KR101253293B1 - DNA markers for detecting increase of porcine meat quality containing SNP in 3' UTR of PPARGC1A gene - Google Patents

Abstract

The present invention relates to a DNA marker for confirming whether meat is increased in pigs including SNPs of the 3 'UTR region of the PPARGC1A gene, and more specifically, 15 to 800 consecutive sequences including the 2956 th C base in the nucleotide sequence of SEQ ID NO: 1. The present invention relates to a DNA marker for confirming whether or not pork is composed of sequences.
According to the present invention, the SNP affects the increase in meat quality by causing an increase in pH, which is a meaty property, or a decrease in free juice, without affecting the muscle fiber composition of pigs. This genetic variation can be used to develop DNA markers useful for improving the quality of pigs and screening superior individuals.

Description

DNA markers for detecting increase of porcine meat quality containing SNP in 3 'UTR of PPARGC1A gene}

The present invention relates to a DNA marker for confirming whether meat is increased in pigs including SNPs of the 3 'UTR region of the PPARGC1A gene, and more specifically, 15 to 800 consecutive sequences including the 2956 th C base in the nucleotide sequence of SEQ ID NO: 1. The present invention relates to a DNA marker for confirming whether or not pork is composed of sequences.

Muscle fiber properties, including muscle fiber composition, are greatly influenced by genetic factors. In particular, pigs have a high heritability of 37-58% for each myofibrillar type and a high genetic correlation with meat-related traits. Larzul, C. et al. 1997. J. Anim Sci. 75: 3126-3137.). Marker Assisted Selection (MAS), a method of selecting DNA markers, increases the accuracy of selection and increases the rate of improvement in livestock breeding and maximizes genetic improvement. have. The fact that the variation of muscle fiber composition is mostly caused by genetic factors implies that the modification with MAS can be very effective.

However, myocyte characteristics are traits that can only be measured in the cartilage of the carcass, and cannot be measured in vivo. Therefore, for selection and improvement based on this, there is a need for a method for predicting the myocyte characteristics of the individual without slaughter. In this regard, the development of DNA markers is fundamentally required, and the development of candidate genes and the securing of genetic resources through gene structure analysis must be essential.

PPARGC1A (Peroxisome proliferator-activated receptor-gamma coactivator-1) gene is a precursor of PGC1, which is deeply involved in animal metabolism by deeply involved in adaptive thermogenesis and adipocyte differentiation. It has been recently discussed as a candidate gene (Erkens, T. et al. 2006. BMC Biotechnol 6: 41). Furthermore, it has been reported that this gene plays a major role in inducing myofibrillar Type I formation (Lin, J. et al. 2002. Nature 418: 797-801), and we have found that the PPARGC1A gene can be used for myofibrillar formation, especially Type I formation. Were selected as candidate genes involved in meat quality and meat quality.

It has been reported that over the last five years, various microRNAs (MicroRNAs, miRNAs) with lengths of 21-25 nucleotides (nts) are expressed from the genomes of all metazoan, including animals and plants. . These miRNAs do not produce proteins, but instead inhibit the expression of sequence complementary target genes (Bartel D. P., 2004, Cell 116: 281-297).

miRNAs were initially produced as immature primary transcripts and subsequently matured by subsequent processing in the nucleus and cytoplasm by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. (Lee Y. et al., 2003, Nature 425: 415-419, Bernstein E. et al., 2001, Nature 409: 363-366). Mature miRNAs enter the RNA-Induced Silencing Complex (RISC) and serve as a guide to find their sequence complementary target mRNAs, resulting in translational repression or degradation of target mRNAs depending on the degree of sequence complementarity. (He L. et al., 2004, Nat Rev Genet. 5: 522-531). In animals, miRNAs have been reported to inhibit translation through binding sites and imperfect base pairing in the 3 'UTR of the target mRNA (Bartel DP, 2004, Cell 116: 281-). 297).

miRNA induces inhibition of expression of the target gene by acting as a guide to the RISC complex binding to the 3 'UTR of the sequence complementary target mRNA. Therefore, if there is a nucleotide sequence change in the target 3 'UTR region, expression inhibition by a specific miRNA may be increased or vice versa. It has already been reported that the change of the nucleotide sequence of the target gene results in a difference in expression regulation by miRNA (Clop A. et al., 2006, Nat Genet 38: 813-818).

