Application of PRTFDC1 gene in promoting porcine skeletal muscle satellite cell differentiation, myotube formation and skeletal muscle growth and development

Abstract

The invention discloses application of a PRTFDC1 gene in promoting porcine skeletal muscle satellite cell differentiation, myotube formation and skeletal muscle development. According to the invention, overexpression and knock-down experiments of the PRTFDC1 are respectively carried out in pig MuSCs, and immunofluorescence, qRT-PCR and Western blot analysis are carried out on cells subjected to induced differentiation, so that the PRTFDC1 is effectively shown to play a positive regulation role in a pig MuSCs differentiation process, and a new marker gene which can be applied to pig molecular breeding and growth performance evaluation is provided; and a novel molecular target and a novel detection tool are provided for muscle development regulation and biological breeding.

Description

Technical Field

[0001] This invention relates to the fields of animal genetics and breeding and molecular biology, specifically to the application of the PRTFDC1 gene in promoting the differentiation of porcine skeletal muscle satellite cells, myotube formation and skeletal muscle growth and development. Background Technology

[0002] Pork is one of the most important sources of meat for people and an important source of high-quality protein. The meat production performance of pigs is closely related to the growth and development of skeletal muscle. The growth and development of skeletal muscle depends on the proliferation, differentiation, and fusion of skeletal muscle satellite cells. Skeletal muscle satellite cells (MuSCs) originate from a subset of myoblast progenitor cells in the muscle tissue. These cells remain undifferentiated in the muscle tissue of newborn individuals, located between the sarcolemma and basement membrane, and possess strong differentiation potential. In pigs, muscle fiber formation is completed around 70 days of embryonic development, and the number of muscle fibers remains almost constant after birth. The growth of skeletal muscle in early childhood mainly relies on the continuous proliferation, differentiation, and fusion of skeletal muscle satellite cells. In adulthood, under normal circumstances, the number and morphology of skeletal muscle remain stable. However, when muscle tissue is damaged, satellite cells, as the main stem cells, are activated to achieve damage repair. After activation, satellite cells differentiate into myoblasts and myocytes, which then fuse to form myotubes. Myotubes further fuse and mature to form new muscle fibers. This process is regulated by a series of strict and complex molecular networks. In recent years, an increasing number of genes and signaling pathways have been discovered to be related to myogenesis and satellite cell differentiation. However, some genes that may be involved in the regulation of myogenesis have not yet been discovered or systematically described. Therefore, discovering new regulatory genes is of great significance for elucidating the mechanisms of skeletal muscle development and improving the production performance of pigs.

[0003] The Phosphoribosyl transferase domain containing 1 (PRTFDC1) gene encodes a protein containing a phosphoribosyltransferase domain, belonging to the purine / pyrimidine phosphoribosyltransferase family. Its structure is highly similar to the classic catalytic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) and exhibits high conservation in mammals. Although PRTFDC1 possesses a phosphoribosyltransferase-like structure, studies suggest it may lack typical catalytic activity and is more likely to function as a regulatory factor or pseudoenzyme, participating in the regulation of protein-protein interactions and signaling pathways. Existing research indicates that PRTFDC1 is closely associated with various neuropsychiatric disorders and stress-related disorders (such as dissociative amnesia and acute stress response), and plays an important role in the occurrence and progression of various tumors. However, current research on the functional involvement of PRTFDC1 in skeletal muscle formation, satellite cell differentiation, and muscle growth remains very limited, particularly regarding its expression patterns and regulatory mechanisms in important livestock species such as pigs.

[0004] The Sujiang pig is a new breed developed through selective breeding of Jiangquhai pigs and Fengjing pigs (Taihu pigs) as the base maternal line and Duroc pigs as the paternal line. It was recognized as a new breed for pig breeding by the China Livestock and Poultry Genetic Resources Committee in 2013. This breed is characterized by its tolerance to roughage, strong adaptability, excellent meat quality, and high litter size, making it valuable for research on meat production and muscle growth and development. Currently, there is very little research on genes related to muscle growth and development in Sujiang pigs, and its meat production regulation mechanism requires further investigation.

[0005] Based on this, deepening the understanding of the molecular regulatory network of porcine skeletal muscle development, and providing a scientific basis for the molecular improvement of the superior breed of Sujiang pig, as well as for the discovery of candidate genes related to muscle growth and the development of molecular marker-assisted breeding, are the problems that this invention urgently needs to solve. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to overcome the limitations of the number of existing disclosed porcine skeletal muscle satellite cell differentiation-related regulatory genes and the shortcomings of application methods, and to provide an application of the PRTFDC1 gene in promoting porcine skeletal muscle satellite cell differentiation and muscle growth.

