggcttctttt cactcaccaa cttatccatt tctctcgaga tctaattgtt 61

gttgttgttg ttgtgggggt gtcttcttat atagggtatt gatattgaag gagagatacg 121

tttttctcac gtgcc 181

<210>2

<211>20

<212> DNA

<213> Chinese cabbage (Brassica rapa spp. pekinnesis)

<400>2

ggcacgtgag aaaaacgtat 20

<210>3

<211>20

<212> DNA

<213> Chinese cabbage (Brassica rapa spp. pekinnesis) <220>

<400>3

gacgagtt gacgaagtaa 20

<210>4

<211>552

<212> DNA

<213>Plasmodiophora brassicae

<400>4

gtggtcgaac ttcattaaat ttgggctctt agaagaagga gaagtcgtaa caaggtttcc 1

gtaggtgaac ctgcagaagg atcattaaca cagtgggcgg ccctagcgct gcatcccaca 61

tcccaacccc acccccatgtg aaccggtgac gtgcggcgta agctgcgtgt ttcattttcg 121

aaccatgcta gccgaaacac aactgaagtt ccatacatac acatgttaca actcttagca 181

atggatatct tggttctcac aacgatgaag aacgcagcga actgcgatac gtagtgcgaa 241

ttgcagaatt cagtgaatca tcaaatcttt gaacgcaagt tgcgcttccg agataccctt 301

ggaagcatgc ctctttgagt gtcggttcct gtttgtttgg gcgccccgcg cgcgcaagac 361

aatgagcttt gctgcggcat ggcttgaact agcgagaccc gggaccgtgc gtgcattgtt 421

gagaacacca tcgagagaat gccggagcgg tccccgccga tctgcgcgca atcgcagaca 481

cacacacacc aa 541

<210>5

<211>30

<212> DNA

<213>Plasmodiophora brassicae

<400>5

gtggtcgaac ttcattaaat ttgggctctt 30

<210>6

<211>30

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<213>Plasmodiophora brassicae

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ttcacctacg gaacgtatat gtgcatgtga 30

Claims (2)

1. a kind of extracting method of brassicaceous vegetable Caulis et Folium Brassicae campestriss plasmodiophora brassicae DNA, is characterized in that extracting plasmodiophora brassicae using diatom local method Spore DNA, concrete steps include：

(1) susceptible for 1g old complaint is flattened, discharge spore, mix with the sterile purified water of 50mL, 16 layers of filtered through gauze, filtrate is again Secondary filtered by Medium speed filter paper, be dispensed in the centrifuge tube of 50mL, filtrate 4500rpm, be centrifuged 10 minutes, supernatant discarded solution, Precipitation continues the distillation water dissolution with 50mL through sterilizing, filtrate 4500rpm, is centrifuged 10 minutes, then filtrate 4500rpm, centrifugation 10 minutes；The sucrose 3100rpm of addition 5mL25% (w/v) concentration in the precipitation that third time centrifugation is obtained, centrifugation 5 minutes, Fall supernatant, precipitation is redissolved in 30mL distilled water, mixes, 3100rpm, is centrifuged 5 minutes；Precipitation 30mL distilled water hangs Floating, that is, obtain pathogen plasmodiophora spore suspension；By the spore suspension 4000rpm of concentration >=108 of preparation, it is centrifuged 5 minutes, Remove supernatant, the precipitation distillation aqueous suspension of 1mL sterilizing, 4 DEG C of preservations, for DNA extraction；

(2) 200 μ L guanidinium isothiocyanate buffer are added to mix in the centrifuge tube containing 1mL pathogen plasmodiophora spore suspension, its In, 50mL guanidinium isothiocyanate buffer guanidinium isothiocyanate containing 0.2M, 0.25gEDTA, 40mM Tris-HCl room temperature places 10 points Clock, 8000rpm, it is centrifuged 30 seconds；

(3) supernatant is proceeded in another centrifuge tube, add 20mg/mL RNase, 37 DEG C of water-baths 30 minutes；

(4) add 30 μ L diatomite adsorption liquid, after concussion uniformly, room temperature is placed 15 minutes, 13000rpm, be centrifuged 30 seconds, discard Clear liquid；In diatomite adsorption liquid, 1g kieselguhr is mixed for 1: 1 (w/v) with 1 × TE buffer ratio, and wherein, 1 × TE buffer contains 10mM Tris-Hcl and 1mM EDTA,

(5) 200 μ L guanidinium isothiocyanate buffer are continuously added in precipitating, after mixing, room temperature is placed 10 minutes, 8000rpm, centrifugation 30 seconds, abandon supernatant；

(6) use lavation buffer solution, washing precipitation 3 times, 300 μ L every time, then precipitated with 200 μ L70% (v/v) washing with alcohol, 13000rpm, is centrifuged 2 minutes, abandons supernatant, is deposited in room temperature and places 1 hour；Wherein, every 500mL lavation buffer solution contains 60% (v/ V) ethanol, 20mM Tris-HCl, EDTA0.25g, 400mM NaCl；

(7) add 30 μ L distilled water in precipitation, turbula shaker fully mixes, 55 DEG C of water-baths 10 minutes, 13000rpm, Centrifugation 5 minutes, supernatant is DNA extraction liquid；

(8) it is utilized respectively molecular marker TCR01 and TC01 and enters performing PCR amplification, with the whether part containing host in amplified production PCR fragment is identifying whether contain host's DNA pollution in sample.

2. a kind of application of brassicaceous vegetable Caulis et Folium Brassicae campestriss plasmodiophora brassicae DNA extraction method as claimed in claim 1, is characterized in that：? Application in crucifer pathogen plasmodiophora biological strain Molecular Identification, anti-clubroot breeding.