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Method for sequencing plant phytoplasma genome

A phytoplasma and genome technology, applied in the field of plant phytoplasma genome sequencing

Active Publication Date: 2019-01-15
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, there is not enough understanding of phytoplasma Paulownia arbuscularis and its genetic requirements, genomic characteristics and evolutionary relationship

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Obtaining phytophyte-enriched Paulownia arbuscular tissue-cultured seedling stems

[0034] Phytoplasma-infected P. fortunei (Paulownia albicans) seedlings were uniformly cultured on 1 / 2 MS medium in a 100 ml triangular flask for 30 days. Paulownia arbuscular at 29℃, 130μmol·m -2 the s -1 Cultivate under light for 16 hours a day, and after 30 days, cut the tender stems of the seedlings, and then immediately freeze the tender stems of the seedlings in liquid nitrogen and store them at -80°C.

Embodiment 2

[0036] Separation and Extraction of DNA Mixtures of Paulownia alba and Phytoplasma Genomes

[0037]Grind the phytoplasma-infected Paulownia alba seedling stems into powder with liquid nitrogen, then add 1000 μL CTAB 65°C to 0.1 sample powder and mix thoroughly, and place in a 60°C water bath for 50 minutes, shaking up and down every 10 minutes.

[0038] Add 1000 μL of solution mixture 1 (a mixture of phenol, chloroform and isoamyl alcohol with a volume ratio of 25:24:1, respectively), mix well for 3 minutes, and then centrifuge at 1,2000 r / min for 1 minute at 25°C. Discard the supernatant, and add about 900 μL of solution mixture II (a mixture of chloroform and isoamyl alcohol with a volume ratio of 24:1), mix thoroughly, and then centrifuge at 1,2000 r / min for 15 min at 25°C. Discard the supernatant, add 1 / 10 volume of NaAc (3mol / L, pH5.2) and 2.5 times the volume of absolute ethanol, shake gently, discard the supernatant, and then centrifuge at 25°C, 1,2000μ / min 15min. The...

Embodiment 3

[0040] The DNA of Paulownia albiflora and Phytoplasma were randomly interrupted with g-TUBE (Covaris, Woburn, MA, USA). Then use 1X magnetic beads to enrich and purify the fragmented DNA, and then perform damage repair and end repair on the fragmented DNA. Use ligase to ligate the two ends of the DNA fragments with adapters; the unligated DNA fragments are excised with exonuclease, then the ligation products are purified, and the library is evaluated with a kit, and finally the mixture of DNA template and enzyme is transferred into the nanopore of the sequencer for real-time single-molecule sequencing.

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Abstract

The invention discloses a method for sequencing plant phytoplasma genome, which comprises the following steps: obtaining Paulownia tomentosa tissue culture seedling stem rich in phytoplasma; Isolationand Extraction of Genomic DNA Mixture from Paulownia Alba and Phytoplasma; DNA Sequencing of Paulownia Alba and Phytoplasma Gene Mixture; Phytoplasma genome sequencing data processing, statistics andassembly and Paulownia tomentosa and phytoplasma transcriptome sequencing to verify the reliability of the genome. The invention obtains the genome of Paulownia arbuscular phytoplasma for the first time, and analyzes the information of interaction between Paulownia arbuscular pathogen and host through the genome. The invention is helpful to increase the information of basic metabolic pathway of phytoplasma, and lays a foundation for revealing the genome information of plant phytoplasma comprehensively.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a method for sequencing plant phytoplasma genomes. Background technique [0002] Phytoplasma is a special pleomorphic bacteria without a cell wall, parasitic in the phloem sieve tube of plants, can be transmitted by insects, and causes hundreds of plant diseases worldwide, including some important economic crops, such as Wheat, corn, peanuts, etc., causing huge economic losses. In addition, many new diseases caused by phytoplasma are emerging. Therefore, it is necessary to carry out the research of phytoplasma genome to analyze the genetic evolution and pathogenic mechanism of phytoplasma. So far, only 6 complete genome sequences of nearly 400 phytoplasma have been reported by pulse electrophoresis and other methods: 'Ca.P.asteris' onion yellow phytoplasma (OY-M) , Aster yellow arbuscularis (AY-WB), maize phytoplasma M3, 'Ca.P.mali' apple phytoplasma (AT), 'Ca.P.austral...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/6869
CPCC12Q1/6869C12Q1/689
Inventor 范国强翟晓巧赵振利曹喜兵
Owner HENAN AGRICULTURAL UNIVERSITY
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