Gene of cortexin-3 receptor of pig melanin and method for detecting polymorphism of mononucleotide

A technology of corticosteroids and nucleotide sequences, used in genetic engineering, plant genetic improvement, botany equipment and methods, etc.

Inactive Publication Date: 2004-03-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Gene of cortexin-3 receptor of pig melanin and method for detecting polymorphism of mononucleotide
  • Gene of cortexin-3 receptor of pig melanin and method for detecting polymorphism of mononucleotide
  • Gene of cortexin-3 receptor of pig melanin and method for detecting polymorphism of mononucleotide

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Experimental program
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Effect test

Embodiment 1

[0032] The Chinese local breed Meishan pig and the exotic (foreign) breed Large White pig were selected as typical experimental materials, and the primers (external primers) were designed according to the conserved region of human and mouse MC3R genes by using comparative genomics methods.

[0033] upstream primer F 1 : 5′-GCC TTC TGT GAG CAG GTC TTC-3′

[0034] downstream primer R 1 : 5′-GAC GGA GTT GCA CAT GAT GAG-3′

[0035] The PCR reaction was carried out in the environment containing 50ng genomic DNA, 15mmol / L Tris-HCl, 50mmol / L KCl (pH8.0), 2mmol / L MgCl 2 , 200μmol / L dNTPs, primer F 1 and R 1 5 μmol / L, 4% dimethyl sulfoxide and 0.5 U TaqDNA polymerase in a 25 μl reaction system, the reaction conditions are 95 ° C for 5 min, 35 cycles (94 ° C 45S, 60 ° C 45S and 72 ° C 90S), 72 ° C for 5 min . The amplified PCR product (783bp) was purified and sequenced directly. DNA sequence with Sequencher TM 3.0 software (Gene Codes Corporation) for alignment and comparison, ...

Embodiment 2

[0037] After DNA sequence determination and sequence comparison analysis, at nucleotide 480 of the MC3R gene PCR amplification product

[0038] A base change (C→T) was found at the bit position, that is, SNP (see Figure 2): in the pig breed Meishan pig is C, while Large White pig is T. Accordingly, the specific internal primers (F 2 : 5'-AAG ATG GTC ATC GTG TGC CTT-3', R 2 : 5'-GGCGAAGAAGATGACGACG-3'), with outer primer (F 1 : 5'-GCCTTCTGTGAGCAGGTCTTC-3', R 1 : 5'-GACGGAGTTGCACATGATGAG-3') together, using Bi-PASA analysis technology for SNP typing. The PCR reaction was carried out in the environment containing 50ng genomic DNA, 15mmol / L Tris-HCl, 50mmol / L KCl (pH8.0), 2mmol / L MgCl 2 , 200μmol / L dNTPs, outer primer F 1 and R 1 5μmol / L, internal primer F 2 and R 2 2.5μmol / L, 4% dimethyl sulfoxide and 0.5 U Taq DNA polymerase in a 25μl reaction system, the reaction conditions are 95℃ for 5min, 35 cycles (94℃ 60S, 65℃ 60S and 72℃ 90S), 72℃ Extend 10min. The PCR product...

Embodiment 3

[0044] In order to determine whether the SNP polymorphism of MC3R gene is related to the difference of pig phenotype, 136 large white pigs×Meishan pig F 2 Substitute as test material, adopt the Bi-PAPS method described in embodiment 2 to carry out polymorphic scanning, MC3R gene CC genotype observation number is 105 (genotype frequency is 0.78), and CT genotype is 9 (genotype frequency is 0.78). 0.06), and the TT genotype was 22 (genotype frequency was 0.16). And using SAS statistical software, 136 Large White×Meishan F 2 Significance tests and multiple comparisons were carried out on carcass and meat traits of different genotypes in different generations. The preliminary results showed that there was no significant difference among the traits of different genotypes of MC3R gene (p>0.05, see Table 2).

[0045] Table 2 Effects of different genotypes of MC3R gene on traits Genotype CC genotype CT genotype TT Shoulder fat thickness 3.11±0.07 2.86±0.25 2.86±0.166-7 rib fat ...

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Abstract

A process for detecting pig melanocortin-3 receptor (MC3R) gene and its single nucleotide polymorphism (SNP) includes extracting DNA from pig blood genom, designing primer based on human and mouse MC3R gene conservative region, PCR amplification, directly sequencing the PCR product, comparing sequence, analyzing and detecting SNP.

Description

technical field [0001] The invention belongs to the technical field of pig molecular biology, and relates to the cloning and sequencing of pig MC3R gene and the detection method of single nucleotide polymorphism. It is related to molecular technology breeding of pigs. technical background [0002] Melanocortin receptor is a member of the √√family of G-protein coupled receptors (GPCRs) superfamily. So far, five subtypes of MCRs (MC1R-MC5R) in human, mouse and chicken have been cloned and characterized, and their tissue distribution and biological functions are different. [0003] MC3R is the latest cloned and identified receptor among the five MCRs. Human, mouse and chicken MC3R genes have been cloned and identified, encoding 360, 323 and 325 amino acids, respectively. The locus encoding human MC3R gene is located on human chromosome 20q, and this locus is linked with controlling body mass index (body mass index), subcutaneous fat mass and fa...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12Q1/68
Inventor 蒋思文彭健刘桂兰熊远著郑嵘
Owner HUAZHONG AGRI UNIV
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