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Cynoglossus semilaevis bacterial disease resistance related gene and application method thereof

A semi-smooth tongue sole, anti-bacterial technology, applied in the direction of anti-bacterial drugs, applications, genetic engineering, etc., can solve the difficult problem of semi-smooth tongue sole's ability to resist bacterial diseases, and achieve the effect of inhibiting proliferation

Active Publication Date: 2019-04-05
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the existence of race-specificity, the proteins encoded by these homologous genes do not necessarily have the function of anti-disease immunity in fishes such as tongue sole, especially the role of resisting specific pathogenic bacteria in tongue sole
In addition, the expression levels and differences of the above immune-related genes in the disease-resistant and susceptible families of half-smooth tongue sole have not been reported, and it is difficult to develop them as marker genes for the resistance to bacterial diseases of half-smooth tongue sole

Method used

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  • Cynoglossus semilaevis bacterial disease resistance related gene and application method thereof
  • Cynoglossus semilaevis bacterial disease resistance related gene and application method thereof
  • Cynoglossus semilaevis bacterial disease resistance related gene and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Cloning and sequence analysis of the full-length cDNA of the anti-bacterial disease-related gene of half-smooth tongue sole

[0041] The applicant Chen Songlin et al. first used conventional methods to carry out whole-genome resequencing and genome-wide association (GWAS) analysis on the previously screened semi-smooth tongue sole resistant to Harvey's vibrio disease families and susceptible families, and found that the half-smooth tongue sole is resistant to Harvey's vibrio disease. SNP markers linked to mycosis traits, and further locate the SNP site to a fragment, and then verify the fragment by RT-PCR and RACE-PCR amplification, and obtain the full-length cDNA sequence of the gene.

[0042] (1) Experimental method: RNA extraction by Trizol method, synthesis of cDNA, PCR amplification

[0043] RNA extraction by Trizol method: Take about 100 mg of tissue pieces, put them into an enzyme-free centrifuge tube with 500 μL Trizol reagent, and grind on ice with a...

Embodiment 2

[0057] Example 2: Analysis of expression levels of anti-bacterial disease-related genes in tongue sole in different tissues of normal fish

[0058] (1) Design quantitative primers and verify the specificity of primers

[0059] Design primers for the quantitative amplification of the target gene according to the anti-bacterial disease-related gene sequence, carry out ordinary PCR amplification, electrophoresis detection, and clone sequencing. Determine that the primer is specific; in addition, perform fluorescent quantitative PCR on the machine to observe the peak pattern, CT value and amplification efficiency. If the fluorescent quantitative PCR finds that the peak pattern is single, and the amplification efficiency of the internal reference gene and the target gene are almost identical, it proves that the primer Specific, can be used for real-time quantitative PCR experiments. The quantitative primers for the Actin gene were derived from published articles by Chen Songlin's ...

Embodiment 3

[0068] Example 3: Relative expression analysis of anti-bacterial disease-related genes of tongue sole in half-smooth tongue sole in immune-related tissues after infection with Vibrio harveyi

[0069] Using the Vibrio harveyii preserved in Chen Songlin's laboratory, the experiment of Vibrio harvelii infection was carried out on tongue sole half smooth. A control group and an experimental group were set up, the control group was injected with 0.01mol / L sterile PBS, and the experimental group was injected with a concentration of 1.0×10 4 For Vibrio harveyi cfu, the injection dose is about 0.1mL / 10g. At six time points of 0h, 12h, 24h, 48h, 72h and 96h after injection, six tissues of liver, spleen, kidney, intestine, gill and skin were collected respectively. The expression levels of anti-bacterial disease-related genes in the above six tissues were detected by fluorescent quantitative PCR. The primers, reagents and experimental methods used are the same as above.

[0070] The ...

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Abstract

The invention provides a bacterial disease resistance related gene screened from cynoglossus semilaevis. The nucleotide sequence of the gene is SEQ ID NO: 1, and the sequence of a coded protein is SEQID NO: 2. The gene provided by the invention is relevant to the bacterial disease resistance of the cynoglossus semilaevis, and the expression amount of the gene in intestine is relevant to the disease resistance of a fish body. When the expression level is higher, the disease resistance of the fish body is higher. An in-vitro recombinant expressed protein thereof can remarkably restrain the multiplication of certain types of gram-negative bacteria, so that the gene has an application value on the aspects of breeding of a cynoglossus semilaevis disease resistance family, disease control, research of feed additives and bactericide and the like.

Description

technical field [0001] The invention belongs to the technical field of aquatic genetics and breeding, and in particular relates to a gene related to resistance to bacterial diseases of tongue sole and an application method thereof. Background technique [0002] Semi-smooth tongue sole (Cynoglossus semilaevis) is mainly distributed in the coastal areas of my country. It is an important marine economically farmed fish in my country. Its meat is delicious and nutritious, and it is loved by consumers. It has good market demand and breeding development potential. However, due to the high-density intensive breeding mode and environmental pollution, the frequent outbreaks of bacterial diseases such as ascites, fin rot, and tail rot in half-smooth tongue sole, especially the skin ulcers caused by Vibrio harveyi are the most serious, resulting in huge The economic loss seriously hinders the development of half-smooth tongue sole aquaculture. At present, the prevention and treatment ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C07K14/46C12Q1/6888A61K38/17A61P31/04
CPCA61K38/00A61P31/04C07K14/461C12Q1/6888C12Q2600/158
Inventor 陈松林扶晓琴陈亚东周茜王磊李仰真李明
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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