Clone and use of molecular marker related to pig growth and meat quality traits
A technology of molecular markers and meat quality traits, applied in the field of livestock genetic engineering, can solve the problems of limited molecular markers affecting growth and meat quality
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Embodiment 1
[0031] Example 1 Cloning of JHDM1A gene
[0032] (1) Primer design:
[0033] Using human JHDM1A gene cDNA (GenBank accession number: NM_012308) as an information probe, using the BLAST tool in NCBI to perform homologous sequence screening in the GenBank pig EST database, a series of ESTs with a homology of more than 80% (the fragment length is greater than 100bp), use ENTREZ (http: / / www.ncbi.nlm.nih.gov / Web / Search / index.html) to query the corresponding sequences of these ESTs in NCBI, and then use the SeqMan program in DNAstar to construct pig EST-contigs; at the same time, using the human NM_012308 sequence to search for the porcine Trace-WGS sequence in GenBank, and according to the homology, it may be the partial sequence information of the corresponding exon of the corresponding porcine gene. Three pairs of primers P1, P2 and P3 were designed to amplify the JHDM1A gene cDNA sequence according to the human-mouse homologous sequence, EST splicing sequence and JHDM1A gene Tr...
Embodiment 2
[0051] Example 2 Physical location of JHDM1A gene:
[0052] (1) The primer sequence used for physical positioning is
[0053] P4: PL 5′-CCGCTGCGACCTTTGATGAC-3′ in intron 20
[0054] PR 5′-AAGGTGACGTTGGCGATGC-3′ in exon 21
[0055] The length of the fragment amplified by the primers is 498bp.
[0056] (2) Experimental materials for physical positioning
[0057] The pig×rodent somatic cell hybrid panel (SCHP) was used for chromosome region mapping, and the pig radiation hybrid panel (INRA-Minnesota porcine radiation hybrid panel, IMpRH) co-constructed by the University of Minnesota, USA was used for chromosome accuracy. Positioning, two sets of somatic cell hybrid plates were kindly donated by Dr. Martin Yerle (Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, France).
[0058] The radiation dose used by IMpRH is 7,000-rad. IMpRH includes 118 pig × hamster radiation hybrid cell lines, as well as hamster and pig genomic DNA positive controls, the identificatio...
Embodiment 3
[0063] Embodiment 3PCR-RFLP diagnostic method is established
[0064] (1) Primer sequence
[0065] P5: PL 5′-CCCAGACTGAGCAGGAACC-3′
[0066] PR 5′-GGACACCACGAGGAGAACC-3′
[0067] The length of the fragment amplified by the primers is 453bp.
[0068] (2) PCR amplification conditions
[0069] The total volume of the PCR reaction is 20μl, including about 100ng of porcine genomic DNA, containing 1×buffer (Promega), 1.5mmol / L MgCl 2 , the final concentration of dNTP is 150 μmol / L, the final concentration of primers is 0.2 μmol / L, and 2U Taq DNA polymerase (purchased from Promega Company). The PCR amplification program was: 94°C for 5min, then cycled 34 times at 94°C for 45s, 57°C for 45s, 72°C for 30s, and finally extended at 72°C for 5min. PCR reaction products were detected by 2% agarose gel electrophoresis and photographed.
[0070] (3) RFLP detection conditions
[0071] The volume of PCR product digestion reaction is 10μl, including 1μl of 10×buffer, 3-5μl of PCR pro...
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