In the present invention, the actual red meat production capacity and meat quality were measured for each pig breed, and PPARGC1A gene was selected as a candidate gene involved in muscle fiber composition, especially type I formation and related to meat quality, and genotyping result in the 3 'UTR region. Based on this, we conducted a correlation analysis. Through this, the SNP was searched in the 3 'UTR region of the PPARGC1A gene, the function related to meat improvement was confirmed, and the utility as a related DNA marker was verified.

Accordingly, a main object of the present invention is to provide a meat growth-related DNA marker for improving meat quality using SNPs present in the 3 ′ UTR region of the PPARGC1A gene of pigs, and a kit for SNP analysis using the same.

It is another object of the present invention to provide a method for confirming whether or not meat is increased by using the DNA markers related to meat quality.

According to an aspect of the present invention, the present invention provides a DNA marker for increasing the meat quality of pigs consisting of 15 to 800 consecutive sequences including the 2956 th C base in the nucleotide sequence of SEQ ID NO: 1.

The present inventors have found a single nucleotide polymorphism (SNP) related to the meat quality of pigs in the 3 'UTR region of the PPARGC1A gene represented by SEQ ID NO: 1, and compared with the meat characteristics and genotypes of the pig individual according to the SNP. Association was analyzed. The DNA marker is part of the 3 'UTR sequence of the PPARGC1A gene, which has recently been studied as a meat-related candidate gene for livestock, and includes a single nucleotide polymorphism (SNP) site where a base is substituted with T or C at a specific site.

The present invention finds an SNP (T → C substitution) site that causes an increase in meat quality in the 3 'UTR region of the PPARGC1A gene and uses it as a "DNA marker for confirming whether the pork meat is increased." It is about.

The PPARGC1A gene is known to be involved in energy metabolism in mammals, and various studies have been conducted as a candidate for meat quality in livestock, and in particular, it is known to play a major role in inducing muscle fiber type I formation in mice. . However, there is no known method for screening individuals having improved meat quality using SNPs at specific sites in the 3 'UTR region of the PPARGC1A gene in pigs, and the inventors first disclosed the SNPs related to meat quality increase.

In the present invention, the DNA fragment is a DNA marker comprising the 2956th C base, which is a new genetic variation (SNP site) of the 3 'UTR region of the PPARGC1A gene related to the meat properties of pigs. This genetic mutation site is replaced by a base T → C difference in the expression of the gene, resulting in improved meat quality. The size of the DNA fragment may be any fragment size including the SNP site, as long as it is not the full length of the entire gene, but preferably 15 to several hundred bases, more preferably 15 to 800 bases. Especially 15 to 30 base can be used as a probe (probe) or primer for detecting the SNP. In an embodiment of the present invention, a DNA fragment of 736 bp (SEQ ID NO: 4) including the SNP site was used as the DNA marker.

In the present invention, the DNA marker is characterized in that the meat quality is increased by increasing the pH and meat free traits (drip loss) without increasing the muscle fiber composition of pigs. As in Example 4 of the present invention, CT and TT genotypes improved by analyzing the correlation analysis of genotypes (CC, CT, TT), muscle fiber and meat quality according to SNP at the 2956 th C base of SEQ ID NO: 1 Was confirmed (see Table 8).

In the present invention, "increase in meat" means that the pH of meat-related traits of pigs is high according to the individual genotype (CC, TC, TT) due to the 2956 th SNP of SEQ ID NO: 1. That is, the 2956 th SNP of SEQ ID NO: 1 means that the CT and TT genotype individuals have increased pH and reduced free juice compared to CC genotype individuals.

Pork's meat quality is classified by grade of meat according to pH, meat color, intramuscular fat, fat color, texture, maturity, etc. Among them, pH and glass broth are important criteria for indicating meat quality of pork. In recent years, since the quality of meat is very important in pigs, it is important to develop high-quality meat with improved quality by selecting, breeding, and producing excellent meat. Therefore, by using the DNA marker of the present invention, it is possible to easily select high-quality meat with improved meat quality by early diagnosis and screening of individuals with improved meat quality.

