A SNP marker detection primer pair related to estrous symptoms of sows and its application
A sow and marking technology, applied in the direction of microbial determination/inspection, recombinant DNA technology, biochemical equipment and methods, etc.
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Embodiment 1
[0024] Download the SULT1C3 gene and the upstream 3000bp sequence from the NCBI website, use the online primer design tool Primer-BLAST to design primers, and amplify the SULT1C3 gene promoter region (-2500~+300, +1 is the transcription start site). Two restriction endonuclease sites, SacI and HindIII, were added to the 5' ends of the upstream and downstream primers respectively. The sequence of the primers is SULT1C3-P1-F / R, see Table 1 for details. Primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd.
[0025] Table 1 Primer Information
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[0028] Table 2 50μL reaction system
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[0031] The reaction program of PCR amplification was: pre-denaturation at 98°C for 30s; cycle amplification stage: denaturation at 98°C for 10s → annealing at 64°C for 30s → extension at 72°C for 45s, cycled 35 times; finally, incubation at 72°C for 7min for final extension. After the amplification reaction, the PCR product was detected by 1...
Embodiment 2
[0033] Example 2 Porcine SULT1C3 Gene Tissue Expression Profile
[0034] Total RNA was extracted from ovary, longissimus dorsi, brain, liver, stomach, myocardium, ileum and spleen tissues of 3 large white pigs by Trizol method. Primers were designed using Primer-BLAST according to the CDS sequence in the mRNA of the SULT1C3 gene on the NCBI website, and the primer information is shown in Table 1. Fluorescent quantitative PCR was used to analyze the expression of SULT1C3 gene in various tissues after reverse transcription. The results showed that the expression of SULT1C3 gene was the highest in the ovary, followed by the ileum, and it was almost not expressed in the longissimus dorsi, brain, and stomach ( figure 2 ).
Embodiment 3
[0035] Example 3 plasmid construction
[0036] According to the results of bioinformatics analysis, the large white pig DNA was used as a template to amplify the SULT1C3 gene promoter region (-2405 / +261), and named SULT1C3-P1 (-2405 / +261), and then its 5' end Deletions of different lengths resulted in SULT1C3-P2(-1238 / +261), SULT1C3-P3(-1084 / +261), SULT1C3-P4(-642 / +261), SULT1C3-P5(-424 / +261), SULT1C3-P6 (-141 / +261) fragment. Two restriction endonuclease cutting sites and protective bases were added to the primer sequence. See Table 1 for specific primer information.
[0037] Each promoter deletion fragment was amplified by PCR, in which LA Taq MasterMix was used for P1-P3, and 2×Taq PCR Mix was used for P4-P6.
[0038] Table 3 50μL reaction system
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[0041]The amplification reaction program is (P1~P3 / P4~P6):
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[0043] After the amplification reaction, the PCR products were detected by electrophoresis and sent to Nanjing Qingke Biotechnol...
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