CAV1 gene molecular marker related to porcine skeletal muscle satellite cell proliferation and application of CAV1 gene molecular marker

Abstract

The invention discloses a CAV1 gene molecular marker related to porcine skeletal muscle satellite cell proliferation and application thereof, and relates to the technical field of molecular genetic markers, the molecular marker is a nucleotide sequence as shown in SEQ ID NO.1 formed by single nucleotide mutation at the + 215th basic group of a 3 'UTR region of a CAV1 gene; the polymorphism of the single nucleotide mutation is represented as T / C polymorphism. The method is used for breeding / assisted breeding of pig varieties or strains related to pig skeletal muscle satellite cell proliferation. The proliferation condition of the pig skeletal muscle satellite cells is identified by judging the polymorphism of the 3 'UTR mutation site of the pig CAV1 gene, an effective method is provided for early-stage seed selection by artificially applying the mutation site, and a new thought is provided for muscle growth and development character 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 the proliferation of porcine skeletal muscle satellite cells and an application thereof. Background Art

[0002] China has rich local pig breed resources. The special breeds bred with local pigs as materials can fully meet the diversified market consumption needs. At the same time, my country is a major producer and consumer of pork. Pork is the main source of protein nutrition for Chinese residents and plays a vital role in residents' lives and economic production. Min pigs are a local pig breed unique to Northeast my country. They have the advantages of cold resistance, excellent meat quality, and high fertility. However, they are significantly lower than introduced pig breeds such as Large White Pigs in some carcass traits, such as lean meat rate, eye muscle area, shear force and other carcass traits.

[0003] Skeletal muscle growth and development is a complex process involving muscle precursor cell proliferation, myoblast differentiation and multinucleated myotube formation. In these developmental events, cell-cell, cell-tissue and cell-extracellular matrix interactions are essential, and this process involves a large amount of cell adhesion and migration. Skeletal muscle satellite cells are a type of myogenic stem cell that play an important role in postnatal muscle development and injury repair in animals, and are closely related to carcass traits, meat yield and meat quality of 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 mainly expressed in endothelial cells, smooth muscle cells and fibroblasts, and it exerts its regulatory effects by regulating membrane trafficking, cholesterol metabolism and cell signaling.

[0005] In recent years, 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 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. CAV1 was knocked down in NIT-1 cells isolated from mice, and pro-apoptotic cytokines were downregulated in the NIT-1 cell line. CAV1 can inhibit tumor growth and metastasis of the mouse model of skin squamous cell carcinoma by regulating the activation of MAPK / AP-1 signaling.

[0006] Zeng Guowei et al. published the article "Research Progress of Caveolin in the Skeletal Muscle System" in the Journal of PLA Medical College in 2015. Although caveolins and caveolins have been studied for more than 50 years, the study of their role in the skeletal and muscular systems is only in its infancy. Many 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 static myocyte precursors. When myocytes are damaged, their mitosis is activated, and satellite cells proliferate, fuse or form new muscle fibers, thus opening the key step of muscle regeneration. Volonte et al. confirmed that the downregulation of Caveolin-1 is indeed a key signal for satellite cell activation in muscle regeneration. Although Caveolin-1 is undoubtedly involved in the process of osteogenesis, bone maturation and bone mass regulation, the specific role and related mechanisms of Caveolin-1 are still unclear. On the one hand, Caveolin-1 acts as an inhibitor of osteogenic 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-sensitive receptors. The physiological significance and related regulatory mechanisms of Caveolins on bone tissue need to be further elucidated.

[0007] Although the article gives the relationship between caveolin CAV1 and skeletal muscle satellite cell proliferation, it does not involve how to use caveolin CAV1 for breeding related to pig skeletal muscle satellite cell proliferation. Therefore, a method is needed to screen out SNPs related to pig skeletal muscle satellite cell proliferation for pig breeding. Summary of the invention

[0008] The present invention screens out a SNP associated with the proliferation of porcine skeletal muscle satellite cells. CAV1 is selected as a candidate gene, and a molecular marker associated with the proliferation of porcine skeletal muscle satellite cells is screened by searching for genetic variations in the 3UTR region of the porcine CAV1 gene. The molecular marker is used for marker-assisted selection and application of the proliferation of porcine skeletal muscle satellite cells.

