KR100415080B1 - Primer related marbling score of Hanwoo and test process of marbling score using thereof - Google Patents

Abstract

The present invention relates to a Korean native beef myofascial fat associated primers and a method for testing Korean bovine myocardial fat using the same. After the synthesis of primers and adapters for AFLP, the primers are reacted with genomic DNA of Hanwoo digested with restriction enzymes to perform PCR. By obtaining specific DNA markers that are differentiated specifically from high and low myocardial fat groups and analyzing the sequencing, the meat quality of Hanwoo was examined using 15mer intramuscular fat related primers. As a result, there is an excellent effect of providing a easier, more economical and accurate method for testing the quality of beef cattle, especially the intramuscular fat score.

Description

Primer related marbling score of Hanwoo and test process of marbling score using

The present invention relates to an intramuscular fat degree associated primer of Hanwoo and a method for testing the intramuscular fatness of Hanwoo using the same. More specifically, the present invention extracts genomic DNA from the blood of Hanwoo, extracts specific DNA markers related to intramuscular adipose from thereafter, and analyzes the sequencing to develop a primer related to intramuscular fat, which is an important factor for determining meat quality. More preferably and easily relates to a method of inspecting the quality of beef cattle, especially intramuscular fat.

Hanwoo (Hanwoo) is a unique prop species of Korea, which has been adapted to the Korean climate for thousands of years and has been fixed in the proper constitution. The amount of foreign meat imported into Korea has increased steadily since the import liberalization, threatening the domestic livestock industry. In order to overcome this situation, it is necessary to improve the quality of beef cattle and selectively raise first-class beef cattle.

In general, the determinants of beef quality include marbling score, color and brightness of meat, moisture retention and fat composition. Among these, muscle fatness is the most important factor that determines meat quality. to be. In order to check the meat quality in the past, butchering the cows could be confirmed, but it is very inefficient. Accordingly, there has been a demand for a method for determining the quality of beef cattle for raising beef cattle of excellent quality grade. The development of DNA-level experimental techniques has been continuously made in accordance with the development of genetic engineering. Based on the development of these experimental techniques, various DNA typing techniques have been developed and used for animal husbandry to improve the genetic characteristics and ability of livestock. Such genetic engineering techniques include random amplified polymorphic DNA (RAPD) using PCR (Polymerase Chain Reaction), Arbitratily Primer-Polymerase Chain Reaction (AP-PCR), and Amplified Fragment Length Polymorphism (AFLP). Representatives of DNA include DNA fingerprinting (DNA typing) and Restriction Fragment Length Polymorphism (RFLP). PCR has the advantage of easy processing method and a large number of sample results in a short time, but shows a problem in reproducibility, using a labeled probe after transferring the DNA fragments generated by the restriction enzyme to the membrane Fingerprinting by RFLP, which detects DNA polymorphism, is not based on PCR, so it is not inconvenient for DNA typing.However, it is difficult to apply various probes to genetic fingerprinting. Redundant mutations can vary from one individual to another, making them vulnerable to cloning and mapping of DNA markers. AFLP, a DNA fingerprinting method developed recently by Vos et al. (1995), combines the advantages of RFLP, a tough technique that relies on Southern blotting, which is conceptually challenging, and RAPD technology, which is very sensitive to reaction conditions and is less reproducible. As a technique, various DNA polymorphisms can be obtained through an easier experimental process than the technique of DNA fingerprinting published by Jeffreys et al. (1985), and it is recognized as an important method for researching new livestock DNA. It is applied by technique. In addition, DNA markers exhibited by DNA polymorphism are not inconvenient to use for DNA typing in DNA fingerprinting, but there are many difficulties in identifying DNA sequences through cloning of DNA markers themselves. However, DNA markers shown through AFLP experiments are known to be widely used because they can be cut on a gel and immediately cloned to sequence DNA structures.

Unlike protein mutations, DNA polymorphism is not affected by the timing and stage of development of livestock, and most DNA in the genome is not expressed as a trait (protein, enzyme, etc.) and is not expressed as a trait. Even DNA can be analyzed at the DNA level, so DNA can be analyzed advantageously and accurately. In particular, probes mainly used to detect DNA mutations are highly sensitive enough to detect differences between individuals. In the present invention, a plurality of economic trait DNA markers were identified through AFLP, and sequencing of these DNA markers was completed to develop some primers.

