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Sheep disease resistance related molecular marker of ISG15 gene and application thereof

A molecular marker and disease resistance technology, applied in the field of molecular marker-assisted selection of sheep, which can solve problems such as inability to eradicate diseases

Active Publication Date: 2014-03-12
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the application of biological products and antibiotics can alleviate animal epidemics to a certain extent, the use of vaccines and drugs can only prevent and control diseases but cannot eradicate them. The fundamental way to solve this problem is to cultivate livestock breeds with high disease resistance

Method used

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  • Sheep disease resistance related molecular marker of ISG15 gene and application thereof
  • Sheep disease resistance related molecular marker of ISG15 gene and application thereof
  • Sheep disease resistance related molecular marker of ISG15 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Cloning of ISG15 gene sequence (preparation example)

[0052] 1. Primer Design

[0053] Using the DNA sequence of bovine ISG15 gene (GenBank accession number: AC_000173.1), a pair of primers for amplifying the DNA sequence of sheep ISG15 gene was designed. The primers were synthesized by the Shanghai Office of Invitrogen Life Technologies Co., Ltd., the United States. The DNA sequences of the primer pairs are as follows

[0054] Forward primer: 5'-CATAATTTCACAATCCCATCAGCAA-3' (corresponding to SEQ ID NO: 2),

[0055] Reverse primer: 5'-GTCTAGTATCTGCATCGTCTGGAA-3' (corresponding to SEQ ID NO: 3).

[0056] 2. Amplification, purification and sequencing of PCR products

[0057] (1) PCR amplification

[0058] Add 2 μL of 50ng / μL DNA template, 2.5 μL of 10×PCR Buffer, 1 μL of 2.5 mM dNTP, 0.1 μL of 10 mM forward and reverse primers, 1 U of Taq enzyme, and double distilled water to 25 μL of the 25 μL reaction system. The PCR reaction program was as follows: pre...

Embodiment 2

[0063] Embodiment 2: The establishment of ISG15PCR-SSCP genotype detection method

[0064] Take 5 μL of the PCR amplification products of the forward and reverse primers, mix them with 10 μL of loading buffer (98% formamide, 0.025% xylene cyanol, 2% glycerol, 0.01mmol·L-1EDTA), centrifuge briefly and put them in PCR Denature in the instrument at 99°C for 10min, and immediately place on ice, after 30min in ice bath, load the sample on 15% non-denaturing polyacrylamide gel, electrophoresis at 4°C, 180V, 200mA for 12-15h. After electrophoresis, the gel was taken for silver staining to determine the genotype.

[0065] SSCP analysis of PCR amplification products found three band types: AA, AB, and BB, among which AA type was wild homozygous type, corresponding to AA genotype; BB type was mutation homozygous type, corresponding to CC genotype; AB was mutation heterozygous type type, corresponding to the AC genotype ( Figure 5 ).

Embodiment 3

[0066] Example 3: Detection of the distribution of ISG15PCR-SSCP polymorphism in Hu sheep

[0067] The genotype and allele frequency of ISG15 mutation in Hu sheep were detected by PCR-SSCP. The results showed that allele C (i.e. mutant 235C) was the main mutation detected in the 5′ flanking region of the ISG15 gene in the Hu sheep population, reaching 0.5306, and the allele frequency of allele A (i.e. wild type 235A) was 0.4694 (Table 1). The information content of SNP polymorphism in the 5'flanking region of ISG15 gene was 0.3741, which belonged to moderate polymorphism, indicating that the genetic variation of SNP in the 5'flanking region of ISG15 gene was relatively large, and more selection effects were expected to be obtained. The results of SNPsχ2 fitness test showed that the Hu sheep population had not yet reached the Hardy-Weinberg equilibrium state at this site (P<0.05).

[0068] Table 1 Genotypes and allele frequencies of mutations in the 5′ flanking region of ISG1...

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Abstract

The invention relates to a sheep disease resistance related molecular marker of ISG15 gene and application thereof. A sheep disease resistance related gene segment applied as a molecular marker is obtained through cloning from sheep ISG15 gene, and the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table; and the gene has a base transversion of A235-C235 at the SEQ ID NO: 1. The sheep disease resistance related molecular marker of ISG15 gene has the following beneficial effect that three gene types of wild isozygoty AA, mutation heterozygosity AC and mutation isozygoty CC are discovered in the hu-sheep flock. The invention also discloses a primer for amplifying the intron region of the ISG15 gene part and a detection method for the molecular marker. The invention provides a new molecular marker for marker assisted-selection of the sheep.

Description

technical field [0001] The invention relates to the technical field of molecular marker-assisted selection of sheep, in particular to an ISG15 gene as a molecular marker related to sheep disease resistance and its application. Background technique [0002] Disease is one of the important factors affecting the production efficiency of livestock and poultry. Modern industrialized feeding methods not only damage animal welfare, but also increase the susceptibility of animals to diseases; at the same time, due to the close contact between humans and livestock, the possibility of mutual infection of zoonotic diseases is increased; and the global trade circulation of livestock and poultry and products and The rapid and smooth traffic has increased the frequency of zoonotic infectious diseases and expanded the scope of infection. Although the application of biological products and antibiotics can alleviate animal epidemics to a certain extent, the use of vaccines and drugs can onl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 宋雪梅蒋永清姜俊芳吴建良郑会超黄新周卫东
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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