[0007] To achieve the above objectives, the present invention provides the application of the PRTFDC1 gene in promoting the differentiation of porcine skeletal muscle satellite cells for non-disease treatment purposes.

[0008] This invention also provides the application of the PRTFDC1 gene in accelerating myotube formation and skeletal muscle growth and development for non-disease treatment purposes.

[0009] Preferably, the pig is a Sujiang pig.

[0010] This invention involves overexpression and knockdown of PRTFDC1 in pig MuSCs (i.e., porcine skeletal muscle satellite cells, or porcine MuSCs), followed by analysis of the induced differentiated cells using immunofluorescence, qRT-PCR, and Western blot. The results showed that the mRNA level of PRTFDC1 was significantly increased in the overexpression group (…). P <0.01, while its number of myotubes increased significantly, the myotubes were longer and the degree of fusion was higher, and its differentiation index was significantly increased ( P <0.05, the transcriptional levels of muscle differentiation marker genes MYOD, MYOG and MYH1 were significantly upregulated; the expression level of PRTFDC1 was significantly reduced in the knockdown group ( P <0.05, its differentiation index decreased significantly ( P (<0.05) Simultaneously, the expression level of MYOG also decreased significantly. This effectively demonstrates that PRTFDC1 plays a positive regulatory role in the differentiation of MuSCs in Sujiang pigs, thus providing a new marker gene applicable to pig molecular breeding and growth performance evaluation, and offering new molecular targets and detection tools for muscle development regulation and bio-breeding. Attached Figure Description

[0011] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a diagram illustrating the in vitro differentiation process of porcine skeletal muscle satellite cells prepared in Example 1; Figure 2 The analysis of PRTFDC1 gene expression in various tissues of Sujiang pigs and during MuSC differentiation was performed in Example 1. Figure 3 Example 2 showed overexpression of PRTFDC1 (PRTFDC1). OE The results of the detection of MuSCs differentiating into myotubes are shown in the figure. Figure 4 This is a graph showing the detection results of PRTFDC1 knockdown (PRTFDC1-siRNA) inhibiting MuSC differentiation into myotubes in Example 3; Figure 5 This is a diagram illustrating the activation of the cGAS-STING pathway in Sujiang pig MuSCs by overexpression of PRTFDC1 in Example 4. Detailed Implementation

[0012] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0013] The Sujiang pigs used in this invention were purchased from Jiangsu Jiangquhai Pig Breeding Co., Ltd.

[0014] The FBS used in this invention is a commercially available product from GeminiBio (West Sacramento, CA, USA); DMEM high-glucose medium, bFGF, DMEM / F12 high-glucose medium, and horse serum are commercially available products from Gibco (Carlsbad, CA, USA); erythrocyte lysis buffer is a commercially available product from Beijing Solarbio Technology Co., Ltd. (Beijing, China); matrix gel is a commercially available product from Corning (NY, USA); Dispase II is a commercially available product from Beijing Coollife Technology Co., Ltd. (Beijing, China); collagenase II is a commercially available product from Sigma-Aldrich (St. Louis, MO, USA); penicillin-streptomycin is a commercially available product from Beyotime Biotechnology (Shanghai, China); psPAX2 and pMD2.G are commercially available products from Addgene (Watertown, MA, USA); and X-tremeGENE 360 is from Merck. KGaA (Darmstadt, Germany)'s regular commercially available products.

[0015] The specific small interfering RNAs targeting PRTFDC1 used in this invention, such as those shown in SEQ ID No:1 and SEQ ID No:2, were synthesized and provided by Genecreate (Wuhan, China).

[0016] In the following specific embodiments of the present invention, RT-qPCR is performed according to the following steps: RNA of the sample is reversed into cDNA according to the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) instructions. qPCR is performed using PerfectStart Green qPCR SuperMix (+Universal Passive Reference Dye) (TransGen Biotech, Beijing, China), with 18S rRNA as an internal reference gene, and 2...−∆∆Ct The relative expression levels of genes were calculated. Primers for MYOG (as shown in SEQ ID No:5 and SEQ ID No:6), MYH1 (as shown in SEQ ID No:7 and SEQ ID No:8), PRTFDC1 (as shown in SEQ ID No:9 and SEQ ID No:10), MYOD (as shown in SEQ ID No:11 and SEQ ID No:12), and PAX7 (as shown in SEQ ID No:13 and SEQ ID No:14), and primer sequences for the internal reference gene 18S rRNA (as shown in SEQ ID No:15 and SEQ ID No:16), are detailed in Table 1. The qPCR reaction system consisted of 20 μL of qPCR SuperMix, 4 μL of cDNA template, 0.4 μL each of forward and reverse primers (10 μM concentration), and 5.2 μL of RNase-free water. The reaction conditions were 94℃ for 30s, followed by 40 cycles of 94℃ for 5s and 60℃ for 30s.

