A cav1 gene molecular marker related to proliferation of porcine skeletal muscle satellite cells and application thereof

Abstract

The application discloses a CAV1 gene molecular marker related to proliferation of pig skeletal muscle satellite cells and application thereof, relates to the technical field of molecular genetic markers, and the molecular marker is a nucleotide sequence shown as SEQ ID NO. 1 formed by single nucleotide mutation at the +215th base of a 3'UTR region of a CAV1 gene; the single nucleotide mutation polymorphism is T / C polymorphism. The application is used for breeding or assisting in breeding pig varieties or strains related to the proliferation of pig skeletal muscle satellite cells. The application identifies the proliferation of pig skeletal muscle satellite cells by judging the polymorphism of a mutation site of a 3'UTR of a pig CAV1 gene, and artificially applies the mutation site to provide an effective method for early breeding and provides a new idea for muscle growth and development trait breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetic markers, and in particular to a CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation and an application thereof. Background Art

[0002] China boasts a rich resource of local pig breeds, and specialized breeds bred from these pigs can fully meet diverse market demands. my country is also a major producer and consumer of pork, and pork is the primary source of protein for Chinese residents, playing a vital role in their daily lives and economic production. The Minzhu, a local pig breed unique to Northeast my country, boasts cold tolerance, excellent meat quality, and high fertility. However, some carcass traits, such as lean meat percentage, loin eye area, and shearing strength, are significantly lower than those of imported breeds such as the Large White.

[0003] Skeletal muscle growth and development is a complex process involving the proliferation of muscle precursor cells, myoblast differentiation, and the formation of multinucleated myotubes. Cell-cell, cell-tissue, and cell-extracellular matrix interactions are essential for these developmental events, involving extensive cell adhesion and migration. Skeletal muscle satellite cells, a type of myogenic stem cell, play a crucial role in postnatal muscle development and injury repair in animals and are closely associated with carcass traits, meat yield, and meat quality in meat-producing livestock.

[0004] Caveolin (Secretory Leukocyte Peptidase Inhibitor, CAV1) is a 22-kDa membrane protein that is essential for the formation of small invaginations (caveolae) in the plasma membrane. The CAV1 gene is primarily expressed in endothelial cells, smooth muscle cells, and fibroblasts, where it exerts its regulatory functions by regulating membrane trafficking, cholesterol metabolism, and cell signaling.

[0005] Recent studies have found that CAV1 plays an important role in life activities. CAV1 plays an important role in the proliferation and differentiation of muscle precursor cells; CAV1 can induce the EMT process through the Wnt / β-catenin pathway and promote the metastasis of liver cancer cells; CAV1 can positively regulate the TGF-β pathway to promote the myogenic differentiation of C2C12 cells; CAV1 in mouse embryonic fibroblasts can regulate TGF-β signaling by mediating TGF-β receptor renewal; knocking down CAV1 in NIT-1 cells isolated from mice, pro-apoptotic cytokines are downregulated in the NIT-1 cell line; CAV1 can inhibit tumor growth and metastasis in a mouse model of cutaneous squamous cell carcinoma by regulating the activation of MAPK / AP-1 signaling.

[0006] Zeng Guowei et al., in their 2015 paper "Research Progress on Caveolins in the Skeletal Muscle System," published in the Journal of PLA Medical College, noted that although caveolins and caveolins have been studied for over 50 years, research into their roles in the skeletal and muscular systems is still in its infancy. Numerous studies have confirmed that caveolin-1 and caveolin-2 are co-expressed in fibroblasts, smooth muscle cells, and osteoblasts. Caveolin-3 is specifically expressed in cardiomyocytes, smooth muscle cells, and skeletal muscle cells. Satellite cells are quiescent myocyte precursors. Upon injury, their mitosis is activated, and satellite cells proliferate, fuse, or form new myofibers, initiating a key step in muscle regeneration. Volonte et al. demonstrated that downregulation of caveolin-1 is indeed a key signal for satellite cell activation in muscle regeneration. While caveolin-1 is undoubtedly involved in osteogenesis, bone maturation, and bone mass regulation, its specific role and underlying mechanisms remain unclear. On the one hand, caveolin-1 acts as an inhibitor of osteoblast differentiation, inhibiting bone maturation and mineralization. On the other hand, caveolin-1 attenuates osteoclast-mediated osteolysis by interacting with the extracellular matrix of osteoblasts and calcium-sensing receptors. The physiological significance and regulatory mechanisms of caveolins' effects on bone tissue require further clarification.

