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A method for sequencing plant phytoplasma genome

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

Active Publication Date: 2021-08-27
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 genomes, which comprises obtaining Paulownia albiflora tissue culture seedling stems rich in phytoplasma; separating and extracting the DNA mixture of Paulownia albaflower and phytoplasma genomes; DNA of Paulownia albiflora and phytoplasma genomes Mixture sequencing; processing, statistics and assembly of phytoplasma genome sequencing data and sequencing of transcriptomes of Paulownia albaflower and phytoplasma to verify the reliability of the genome. The present invention obtains the genome of the Paulownia arbuscularis phytoplasma for the first time, and analyzes the information of the interaction between the Paulownia arbuscularis pathogen and the host through the genome. Genomic information on phytoplasma lays the foundation.

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 Patents(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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