[0017] Immunofluorescence analysis was performed as follows: Cultured cell samples were washed twice with PBS and fixed with 4% paraformaldehyde for 20 min. After fixation, the samples were washed three times with PBS. Immunofluorescence blocking buffer was added to the culture wells and incubated at room temperature for 1 h. The blocking buffer was aspirated, primary antibody was added, and the samples were incubated overnight at 4°C. After incubation, the samples were washed three times with PBS. Secondary antibody was added, and the samples were incubated at room temperature in the dark for 1 h. The samples were washed three times with PBS in the dark. Mounting buffer containing DAPI was added to the culture wells in the dark to cover the cells. The samples were stored at 4°C in the dark for microscopic examination. The following primary antibodies were used according to the testing requirements: mouse anti-PAX7 (1:100, Developmental Studies Hybridoma Bank (DSHB), Lowa City, IA, USA), mouse anti-MYOD (1:100, DSHB), mouse anti-MYOG (1:100, DSHB), mouse anti-myosin heavy chain (MYHC, 1:100, DSHB), and mouse anti-HIS tag (1:100, Solarbio, Beijing, China). The following secondary antibodies were used: Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa fluo 488, and Alexa fluo 555 (1:400, Thermo Fisher Scientific, Waltham, MA, USA).

[0018] Western blotting was performed as follows: Cultured cell samples were collected, and total protein was extracted using RIPA protein lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1% PMSF (Beyotime Biotechnology, Shanghai, China). SDS-PAGE electrophoresis was performed using a 4%–20% precast gel (Yeasen Biotechnology, Shanghai, China). After electrophoresis, the protein was transferred to a PVDF membrane using a transfer program of 300 mA for 1 hour. After blocking at room temperature for 1.5 hours, primary antibody was added, and the membrane was incubated overnight at 4°C. The membrane was washed three times with TBST, then incubated with secondary antibody at room temperature for 1 hour, followed by three more washes with TBST. Development was then performed after completing these steps. The following primary antibodies are used according to the detection requirements: mouse anti-PAX7 (1:1000, DSHB), mouse anti-MYOD (1:1000, DSHB), mouse anti-MYOG (1:1000, DSHB), mouse anti-MYHC (1:1000, DSHB), mouse anti-HIS tag (1:1000, Solarbio, Beijing, China), rabbit anti-PRTFDC1 (1:1000, Bioragon, Suzhou, Jiangsu, China), mouse anti-β-TUBULIN (1:5000, Abcam, Cambridge, UK), and mouse anti-GAPDH (1:5000, Beyotime Biotechnology, Shanghai, China). The antibodies used in the cGAS-STING pathway detection process are from the mouse-reactive STING pathway antibody sampler kit (1:1000, Cell Signaling Technology, Danvers, MA, USA). The following secondary antibodies were used: horseradish peroxidase (HRP) conjugated anti-mouse IgG and HRP conjugated anti-rabbit IgG (1:5000, Beyotime Biotechnology, Shanghai, China).

[0019] Table 1 Preparation Example 1: Isolation, culture, and induced differentiation of skeletal muscle satellite cells from *Scutellaria baicalensis* pigs.

[0020] Porcine skeletal muscle satellite cells were isolated from the gastrocnemius muscle of Sujiang pigs (11 days old, female, n=3).

[0021] The specific procedures are as follows: After anesthetizing and euthanizing the pigs, the skin surface was disinfected with 75% alcohol. The skin was then incised, and muscle tissue was cut off and washed three times with PBS. The muscle was then minced into small pieces using sterile scissors and transferred to a cell separation digestion solution for digestion at 220 rpm and 37°C for 1 hour on a shaker. After complete digestion, digestion was terminated with DMEM high-glucose medium containing 3% FBS, and the cells were filtered through 100μm and 40μm pore sizes to remove tissue debris and obtain a cell suspension. The cell suspension was treated with erythrocyte lysis buffer for 5 minutes and then centrifuged at 1500 rpm for 5 minutes to obtain satellite cell pellet. The satellite cell pellet was resuspended in complete growth medium and transferred to a T25 culture flask treated with Matrigel, and cultured at 37°C and 5% CO2. All culture dishes used during the culture process were treated with Matrigel. At the first passage, fibroblasts were removed by differential adhesion for 1 hour. When the confluence of porcine skeletal muscle satellite cells reaches more than 90%, the differentiation medium is replaced to induce differentiation, and the differentiation of porcine skeletal muscle satellite cells is observed within 1 to 5 days.

