MYL2 gene SNP (Single Nucleotide Polymorphism) molecular marker related to porcine skeletal muscle satellite cell differentiation and application of MYL2 gene SNP molecular marker

Abstract

The invention discloses an MYL2 gene SNP molecular marker related to porcine skeletal muscle satellite cell differentiation and application of the MYL2 gene SNP molecular marker, relates to the technical field of molecular genetic markers, and screens out an MYL2 gene SNP marker related to porcine skeletal muscle satellite cell differentiation. MYL2 is selected as a candidate gene, a molecular marker related to pig skeletal muscle satellite cell differentiation is screened by searching genetic variation of a 3'untranslated region of the pig MYL2 gene, and the molecular marker is used for marker-assisted selection and application of pig skeletal muscle satellite cell differentiation. Experiments find that the expression of MYL2 is remarkably increased by C-to-T mutation of the * 57 site of the porcine MYL2 gene promoter region in an in-vitro test, and the difference between the C-to-T mutation and the MYL2 expression is remarkable, so that the method for detecting the molecular marker associated with mutation is simple, convenient and rapid, is not influenced by the environment, and can realize early-stage seed selection.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetic markers, and in particular to a MYL2 gene SNP marker associated with the differentiation ability of pig skeletal muscle satellite cells and an application thereof. Background Art

[0002] Pig farming is a key pillar of my country's animal husbandry sector and plays a vital role in ensuring the safety of the meat supply. Lean meat percentage and growth rate have long been the goals of pig breeders and producers and are also key determinants of the economic benefits of pig production. Myosin, the primary protein constituting muscle fibers, consists of two heavy chains and multiple light chains. Myosin light chain 2 (MYL2), a key member of the myosin light chain family, is primarily expressed in cardiac and skeletal muscle, playing a crucial role in maintaining normal growth and development of both. Mutations in the MYL2 gene lead to myocardial hyperplasia and hypertrophy, while MYL2 knockout mice exhibit abnormal growth and even death. Other studies have shown that MYL2 can influence muscle growth by regulating myofiber type conversion. Therefore, MYL2 is an important candidate gene for regulating skeletal muscle development in livestock.

[0003] Pork production is determined by the growth and development of skeletal muscle during the early embryonic period and the proliferation and differentiation of skeletal muscle satellite cells after birth. Satellite cells are the primary stem cell population in skeletal muscle, responsible for its growth and regeneration. Embryonic growth and development in mammals determine the number and type of skeletal muscle fibers. After birth, the number of skeletal muscle fibers remains constant, primarily through the proliferation, differentiation, and fusion of skeletal muscle satellite cells into the muscle fibers, promoting muscle development and growth. Analyzing the molecular regulatory mechanisms of skeletal muscle satellite cell proliferation and differentiation and identifying molecular markers will help improve pig growth rate and meat production and promote the development of new breeds. Summary of the Invention

[0004] This study identified a MYL2 gene SNP marker associated with porcine skeletal muscle satellite cell differentiation. MYL2 was selected as a candidate gene, and by identifying genetic variations in the 3' untranslated region of the porcine MYL2 gene, a molecular marker associated with porcine skeletal muscle satellite cell differentiation was screened. This molecular marker was then used for marker-assisted selection and application of porcine skeletal muscle satellite cell differentiation.

[0005] The present invention provides a MYL2 gene SNP marker associated with porcine skeletal muscle satellite cell differentiation, wherein the SNP marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by a single nucleotide mutation at the base at position 57 (*57) downstream of the MYL2 gene mRNA stop codon; the SNP marker is a single nucleotide mutation at the *57th base in the nucleotide sequence as shown in SEQ ID NO.1, and the polymorphism of the single nucleotide mutation is a C / T polymorphism.

[0006] The present invention discloses a MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation. The SNP marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by a single nucleotide mutation at the base 57 positions downstream of the MYL2 gene mRNA stop codon; the polymorphism of the single nucleotide mutation is a C / T polymorphism.

[0007] Furthermore, the C / T polymorphism affects the differentiation of porcine skeletal muscle satellite cells.

[0008] Furthermore, the influencing of pig skeletal muscle satellite cell differentiation is achieved by influencing the expression level of myosin light chain 2 gene MYL2.

[0009] Furthermore, the C / T polymorphism is a mutation of C>T; wherein, individuals with the TT genotype have a higher efficiency of skeletal muscle satellite cell differentiation than individuals with the CC genotype.