[0009] The invention discloses a CAV1 gene molecular marker associated with the proliferation of porcine skeletal muscle satellite cells. 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 invention discloses an application of a CAV1 gene molecular marker related to the proliferation of pig skeletal muscle satellite cells. The molecular marker is used for breeding / assisted breeding of pig breeds or strains related to the proliferation of pig skeletal muscle satellite cells.

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

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

[0017] Second, using the above-mentioned pig 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 above amplified products by agarose gel electrophoresis;

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

[0022] Fifth, pigs with CC genotype at position +215 in 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 pig skeletal muscle satellite cells, the primer sequence 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 there are significant differences in the expression level of caveolin-1 (CAV1) gene in the longissimus dorsi muscle at different developmental stages, suggesting that CAV1 plays an important role in muscle development.

[0029] The present invention further conducted a genetic polymorphism analysis on the CAV1 gene, and found that there was a C to T mutation at the 3'UTR+215 site of the pig CAV1 gene through direct sequencing of PCR products. The mutation existed at a higher 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 conducted an in-depth analysis of the genetic effect of the C>T polymorphism at the +215 site using a luciferase reporter gene experiment, and found that the C to T mutation at the +215 site changed the activity of the CAV1 3'UTR in an in vitro experiment. After the mutation of the CAV1 gene +215 site (C mutated to T), the activity of the luciferase reporter gene increased significantly, indicating that the point mutation has the function of promoting the transcriptional expression of the CAV1 gene;

[0030] The CAV1 gene +215 locus was TT genotype individuals, and the CAV1 gene expression was significantly higher than that of CC genotype individuals; CCK8 and EdU staining analysis determined that CAV1 had the effect of inhibiting cell proliferation. It can be seen that the detection of molecular markers associated with mutations is not only simple and fast, but also not affected by the environment, and can achieve early selection. Therefore, the selection of CAV1 gene +215 locus as the C gene for breeding has important breeding significance for improving the muscle development of pigs (especially Min pigs).

[0031] The present invention experimentally found that the mutation from C to T at the +215 position of the 3'UTR region of the pig CAV1 gene significantly increased the activity of the CAV1 3'UTR in an in vitro test, and 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 not affected by the environment, and can achieve early seed selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The sequencing results of SNP sites in the 3'UTR region of the pig CAV1 gene; Figure A is a TT type individual, and Figure B is a CC type individual;

[0033] Figure 2 The figure shows the results of restriction enzyme digestion identification of reporter gene vectors psicheck2-CAV1-T and psicheck2-CAV1-C; lanes 1 and 3 are DL5000 markers, lane 2 shows the results of single restriction enzyme digestion (XhoⅠ) of psicheck2-CAV1-C plasmid, and lane 4 shows the results of single restriction enzyme digestion (XhoⅠ) of psicheck2-CAV1-T plasmid;

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

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

[0037] Figure 6 This is the diagram of EdU analysis of the effect of CAV1 on the proliferation of porcine skeletal muscle satellite cells, ** indicates extremely significant difference, p<0.01;

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

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

[0040] Fig. 9 This is a diagram 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 by 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 them according to 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: The dorsal muscles of Min pigs were taken and genomic DNA was extracted 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 sequence of the porcine CAV1 gene. The primer sequences are as follows:

[0047]

[0048] 3. Identification of polymorphic sites: 10 pigs were selected from the Min pig population, and their mixed genomic DNA was used as a template. PCR amplification was performed with primers CAV1_F / R, and the PCR amplification products were sequenced. The sequencing results were compared with the known sequence (NM_001348935.1) using Snap Gene software, and the sequencing peaks were manually checked using chromas software to identify polymorphic sites. One SNP was detected in this region, which was a SNP (+215C>T) site.

[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, and the base composition of the SNP+215C>T site was detected by direct sequencing of PCR products. The chromas sequencing peaks were visually checked to identify homozygotes and heterozygotes, 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, with a genotype frequency of 0.18, 4 individuals with TT genotype, with a genotype frequency of 0.12, 23 individuals with CT genotype, with a 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, with a genotype frequency of 0.53, 0 individuals with TT genotype, with a genotype frequency of 0, and 8 individuals with CT genotype, with a genotype frequency of 0.47, C allele frequency of 0.76, and T allele frequency of 0.24. The sequencing peaks of individuals with different genotypes are shown in Figure 1 .