According to another aspect of the present invention, the present invention provides a primer for searching for a SNP related to meat quality in the 3 'UTR region of the porcine PPARGC1A gene, consisting of oligonucleotides represented by SEQ ID NO: 2 and SEQ ID NO: 3. In the embodiment of the present invention, a primer was prepared to search for SNP present in the 3 'UTR of the PPARGC1A gene of the present invention based on the gene sequence registered in Genebank (accession no. AB106108). The primer of the present invention is for amplifying a gene fragment containing a SNP, which is not known in the prior art, to prepare a gene fragment containing a SNP at a specific site (SEQ ID NO: 1 to 2956 base) of the 3 'UTR sequence of the porcine PPARGC1A gene. It allows you to.

The nucleotide sequence of the gene fragment prepared using the primer can be determined by various methods known in the art. For example, by determining the nucleotide sequence of a nucleic acid directly by the dideoxy method, or by hybridizing a probe comprising the sequence of the SNP site or a probe thereof to the DNA and measuring the degree of hybridization obtained therefrom. Methods for determining the nucleotide sequence may be used. The degree of hybridization can be determined, for example, by marking a detectable label on the target DNA and specifically detecting only the hybridized target DNA, but other electrical signal detection method and the like can be used.

Specifically, allele-specific probe hybridization, allele-specific amplification, sequencing, 5 'nuclease digestion, and 5' Molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism, and the like. have.

In the embodiment of the present invention, a DNA fragment comprising the SNP site of the present invention was prepared and genotype was determined by determining the nucleotide sequence of the SNP site using direct sequencing. For example, genotypes could be divided into CC, CT, and TT using DNA fragments (736 bp) amplified by primers of SEQ ID NO: 2 and SEQ ID NO: 3 (see FIG. 3).

According to another aspect of the present invention, the present invention provides a kit for SNP analysis related to the increase in meat quality of a pig comprising a probe that binds complementarily to the DNA marker. Probe (probe) in the present invention means a hybridization probe, it means an oligonucleotide capable of sequence-specific binding to the complementary strand of the nucleic acid. The probe of the present invention can be used for a method for detecting SNPs. The detection method includes detection methods based on hybridization of nucleic acids such as Southern blotting, or the like, and may be provided in a form that is pre-coupled to a substrate of a microarray in a method using a microarray.

According to another aspect of the present invention, the present invention provides a method for checking whether the pork is increased in meat, comprising the following steps:

a) separating genomic DNA from pigs;

b) amplifying a specific site including the 2956 base of the 3 ′ UTR region of the PPARGC1A gene represented by SEQ ID NO: 1 using the DNA of step a) as a template;

c) analyzing the base sequence of the DNA amplified in step b); And

d) confirming whether or not the 2956 base of SEQ ID NO: 1 is replaced by the T base to the C base from the base sequence result of step c).

In the method of the present invention, characterized in that the amplification using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 in step b). The DNA amplification of step b) may be any primer capable of amplifying a specific region including SNP, which is the 2956 base of the 3PAR UTR region of the PPARGC1A gene, but is preferably an oligonucleotide represented by SEQ ID NO: 2 and SEQ ID NO: 3 Can be amplified using a primer consisting of.

In the method of the present invention, a DNA fragment containing the SNP region may be prepared, and DNA fragments may be genotyped by determining the nucleotide sequence of the SNP region using direct sequencing. According to the above method, genotypes can be divided into CC, CT, and TT by identifying the bases of the SNP sites present in the DNA fragments amplified by the primers of SEQ ID NO: 2 and SEQ ID NO: 3, and by comparing meat quality according to each genotype. It is possible to determine whether there is an increase in meat quality in pig subjects (see Table 8).