[0010] The present invention discloses an application of a MYL2 gene SNP molecular marker associated with pig skeletal muscle satellite cell differentiation, and an application of the SNP marker in identifying pig muscle development traits.

[0011] Furthermore, the SNP marker detects the expression of MYL2 gene through the primer of MYL2 gene SNP marker to identify the muscle development traits of Min pigs; the primer is

[0012] Primer MYL2_F1: 5′-TCTGACACATGATAAGGGCAATAT-3′;

[0013] Primer MYL2_R1: 5′-ACTTGACCTCCTGTTTATTTGAACAT-3′.

[0014] The present invention discloses an application of a MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation, and an application of the SNP marker in breeding / assisted breeding of porcine breeds or strains for porcine skeletal muscle satellite cell proliferation.

[0015] Furthermore, the method for selecting / assisting in the selection of pig breeds or strains for the proliferation of pig skeletal muscle satellite cells is:

[0016] For the SNP marker of the MYL2 gene, pigs with a genotype of TT at position 57 downstream of the stop codon in the 3' untranslated region of the SLPI gene are selected as the screened targets, and the screened target pigs are bred to complete the method of selecting / assisted selecting pig breeds or strains for differentiation of pig skeletal muscle satellite cells.

[0017] The present invention provides a primer pair for identifying the MYL2 gene SNP marker associated with pig skeletal muscle satellite cell differentiation, wherein the primer pair is

[0018] Primer MYL2_F1: 5′-TCTGACACATGATAAGGGCAATAT-3′;

[0019] Primer MYL2_R1: 5′-ACTTGACCTCCTGTTTATTTGAACAT-3′.

[0020] The invention relates to a kit comprising the primer pair for identifying MYL2 gene SNP markers related to pig skeletal muscle satellite cell differentiation.

[0021] The present invention has the following beneficial effects:

[0022] Myosin is the primary protein component of muscle fibers, accounting for 60% of the total myofibril protein. It is composed of two heavy chains and multiple light chains. Members of the myosin light chain family are primarily expressed in cardiac and skeletal muscle. Myosin light chain 2 (MYL2) is a key member of this family, playing an important role in myofibril development and muscle growth.

[0023] The present invention conducted a genetic polymorphism analysis of the 3' untranslated region of the porcine MYL2 gene. Direct sequencing of PCR products revealed a C-to-T mutation at position 57 downstream of the MYL2 mRNA stop codon (*57). This mutation is present at a high frequency in Min pigs. Among 115 Min pigs, the C and T allele frequencies were 0.87 and 0.13, respectively. In contrast, no T allele was found in the 20 Large White pigs tested. The present invention further analyzed the genetic effects of the C>T mutation at *57 using a luciferase reporter gene assay. The C-to-T mutation altered the activity of the SLPI promoter in vitro. Mutating the MYL2 gene at position *57 (C to T) significantly increased luciferase reporter gene activity, suggesting that this point mutation promotes MYL2 gene transcriptional expression.

[0024] By overexpressing the MYL2 gene in primary cultured porcine skeletal muscle satellite cells, the present invention confirms that MYL2 promotes the differentiation of porcine skeletal muscle satellite cells. This indicates that detecting molecular markers associated with mutations is not only simple and rapid, but also unaffected by environmental factors and allows for early selection. Therefore, using molecular markers to select a T-gene-forming base population for breeding has important implications for improving muscle development in pigs, particularly Min pigs.

[0025] Through experiments, the present invention found that the mutation from C to T at the *57 site in the promoter region of the porcine MYL2 gene significantly increased the expression of MYL2 in in vitro experiments. The difference between the two was significant. It can be seen that the detection of molecular markers associated with mutations is not only simple and rapid, but also unaffected by the environment and can achieve early breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the electrophoresis diagram of the 3' untranslated region of the porcine MYL2 gene amplified by PCR; in the figure, lane 1 is the DL2000 marker and lane 2 is the target band;

[0027] Figure 2 The sequencing results of the SNP sites in the 3' untranslated region of the porcine MYL2 gene; Figure A shows the three genotypes at the *19 site, and Figure B shows the three genes at the *57 site;

[0028] Figure 3 Figure 2 shows the sequencing results of reporter gene vectors psi-MYL2-WT, psi-MYL2-*19G, and psi-MYL2-*57T;