At present, the standard grade for determining the quality of Korean beef is divided into 1+ grade, 1 grade, 2 grade and 3 grade according to intramuscular fat. In the present invention, high grade (1+ or 1 grade) individuals and low grade ( Level 3) individuals can find the DNA markers with valid differences among the PCR methods using AFLP DNA analysis technology, and then analyze the nucleotide sequences of these DNA markers to easily identify markers related to intramuscular fat within these nucleotide sequences. Primers were developed. This invention is to use DNA (gene) is invariant characteristics according to the development stage of the cow was carried out to substantially improve the income of farmers by discriminating and selectively breeding young Hanwoo calf individuals using the primer of the present invention. In particular, the developed primer of the present invention has a meaning as a development of the DNA marker associated with the economic traits of Korean native varieties of Korean native cattle, which is an essential condition to secure an advantage in competition with foreign cattle.

Accordingly, an object of the present invention is to provide a nucleotide sequence of a specific DNA marker associated with intramuscular fat extracted from Hanwoo genomic DNA. Another object of the present invention is to provide a primer and its base sequence associated with intramuscular fat that can determine the meat quality of Hanwoo cultivar. Another object of the present invention to provide a method for testing the intramuscular fat score of Hanwoo using the primer of the present invention.

The above object of the present invention is to isolate the genomic DNA from the leukocytes of Hanwoo, and add double Taq I and Eco Rl restriction enzyme to perform DNA double immersion and ligated a double-stranded adapter at the end of the DNA to prepare Hanwoo DNA, AFLP After synthesizing the primers to be used, AFLP-PCR was performed by reacting with the DNA reagent. Among them, DNA markers showing specific reactions in the high and low myocardial fat scores were isolated and the sequencing was analyzed. Hanwoo intramuscular fat was also achieved by developing an associated primer and providing a method for the evaluation of intramuscular fat using Hanwoo.

Hereinafter, the configuration of the present invention will be described in detail.

1 shows AFLPEcoRⅠ 9 /TaqI PCR results showed specific DNA markers differentiated between high and low populations of Hanwoo (Korea cattle) using 2 primers.

Figure 2 shows the nucleotide sequence of the 139 bp DNA specific marker associated with intramuscular fat, which is differentiated from the high and low myopia fat score groups by PCR using the Eco RI9 / Taq I2 primer of AFLP.

Figure 3 shows a primer prepared by analyzing the nucleotide sequence of the 139 bp DNA marker associated with the intramuscular fat diagram of Figure 2 identified as Eco RI9 / Taq I2 primer.

FIG. 4 shows the results of PCR using primers designed based on the nucleotide sequence of the 139 bp DNA-specific marker associated with intramuscular fat in FIG. 2.

The present invention comprises the steps of extracting white blood cells, removing proteins and condensing DNA with ethanol by centrifuging the blood collected from the Hanwoo meat quality assessment competition in 1997 and 1999; The Taq Ⅰ restriction enzyme (Taq Ⅰ restriction enzyme) and Eco RⅠ restriction enzymes (Eco RⅠ restriction enzyme) was added to the DNA double dipping the double-stranded adapter (double strand adapter) to the DNA ends and then subjected to a (double digestion) to the DNA Ligation; Synthesizing an adapter and a primer to be used for AFLP (Amplified Fragment Length Polymorphism) PCR, and then performing PCR by mixing the prepared DNA, Taq I primer, Eco R I primer Taq polymerase, and the like; Electrophoresis of the PCR reaction product on a 1% agarose gel to confirm the presence of the reaction, and after the gel is hardened, pre-run; Adding and denature the STR 3X loading buffer to each PCR reaction product, and loading the reagents stored in ice into each well of the electrophoretic plate, followed by electrophoresis; Fixing and washing the plate and then staining and developing the plate; Cutting out specific DNA markers having polymorphism on acrylamide gel and performing PCR with the same primers, followed by electrophoresis of the reaction product to identify single bands and analyzing nucleotide sequences; Designing a probe and a primer that can determine the economic traits of Hanwoo based on the base sequence to develop the present invention Hanwoo intramuscular fat associated primers and to examine the quality of Hanwoo beef using the prepared intramuscular fat associated primers It consists of steps. Part of the base sequence of the 15mer primer that can examine the meat quality of the Hanwoo developed in the present invention by substituting with another base, or changing the position and orientation of some base sequences will also fall within the scope of the primer of the present invention.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Preparation of the Primer of the Invention

The present invention was performed on the marbling score of 168 long ribs of the Hanwoo meat quality assessment competition in 1997 and 1999.

First step. Extraction of DNA from Hanwoo Leukocytes

DNA was extracted from the leukocytes of the present invention.