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

[0052]

[0053] Note: The numbers in brackets are 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 the longissimus dorsi muscle of the pig in our laboratory and the published pig mRNA sequence (NM_001348935.1), the 3'UTR region sequence of the CAV1 gene was determined. Primer Premier 5.0 was used to design primers CAV1_F1 and CAV1_R1 and homology arms were introduced at the 5' end. The CAV1_F1 / R1 primers were used to amplify the 1868-length fragment of the pig CAV1 3'UTR, and psiCHECK2 was used as the backbone to construct the CAV1 gene 3'UTR reporter gene vector. The specific method is as follows:

[0058]

[0059] Note: Underlines indicate Xho1 restriction sites.

[0060] 3. PCR amplification: This example uses conventional PCR to amplify the 1868 fragment of the 3'UTR region of the Min pig CAV1 gene (the nucleotide sequence of the fragment is shown in the sequence list 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 Master Mix enzyme, and ddH2O to make up to 50 μL.

[0062] The amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60.2°C for 15 s, extension at 72°C for 30 s, 35 cycles; 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 completed, prepare a 1.0% agarose gel to detect the PCR amplification product. The setting conditions are: 100V, electrophoresis for 30 minutes. After the electrophoresis is completed, observe the PCR amplification results on the gel imaging system. Observe the single, bright and correct length target fragments, and perform gel cutting on the target fragment area. Perform gel recovery test according to the instructions of the gel recovery kit of BioTeke Corporation. After agarose gel electrophoresis, the gel recovery products are uniformly stored in a -20℃ refrigerator.

[0065] 5. Enzyme digestion and ligation: Use restriction endonuclease Xho1 to digest the psiCHECK2 empty vector.

[0066] Enzyme digestion system: QuickCut TM XhoⅠ, 1μL; 10×QuickCut Buffer, 5.0μL; Plasmid, 1μg; ddH2O to make up to 50μL; reaction conditions: 37℃ water bath, 1 hour.

[0067] After enzyme digestion, the empty vector was purified by 1.0% agarose gel electrophoresis according to the instructions using the agarose gel purification kit of BioTeke Corporation. The purified empty vector and PCR product were connected using the homologous recombinase of TaKaRa. The connection system and reaction conditions were strictly in accordance with the instructions.

[0068] 6. Transformation of recombinant psiCHECK2 plasmid: According to the DH5α instruction manual of 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 min. Pipette 30 μL of competent cells and add them to 10 μL of ligation product. Mix gently and place on ice for 30 min.

[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 the 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.

[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 plate.

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

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

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

[0079] Example 3

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

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

[0082]

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

[0084] 2. The method for site-directed mutagenesis was overlap extension PCR, which included two rounds of reactions. In the first round, primer pairs CAV1_F1 / CAV1_R2 and CAV1_F2 / CAV1_R1 were used with psicheck2-CAV1-215C vector as templates to amplify fragments with point mutations at both ends and overlapping with each other. In the second round, appropriate dilutions of the PCR products of the above two reactions were used as templates, and CAV1_F1 / CAV1_R1 was used as primers for PCR amplification to introduce point mutations into the interior of the PCR product.

[0085] The first round of PCR amplification system was: psiCHECK2-CAV1-215C vector DNA 100ng, 2× PrimeSTARMaxPremix 25μL, upstream and downstream primers 20μM each, ddH2O to 50μL. The PCR amplification program was: 98℃ denaturation 10s, 54℃ annealing 15s, 72℃ extension 30s, 35 cycles.

[0086] The second round of PCR amplification system was: 1 μL of each of the two PCR product dilutions from the first round of reaction, 25 μL of 2× PrimeSTARMax Premix, 10 μM of each of the upstream and downstream primers, and ddH2O to make up to 50 μL. The PCR amplification program was: denaturation at 98°C for 10 s, annealing at 60.2°C for 15 s, extension at 72°C for 2 min, and 35 cycles.