[0007] Although the article provides a link between caveolin CAV1 and skeletal muscle satellite cell proliferation, it does not address how to use caveolin CAV1 for pig skeletal muscle satellite cell proliferation-related breeding. Therefore, a method is needed to screen for SNPs related to pig skeletal muscle satellite cell proliferation for pig breeding. Summary of the Invention

[0008] The present invention screened for a single nucleotide polymorphism (SNP) associated with porcine skeletal muscle satellite cell proliferation. CAV1 was selected as a candidate gene. By identifying genetic variations in the 3'UTR region of the porcine CAV1 gene, a molecular marker associated with porcine skeletal muscle satellite cell proliferation was screened. This molecular marker was then used for marker-assisted selection and application of porcine skeletal muscle satellite cell proliferation.

[0009] The present invention discloses a CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation. The molecular marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by a single nucleotide mutation at the +215th base in the 3'UTR region of the CAV1 gene; the polymorphism of the single nucleotide mutation is T / C polymorphism.

[0010] Furthermore, the T / C polymorphism affects pig muscle development.

[0011] Furthermore, the T / C polymorphism is a mutation from C to T; wherein, the CAV1 gene expression level of individuals with TT genotype is significantly higher than that of individuals with CC genotype.

[0012] The present invention discloses an application of a CAV1 gene molecular marker related to the proliferation of porcine skeletal muscle satellite cells. The molecular marker is used for breeding / assisted breeding of pig breeds or strains related to the proliferation of porcine skeletal muscle satellite cells.

[0013] Furthermore, the breeding / assisted breeding is to select pigs with CC genotype at position +215 in the 3'UTR region of the CAV1 gene as target pigs for breeding.

[0014] Furthermore, the pig is a Min pig or a Large White pig.

[0015] Furthermore, the breeding / assisted breeding is to select pigs with CC genotype at position +215 in the 3'UTR region of the CAV1 gene as target pigs for breeding. The specific operation is as follows:

[0016] 1. Obtain genomic DNA from the pig to be tested;

[0017] Second, using the above-mentioned porcine genomic DNA as a template, PCR amplification was performed using the following primer pairs to obtain a gene fragment of the 3'UTR region of the CAV1 gene containing the SNP site;

[0018] Primer CAV1_F1: 5′-GTGACATTTCAAGGGTATAAGTAT-3′;

[0019] Primer CAV1_R1: 5′-AACTGTTAAACATTTTTATTGTGC-3′;

[0020] 3. Detecting the amplified product by agarose gel electrophoresis;

[0021] 4. Sequencing and analyzing the PCR products;

[0022] Fifth, pigs with CC genotype at position +215 of the 3'UTR region of the CAV1 gene were selected as the screening targets;

[0023] 6. Breed the selected target pigs and the process is complete.

[0024] The primer pair of the present invention for detecting the CAV1 gene molecular marker associated with the proliferation of porcine skeletal muscle satellite cells is as follows:

[0025] Primer CAV1_F1: 5′-GTGACATTTCAAGGGTATAAGTAT-3′;

[0026] Primer CAV1_R1: 5′-AACTGTTAAACATTTTTATTGTGC-3′.

[0027] A kit comprising the primers described above.

[0028] The present invention uses high-throughput sequencing technology to analyze the genetic basis of pig muscle development and finds that the expression level of the caveolin-1 (CAV1) gene in the longissimus dorsi muscle at different developmental stages is significantly different, suggesting that CAV1 plays an important role in muscle development.

[0029] The present invention further analyzed the genetic polymorphism of the CAV1 gene. Direct sequencing of PCR products revealed a C-to-T mutation at the +215 site of the 3'UTR of the porcine CAV1 gene. This mutation was found at a high frequency in Min pigs. In 33 Min pigs, the C and T allele frequencies were 0.53 and 0.47, respectively, while in 17 Large White pigs, the C and T allele frequencies were 0.76 and 0.24, respectively. The present invention further analyzed the genetic effects of the C>T polymorphism at the +215 site using a luciferase reporter gene assay. It was found that the C-to-T mutation at the +215 site altered the activity of the CAV1 3'UTR in vitro. After the CAV1 gene +215 site was mutated (C to T), the activity of the luciferase reporter gene increased significantly, suggesting that this point mutation has the function of promoting the transcriptional expression of the CAV1 gene.