[0029] Figure 4 The expression changes of reporter genes detected by luciferase reporter system before and after mutation of *19 and *57 sites; **, p<0.01; ***, p<0.001;

[0030] Figure 5 This is a diagram showing the effect of overexpression of MYL2 gene in pig skeletal muscle satellite cells detected by western blotting method;

[0031] Figure 6 This figure shows the effect of MYL2 on the differentiation of porcine skeletal muscle satellite cells; ** indicates extremely significant difference, p < 0.01; * indicates significant difference, p < 0.05; scale bar is 50 μm. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the contents of the present invention based on the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0033] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0034] Example 1

[0035] Identification of polymorphic sites and establishment of genotyping technology

[0036] 1. Genomic DNA extraction: Take pig ear tissue samples and extract genomic DNA using the conventional phenol-chloroform method.

[0037] 2. Primer design: Based on the porcine MYL2 sequence (GeneID: 396690) provided by GenBank, primers MYL2_F1 and MYL2_R1 were designed using PrimerPremier 5.0 to amplify the 3' end sequence of the porcine MYL2 gene. The primer sequences are as follows:

[0038]

[0039] 3. Identification of polymorphic sites: Ten Min pigs were selected and their mixed genomic DNA was used as template for PCR amplification using primers MYL2_F1 / R1. The PCR amplification system and reaction conditions were as follows:

[0040] The amplification system was as follows: 5 μL of genomic DNA template, 2 μL of 10 μM upstream and downstream primers, 25 μL of 2× RapidMaster Mix enzyme, and ddH2O was added to make up to 50 μL.

[0041] The amplification program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s; final extension at 72°C for 5 min; and storage at 4°C.

[0042] After the amplified product was detected by agarose gel electrophoresis, it was sent to Heilongjiang Jiansu Gene Technology Co., Ltd. for sequencing. The sequencing results were compared with the known sequence (NC_010459.5) using Snap Gene software, and the sequencing peak graph was manually checked using chromas software to identify the polymorphic sites. Two SNPs were detected in this region, located in the 3' untranslated region of the MYL2 mRNA sequence (transcript_id: NM_213791.2), named SNP*19A>G and SNP*57C>T sites (* represents the stop codon, and the present invention identifies the position of the first base after the stop codon as 1). The electrophoresis detection results are shown in Figure 1 .

[0043] The mRNA sequence of the MYL2 gene containing the SNP molecular marker of the present invention is as follows:

[0044]

[0045]

[0046] 4. Genotype and gene frequency analysis

[0047] The MYL2 gene 3' end sequence was amplified using primers MYL2_F1 / R1, respectively. The base composition of the SNP*19A>G and SNP*57C>T sites was determined by direct sequencing of the PCR products. Homozygotes and heterozygotes were identified by visual inspection of chromatin peaks, and the gene and genotype frequencies were calculated. As shown in Tables 1 and 2, SNP*19A>G and SNP*57C>T were present at high frequencies in Min pigs but not in Large White pigs. Among 115 Min pigs, 47 pigs had the AA genotype, 59 had the AG genotype, and 9 had the GG genotype. The genotype frequencies of the SNP*19A>G were 0.41, 0.51, and 0.08, respectively. The A and G allele frequencies were 0.67 and 0.33, respectively. Among 115 Min pigs, 88 pigs had CC genotype, 24 pigs had CT genotype, and 3 pigs had TT genotype. The genotype frequencies were 0.76, 0.21, and 0.03, respectively. The C and T allele frequencies were 0.87 and 0.13, respectively. Figure 2 .

[0048] Table 1 Genotype frequency and gene frequency of pig MYL2 gene SNP*19A>G in the population

[0049]

[0050] Note: The numbers in brackets are individuals.

[0051] Table 2 Genotype frequency and gene frequency of pig MYL2 gene SNP*57C>T in the population

[0052]

[0053]

[0054] Note: The numbers in brackets are individuals.

[0055] Example 2

[0056] Construct the wild-type luciferase reporter gene vector of MYL2 gene.

[0057] 1. Genome extraction: see Example 1.