The blood of Hanwoo, collected with a 0.5M EDTA-treated syringe, was hemolysed with 0.2% NaCl solution and centrifuged at 1000 rpm for 5 minutes to separate the leukocyte layer, followed by 0.16M NaCl / 1mM EDTA and 0.5% N-lauroylsarcosine solution. And Proteinase K (10 mg / ml, Promeag Co. USA). The same amount of TE: phenol (1: 1) and phenol: chloroform: iso-amylalchol (25: 24: 1) was added once to remove protein, followed by 3M sodium Sodium acetate (pH5.2) was added, and 100% ethanol was added to condense DNA. The DNA was dissolved in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA) and stored at 4 ° C.

Second step. DNA double digestion and ligation

Was added to 7.5 units Taq Ⅰ restriction enzyme (Taq Ⅰ restriction enzyme 7.5 unit) in the prepared DNA 500ng total volume (total volume) such that 25㎕, it was immersed (digestion) for 1 hour at 65 ℃. Thereafter, 2.5 units of Eco Rl restriction enzyme were added to the total volume to 50 µl, and soaking was performed at 37 ° C for 1 hour.

5 pmol Taq I double strand adapter, 5 pmol Eco Rl double strand adapter, 1 mM ATP, 1 unit T4 DNA ligase to ligate a double stand adapter to the double immersed DNA fragment ends 10 L of the double immersed sample was allowed to have a total volume of 50 L and then ligated at 37 ° C for 3 hours. After the ligation was completed, dilution solution (10 mM Tris-HCl, 0.1 mM EDTA) was added to dilute.

Third step. Perform PCR

Adapters and primers to be used for the PCR reaction of AFLP (Vos et al., 1995) were used by Ajmone-Marsan et al. (1997) and Basedow et al. (1998). It was synthesized from. (Korea). The base sequences of the adapters and primers are shown in Table 1.

DNA sequences of adapters and primers for AFLP-PCR designation order Adapter EcoR I Eco top strand 5′- CTCGTAGACTGCGTACC Eco bottom strand 5′- AATGGTACGCAGTCTAC Adapter Taq Ⅰ Taq top strand 5′- GACGATGAGTCCTGAC Taq bottom strand 5′- CGGTCAGGACTCAT Primer EcoR I EP 1 5′- GACTGCGTACCAATTCA EP 2 5′- GACTGCGTACCAATTCAAC EP 3 5′- GACTGCGTACCAATTCAAG EP 4 5′- GACTGCGTACCAATTCACA EP 5 5′- GACTGCGTACCAATTCACT EP 6 5′- GACTGCGTACCAATTCAGA EP 7 5′- GACTGCGTACCAATTCAGT EP 8 5′- GACTGCGTACCAATTCATC EP 9 5′- GACTGCGTACCAATTCATG Primer Taq Ⅰ TP 1 5′- GATGAGTCCTGACCGAA TP 2 5′- GATGAGTCCTGACCGAAAC TP 3 5′- GATGAGTCCTGACCGAAAG TP 4 5′- GATGAGTCCTGACCGAACA TP 5 5′- GATGAGTCCTGACCGAACT TP 6 5′- GATGAGTCCTGACCGACAC TP 7 5′- GATGAGTCCTGACCGACAG TP 8 5′- GATGAGTCCTGACCGACAT TP 9 5′- GATGAGTCCTGACCGACCA EP: EcoR I Primer TP: Taq I Primer

After immersion and ligation, 3 μl of the diluted DNA sample was mixed with 75 ng Taq I primer, 75 ng Eco R I primer, 0.2 mM dNTP, 1 mM MgCl _2, 0.1-fold 10 × buffer and 1 unit Taq polymerase. Sterile distilled water was added to bring the total volume to 25 μl. The reaction was carried out in a T-Gradient thermal cycler (Biometra, Germany) after 35 cycles of 30 seconds (denaturation) at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C, followed by 72 ° C. One run for 10 minutes at.

Fourth step. Electrophoresis

Electrophoresis was performed on the DNA PCR reaction product.

The PCR product was electrophoresed on a 1% agarose gel to confirm the presence of the reaction, and a 6% denaturing polyacrylamide gel (6% acryamide solution, 7Murea, 1X TBE buffer) was prepared and 10% with TEMED (Biorad, USA). Ammonium persulfate was added to solidify the gel and pre-run the gel plate to about 50 ° C. in 6 μl of each PCR product in STR 3X loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% 3 μl of xylene cyanol FF) was added, denaturated at 95 ° C. for 2 minutes, immediately placed on ice, and 6 μl of which was loaded into each well. The SQ3 sequencer (Hoefer Pharmacia Biotech) Igc., USA).