[0087] The second round of PCR products were purified by agarose gel electrophoresis and connected to the psiCHECK2 vector. The constructed vector plasmid was digested with XhoI restriction enzyme and then sent to Arrow Gene Co., Ltd. for sequencing, which confirmed that the C mutation at the +215 site of the CAV1 gene was changed to T. The digestion results are shown in Figure 2 .

[0088] Example 4 Dual luciferase reporter gene analysis

[0089] 1. Extraction of endotoxin-free plasmid: Inoculate the correctly sequenced bacterial solution containing the luciferase reporter gene into LB liquid culture medium containing 50μg / mL ampicillin at a ratio of 1:500, and shake vigorously at 200rpm for 16h. Collect the bacteria by centrifugation at room temperature at 12000rpm, and extract the plasmid using Tiangen's endotoxin-free plasmid extraction kit. The specific operation is carried out strictly according to the instructions, and the plasmid concentration is measured using a UV spectrophotometer.

[0090] 2. Cell culture and transfection: PK-15 and 293T cells were inoculated on 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 confluence of the monolayer cells reached 50-70% under a microscope. The transfection was performed according to the instructions of the Lipofectamine 8000 transfection kit of 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 a blank control was set up. Each group was set up with 3 replicates.

[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 min.

[0093] (3) Take the cell culture dish out of the 37°C CO2 incubator, wash the dish twice with PBS, add 50 μL of the 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] 3. Luciferase activity detection: Use the Dual Luciferase Reporter Gene Assay Kit of Beyotime to perform dual luciferase activity detection. 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 1× Cell Lysis Buffer to each well of the 24-well plate, let stand at room temperature for 5 min, and then gently shake for 15 min to lyse the cells. The cell lysate was pipetted and transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm in a normal 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 activity of the Firefly luciferase reporter gene in a luminescence detector.

[0098] (3) Renilla luciferase reaction test: Add 15 μL of Renilla substrate working solution to the mixed solution 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 Renilla relative to Firefly.

[0100] The results of dual luciferase assay were as follows Figure 3As shown in the figure, 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 the mutation of this site, the activity of 3'UTR was significantly increased (p<0.01), indicating that this point mutation affects the expression of CAV1.

[0101] Example 5

[0102] Construction of 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 operations of freezing, thawing and subculturing porcine skeletal muscle satellite cells in this laboratory are as follows:

[0104] (1) Cell recovery

[0105] The frozen cells were taken out from liquid nitrogen, quickly placed in 37°C water, shaken to promote melting, and the cell suspension was obtained. The cell suspension was resuspended in 10 ml PBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cell suspension was resuspended in complete medium, and the cell suspension was transferred to a 6 cm culture dish and cultured in a 37°C, 5% CO2 cell culture incubator.

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

[0107] 2. Overexpression vector construction

[0108] (1) Primer design: Based on the cDNA sequence of the CAV1 gene (NM_213870.1), primers were designed using Primer premier 5.0 software to clone the full-length coding region, and the EcoRⅠ restriction site was introduced. The homologous recombination method 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 site.

[0111] (2) RT-PCR amplification: The coding region was amplified using the reverse transcription (RT)-PCR method. Total RNA from pig back muscle tissue was extracted using Trizol (TaKaRa), and reverse transcription was performed using the instructions of Vazyme's HiScript III 1st strand cDNA synthesis kit to synthesize cDNA.

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

[0113] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 15 s, 35 cycles; final extension at 72°C for 5 min; and storage at 4°C.

[0114] (3) RT-PCR product purification: using 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 open using the restriction endonuclease recognition site (EcoRⅠ) introduced on the primer.

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

[0117] After enzyme digestion, the pCMV-HA vector was purified by 1.0% agarose gel electrophoresis according to the instructions using the agarose gel purification kit of BioTeke Corporation, and the purified RT-PCR product was connected. The connection reaction was carried out using the homologous recombinase of TaKaRa Company. The connection system and reaction conditions were strictly carried out according to 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. The total RNA extraction and reverse transcription reaction are shown in the above "RT-PCR amplification". The real-time PCR detection was performed using the AceQ Universal SYBR qPCR Master Mix kit of Vazyme Company 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 detection

[0120] Pig skeletal muscle satellite cells were seeded in a 96-well plate at a density of 2000-3000 cells / well. On the second day, the plasmid was transfected into the cells. The transfection method was shown in Example 1. There were 3 replicates in 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). The results are shown in Figure 5 .