[0058] 2. Primer Design: Based on the porcine MYL2 gene mRNA sequence provided by GenBank (transcript_id: NM_213791.2), primers SLPI_F2 and SLPI_R2 were designed using Primer Premier 5.0. Restriction endonuclease recognition sites were introduced at the 5' end to amplify the porcine MYL2 gene 3' untranslated region (3' UTR). The fragment was inserted into the psiCHECK2 vector to construct a MYL2 gene 3' UTR reporter gene vector. The specific method is as follows:

[0059]

[0060] The underlines indicate the recognition sites of restriction endonucleases Xho I and Not I, respectively.

[0061] 3. PCR amplification: see Example 1.

[0062] 4. PCR Product Detection and Gel Recovery and Purification of Target Fragments: After the PCR reaction is complete, examine the PCR amplification products on a 1.2% agarose gel using electrophoresis at 100V for 30 minutes. After electrophoresis, visualize the PCR amplification results using a gel imaging system. If a single, bright band of the target fragment of the correct length is observed, excise the target fragment from the gel. Perform gel recovery according to the BioTeke Corporation gel recovery kit instructions. After gel recovery, store the recovered products at -20°C.

[0063] 5. Enzyme digestion and ligation: Use restriction endonucleases Xho I and Not I to double-digest the psiCHECK2 empty vector and PCR product, respectively.

[0064] Enzyme digestion system: Xho I, 1 μL; Not I, 1 μL; 10× Buffer, 1.0 μL; DNA, 1 μg; ddH2O to 20 μL; reaction conditions: 37°C water bath, 1 hour.

[0065] After enzyme digestion, the fragment was identified by 1.2% agarose gel electrophoresis. The target fragment was purified using the agarose gel purification kit from BioTeke Corporation according to the instructions. The purified empty vector and the PCR product were ligated using T4 DNA ligase from TaKaRa. The ligation system and reaction conditions were strictly in accordance with the instructions.

[0066] 6. Recombinant plasmid transformation: According to the instructions of Qingke Biotechnology Co., Ltd. DH5α, the specific steps are as follows:

[0067] (1) Take out the competent cells from the -80℃ refrigerator and thaw them in an ice bath for 5 minutes.

[0068] (2) Pipette 30 μL of competent cells and add them to 10 μL of ligation product, mix gently, and let it stand on ice for 30 minutes.

[0069] (3) Heat shock in a 42°C water bath for 1 min 30 s, then quickly transfer to an ice bath and let stand for 1-2 min.

[0070] (4) Add 400 μL of resistance-free LB culture medium to the centrifuge tube and resuscitate at 37°C / 200 rpm for 2 h.

[0071] (5) Take an appropriate volume of resuscitation solution and evenly spread it on the culture medium containing the corresponding antibiotics, and culture it upside down in a 37°C incubator overnight.

[0072] 7. Screening and identification of positive clones:

[0073] (1) In a clean bench, use a sterilized pipette tip to pick up a single colony from a glass dish. Place the pipette tip into a 1.5 mL EP tube (1 mL of LB liquid medium supplemented with ampicillin is added to the tube). Pick 5-12 colonies from each dish.

[0074] (2) Place the EP tube in a 37°C constant temperature air bath shaker at 200 rpm for 2 h.

[0075] (3) PCR amplification was performed using the bacterial suspension as a template. The amplification system was as follows: 5 μL of 2× RapidMaster Mix, 0.4 μL of upstream and downstream primers, 1 μL of bacterial suspension DNA template, and dd H2O for saturation. PCR amplification products were detected by electrophoresis on a 1.2% agarose gel.

[0076] (4) A single plasmid of the correct length was selected and, after identification by enzyme digestion, the plasmid was sent to Arrow Gene Co., Ltd. for sequencing. The wild-type pGL3-MYL2 vector (with A and C at sites *19 and *57, respectively) was obtained and named psi-MYL2-WT.

[0077] Example 3

[0078] Construct a MYL2 gene mutant luciferase reporter gene vector.

[0079] 1. Using psi-MYL2-WT constructed in Example 2 as a template, site-directed mutagenesis was performed on the two SNP sites by overlap extension PCR to construct vectors in which the nucleotides at these sites were G and T, respectively. The primer sequences were:

[0080]

[0081] Note: The underline indicates the mutated base.