5th step. Silver staining

The electrophoretic gel plate was fixed in a fixed solution (fix solution; 10% acetic acid) for 20 minutes, washed three times for 2 minutes with distilled water, and then dyed for 30 minutes with a dye solution (2 g silvet nitrate, 3 ml 37% formaldehyde). Dye After 10 seconds of washing with about 3 liters of distilled water, develop with developer solution (60g sodium carbonate, 3ml 37% formaldehyde, 400μl-10mg / ml sodium tiosulfate), pour fixed solution, fix phase and wash with distilled water for 2 minutes. The glass plate was dried.

As a result, DNA markers that were differentiated at 402 bp in the high group and 179 bp and 139 bp in the low group were identified. The results are shown in FIG.

Step 6. sequence analysis

The nucleotide sequence of the specific 139 bp DNA marker shown in the low intramuscular fat score isolated from the acrylamide gel was analyzed.

The 139 bp DNA markers of the 402 bp, 179 bp, and 139 bp DNA markers having the polymorphism were cut out on an acrylamide gel, and PCR was performed using the same primers.The PCR product was subjected to 1% agarose. Electrophoresis on the gel confirmed a single band. The sequencing was performed using ABI PRISM 377 automatic sequencer (Perkin-Elmer, USA) with the pure product to be obtained with QIA quick ^TM PCR Purification Kit (QIAGEN, Germany, Cat. No. 28104). . The sequencing results are shown in FIG. 2.

Step 7. Preparation of primers and probes

50% or more of probe or GC components using a unique repeat sequence that can determine the economic traits related to the weight gain and intramuscular fat of Hanwoo using the primer design program based on the identified sequence results, and there is no secondary structure By designing primer that shows accurate results by satisfying the condition that melting point [melting temperature (Tm)] is over 50 ℃, it can be used as a means for improving the ability of Hanwoo by a quick and simple technology that can be applied uniquely in Hanwoo It was. The primers produced by analyzing the nucleotide sequence of the 139 bp DNA marker related to the intramuscular fat identified by the Eco RI9 / Taq I2 primer are shown in ① and ② of FIG. 3. Where ① represents MS139L (forward primer; 15mer) and ② represents MS 139R (reverse primer; 15mer).

Example 2 implementation of the Hanwoo muscle fat score test using the present invention primer

Hanwoo DNA samples were extracted and subjected to general PCR using the primers of the present invention.

PCR of all 168 subjects using MS139R of the forward primer AAGTGTGGCGGTGGA and reverse primer CGGACACTGTGCTTC designed from the sequence of the 139 bp marker was shown in FIG. 4, and the analysis results are shown in Table 2.

Isolation of MS139R-Specific DNA Markers for Intramuscular Fat Test of Korean Cattle In band No band Total Grade 3 1 (3.1%) 1 (0.7%) 2 (1.2%) Grade 2 11 (34.4%) 27 (19.9%) 38 (22.6%) Grade 1 7 (21.9%) 37 (27.2%) 44 (26.2%) 1+ grade 13 (40.6%) 71 (52.2%) 84 (50.0%) 1st grade (1st grade) + (1+ grade) 20 (62.5%) 108 (79.4%) 128 (76.2%) total individuals (%) 32 (100%) 136 (199%) 168 (100%)

As a result of testing using the primers MS139L and MS139R of the present invention, the results of the group having the band showed 62.5% of the total grade 1 appearance, and the grade 1 appearance rate of the group without the marker showed 79.4%, and the overall result was 17% or more. We believe that a new primer has been developed to increase muscle fat. Using the designed primer of the present invention, it is possible to check the result simply and quickly as PCR rather than the experimental method of AFLP which can see the result through a complicated step, and can be used as a very useful method for predicting intramuscular fat in advance. .

As is apparent from the above examples, the present invention is to analyze the nucleotide sequence of DNA markers that are differentiated in the high and low myopia scores by reacting with the AFLP Eco R9 / Taq I2 primer prepared by the genomic DNA of Hanwoo From there, it is effective to develop and provide primers and probes related to the major traits of Korean cattle, especially the intramuscular fat of Korean cattle. In addition, since the use of the primer has excellent effect of providing a test method for the quality of the beef, especially the intramuscular fat, which makes it possible to discriminate more preferably, easily and easily, beef cattle having excellent intramuscular fat and excellent meat quality regardless of the stage of Hanwoo development. It is a very useful invention.

Claims (3)

Specific DNA marker sequences associated with intramuscular fat derived from Hanwoo gene as follows. MS139L or MS 139R of Hanwoo muscle fat determination primer of the following sequence prepared by analyzing the specific DNA marker nucleotide sequence related to myocardial fat derived from the Hanwoo gene of claim 1. delete