CYP3A88-molecular-marker breeding method for sorting porcine reproductive and respiratory syndrome (PRRS)-resistant pigs and application thereof

A PRRS, targeted technology, applied in the field of molecular genetics, can solve problems such as differences in gene expression, immune function genes and cytokine expression levels

Active Publication Date: 2013-06-05
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Petry et al. (2005) compared the differences between Nebraska index line and Hampshire-Duroc hybrid pigs after PRRSV infection, and detected the changes in body weight, rectal temperature and virus titer of these two pigs. The results showed that the Nebraska index line had strong resistance; Petry et al. (2007) also found that in these two pigs, there were differences in the expression levels of specific immune function genes and cytokines in lung and lymph node tissues; Bates et al. (2008) used gene chip technology to screen differentially expressed genes in the lungs and lymph nodes of these two pigs, and found that there were differences in the expression of a large number of genes

Method used

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  • CYP3A88-molecular-marker breeding method for sorting porcine reproductive and respiratory syndrome (PRRS)-resistant pigs and application thereof
  • CYP3A88-molecular-marker breeding method for sorting porcine reproductive and respiratory syndrome (PRRS)-resistant pigs and application thereof
  • CYP3A88-molecular-marker breeding method for sorting porcine reproductive and respiratory syndrome (PRRS)-resistant pigs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1Genome walker method amplifies the 5' regulatory region of CYP3A88 gene

[0025] Genome extraction: Pig ear tissue was taken, and genomic DNA was extracted according to the instructions of the TIANamp genomic DNA (Tiangen) kit.

[0026] This experiment uses Clontech's GenomeWalker TM Universal Kit, its main principle is to design two gene-specific primers (GSP) in the same direction and higher annealing temperature according to the known DNA sequence, and the two uniquely designed primers (GSP) provided in the kit have a lower annealing temperature The degenerate primers, namely AP1 and AP2, were used for landing PCR reaction. The chromosomal primers for the 5′ regulatory region of the pig CYP3A88 gene are shown in the table below:

[0027]

[0028] Drop-down PCR amplification system:

[0029]

[0030] Perform touchdown PCR as follows

[0031]

[0032] The results of three Genome walker electrophoresis of pig CYP3A88 promoter are as follows: fig...

Embodiment 2

[0039] Example 2 Construction of CYP3A88 promoter vector and deletion vector, luciferase reporter system to detect its activity

[0040] Design primers CYP3A88F2 (R2) and CYP3A88F3 (R3) to PCR amplify Dudachang hybrid pig P2801 and P27 fragments respectively, CYP3A88F2, whose sequence is shown in SeqID No: 11; CYP3A88R2, whose sequence is shown in SeqID No: 12; CYP3A88F3, its sequence is shown in Seq ID No: 13; CYP3A88R3, its sequence is shown in Seq ID No: 14. Among them, MluI restriction site and HindIII restriction site were added at the 5′ of CYP3A88F2 and CYP3A88F3 respectively.

[0041]

[0042] The amplification system is:

[0043]

[0044] The PCR program was pre-denaturation at 95°C for 5 min, followed by 35 cycles of 98°C for 10 sec, 66°C for 15 sec, 72°C for 4 min, and 72°C for 10 min. PCR products were detected by 1% TBE agarose gel electrophoresis. The PCR product was recovered with a Promega gel recovery kit. The PCR recovered product was reacted in a w...

Embodiment 3

[0059] Example 3 Site-directed mutation of the 5' regulatory region -78 of the pig CYP3A88 gene and detection of luciferase activity

[0060] According to the results of the deletion experiment in Example 2, the TESS prediction software was used to analyze the 5′ regulatory region-695-+76 segment of the CYP3A88 gene, and combined with polymorphism analysis, it was found that the change from T to A at the important regulatory region-78 site caused the transcription factor Combination of YY1. The P694* vector for site-directed mutation of the YY1 binding site was constructed by using the P694 vector as the backbone vector by PCR. Primer sequences CYP3A88PF and CYP3A88PR of the P694* vector

[0061]

[0062] Amplification conditions are: 25μl system includes 2×PrimeSTAR GC Buffer (Mg 2+Plus) 12.5 μl, dNTP Mixture (2.5 mM each) 2 μl, primers (10 μM) 0.5 μl each, PrimeSTARTM HS DNA Polymerase (5 U / μl) 0.25 μl, carrier DNA 0.5 μl. The P694* vector was amplified by 16 cycles of...

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PUM

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Abstract

The invention relates to the field of molecular genetics, in particular to the application of a molecular marker method in pig breeding for disease resistance, wherein according to the molecular marker method, the molecule at a mutation site in a CYP3A88 gene 5' regulatory region of a pig is marked. The inventor of the method discovers that the CYP3A885' regulatory region of a large Chinese streaky-head pig and the CYP3A885' regulatory region of a Duroc long hybrid pig have a plurality of differences, wherein an A-to-T mutation exists at the -78 site, through a luciferase reporter gene system, the fact that promoter activity of the -78 site is lowered remarkably after an A at the -78 site is mutated into a T in a site-directed mutagenesis mode is found, and the promoter activity of the -78 site is the same as the low level of messenger ribonucleic acid (mRNA) expression of a CYP3A88 gene of the porcine reproductive and respiratory syndrome (PRRS)-resistant large Chinese streaky-head pig. Therefore, through genotype detection of the -78 site of the CYP3A88 regulatory region in a pig genome, the genotype of the -78 site of the CYP3A88 regulatory region can be used as a modular marker associated with traits of the PPRS, the molecular marker method not only is simple, convenient and rapid, but also cannot be affected by the environment, and early selection for breeding can be realized.

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 PRRS, by judging the polymorphism of the mutation site in the 5' regulatory region of the pig CYP3A88 gene to select the breed, and artificially applying the mutation The site realizes the breeding of pigs resistant to PRRS. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a porcine reproductive and respiratory disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection of porcine lung macrophages. The leading infectious disease affecting the swine industry worldwide (Neumann et al. 2005). The disease mainly causes reproductive disorders such as fever, premature birth, mummified fetuses and weak piglets in pregnant sows, as well as respiratory symptoms and high mortality in pigs of all ages (Rossow1998). The disease resista...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 姜运良蒋成兰李艳平王鹏飞康丽姜伟王力圆
Owner SHANDONG AGRICULTURAL UNIVERSITY
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