[0030] Individuals with the TT genotype at the +215 position of the CAV1 gene showed significantly higher CAV1 gene expression than those with the CC genotype. CCK8 and EdU staining confirmed that CAV1 inhibited cell proliferation. 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 breeding population that establishes a C gene at the +215 position of the C gene has important implications for improving muscle development in pigs, particularly Min pigs.

[0031] The present invention experimentally found that the mutation from C to T at position +215 in the 3'UTR region of the porcine CAV1 gene significantly increased the activity of the CAV1 3'UTR in in vitro tests. 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

[0032] Figure 1 For Pigs CAV1 Sequencing results of SNP sites in the 3'UTR region of the gene; Figure A shows a TT type individual, and Figure B shows a CC type individual;

[0033] Figure 2 The results of enzyme digestion identification of reporter gene vectors psicheck2-CAV1-T and psicheck2-CAV1-C are shown in Figure 1. Lanes 1 and 3 are DL5000 markers, and lane 2 represents single enzyme digestion of psicheck2-CAV1-C plasmid ( Xho Ⅰ) Identification results, lane 4 shows the single enzyme digestion of psicheck2-CAV1-T plasmid ( Xho Ⅰ) Identification results;

[0034] Figure 3 Figure 3 is a graph showing the changes in reporter gene expression detected by the luciferase reporter system before and after the +215 site mutation; A is the result of PK15 cells; B is the result of 293T cells, ** indicates extremely significant differences. p <0.01, * indicates significant difference, p <0.05;

[0035] Figure 4 This is a graph showing the effect of overexpression of the CAV1 gene in pig skeletal muscle satellite cells detected by real-time PCR. ** indicates extremely significant differences. p <0.01;

[0036] Figure 5 This is a diagram of CCK8 analysis of the effect of CAV1 on the proliferation of porcine skeletal muscle satellite cells. ** indicates extremely significant differences. p <0.01;

[0037] Figure 6 This is the EdU analysis of the effect of CAV1 on the proliferation of pig skeletal muscle satellite cells. ** indicates a very significant difference. p <0.01;

[0038] Figure 7 This is a flow cytometry analysis of the effect of CAV1 on the proliferation of pig skeletal muscle cells. ** indicates a very significant difference. p <0.01;

[0039] Figure 8 This is a graph showing the effect of CAV1 on the expression of porcine skeletal muscle satellite cell proliferation marker genes detected by Real Time PCR. ** indicates extremely significant differences. p <0.01;

[0040] Figure 9 This is a graph showing the effect of CAV1 on the expression of porcine skeletal muscle satellite cell proliferation marker genes detected by Western blotting. ** indicates extremely significant differences. p <0.01. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] Example 1

[0044] Identification of polymorphic sites and establishment of genotyping technology

[0045] 1. Genomic DNA extraction: Genomic DNA was extracted from the back muscles of Min pigs using the conventional phenol-chloroform method.

[0046] 2. Primer design: Primer Premier 5.0 was used to design primers CAV1_F and CAV1_R to amplify the 3'UTR region of the porcine CAV1 gene. The primer sequences are as follows:

[0047]

[0048] 3. Identification of Polymorphic Sites: Genomic DNA from 10 pigs was pooled and used as a template for PCR amplification using primers CAV1_F / R. The PCR amplification products were sequenced. Sequencing results were compared with the known sequence (NM_001348935.1) using SnapGene software. Sequencing peaks were manually verified using Chroma software to identify polymorphic sites. One single nucleotide polymorphism (SNP) (+215C>T) was detected in this region.

