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Plasmodiophora brassicae woronin DNA extraction method

The invention relates to a technology for the extraction method of P. brassicae, which is applied in the fields of biotechnology and DNA extraction, and can solve the problems of low DNA purity of P.

Pending Publication Date: 2020-02-21
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, the DNA of Plasmodium brassicae needs to be extracted to study the genetic diversity of Plasmodium brassicae. In the existing DNA extraction method of Plasmodium brassicae, the extracted DNA is mixed with a large amount of host Chinese cabbage. , bacterial and fungal DNA, resulting in lower purity of Plasmodium brassica DNA

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  • Plasmodiophora brassicae woronin DNA extraction method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The collected LNXM-1 root nodules were cleaned, peeled, soaked in 10% sodium hypochlorite solution for 20 minutes, and then rinsed with sterile water several times until there was no sodium hypochlorite taste. Put the sterilized nodules in 100 μg / ml streptomycin, 100 μg / ml carbenicillin, 400 μg / ml timentin, 100 μg / ml vancomycin, 50 μg / ml hygromycin B and 400 μg / ml natamycin The mixed solution was treated at room temperature for 24 hours to make the zoospores in the root nodules become dormant spores. After the treatment, the Plasmodium was washed with sterile water for 3 times.

[0023] Plasmodium is a symbiotic bacterium and cannot be cultured in vitro. Before extracting DNA, the dormant spores of Plasmodium should be extracted to reduce the host DNA. The present embodiment adopts 50% sucrose solution suspension method to extract Plasmodium dormant spores (equipment 50ml centrifuge tube, gauze, mica cloth, pipette tip, etc. used in the test all need autoclaving) method...

Embodiment 2

[0027] Detection of DNA of Plasmodium brassicae

[0028] The concentration and quality of Plasmodium brassica DNA were detected. Use electrophoresis to detect the DNA concentration (1% agarose gel, 1×TAE buffer, 100V, electrophoresis for 20 min), and use 1 μl LoadingBuffer, 1 μl DNA and 4 μl sterile water as a mixed sample to detect the DNA concentration.

[0029] Such as figure 1As shown, the fourth band represents the known DNA concentration of 50ng / μl, and its concentration is indicated by the brightness of the band. Among them, the concentration of band 1 is higher, much higher than the known 50ng / μl. The electrophoresis strip of the target band Single band, no degradation and RNA contamination, Table 1 is the DNA test results, combined with the test results in Table 1, it can be seen that the quality and concentration of DNA extraction are relatively high.

[0030] Table 1 DNA quality test results

[0031] sample name Concentration,ng / L Volume, μl 260 / 2...

Embodiment 3

[0033] Perform electrophoresis detection (1.5% agarose gel, 1×TAE buffer solution, 120V, electrophoresis for 20 min) of the DNA PCR reaction result of Plasmodium bacterium, and observe the electrophoresis detection result and concentration. The detected Plasmodium DNA was sent in for DNA quality evaluation, next-generation sequencing, and genome sequence comparison based on the known whole genome of Plasmodium.

[0034] Using NCBI's next-generation sequencing genome as a reference, the sequencing data was quality-controlled (quality control conditions: 1. Remove reads containing adapter, 2. Remove reads with N ratio greater than 10%, 3. Remove low-quality reads, quality The number of bases with a value Q≤10 accounts for more than 50% of the entire read), high-quality reads will be compared to the reference genome by bwa mem, a total of 17,705,482 reads, and 74.38% of the reads are compared to the reference of Plasmodium Genome, 8.04% of the reads are compared to the reference ...

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Abstract

The invention provides a plasmodiophora brassicae woronin DNA extraction method which comprises the following operation steps of (1) putting sterilized root nodules into an antibiotic mixed water solution to be subjected to soaking treatment for 20 to 30 hours, changing zoospores in the root nodules into resting spores, and flushing the treated root nodules by sterile water; (2) extracting plasmodiophoromycetes resting spores by using a 40-60-percent saccharose water solution by a suspension method; and (3) adding liquid nitrogen into the extracted plasmodiophoromycetes resting spores, performing sufficient grinding, then, putting the obtained material into a precooled centrifugal tube, adding a urea extracting solution, and after extraction, adding extracted plasmodiophora brassicae woronin DNA into a TE buffer solution for storage. The plasmodiophora brassicae woronin DNA extraction method provided by the invention has the advantages of short time consumption, low sample consumptionand low cost; the extracted plasmodiophora brassicae woronin DNA has high purity and few impurities; and important significance is realized for researching the genetic diversity of the plasmodiophorabrassicae woronin.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and DNA extraction, and in particular relates to a DNA extraction method of Plasmodium brassicae. Background technique [0002] Clubroot is a worldwide soil-borne disease caused by Plasmodium brassicae infection, which causes serious economic losses to cruciferous crops such as rapeseed, Chinese cabbage, and cabbage. Plasmodiophora belongs to the Plasmodiophora genus of the Protozoa kingdom Plasmodiophora, which obligately parasitizes cruciferous plants. The pathogen enters the cortex by infecting the root hairs of living plants, causing the parenchyma cells in the roots to expand and form tumors. Once the host plant is infected by Plasmodium, it will affect its absorption of water and nutrients, and in severe cases, the plant will die. Existing studies have found that there is genetic differentiation of Plasmodium, and the pathogenicity of Plasmodium from different sources to hosts is si...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 李晓楠马坡朴钟云张丹
Owner SHENYANG AGRI UNIV
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