[0121] 4. EdU Detection

[0122] The cells were inoculated into a 96-well plate. When the cell confluence reached 30%, the plasmid was transfected into the cells. The transfection method was shown in Example 1. The blank group, control group, and experimental group were set up with 3 replicates. After 24 hours, the BeyoClick EdU-555 cell proliferation detection kit of Beyotime was used to detect cell proliferation. The results showed that compared with the control group, the number of skeletal muscle satellite cells increased significantly after overexpression of CAV1 (p<0.01). Figure 6 .

[0123] 5. Flow cytometry

[0124] The cells were inoculated into a 6-well plate. 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 digested with trypsin 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 machine. The experimental results were fitted with the cycle data using the software provided by the flow cytometer. The results showed that compared with the control group, overexpression of CAV1 could inhibit 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). The results are shown in Figure 7 .

[0125] 6. Real-time PCR level detection

[0126] The cells were seeded into a 12-well plate, and the cell confluence reached about 70%. 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. The cells were collected after culturing for 24 hours, and the total RNA was extracted using the Trizol method. Cdna was synthesized according to the instructions of Vazyme's reverse transcription kit HiScript IIIQ RT SuperMix for qPCR (+gDNAwiper), and the proliferation marker genes PCNA, MKI, CDK4, CCNB1, and CCND1 were detected by qRT-PCR. The results showed that compared with the control group, after overexpression of CAV1, the expression levels of cell proliferation marker genes PCNA, MKI, CDK4, CCNB1, and CCND1 were significantly upregulated (p<0.01). The results are shown in Figure 8 .

[0127] 7. Western Blotting

[0128] The cells were inoculated into a 6-well plate. When the cell confluence reached 60%, the plasmid was transfected into the cells. The transfection method was shown in Example 4. One well was transfected in each of the control group and the experimental group. After 48 hours of culture, the cells were collected and the 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, after overexpression of CAV1, the expression level of the cell proliferation marker protein PCNA was significantly reduced (p<0.01). Fig. 9 .

Claims

1. A CAV1 gene molecular marker associated with the proliferation of porcine skeletal muscle satellite cells, characterized in that The molecular marker is a nucleotide sequence 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.

2. The CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation according to claim 1, characterized in that The T / C polymorphism affects pig muscle development.

3. A CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation according to claim 1 or 2, 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.

4. The use of a CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation as claimed in claim 1, characterized in that The molecular marker is used for breeding / assisted breeding of pig breeds or strains related to the proliferation of pig skeletal muscle satellite cells.

5. The use according to claim 4, characterized in that The breeding / assisted breeding is to select pigs with CC genotype individuals at position +215 in the 3'UTR region of the CAV1 gene as target pigs for breeding.

6. The use according to claim 4 or 5, characterized in that The pig is a Min pig or a Large White pig.

7. The use according to claim 4 or 5, characterized in that The breeding / assisted breeding is to select pigs with CC genotype individuals at position +215 in the 3'UTR region of the CAV1 gene as target pigs for breeding. The specific operation is as follows:

1. Obtain genomic DNA of the pig to be tested; Second, using the above-mentioned pig 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; Primer CAV1_F1: 5′-GTGACATTTCAAGGGTATAAGTAT-3′; Primer CAV1_R1: 5′-AACTGTTAAACATTTTTATTGTGC-3′; 3. Detecting the above amplified products by agarose gel electrophoresis; 4. Sequencing and analyzing the PCR products; 5. Select the pigs with CC genotype at the +215th position of the 3'UTR region of the CAV1 gene as the selected target; 6. Breed the selected target pigs, and the process is completed.

8. A primer pair for detecting the CAV1 gene molecular marker associated with porcine skeletal muscle satellite cell proliferation as claimed in claim 1, characterized in that The primer sequences are as follows: Primer CAV1_F1: 5′-GTGACATTTCAAGGGTATAAGTAT-3′; Primer CAV1_R1: 5′-AACTGTTAAACATTTTTATTGTGC-3′.

9. A kit comprising the primer according to claim 8.

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