[0049] 4. Genotype and gene frequency analysis

[0050] The CAV1 3'UTR sequences of 33 Min pigs and 23 Large White pigs were amplified using CAV1_F / R primers. The base composition of the SNP +215C>T site was detected by direct sequencing of PCR products. The homozygotes and heterozygotes were identified by visual inspection of chromas sequencing peaks, and the gene frequency and genotype frequency were calculated. As shown in Table 1, among the 33 Min pigs, there were 6 individuals with CC genotype and genotype frequency of 0.18, 4 individuals with TT genotype and genotype frequency of 0.12, 23 individuals with CT genotype and genotype frequency of 0.70, C allele frequency of 0.53, and T allele frequency of 0.47. Among the 17 Large White pigs, there were 9 individuals with CC genotype and genotype frequency of 0.53, 0 individuals with TT genotype and genotype frequency of 0, 8 individuals with CT genotype and genotype frequency of 0.47, C allele frequency of 0.76, and T allele frequency of 0.24. The sequencing peaks of different genotype individuals are shown in the figure. Figure 1 .

[0051] Table 1 Genotype frequency and gene frequency of the pig CAV1 gene SNP +215C>T in the Min pig population

[0052]

[0053] Note: The numbers in brackets are the sample numbers.

[0054] Example 2

[0055] Construction of CAV1 3'UTR reporter gene vector

[0056] 1. Genome extraction: see Example 1.

[0057] 2. Primer Design: Based on the high-throughput sequencing results of porcine longissimus dorsi muscle in our laboratory and the published porcine mRNA sequence (NM_001348935.1), the 3'UTR region of the CAV1 gene was determined. Primers CAV1_F1 and CAV1_R1 were designed using Primer Premier 5.0, and homology arms were introduced at the 5' end. The 1868-byte fragment of the porcine CAV1 3'UTR was amplified using CAV1_F1 / R1 primers. A CAV1 gene 3'UTR reporter gene vector was constructed using psiCHECK2 as the backbone. The specific method is as follows:

[0058]

[0059] Note: The underlined characters represent Xho1 Restriction site.

[0060] 3. PCR amplification: This example uses conventional PCR to amplify the 1868 segment of the 3'UTR region of the Min pig CAV1 gene (the nucleotide sequence of this segment is shown in the sequence listing CAV1 gene (NM_001348935.1).

[0061] 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× Rapid MasterMix enzyme, and ddH2O was added to make up to 50 µL.

[0062] The amplification program was as follows: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 60.2°C for 15 s, and extension at 72°C for 30 s; final extension at 72°C for 5 min; and storage at 4°C.

[0063] The amplified products were detected by agarose gel electrophoresis.

[0064] 4. PCR Product Detection and Gel Recovery and Purification of Target Fragments: After the PCR reaction is complete, examine the PCR products on a 1.0% agarose gel using electrophoresis at 100 V 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.

[0065] 5. Enzyme digestion and ligation: using restriction endonucleases Xho1 The psiCHECK2 empty vector was digested with enzymes.

[0066] Enzyme digestion system: QuickCut TM Xho I, 1 μL; 10× QuickCut Buffer, 5.0 μL; Plasmid, 1 μg; ddH2O to 50 μL; Reaction conditions: 37°C water bath, 1 hour.

[0067] After enzyme digestion, the product was identified by 1.0% agarose gel electrophoresis. The empty vector 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 the homologous recombinase from TaKaRa. The ligation system and reaction conditions were strictly in accordance with the instructions.

[0068] 6. Transformation of recombinant psiCHECK2 plasmid: Follow the instructions for DH5α from Qingke Biotechnology. The specific steps are as follows:

[0069] (1) Take out the competent cells from the −80°C refrigerator and place them on ice to thaw.

[0070] (2) Place the ligation product on ice for 5 minutes. Pipette 30 µL of competent cells and add them to 10 µL of ligation product. Mix gently and place on ice for 30 minutes.

[0071] (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.

[0072] (4) Add 400 μL of resistance-free LB culture medium to the centrifuge tube and resuscitate at 37°C / 200 rpm for 1 h.

[0073] (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℃ incubator overnight.

[0074] 7. Screening and identification of positive clones:

[0075] (1) In a clean bench, use a sterilized pipette tip to pick up a single colony from a glass plate. Place the pipette tip into a 1.5 mL EP tube (1 mL of LB liquid culture medium supplemented with ampicillin is added to the tube). Pick 5-12 colonies from each dish.

[0076] (2) Place the EP tube in a 37°C constant temperature air bath shaker at 200 r / min and incubate for 2 h.

