CXCL13 molecular marker breeding method for screening anti-porcine circovirus disease pigs and application thereof
A porcine circovirus disease and molecular marker technology, applied in the field of molecular genetics, can solve problems such as large confidence intervals, difficult breeding, and inability to determine mutation sites, and achieve the effect of being unaffected by the environment, and the method is simple and fast
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Embodiment 1
[0048] Example 1: Cloning of the 5' regulatory region sequence of pig CXCL13 gene
[0049] Genome extraction: The large and long hybrid pig ear tissue was taken, and the genomic DNA was extracted according to the instructions of the TIANamp Genomic DNA Kit of TIANGEN Company.
[0050] According to the sequence of the 5' regulatory region of CXCL13 gene (NCBI Reference Sequence: NC_010450.4), DNAMAN 7.0.2.176 was used to design as follows: the sequence of primer CXCL13PF is shown in SEQ ID NO.1, and the sequence of CXCL13PR is shown in SEQ ID NO.2. See Table 1 for details.
[0051] Table 1:
[0052]
[0053] Xho I and Hind III restriction endonuclease sites and their protective bases (italics) were added to the primers, respectively. CXCL13PF and CXCL13PR primers were used to amplify the CXCL13 5'regulatory region-3407bp to +79bp fragment, which included the range from 79bp downstream to 3407bp upstream of the transcription start site. The amplification system was 2×Prime...
Embodiment 2
[0054] Embodiment 2: Construction of pig CXCL13 promoter vector
[0055] 1) Construct the CXCL13 promoter vector with pGL3-basic as the backbone vector. The pGL3-basic vector and the CXCL13 promoter fragment obtained in Example 1 were digested with Xho I and Hind III restriction enzymes, respectively. Enzyme digestion system: 10× Green Buffer 5 μL, Xho I endonuclease 2 μL, Hind III endonuclease 2 μL, pGL3-basic vector 4 μL (CXCL13 promoter fragment 20 μL), add ddH 2 O to a total volume of 50 μL. Digest at 37°C for 3h. The product was loaded on 1% TAE agarose gel, electrophoresed at 180V for 15 minutes, and then the gel was cut to recover each fragment, and the purified product was obtained according to the purification instructions of the Axygen agarose gel purification kit.
[0056] 2) Ligation and transformation of DNA. The ligation system is: 10 μL system includes 2 μL of 5×Rapid ligation buffer, 0.5 μL of T4DNA ligase (5U / μL), 1 μL of vector, 3 μL of insert fragment...
Embodiment 3
[0058] Example 3: Cloning of pig CXCL13 promoter deletion fragment
[0059] According to the promoter fragment sequence on the pGL3-CXCL13 (-3407 / +79) vector obtained in Example 2, different upstream primers were designed with DNAMAN, the CXCL13F1 sequence is shown in SEQ ID NO.3, and the CXCL13F2 sequence is shown in SEQ ID NO.4 As shown, the sequence of CXCL13F3 is shown in SEQ ID NO.5, the sequence of CXCL13F4 is shown in SEQ ID NO.6, the sequence of CXCL13F5 is shown in SEQ ID NO.7, and the downstream primer is still CXCL13PR. See Table 2 for details.
[0060] Table 2:
[0061]
[0062] The Xho I restriction endonuclease cutting site and its protective bases (italics) were added to the primers respectively. CXCL13F1, CXCL13F2, CXCL13F3, CXCL13F4, CXCL13F5, and CXCL13PR primers were used to amplify the CXCL13 5' regulatory region -2666bp, -2205bp, -1532bp, -1089bp, -589bp to +79bp fragments, which respectively contained fragments from the transcription start site The ...
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