[0022] Immunofluorescence and Western blotting were performed on cells before differentiation (labeled GM), at day 1 of differentiation (labeled DM1), at day 2 of differentiation (labeled DM2), and at day 4 of differentiation (labeled DM4). The marker proteins used for detection included PAX7, a marker of satellite cell proliferation, MYOD and MYOG, markers of differentiation, and MYHC, a constitutive protein of myotubules. Results are as follows: Figure 1 As shown, satellite cells in the proliferative phase highly express PAX7, and the percentage of PAX7-positive cells gradually decreases as differentiation progresses. MYOD, MYOG, and MYHC are expressed at low levels in satellite cells during the proliferative phase, but their expression significantly increases after differentiation. These results indicate that the isolated skeletal muscle satellite cells from *Sinonovacula contorta* can be successfully induced to differentiate. Figure 1 Figure A shows the expression of key biomarkers in pig MuSCs during in vitro differentiation, identified by PAX7, MYOD, MYOG, and MYHC immunofluorescence staining. Figure 1 Figure B in the middle shows the changes in the expression levels of PAX7, MYOD, MYOG, and MYHC proteins during the in vitro differentiation of pig MuSCs, detected by Western blotting. GAPDH was used as an internal control.

[0023] The cell separation and digestion solution used above contains 2 mg / mL -1 Dispase II and 4 mg / mL -1 Collagenase II was prepared in DMEM high-glucose medium. The complete growth medium was DMEM / F12 high-glucose medium containing 20% ​​FBS, 1% penicillin-streptomycin, and 5 ng / mL bFGF. The differentiation medium was DMEM high-glucose medium supplemented with 2% horse serum and 1% penicillin-streptomycin. Preparation Example 2: Construction and Packaging of PRTFDC1 Overexpression Lentiviral Vectors and Interference Vectors

[0024] The PRTFDC1 overexpression recombinant plasmid PLVX-IRES-ZsGreen1-PRTFDC1 (C-terminally fused with a 6×His tag) and its control plasmid PLVX-IRES-ZsGreen1-Scramble were constructed by Suzhou Genewiz Biotechnology Co., Ltd. Specifically, the conventionally synthesized gene PRTFDC1, the vector PLVX-IRES-ZsGreen1 (Ampicillin), and the random sequence Scramble were all provided by Suzhou Genewiz Biotechnology Co., Ltd.

[0025] The recombinant plasmid PLVX-IRES-ZsGreen1-PRTFDC1 and the control plasmid PLVX-IRES-ZsGreen1-Scramble were constructed using recombinant plasmid construction methods conventionally understood in the art. Specifically, in this invention, they were constructed using the following method:

[0026] Control plasmid PLVX-IRES-ZsGreen1-Scramble: A random sequence (denoted as Scramble, the specific sequence is shown in SEQ ID No:18, specifically: CTCGAGACTAGTTCTAGA) was synthesized, and 5' (EcoRI) and 3' (NotI) were added. The synthesized random sequence was cloned into the vector PLVX-IRES-ZsGreen1(Ampicillin) through 5' EcoRI and 3' NotI to construct PLVX-IRES-ZsGreen1-Scramble.

[0027] 5×10 6 HEK293T cells were seeded in 10 mm culture dishes and cultured for 24 h. Transfection was performed when cell confluence reached 80%-90%. The PRTFDC1 overexpression plasmid or control plasmid was mixed with helper plasmids psPAX2 and pMD2.G at a mass ratio of 3:2:1 and co-transfected. The amount of plasmid used in each 10 cm culture dish was 10.5 μg, 7 μg, and 3.5 μg, respectively. Transfection was performed according to the standardized procedure of PolyJet in vitro DNA transfection reagent (SignaGen Laboratories, Rockville, MD, USA), with a reagent-to-plasmid ratio of 2:1. After transfection, the cells were incubated for 6 h. The original culture medium was discarded and replaced with 10 mL of fresh culture medium. The supernatant was collected after 72 h. The supernatant was centrifuged at 5000×g for 10 min, then filtered through a 0.45 μm filter membrane, and finally concentrated using an Amicon Ultra filter (100 kDa MWCO, Merck KGaA, Darmstadt, Germany). The resulting viral concentrate was aliquoted into 1.5 mL EP tubes and stored at -80 °C. The viral concentrate obtained after transfection and culture with the PRTFDC1 overexpression recombinant plasmid was designated as PRTFDC1 overexpression lentivirus; the viral concentrate obtained after transfection and culture with the control plasmid was designated as control virus. Example 1: In vitro PRTFDC1 overexpression