[0077] (3) Perform PCR amplification using the bacterial suspension as a template. The amplification system is as follows: 5 µL of 2× Rapid Master Mix, 0.4 µL of upstream and downstream primers, 1 µL of bacterial suspension DNA template, and dd H2O to make up to 10 µL of the system. PCR amplification products are detected by 1.0% agarose gel electrophoresis.

[0078] (4) Select the PCR amplification positive bacterial solution, extract the plasmid, and after enzyme digestion and identification, send it to Arrow Gene Co., Ltd. for sequencing identification.

[0079] Example 3

[0080] Construction of a reporter gene vector for site-directed mutation of the porcine CAV1 gene 3'UTR SNP +215C>T

[0081] 1. Using the psiCHECK2-CAV1-215C vector constructed in Example 1 as a template, perform site-directed mutagenesis at the +215 site by overlap extension PCR to construct a mutant vector psicheck2-CAV1-215T with a T genotype at this site. The primer sequences are:

[0082]

[0083] Note: Underlined and emphasized bases indicate mutated bases.

[0084] 2. Site-directed mutagenesis was performed using overlap extension PCR, consisting of two rounds of reactions. The first round used the CAV1_F1 / CAV1_R2 and CAV1_F2 / CAV1_R1 primer pairs, respectively, using the psicheck2-CAV1-215C vector as templates to amplify overlapping fragments containing the point mutation at both ends. The second round used appropriate dilutions of the PCR products from these two reactions as templates and primers CAV1_F1 / CAV1_R1 to amplify the point mutation within the PCR product.

[0085] The first-round PCR amplification system consisted of 100 ng of psiCHECK2-CAV1-215C 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 54°C for 15 s, and extension at 72°C for 30 s, for 35 cycles.

[0086] 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 s, annealing at 60.2°C for 15 s, and extension at 72°C for 2 min, for 35 cycles.

[0087] The second round of PCR products were purified by agarose gel electrophoresis and connected to the psiCHECK2 vector. Xho After single enzyme digestion identification with I restriction endonuclease, the gene was sent to Arrow Gene Co., Ltd. for sequencing, which confirmed that the C at position +215 of the CAV1 gene was mutated to T. The enzyme digestion identification results are shown in Figure 2 .

[0088] Example 4 Dual luciferase reporter gene analysis

[0089] 1. Endotoxin-Free Plasmid Extraction: Inoculate a correctly sequenced bacterial cell containing the luciferase reporter gene at a ratio of 1:500 into LB liquid medium containing 50 µg / mL ampicillin. Shake vigorously at 200 rpm for 16 hours. Collect the cells by centrifugation at 12,000 rpm at room temperature. 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.

[0090] 2. Cell culture and transfection: PK-15 and 293T cells were seeded into 24-well cell culture plates and cultured in complete medium (DMEM containing 10% fetal bovine serum and 1% double-antibody (penicillin + streptomycin)). DMEM was purchased from SEVEN and cultured in a 5% CO2 incubator at 37°C. Transient transfection was performed when the monolayer cell confluence reached 50-70% under a microscope. The transfection procedure was described in the instructions for the Lipofectamine 8000 transfection kit from Beyotime.

[0091] (1) 1.5 μg of recombinant plasmids psiCHECK2-CAV1-215C, psiCHECK2-CAV1-215T 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.

[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) Remove the cell culture dish from the 37°C CO2 incubator, wash the dish twice with PBS, add 50 μL of pre-mixed transfection reagent / DNA complex and 200 μL of DMEM culture medium to each well.

[0094] (4) Place the cell culture dish in a 5% 37°C carbon dioxide incubator, take it out after 6 hours, add 250 μL of DMEM culture medium to each well, and collect the cells 24 hours after transfection to detect dual luciferase activity.

[0095] 3. Luciferase activity assay: Dual luciferase activity assay was performed using the Beyotime Dual Luciferase Reporter GeneAssay Kit. 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 minutes, and then gently shake it for 15 minutes. The cell lysate was blown off and transferred to a 1.5 mL centrifuge tube. Centrifuge at 12,000 rpm for 5 minutes in a normal temperature centrifuge. 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 lysis supernatant into it, quickly mix evenly, 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 detect immediately. Renilla Luciferase reporter gene activity.

