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Avian leukemia resistance molecular marker tva507A > G in chicken subgroup A and application of marker

A technology of molecular markers and avian leukemia, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc., to achieve good application and promotion value

Pending Publication Date: 2019-05-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiency that the prior art cannot fully control the occurrence and prevalence of A-subgroup avian leukemia in China, and provide a molecular marker tva for chicken A-subgroup avian leukemia resistance 507A＞G and its application

Method used

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  • Avian leukemia resistance molecular marker tva507A > G in chicken subgroup A and application of marker
  • Avian leukemia resistance molecular marker tva507A > G in chicken subgroup A and application of marker
  • Avian leukemia resistance molecular marker tva507A > G in chicken subgroup A and application of marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Genetic variation analysis of tva receptor gene

[0057] 1. Experimental method

[0058] 1. Design of primers for tva receptor gene

[0059] Taking the DNA sequence of the chicken tva gene in the NCBI database (GenBank accession number: AY531262.1) as a reference, 3 pairs of primers were designed and divided into 3 fragments to PCR amplify the full-length sequence of tva gene 3607bp (section A, section B and section C), The primer sequences, positions and PCR amplified fragment sizes are shown in Table 1.

[0060] Table 1 PCR amplification information of the full-length sequence of tva receptor gene:

[0061]

[0062] 2. PCR reaction

[0063] Genomic DNA from blood samples of different Chinese chicken breeds (including local chicken breeds and commercial strains) was extracted, and the full-length sequence of the tva gene was amplified by PCR with the three pairs of primers.

[0064] PCR reaction system composition: DNA template 1 μL, 10× buffer 2.5 μL, dNTPs 2 μ...

Embodiment 2

[0072] tva 507A＞G Mutation causes host resistance to ALV-A infection

[0073] 1. Experimental method

[0074] The RCASBP(A)-EGFP expression plasmid (ALV-A reporter vector) was successfully constructed, see image 3 . The RCASBP(A)-EGFP expression plasmid was transfected into DF-1 cells, and 7 days after the transfection, the RCASBP(A)-GFP virus in the cell supernatant (that is, the ALV-A reporter virus, which can infect chickens) was collected. Fibroblast CEF). tva infected with RCASBP(A)-GFP virus 507A＞G Mutation site wild type tva 507A / A , heterozygous mutant tva 507A / G and homozygous mutant tva 507G / G CEF (using the chickens detected in Example 1 to prepare), 1, 2, 4, and 7 days after infection, utilize flow cytometry to detect RCASBP (A)-GFP virus infection tva 507A＞G The situation of CEF with different genotypes at the mutation site.

[0075] 2. Experimental results

[0076] The result is as image 3 As shown, wild-type tva 507A / A CEF and heterozygous mutant t...

Embodiment 3

[0078] A kit for detecting the infection resistance of chicken ALV-A

[0079] 1. Composition

[0080] Detection primers and PCR amplification reagents,

[0081] Wherein, the nucleotide sequence of the detection primer is shown in SEQ ID NO: 1,

[0082] PCR amplification reagents include: 10×buffer, dNTPs, KOD-FX, ddH 2 O.

[0083] 2. How to use

[0084] 1. Extract the genomic DNA of the sample to be tested;

[0085] 2. PCR detection

[0086] PCR reaction system composition: DNA template 1 μL, 10×buffer 2.5 μL, dNTPs 2 μL, upstream and downstream detection primers (the nucleotide sequence of which is shown in SEQ ID NO: 1) 1 μL, KOD-FX 0.5 μL, ddH 2 O to 25 μL.

[0087] PCR reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 90 s, a total of 35 cycles; extension at 72°C for 10 min, and finally storage at 4°C.

[0088] 3. After detection by 2% agarose gel electrophoresis, the PCR amplifica...

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Abstract

The invention discloses an avian leukemia resistance molecular marker tva507A > G in a chicken subgroup A and an application of the marker. The invention analyzes the genetic variation of the Chinesechicken species tva gene, and finds that A > G base mutation exists in a 507th position of a Chinese chicken species tva gene DNA sequence (GenBank login number: AY531262.1), and the inventor furtherstudies and proves that the natural mutation of the tva gene can cause the host to generate genetic resistance to ALV-A infection. A detection kit for screening the avian leukemia resistant chickens in the subgroup A is prepared by using the molecular marker, can accurately and quickly determine whether a detection sample is the avian leukosis resistant chickens in the subgroup A or susceptible chickens, and can be applied to screening breeding materials of the avian leukosis genetic resistant chicken varieties (strains) in the subgroup A, and thus breeding of the avian leukemia genetic resistant chicken varieties (strains) in the subgroup A is carried out, so that the marker has good application and popularization value.

Description

technical field [0001] The invention belongs to the technical field of disease-resistant variety breeding, and more specifically relates to a tva related to the resistance of chicken A subgroup to avian leukemia 507A＞G Molecular markers and their applications. Background technique [0002] A subgroup avian leukemia is an immunosuppressive neoplastic infectious disease of chickens caused by Avian Leukosis Virus subgroup A (ALV-A). ALV-A mainly invades lymphocytes of chickens, and transfers to other organs such as liver, kidney, spleen, etc., leading to lymphocytoma and other malignant tumors. ALV-A can cause immunosuppression in infected chickens, decline in production performance, and even death due to characteristic tumors, causing huge economic losses to the poultry industry. So far, there is no commercial vaccine and effective treatment for the disease, and it is mainly prevented by eliminating positive chickens to purify breeder flocks and biosafety measures. However,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
Inventor 谢青梅陈伟国张翔宇张新珩廖立钦
Owner SOUTH CHINA AGRI UNIV
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