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Ribosomal RNA sequence of pathogenic bacterium Septoria sp of oak abnormally withered disease and application thereof

A technology of pathogenic bacteria and ribosomes, applied in the direction of DNA/RNA fragments, applications, recombinant DNA technology, etc., can solve the problems of uncertain pathogenic fungal species, fungal species research, high variability of ITS segments, etc., and achieve good application foreground effect

Active Publication Date: 2019-04-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, based on the rDNA sequences of 5.8S, 18S, and 28S, it is difficult to study the classification of fungal species, and the species of pathogenic fungi cannot be determined. ITS sequences are often used to identify species (spieces) level
However, the high variability of the ITS segment presents many problems in application

Method used

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  • Ribosomal RNA sequence of pathogenic bacterium Septoria sp of oak abnormally withered disease and application thereof
  • Ribosomal RNA sequence of pathogenic bacterium Septoria sp of oak abnormally withered disease and application thereof
  • Ribosomal RNA sequence of pathogenic bacterium Septoria sp of oak abnormally withered disease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Detection of the acquisition of the RNA sequence of the oak early drying disease pathogen Septoria sp

[0060] 1. Experimental method

[0061] (1) High-throughput sequencing

[0062] Randomly search for leaves with typical early drying disease lesions in the oak tree garden where the disease occurs, collect them, cut off the lesions in the oak leaves, cut off the material of the lesions and grind them fully with liquid nitrogen. The total DNA was extracted using Kangwei Century Fungus DNA extraction kit, specifically follow its operating instructions, and the extracted total DNA is stored at -20°C; according to the Illumina DNA library construction process, the total DNA is constructed as a paired-end high-throughput sequencing library with a fragment size of 500bp; Illumina Hiseq2500 The sequencer performed high-throughput sequencing on the constructed DNA library, and a total of 18.13M sequencing fragments were measured. The sequencing read length was 125bp...

Embodiment 2

[0072] Verification experiment of embodiment 2 ribosome assembly result

[0073] 1. Experimental method for verification of ribosome assembly results

[0074] (1) PCR amplification reaction

[0075] Randomly search for leaves with typical black diseased spots in the affected oak garden, collect them, cut out the diseased areas in the oak leaves, cut off the material of the diseased spots and use liquid nitrogen to fully grind, extract the total DNA of the diseased oak leaves, and store in- 80°C;

[0076] Design 4 pairs of primers for verification of ribosome assembly results (primer pair YK26-f1 / YK1570-r1, primer pair YK1461-f2 / YK3455-r2, primer pair YK3100-f3 / YK4500-r3 and primer pair YK4282-f4 / YK5737 -r4), the nucleotide sequences of 4 pairs of primers are shown in table 1 respectively; Then take the total DNA of oak leaf disease as template, apply above-mentioned 4 pairs of primers to carry out PCR amplification, PCR amplification reaction system is as shown in table 2 ....

Embodiment 3

[0090] Embodiment 3 detects the method for oak tree early drying disease pathogenic bacteria Septoria sp

[0091] 1. Detection method

[0092] (1) High-throughput sequencing

[0093] Randomly search for leaves with typical early drying disease lesions in the oak tree garden where the disease occurs, collect them, cut off the lesions in the oak leaves, cut off the material of the lesions and grind them fully with liquid nitrogen. The total DNA was extracted using Kangwei Century Fungus DNA extraction kit, specifically follow its operating instructions, and the extracted total DNA is stored at -20°C; according to the Illumina DNA library construction process, the total DNA is constructed as a paired-end high-throughput sequencing library with a fragment size of 500bp; use Illumina Hiseq The 2500 sequencer performed high-throughput sequencing on the constructed DNA library, and a total of 18.13M sequencing fragments were measured. The sequencing read length was 125bp at both end...

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Abstract

The invention discloses a ribosomal RNA sequence of a pathogenic bacterium Septoria sp of an oak abnormally withered disease and application thereof. The nucleotide sequence of the ribosomal RNA geneof the pathogenic bacterium Septoria sp of the oak abnormally withered disease is shown as SEQ ID NO.1, and the sequence can be utilized to detect the pathogenic bacterium Septoria sp of the oak abnormally withered disease and can also be applied to research on the classification of fungal species. In addition, the invention also provides a primer for detecting the pathogenic bacteria Septoria spof the oak abnormally withered disease. The primer has good specificity, a specific detection method and a specific detection kit are established based on the primer, the required detection time is short, the detection is rapid and efficient, and the primer has a good application prospect in detection of the pathogenic bacteria Septoria sp of the oak abnormally withered disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a ribosomal RNA sequence of Septoria sp, a pathogenic bacterium of eucalyptus early blight, and an application thereof. Background technique [0002] In the existing fungal research methods, ribosomal DNA (ribosome DNA, rDNA) sequences are often sequenced and compared for the identification of fungi. Ribosomes have important functions in cells, and many genes encoded by rDNA are closely related to the reaction process of protein synthesis and play a decisive role in protein biosynthesis. The rDNA sequence is divided into a transcribed region and a non-transcribed region. The transcribed region consists of genes encoding ribosomal 5.8S, 18S, and 28S protein structures and two intergenic transcribed spacers (Internal Tanscribed Spacer, ITS) ITS1 and ITS2. form a transcription unit. [0003] The rDNA sequences encoding 5.8S, 18S, and 28S in rDNA are relatively conserve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/31C12N15/11
CPCC07K14/37C12Q1/6895
Inventor 刘吉平王狗旦孙勋勋
Owner SOUTH CHINA AGRI UNIV
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