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A kind of serpina1 molecular marker breeding method for screening pigs resistant to porcine circovirus disease and its application
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A technology for porcine circovirus disease and porcine circovirus, which is applied in the field of molecular genetics and can solve the problems of decreased serum content, increased serum content, and aggravated inflammatory response of lung tissue.
Active Publication Date: 2019-01-22
SHANDONG AGRICULTURAL UNIVERSITY
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However, the serum level of SERPINA1 decreased significantly (Pfigure 1 ), aggravated the inflammatory response of lung tissue
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Embodiment 1
[0028] Example 1 Cloning of porcine SERPINA1 coding region sequence and search for mutation sites
[0029] RNA extraction and reverse transcription: The lung tissues of Laiwu pigs and Dachang hybrid pigs were taken, and the total RNA of lung tissues was extracted according to the instructions of RNAsimpleTotal RNA Kit from TIANGEN Company. Then follow the TaKaRa PrimeScript TM RTreagent Kit with gDNA Eraser (Perfect Real Time) kit instruction manual for reverse transcription to obtain cDNA.
[0030] According to the mRNA sequence of SERPINA1 gene (NCBI Reference Sequence: NM_214395.1) design following primer SERPINA1CF gene sequence as shown in Seq ID No:1, SERPINA1CR gene sequence as shown in Seq ID No:2, it has included the SERPINA1 gene mRNA sequence The range from the 8th base to the 1273rd base (the sequence is shown in Seq ID No: 5).
[0031]
[0032] Use SERPINA1CF and SERPINA1CR primers to amplify the fragment from the 8th base to the 1273rd base of the SERPINA1 ge...
Embodiment 2
[0034] Embodiment 2 ELISA detects the SERPINA1 serum content of different genotypes
[0035] Take Laiwu pigs and Dachang hybrid pigs at 0, 4, 7, 10, 14, 21, 28 and 35 days after infection with PCV2 Serum, according to the porcine serine protease inhibitor 1 (SERPINA1 / A1AT) enzyme-linked immunosorbent assay kit Instructions for detecting serum levels of SERPINA1. Test results such as Figure 4 and Figure 5 Shown: In a healthy state, the serum level of AA-type individuals is the highest, followed by that of AG-type individuals, and the content of GG-type individuals is the lowest (P Figure 4 ). However, after infection with PCV2, the SERPINA1 serum level of GG individuals significantly increased at 4dpi (P Figure 5 ).
Embodiment 3
[0036] Example 3 Population Detection of Polymorphism at Locus 445 and Marker-Assisted Breeding
[0037] According to the gene sequence of SERPINA1 (NCBI Reference Sequence: NC_010449.4), the following primers were designed: the SERPINA1MF gene sequence is shown in Seq ID No: 3, and the SERPINA1MR gene sequence is shown in Seq ID No: 4. The range from the 4760th position to the 5629th position.
[0038]
[0039] Use SERPINA1MF and SERPINA1MR primers to amplify the fragment from position 4760 to position 5629 of the complete sequence of SERPINA1 gene. The amplification system is: 2×PrimeSTAR GC Buffer (Mg 2+ Plus) 10 μL, dNTPs 1.6 μL, each primer (10 μM) 0.5 μL, PrimeSTAR TM HS DNA Polymerase (5U / μL) 0.2μL, Genomic DNA 1μL, add ddH 2 O to a total volume of 25 μL. The PCR reaction conditions were 94°C for 5 min; 34 cycles of 98°C for 10 s, 56°C for 30 s, and 72°C for 1 min; 72°C for 8 min. The PCR product 870bp was obtained, and the amplified product was mixed with 6×Loa...
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Abstract
The invention relates to the field of molecular genetics, in particular to a molecular marking method for a mutation site of a coding region of a porcine SERPINA1 gene and application of the method to anti-PCV (porcine circovirus) pig breeding. The inventor finds that SERPINA1 coding regions of different varieties of pigs have multiple mutations, wherein a G>A mutation exists at the 445th base of the coding region to further cause a missense mutation: glutamic acid>lysine. Enzyme linked immunosorbent assay shows that an AA type individual has highest SERPINA1 serum content, an AG type individual has the second highest SERPINA1 serum content and a GG type individual has the lowest SERPINA1 serum content in a healthy state. After infection with PCV2, the SERPINA1 serum content of the GG type individual rapidly increases, while the content of the AG type and AA type individuals rapidly decreases, and such a result is consistent with that a Laiwu pig population with a higher anti-PCV capability is more likely to comprise GG type pigs compared with a western population and an mRNA expression level of the SERPINA1 gene is high. Obviously, a genotype of the 445th site of a coding region of an SERPINA1 gene in a porcine genome can be detected as a molecular marker associated with an anti-PCV character of a pig, so that the method is convenient, rapid and free of environmental influence, and early breeding can be implemented.
Description
technical field [0001] The present invention relates to the field of molecular genetics, in particular to a breeding method for screening pigs resistant to porcine circovirus disease, selecting species by judging the polymorphism of the mutation site in the coding region of the SERPINA1 gene of pigs, and artificially applying the mutation Loci to achieve excellent breeding. Background technique [0002] Since Tischer et al. discovered porcine circovirus (Porcine circovirus, PCV) in PK15 cells in 1982, the research on it has a history of about 34 years. PCV is divided into two subtypes, the non-pathogenic PCV1 and the highly pathogenic PCV2. PCV2 mainly infects pigs aged 5-15 weeks, with a lethality rate of about 15%. It is the main pathogen causing post-weaning piglet multisystem failure syndrome (PWMS), respiratory disease syndrome (PRDS) and porcine dermatitis nephritic syndrome (PDNS) (Stevenson et al., 2001). After PCV2 infects the body, it can cause immunosuppression...
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