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

[0100] The results of dual luciferase assay were as follows Figure 3 As shown, in PK15 and 293T cells, the activity of psiCHECK2-CAV1-215T plasmid was significantly higher than that of psiCHECK2-CAV1-215C ( p <0.05), that is, after mutation of this site, 3'UTR activity increased significantly ( p <0.01). This indicates that this point mutation affects the expression of CAV1.

[0101] Example 5

[0102] Construction of a CAV1 gene overexpression vector and analysis of the effect of CAV1 on the proliferation of porcine skeletal muscle satellite cells using overexpression technology

[0103] 1. The specific procedures for freezing, thawing, and subculturing porcine skeletal muscle satellite cells in our laboratory are 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 resuspended in 10 ml of PBS and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the suspension was resuspended in complete medium. The cells were transferred to a 6 cm culture dish and cultured in a cell culture incubator at 37°C and 5% CO2.

[0106] (2) On the second day, observe the cell density and contamination. If there is no contamination, continue culturing with complete culture medium and subculture when the cell density reaches 90%.

[0107] 2. Overexpression vector construction

[0108] (1) Primer design: Based on the CAV1 gene cDNA sequence (NM_213870.1), primers were designed using Primer premier 5.0 software to clone the full-length coding region and introduce EcoR I restriction enzyme site, homologous recombination was used to amplify the full-length coding region of the CAV1 gene using CAV1-CDS-F / R primers. The sequence is as follows:

[0109]

[0110] Note: The underlined site is the EcoRⅠ restriction enzyme cutting site.

[0111] (2) RT-PCR amplification: The coding region was amplified using the reverse transcription (RT)-PCR method. 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 according to the instructions to synthesize cDNA.

[0112] The PCR amplification system was as follows: 5 µL of cDNA template, 2 µL of 10 µM upstream and downstream primers, 25 µL of 2× Rapid Master Mix enzyme, and ddH2O was added to make up to 50 µL.

[0113] The PCR amplification program was as follows: initial 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.

[0114] (3) RT-PCR product purification: Use the agarose gel purification kit from BioTeke Corporation according to the instructions.

[0115] (4) Overexpression vector construction: The modified pCMV-HA empty vector was cut using the restriction endonuclease recognition site (EcoRI) introduced on the primer.

[0116] Enzyme digestion system: EcoRI, 1 μL; 10× H Buffer, 1.0 μL; Plasmid, 1 μg; 0.1% BSA, 1 μL, made up to 10 μL with ddH2O; reaction conditions: 30°C water bath, 8 hours.

[0117] After enzyme digestion, the pCMV-HA vector was purified by 1.0% agarose gel electrophoresis and the purified RT-PCR product was ligated using the homologous recombinase from TaKaRa. The ligation system and reaction conditions were strictly in accordance with the instructions.

[0118] (5) Overexpression effect detection: The constructed recombinant plasmid was transfected into porcine skeletal muscle satellite cells. The transfection method is shown in Example 2. Total RNA extraction and reverse transcription reaction are shown in the above "RT-PCR amplification". Real-time PCR detection was performed using Vazyme's AceQ UniversalSYBR qPCR Master Mix kit to determine the overexpression efficiency of the recombinant plasmid. It was found that the recombinant plasmid can significantly increase the mRNA level of CAV1 in porcine skeletal muscle cells. The results are shown in Figure 4 , the horizontal axis in the figure represents the name of the transfected plasmid.

[0119] 3. CCK-8 Assay

[0120] Pig skeletal muscle satellite cells were seeded in a 96-well plate at a density of 2000-3000 cells / well. The next day, the plasmid was transfected into the cells. The transfection method is shown in Example 1. Three replicates were set up for each of the blank group, control group, and experimental group. 10 μL of enhanced CCK-8 solution was added after 0, 24, 48, 72, 96, and 120 hours of culture, and the cells were incubated for 2 hours. The absorbance was measured at 450 nm, and statistical analysis was performed based on the obtained values ​​to draw a cell growth activity curve. The results showed that compared with the control group, the activity of skeletal muscle satellite cells was significantly reduced after overexpression of CAV1 (p < 0.01). Figure 5 .