As described above, according to the present invention, the SNP of the 3 ′ UTR region of the porcine PPARGC1A gene may be determined to affect the increase in meat quality by increasing the pH of the meat properties and decreasing the free juice without causing a difference in muscle fiber composition. Can be. Therefore, the genetic variation (SNP) of the present invention has a function related to the meat quality of pigs and can be usefully used as a DNA marker related to meat quality improvement through such characteristics.

Figure 1 shows the results of analyzing the nucleotide sequence of the pig PPARGC1A gene 3 'UTR. The 2956th base (T → C substitution) is the newly discovered single base mutation site (SNP).
Figure 2 is an example of a DNA marker according to the present invention, shows a DNA fragment used for direct sequencing using a primer according to the present invention.
Figure 3 shows a method for genotyping using the difference in the peak according to the base by performing PCR and direct sequencing (Sequencing) for the SNP according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1.Sequence analysis and mutation search for 3 'UTR of PPARGC1A gene

For information on porcine PPARGC1A, using the National Center for Biotechnology Information (NCBI) homepage (http://www.ncbi.nlm.nih.gov/), the cDNA sequence of the previously known porcine PPARGC1A gene (AB106108, http: //www.ncbi.nlm.nig.Gov/BLAST) to obtain the sequence of the 3 'UTR region of the PPARGC1A gene. In order to search for the SNP of the 3PAR UTR of the PPARGC1A gene, sequences were analyzed for four pigs (Yorkshire, Landrace, Duroc, Berkshire) using seven amplicons. Seven primer sets capable of PCR of the amplicon were prepared (Table 1). The composition and reaction conditions of the PCR mixture are shown in Tables 2 and 3 below, and the region subjected to sequencing in the present invention is shown in SEQ ID NO: 1. Sequencing of the sequencing results by cultivars (SNP) was found. In the 3 'UTR of the porcine PPARGC1A gene represented by SEQ ID NO: 1, it was confirmed that the 2956 th T base was SNP substituted with C base.

Example 2 Extraction of Genomic DNA from Pigs

Isolation of genomic DNA for gene polymorphism analysis was performed according to the method of Sambrook et al. (1989). After collecting 10 ml of blood from the jugular vein, 2 ml of ACD solution (citric acid 0.48 g, sodium citrate 1.32 g, glucose 1.47 g to H ₂ O 100 ml) was added and maintained at 4 ° C. Transported to the lab. Transfer to a 50 ml centrifuge tube and centrifuge at 1,300 g for 15 minutes to collect buffy coat and collect 15 ml of DNA extraction buffer (10 mM Tris-Cl, pH 8.0; 0.1 M EDTA, pH 8.0; 0.5% SDS; 20% μg / ml) was mixed well and incubated for 1 hour in a 37 ℃ constant temperature water bath. After incubation, Proteinase K (200 μg / ml, BRL) was mixed and reacted in a 37 ° C. constant temperature water bath for 12 hours. Phenol (Phenol) prepared with the same amount of 0.5M Tris-Cl (pH 8.0) was added to the mixture for 30 minutes, extracted, and centrifuged at 5,000 g for 15 minutes at room temperature to form a layer. After repeating this procedure 3 times, the supernatant was taken with a pipette with a diameter of 0.3 cm and dialyzed at 4 ° C in 4 L of 50 ml Tris-Cl (pH 8.0) and 10 mM EDTA (pH 8.0) for 18 hours to obtain chromosomal DNA was isolated. The isolated DNA purity was measured by a spectrophotometer (Beckman, USA) at a ratio of absorbance of A260 to A280, and the molecular weight of DNA was confirmed by 0.8% agarose gel electrophoresis.