[0082] 2. Site-directed mutagenesis was performed using overlap extension PCR, consisting of two rounds of reactions. The first round of reactions at the SNP*19A>G site used primers MYL2_F2 / MYL2_R3 and MYL2_F3 / MYL2_R2, using the psi-MYL2-WT vector as template. This yielded overlapping fragments containing the point mutation at both ends. The second round of PCR amplification used appropriate dilutions of the PCR products from these two reactions as templates and primers MYL2_F2 / MYL2_R2 to introduce the point mutation internally into the PCR product.

[0083] The mutation method for the SNP*57C>T site was the same as above. The first-round reaction used primers MYL2_F2 / MYL2_R4 and MYL2_F4 / MYL2_R2, and the template was the psi-MYL2-WT vector. The amplified product from the first round was appropriately diluted and used as a template for overlap extension PCR using primers MYL2_F2 / MYL2_R2 to obtain a fragment containing the point mutation.

[0084] The first-round PCR amplification system consisted of 100 ng of psi-MYL2-WT vector DNA, 25 μL of 2× PrimeSTARMax Premix, 20 μM of each upstream and downstream primers, and ddH2O to a final volume of 50 μL. The PCR amplification program was as follows: denaturation at 98°C for 10 s, annealing at 62°C for 15 s, and extension at 72°C for 30 s, for 35 cycles.

[0085] The second-round PCR amplification system consisted of 1 μL of each of the two PCR product dilutions from the first-round reaction, 25 μL of 2× PrimeSTARMax Premix, 10 μM of each upstream and downstream primers, and ddH2O to a final volume of 50 μL. The PCR amplification program was as follows: denaturation at 98°C for 10 seconds, annealing at 62°C for 15 seconds, and extension at 72°C for 1 minute, for 35 cycles.

[0086] 3. After the second round of PCR products were recovered and purified by agarose gel electrophoresis, they were ligated to the psiCHECK2 vector. The constructed vector plasmid was double digested with HindIII and NcoI restriction enzymes for identification and sent to Arrow Gene Co., Ltd. for sequencing to confirm the point mutations. The mutant vectors psi-MYL2-*19G and psi-MYL2-*57T were obtained. The sequencing results of the mutant vectors are shown in Figure 3 .

[0087] 4. Inoculate the correctly sequenced bacterial suspension containing the luciferase reporter gene at a ratio of 1:500 into LB liquid medium containing 50 μg / mL ampicillin and shake vigorously at 200 rpm for 16 hours. Harvest the cells by centrifugation at 6000 rpm at 4°C. Extract the plasmid using the Tiangen endotoxin-free plasmid extraction kit, strictly following the manufacturer's instructions. Measure the plasmid concentration using a UV spectrophotometer.

[0088] Example 4

[0089] Dual-luciferase reporter gene assay.

[0090] 1. Cell culture and transfection: PK-15 / HEK293T cells were seeded on 24-well cell culture plates and cultured with complete medium (DMEM containing 10% fetal bovine serum and 1% double-streptomycin) purchased from SEVEN. The cells were cultured in a 5% CO2 incubator at 37°C. Transient transfection was performed when the confluence of the monolayer cells reached 70-90% under a microscope. The transfection procedure was performed according to the instructions of the Lipofectamine 8000 transfection kit from Beyotime.

[0091] (1) 1.5 μg of recombinant plasmids psi-MYL2-WT, psi-MYL2-*19G, psi-MYL2-*57T and empty plasmid psiCHECK2 were added to 75 μL DMEM culture medium, and blank controls were set up. Three replicates were set up for each group, and cells transfected with pRL-TK plasmid were used as internal controls.

[0092] (2) Add 4.5 μL of liposomes to 75 μL of DMEM medium, gently mix the transfection reagent / DNA complex, and let it stand at room temperature for 15 minutes.

[0093] (3) Take the cell culture dish out of the 37°C CO2 incubator, wash the dish twice with PBS, add 50 μL of premixed transfection reagent / DNA complex and 200 μL of DMEM medium to each well.

[0094] (4) The cell culture dish was placed in a 5% 37°C carbon dioxide incubator and taken out after 6 hours. Each well was supplemented with 250 μL of DMEM medium. The cells were collected 24 hours after transfection to detect dual luciferase activity.

