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Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof

A sheep and eye muscle technology, applied in the field of bioengineering, can solve the problems of less research on CAST gene, inability to know clearly, marker-assisted selection of sheep and breeding obstacles, etc.

Inactive Publication Date: 2011-02-16
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is currently known that there are four transcripts of the CAST gene in cattle, but there are few studies on the CAST gene in sheep, and the sequence on GenBank cannot clearly know which transcript it belongs to.
The research on the detection and functional verification of sheep CAST gene SNP is still blank, which brings great obstacles to the marker-assisted selection and breeding of sheep

Method used

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  • Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof
  • Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof
  • Single nucleotide polymorphism (SNP) related to sheep eye muscle property and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Fully cut up about 0.3 g of muscle tissue with ophthalmic scissors, put it in a 1.5 ml centrifuge tube, and dry it for 20 minutes.

[0019] (2) Add 500 μl of tissue DNA extraction solution to the above centrifuge tube.

[0020] (3) Add RNase solution to the above centrifuge tube to a final concentration of 20 μg / ml, mix thoroughly, and digest at 37°C for 1-2 hours.

[0021] (4) Add proteinase K to the above centrifuge tube to a final concentration of 150 μg / ml, mix thoroughly, and digest at 55°C for 12-24 hours.

[0022] (5) Add an equal volume of Tris saturated phenol to the above centrifuge tube, slowly invert the centrifuge tube for 10 minutes to fully mix the two phases of the solution at 4°C, centrifuge at 12000rpm for 10 minutes, then transfer the supernatant to a clean centrifuge tube . repeat.

[0023] (6) Add an equal volume of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1), mix well, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[00...

Embodiment 2

[0030] 1. PCR amplification

[0031] The PCR primers for PCR-SSCP are shown in the table below.

[0032] Table 1 5 pairs of specific primer sequences, annealing temperature and target fragment length

[0033]

[0034]

[0035] The PCR reaction amplification system is 25 μl, of which 10× contains Mg 2+ 2.5 μl of PCR buffer, 2 μl of dNTPs (2.5 mmol / L each), 0.4 μl (20 μmol / L) of each upstream primer and downstream primer, 0.3 μl Taq enzyme (5 U / μl), 2 μl template DNA (cDNA), ultrapure water Make up to 25μl.

[0036] The PCR reaction conditions are: pre-denaturation at 94°C for 3 minutes; enter the following cycle: denaturation at 94°C for 30 sec, the annealing temperature of each pair of primers is shown in the above table, annealing for 30 sec, extension at 72°C for 60 sec, after 35 cycles; extension at 72°C for 10 min; 4°C save. 6 μl of the PCR product was electrophoresed on a 1.5% agarose gel to detect the quality of the PCR product.

[0037] The results showed tha...

Embodiment 3

[0056] According to the method described in Example 2, PCR amplification was performed on the total DNA of 229 offspring of Dorset sheep×Hu sheep and 71 offspring of Dorset sheep×Hu sheep. Since the Nsi I enzyme can recognize the mutation at the 73 position, the Nsi I enzyme is used to carry out restriction typing after PCR restriction ( image 3 ).

[0057] The total system of enzyme digestion reaction is 20 μl, which contains 10 μl of PCR product, 2 μl of 10×Buffer (restriction enzyme specific buffer), 4-5 U of restriction enzyme, and high-pressure sterilized double distilled water to make up to 20 μl. After mixing, digest at 37°C for about 4 hours according to the instructions of the enzyme. The digested products were electrophoresed on 2.0% agarose gel at 5V / cm, observed under ultraviolet light, and photographed.

[0058] The linear model software in SASv8.02 (Statistical Analysis System) was used to analyze the correlation between different genotypes of SNPs polymorphic...

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Abstract

The invention relates to the field of biological engineering, in particular to a method for predicting a sheep eye muscle property by single nucleotide polymorphism (SNP). Polymerase chain reaction (PCR) amplification is performed on the total deoxyribonucleic acid (DNA) of sheep by primers shown as SEQ ID No.2 and SEQ ID No.3, the SNP of a PCR amplification product is detected, the 73rd basic group at the 5' end of SEQ ID No.1 in a sequence table is determined as G or A and an individual with a G allelic gene has a large eye muscle area and a large eye muscle width. Due to the detection of a polymorphic site, a new material is provided for molecular breeding and scientific basis is provided for the marker-assisted selection of the sheep eye muscle property.

Description

technical field [0001] The invention relates to a method for predicting sheep eye muscle traits by using single nucleotide polymorphism in the field of bioengineering. Background technique [0002] Designing degenerate primers based on known gene sequences of one species is a way to obtain unknown genes of another species. After the gene is obtained, the function of the new gene can be predicted by means of bioinformatics, and the tissue expression of the gene can be analyzed by semi-quantitative RT-PCR and fluorescent quantitative PCR, and verified by polymorphism analysis. Calpastatin (Calpastatin, CAST) gene has different transcripts in species such as mouse, human, rabbit and cattle, and the expression of different transcripts in different tissues is different (Emori et al., 1987, Proc Natl Acad Sci USA 84(11):3590-3594; Killefer and Koohmaraie, 1994, J Anim Sci 72(3):606-614; Takano et al., 2000, J Biochem 128(1):83-92). Zhu et al. found that there is a new subtype of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 杜立新魏彩虹张莉张菊路国彬
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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