Example 3. Genotyping by PCR and Direct Base Sequence Analysis

For genotyping of SNPs identified in the 3 'UTR region of the precursor porcine PPARGC1A gene, the RFLP method was used, but since there was no restriction enzyme capable of recognizing the SNPs of the present invention, the present invention includes a newly discovered SNP. Individual genotyping was performed by PCR and direct sequencing of the regions. For PCR reaction and direct sequencing, Forward primer (5'-CAC AGG TTC TGC GTT ACG AC-3 ', 20mer; SEQ ID NO: 2) and Reverse primer (5'-AAA GCA CCA GTT CGG GTT AC-3' A primer pair consisting of 20mer; SEQ ID NO: 3) was prepared (Table 4). For PCR analysis, genomic DNA was prepared by adding 2.0 μl (10 μM) of each primer and 10 × PCR reaction buffer (100 mM Tris-Cl; pH 8.3, 500 mM KCl, 15 mM Mg ₂ Cl), 5.0 μl of dNTP (2 mM each), 0.5 μl of Taq DNA polymerase (5 units / μl, Enzynomics), 2.0 μl of Template (genomic) DNA (approximately 100 ng) and 33.5 μl of ddH ₂ O 50.0 μl was reacted. The reaction conditions were 10 min initial denaturation at 94 ° C, 1 min denaturation at 94 ° C, 1 min annealing at 60.0 ° C, 1 cycle extension at 74 ° C, 30 cycles, and final extension at 72 ° C. After the PCR reaction was terminated (see Table 5 and Table 6).

Thus obtained PCR product (736 bp; SEQ ID NO: 4) through direct sequencing was found the base configuration of 2956 bp position in the 3 'UTR of the PPARGC1A gene represented by SEQ ID NO: 1 (see Fig. 1).

Figure 3 shows the base composition of the 2956 bp position in the 3 'UTR region of porcine PPARGC1A. In the case of CC genotype (C homozygote) and TT genotype (T homozygote), the base peak is shown as one, but in the case of CT genotype (T / C heterozygote), it can be seen that two peaks.

In the present invention, 441 genotypes of SNPs were analyzed for the test pigs consisting of three varieties of Berkshire, Landrace, and Yorkshire, and genotypes and gene frequencies are shown in Table 7 below. Individuals with TT and TC genotypes were found at the SNP position, and T allele was also highly distributed in gene frequency.

Example 4 Identification of Muscle Fiber Composition and Meat Difference by Marker Type

1) muscle fiber characteristics analysis

For muscle fiber characterization, 45 minutes post mortem muscle samples of the dorsum muscles of the thoracic vertebrae were collected, cut into pieces of 0.5- × 0.5- × 1.0-cm, quickly frozen with liquid nitrogen, and -80 ° C until analysis. Were kept at. The cross section (10 μm thick) was prepared on a cryostat (CM1850, Leica, Germany) machine at 20 ° C. and preincubated with type I, IIa and IIb fibers for myofiblot identification analysis (acid preincubation, pH 4.35). ) Was stained and classified using the actomyosin ATPase method. All samples were characterized by muscle fiber characterization using a CCD camera (charge-coupled device color camera; IK-642K, Toshiba, Japan) and an image analysis system (Image-Pro Plus, Media Cybernetics, USA). .

2) meat analysis

Muscle pH was measured at spine 13/14 at 45 minutes post mortem using a spear-type electrode. Lightness was measured at 45 minutes post mortem muscle of the sirloin using a colorimeter (CR-300, Minolta Camera Co., Japan). The glass drip loss was analyzed by measuring the difference in the amount of muscle sample on the surface in a plastic bag for 48 hours at 2 ° C (Honikel, KO (1987). Martinus Nijhoff, In PV Tarrant, G. Eikelenboom, & G. Monin (Eds.): 129142). The results are shown in Table 8 below.

3) Analysis of association between genotype and trait

As shown in FIG. 3, three genotypes of CC, CT, and TT can be identified, and a correlation analysis between genotype, muscle fiber characteristics, and meat quality according to SNP was performed. For statistical analysis program, SAS / GLM procedure provided in SAS 9.13 package was used. In this model, breed, gender and slaughter order were analyzed as fixed effects. The results of the analysis were expressed as Least Squares Means and standard errors. Table 8 below is a result of analyzing the association between the SNP and muscle fiber properties and meat quality in the 3 'UTR region of the porcine PPARGC1A gene.

Existing studies have reported that the major influence on meat quality among muscle fibers is the composition of muscle fiber number and area (Klont, RE et al. 1998. Meat Science 49: S219-S229), especially slow oxidation. Is known to have a positive effect on meat quality when muscle fiber type I is high.