[0095] 2. Luciferase activity assay: Dual luciferase activity assay was performed using the Dual Luciferase Reporter Gene Assay Kit from Beyotime. The steps are as follows:

[0096] (1) Cell lysis: Remove the cell culture plate, discard the culture medium, wash twice with cold PBS, add 100 μL of 1× Cell Lysis Buffer to each well of the 24-well plate, let it stand at room temperature for 5 min, and then gently shake it for 15 min. The cell lysate was pipetted and transferred to a 1.5 mL centrifuge tube, and centrifuged at 12,000 rpm in a room temperature centrifuge for 5 min. The supernatant was used for subsequent experiments.

[0097] (2) Firefly luciferase reaction test: Add 15 μL of Luciferase Substrate that has been equilibrated to room temperature to a 1.5 mL EP tube, then carefully pipette 10 μL of cell lysate supernatant into the tube, quickly mix well, and immediately detect the Firefly luciferase reporter gene activity in a luminescence detector.

[0098] (3) Renilla luciferase reaction test: Add 15 μL of Renilla substrate working solution to the mixture in the previous step, mix quickly and evenly, and immediately detect the activity of the Renilla luciferase reporter gene.

[0099] (4) Calculate the luciferase activity of Firefly relative to that of Renilla.

[0100] The results of dual luciferase assay were as follows Figure 4 As shown, compared with psi-MYL2-WT, the activity of the psi-MYL2-*19G reporter gene did not change (p>0.05), while the activity of the psi-MYL2-*57T reporter gene was significantly increased (p<0.05), indicating that the SNP*57C>T mutation positively regulates the expression of the MYL2 gene.

[0101] Example 5

[0102] Constructing an overexpression vector of the MYL2 gene and analyzing the effect of MYL2 on the differentiation of porcine skeletal muscle satellite cells by overexpression

[0103] 1. Skeletal muscle satellite cells: Isolated and preserved in the laboratory. Cell recovery, culture, and differentiation induction were performed as follows:

[0104] (1) Cell recovery

[0105] The frozen cells were removed from liquid nitrogen and quickly placed in 37°C water. The cells were shaken to promote thawing to obtain a cell suspension. The suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the suspension was resuspended in complete medium, and the cells were transferred to a 6 cm culture dish. The suspension was cultured in a 37°C, 5% CO2 cell culture incubator.

[0106] (2) Subculture

[0107] On the second day of culture, observe the cell density and contamination. If there is no contamination, continue culture with complete medium and subculture when the cell density reaches 90%.

[0108] (3) Induced differentiation

[0109] When the cell density increases to 90%, the culture medium is replaced with 2% horse serum differentiation medium. The differentiation medium is replaced every two days.

[0110] 2. Overexpression vector construction

[0111] (1) Primer design: Based on the MYL2 gene mRNA sequence (transcript_id: NM_213791.2), primers were designed using Primer premier 5.0 software to clone the full-length coding region and introduce restriction endonuclease recognition sites. The overexpression vector was constructed using pCMV-HA as the vector backbone. The primer sequences are as follows:

[0112]

[0113] Note: The underlined lines are EcoRI and XhoI restriction sites respectively.

[0114] (2) RT-PCR amplification of the full-length coding region of the MYL2 gene: The coding region was amplified using reverse transcription (RT)-PCR. Total RNA from pig dorsal muscle tissue was extracted using Trizol (TaKaRa), and reverse transcription was performed using the HiScript III 1st strand cDNA synthesis kit from Vazyme to synthesize cDNA.

[0115] The PCR amplification system was as follows: 5 μL of cDNA template, 2 μL each of 10 μM upstream and downstream primers (MYL2-CDS-F / R), 25 μL of 2× RapidMaster Mix enzyme, and ddH2O was added to make up to 50 μL.

[0116] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 10 s; final extension at 72°C for 5 min; and storage at 4°C.

[0117] (4) PCR product purification: Agarose gel purification kit from BioTeke Corporation was used according to the instructions.

[0118] (5) Overexpression vector construction: The full-length coding region of the MYL2 gene and the pCMV-HA vector were double-digested with EcoRI and XhoI, respectively, and the target fragments were recovered and ligated using TaKaRa's T4 DNA ligase. The enzyme digestion system, ligation system, and reaction conditions were strictly in accordance with the instructions.