[0121] 4. EdU Detection

[0122] The cells were seeded into 96-well plates. When the cell confluence reached 30%, the plasmid was transfected into the cells. The transfection method was as shown in Example 1. The blank group, control group, and experimental group were each set up with 3 replicates. After 24 hours, the cell proliferation was detected using the BeyoClick EdU-555 cell proliferation detection kit of Biyuntian Company. The results showed that compared with the control group, the number of skeletal muscle satellite cells was significantly reduced after overexpression of CAV1 (p < 0.01). Figure 6 .

[0123] 5. Flow Cytometry

[0124] The cells were seeded into 6-well plates. When the cell confluence reached 60%, the plasmid was transfected into the cells. The transfection method is shown in Example 1. One well was transfected in each of the control group and the experimental group. After 24 hours of culture, the cells were trypsinized and the cell samples in the logarithmic growth phase were collected. The cells were stained using the Cell Cycle Staining Kit from Lianke Biotech and tested on the flow cytometer. The experimental results were fitted with cycle data using the software provided by the flow cytometer. The results showed that compared with the control group, overexpression of CAV1 could inhibit the pig skeletal muscle cells from entering the S phase from G1, thereby reducing the number of cells in the S and G2 phases and inhibiting the proliferation of skeletal muscle cells (p < 0.01). Figure 7 .

[0125] 6. Real-time PCR level detection

[0126] Cells were seeded into 12-well plates until the confluence reached approximately 70%. Plasmids were transfected into the cells using the transfection method described in Example 1. One well was transfected in each of the control and experimental groups. After 24 hours of culture, cells were collected and total RNA was extracted using the Trizol method. CDNA was synthesized according to the instructions of the Vazyme reverse transcription kit HiScript III Q RT SuperMix for qPCR (+gDNA wiper). qRT-PCR was used to detect the proliferation marker genes PCNA, MKI, CDK4, CCNB1, and CCND1. The results showed that compared with the control group, overexpression of CAV1 significantly downregulated the expression of the cell proliferation marker genes PCNA, MKI, CDK4, CCNB1, and CCND1 ( p <0.01) Results are shown in Figure 8 .

[0127] 7. Western Blotting

[0128] Cells were seeded into 6-well plates. When the cell confluence reached 60%, the plasmid was transfected into the cells. The transfection method was as described in Example 4. One well was transfected in each of the control and experimental groups. After 48 hours of culture, the cells were collected and protein was extracted using a protein extraction kit from Solaibio. Western blot was used to detect the effect of the CAV1 gene on the cell proliferation marker protein PCNA. The results showed that compared with the control group, the expression level of the cell proliferation marker protein PCNA was significantly reduced after overexpression of CAV1 ( p <0.01) Results are shown in Figure 9 .

Claims

1. A SNP molecular marker associated with muscle development in pigs, characterized in that: The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO.1, and the 215th base of the nucleotide sequence shown in SEQ ID NO.1 shows a T / C polymorphism.

2. A SNP molecular marker related to pig muscle development according to claim 1, characterized in that: The T / C polymorphism is a mutation from C to T; wherein, the CAV1 gene expression level of individuals with TT genotype is significantly higher than that of individuals with CC genotype.

3. Use of a SNP molecular marker primer related to muscle development of Min pigs as claimed in claim 1 in breeding / assisted breeding of pig breeds or strains related to muscle development of Min pigs, characterized in that: The breeding / assisted breeding is to select Min pigs with CC genotype individuals at position 215 of the nucleotide sequence shown in SEQ ID NO.1 as target pigs for breeding.

4. The use according to claim 3, characterized in that The specific operations are as follows:

1. Obtain genomic DNA of the pig to be tested; Second, using the above-mentioned Min pig genomic DNA as a template, PCR amplification was performed using the following primer pairs to obtain a gene fragment containing a SNP molecular marker; Primer CAV1_F1: 5′-GTGACATTTCAAGGGTATAAGTAT-3′; Primer CAV1_R1: 5′-AACTGTTAAACATTTTTATTGTGC-3′; 3. Detecting the amplified product by agarose gel electrophoresis; 4. Sequencing and analyzing the PCR products; 5. Select the Min pig with the genotype of CC at the 215th base of the nucleotide sequence shown in SEQ ID NO.1 as the screened target pig; 6. Breed the selected target pigs and the process is complete.

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