In the results of Table 8, no significant differences were observed between genotypes in total fiber number, mean CSA of fibers, and the density of total fibers. . In particular, no significant difference was observed between genotypes in the fiber composition. However, the meat quality traits were CT, and TT genotype have higher appears (P <0.01) glass broth (Drip loss) was low muscle pH (pH _45min) after 45 minutes after slaughter than the CC genotype (P <0.001), it Through CT, TT genotype can be judged that the meat quality is superior to the CC genotype. In addition, the TT genotype showed lower lightness ( L ^* ) compared to the CC genotype ( P <0.10), indicating that the meat quality was excellent, and especially, the lower drip compared to the CT genotype that showed good meat quality together. ( P <0.001) meat quality is excellent.

As such, in the case of the SNP of the present invention, despite the fact that there was no significant difference in cross-sectional composition by muscle fiber type, the difference in meat quality was not related to meat improvement according to muscle fiber type I area composition, but PPARGC1A involved in fat metabolism and energy metabolism. It can be judged that meat quality is improved by other mechanisms depending on the function of the gene.

Taken together, the SNPs in the 3 'UTR of porcine PPARGC1A (substituted from the 1956 to 2956 T-bases for C bases), finally, have no difference in muscle fiber composition, SNPs affecting meat quality were identified.

<110> Korea University Industrial and Academic Collaboration Foundation <120> DNA markers for detecting increase of porcine meat quality          containing SNP in 3 'UTR of PPARGC1A gene <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 3771 <212> DNA <213> Sus scrofa <400> 1 atgactttga tcctgcttcc accaagagca agtatgactc tctggatttc gatagtttac 60 tgaaagaagc tcagagaagc ttgcgcaggt aacatgttcc ctggctgagg atgacagagg 120 gacggtgaat acctcacggg acagcgcgtc cttccctaac agactcttgc aagtcatact 180 taggaatttc tcctacttta cactctctgt acaaaaacaa aacacaacaa caacaataca 240 acaagaacaa caataatggt ttacatgaac acagctgctg aagaggcaag agacaggatg 300 gatatccagt aagcacatgt ttattcatgg gtgtcagctt tgctttccct gagtctcttg 360 gtgatggagt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtga gtgcgcgtgt 420 gtgcttggtt taggggaagt atgtgtgggt acatgtgagg actgggggca cctgaccaga 480 atgcgcaagg gcaaaccatt tcaaatggca gcagttccat gaagacacgc ttaaaaccta 540 gaacttcaaa atgttcgtat tctattcaaa aggaaaaaaa aaaaaaaaaa aaaaaaaaaa 600 agcataaatt caaaaggaaa 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Claims (6)

In the nucleotide sequence of SEQ ID NO: 1, DNA marker for checking whether meat is increased in meat consisting of 15 to 800 consecutive sequences including the 2956 th C base.
According to claim 1, wherein the DNA marker is a DNA label, characterized in that to increase the meat quality by increasing the pH, meat-related traits and reducing free loss (drip loss) without making a difference in the muscle fiber composition of pigs factor.
A primer for searching for a DNA marker according to claim 1, wherein the oligonucleotide represented by SEQ ID NO: 2 and SEQ ID NO: 3 is related to whether meat quality is increased in the 3 'UTR region of the porcine PPARGC1A gene.
The kit for analyzing a DNA marker according to claim 1, related to whether or not meat is increased in a pig comprising a probe complementarily binding to the DNA marker of claim 1.
A method for confirming whether meat is increased in meat, comprising the following steps:
a) separating genomic DNA from pigs;
b) amplifying a specific site including the 2956 base of the 3 ′ UTR region of the PPARGC1A gene represented by SEQ ID NO: 1 using the DNA of step a) as a template;
c) analyzing the base sequence of the DNA amplified in step b); And
d) confirming whether or not the 2956 base of SEQ ID NO: 1 is replaced by the T base to the C base from the base sequence result of step c).
The method of claim 5, wherein the amplification using the primers represented by SEQ ID NO: 2 and SEQ ID NO: 3 in step b).

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