[0119] (6) Overexpression effect detection: The constructed recombinant plasmid was transfected into porcine skeletal muscle satellite cells. The transfection method is shown in Example 4. Western blotting was used to confirm that the overexpression vector was successfully constructed. The main methods of western blotting are as follows:

[0120] 48 hours after transfection, cells were collected and the sample concentration was detected using the BCA protein concentration assay kit from Yazyme. 20 μg of total protein was loaded onto a polyacrylamide gel (5% stacking gel, 10% separation gel) for electrophoresis. The bands were transferred to a PVDF membrane, blocked with 5% skim milk, and incubated overnight at 4°C with an anti-HA tag primary antibody (anti-HA). A fluorescent secondary antibody (goat anti-rabbit IgG antibody) was used for incubation and visualization. Results are shown in Figure 5 .

[0121] (7) Immunofluorescence detection

[0122] Skeletal muscle satellite cells were transfected with a MYL2 overexpression vector and induced to differentiate. After 5 days of differentiation, the cells were collected and immunofluorescence staining was performed. The main methods are as follows:

[0123] The cells were fixed in 4% paraformaldehyde for 30 minutes (room temperature); after washing, 0.5% Triton X-100 was added to permeabilize the membrane for 30 minutes; after washing again, 5% BSA (bovine serum albumin in PBS) was added to block the cells at room temperature for 30 minutes to 1 hour; anti-MYHC antibody (1:250 dilution) was added and incubated overnight at 4°C; fluorescent secondary antibody (goat anti-rabbit IgG antibody; 1:500 dilution) was added and incubated in the dark at room temperature for 40 minutes, followed by the addition of DAPI and an additional 15 minutes of incubation; the cells were observed and counted under an inverted microscope.

[0124] (8) Calculation of cell fusion index

[0125] The cell fusion index was calculated as follows: the number of 2 or more nuclei in the myotube / the total number of nuclei × 100%.

[0126] Compared with the control group, overexpression of MYL2 significantly increased the cell fusion index, indicating that MYL2 can promote the differentiation of skeletal muscle satellite cells. Figure 6 .

Claims

1. A MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation, characterized in that: The SNP marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by a single nucleotide mutation at the base 57 positions downstream of the MYL2 gene mRNA stop codon; the polymorphism of the single nucleotide mutation is expressed as a C / T polymorphism.

2. The MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation according to claim 1, characterized in that: The C / T polymorphism affects the differentiation of porcine skeletal muscle satellite cells.

3. The MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation according to claim 2, characterized in that: The described influence on the differentiation of pig skeletal muscle satellite cells is achieved by influencing the expression level of the myosin light chain 2 gene MYL2.

4. The MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation according to claim 1, characterized in that: The C / T polymorphism is a mutation of C>T; wherein, individuals with the TT genotype have a higher efficiency of skeletal muscle satellite cell differentiation than individuals with the CC genotype.

5. The use of a MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation according to claim 1, characterized in that: The application of the SNP marker in identifying muscle development traits of Min pigs.

6. The use according to claim 5, characterized in that The SNP marker detects the expression of MYL2 gene through the primer of MYL2 gene SNP marker, thereby identifying the muscle development traits of Min pigs; the primer is Primer MYL2_F1: 5′-TCTGACACATGATAAGGGCAATAT-3′; Primer MYL2_R1: 5′-ACTTGACCTCCTGTTTATTTGAACAT-3′.

7. The use of a MYL2 gene SNP molecular marker associated with porcine skeletal muscle satellite cell differentiation according to claim 1, characterized in that: The SNP marker is used in the selection / assisted selection of pig breeds or lines for the proliferation of pig skeletal muscle satellite cells.

8. The use according to claim 1, characterized in that The method for selecting / assisting in the selection of pig breeds or strains for the proliferation of pig skeletal muscle satellite cells is as follows: For the SNP marker of the MYL2 gene, pigs with a genotype of TT at position 57 downstream of the stop codon in the 3' untranslated region of the SLPI gene are selected as the screened targets, and the screened target pigs are bred to complete the method of selecting / assisted selecting pig breeds or strains for differentiation of pig skeletal muscle satellite cells.

9. A primer pair for identifying the MYL2 gene SNP marker associated with porcine skeletal muscle satellite cell differentiation, characterized in that The primer pair is Primer MYL2_F1: 5′-TCTGACACATGATAAGGGCAATAT-3′; Primer MYL2_R1: 5′-ACTTGACCTCCTGTTTATTTGAACAT-3′.

10. A kit comprising the primer pair according to claim 9 for identifying SNP markers of the MYL2 gene associated with porcine skeletal muscle satellite cell differentiation.

Images