[0028] When the porcine skeletal muscle satellite cells isolated and cultured in Preparation Example 1 reached 80% confluence, they were inoculated with the PRTFDC1 overexpressing lentivirus prepared in Preparation Example 2 at a MOI of 0.01. After 48 hours of transduction, the medium was replaced with induction differentiation medium (i.e., the differentiation medium from Preparation Example 1), and cells were cultured for another 2 days before being collected. The mRNA and protein levels of PRTFDC1 were analyzed by qRT-PCR and Western blot to verify the overexpression efficiency. Example 2: In vitro PRTFDC1 knockdown experiment

[0029] Porcine skeletal muscle satellite cells were transfected with specific small interfering RNAs (PRTFDC1-siRNA-822, SEQ ID No:1: 5′-GCAGAUAAUUGGAGGCGAATT-3′, SEQ ID No:2: 5′-UUCGCCUCCAAUUAUCUGCTT-3′) targeting PRTFDC1, as shown in SEQ ID No:1 and SEQ ID No:2. Transfection was performed simultaneously with changing the differentiation induction medium (i.e., the differentiation medium used in Preparation Example 1), and cells were collected 4 days after induction. The mRNA and protein levels of PRTFDC1 were assessed by qRT-PCR and Western blot to evaluate the knockdown effect. Comparative Example 1

[0030] The procedure was performed according to Example 1, except that the virus used for inoculation was not a PRTFDC1 overexpressing lentivirus, but a control virus. Comparative Example 2

[0031] The procedure was performed according to Example 2, except that the specific small interfering RNA targeting PRTFDC1 was replaced with the negative control siRNA (sense strand SEQ ID No:3: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense strand SEQ ID No:4: 5′-ACGUGACACGUUCGGAGAATT-3′) as shown in SEQ ID No:3 and SEQ ID No:4. Detection Example 1

[0032] The expression profile of PRTFDC1 in various tissues of 11-day-old Sujiang pigs was detected by RT-qPCR. The expression level in heart tissue was used as a reference (set as 1). Figure 2 As shown, the results indicate that PRTFDC1 was expressed in all tissues examined, exhibiting a broad expression pattern. Among them, Figure 2 Figure A shows that the relative expression level of PRTFDC1 in different tissues of Sujiang pigs was detected by qRT-PCR, with 18S rRNA as an internal reference gene and 3 biological replicates per group. Figure 2 Figure B shows the changes in PRTFDC1 protein expression levels during the in vitro induced differentiation of Sujiang pig MuSCs using Western blot analysis, with GAPDH as an internal control. Figure 2 The subcellular localization of PRTFDC1 in Sujiang pig MuSCs was analyzed using immunofluorescence staining (Figure C). The primary antibody was mouse anti-HIS tag, and the secondary antibody was goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa fluo 555. Scale bar = 20 μm.

[0033] Among them, higher expression levels were observed in the liver, brain, and large intestine, while clear expression was also detected in skeletal muscle tissue (e.g., Figure 2 (As shown in Figure A). Further analysis of PRTFDC1 protein expression dynamics during pig MuSCs differentiation using Western blotting revealed a significant decrease in protein expression levels during the early stages of differentiation (e.g., ...). Figure 2 As shown in Figure B, the results were consistent with those of transcriptome sequencing and RT-qPCR. However, the protein level of PRTFDC1 showed an upward trend in the later stages of differentiation. This dynamic change suggests that PRTFDC1 may play different regulatory roles in different differentiation stages of pig MuSCs. In addition, to clarify its subcellular localization, a recombinant vector for PRTFDC1 overexpression carrying a 6×His tag (PLVX-IRES-ZsGreen1-PRTFDC1) was constructed and transduced into MuSCs via a lentiviral system. Immunofluorescence detection using His protein antibody showed that PRTFDC1 protein was mainly localized in the nucleus (e.g., ...). Figure 2 (As shown in Figure C). Detection Example 2

[0034] Example 1 (corresponding to) Figure 3 PRTFDC1 OE ) and Comparative Example 1 (corresponding to Figure 3 Cells collected after culture in NC (Neuro-Nutritional Cell) were analyzed by qRT-PCR, Western blot, and MyHC immunofluorescence staining, and their differentiation index was statistically analyzed. The results are as follows: Figure 3 As shown. Among them, Figure 3 Figure A shows the detection of PRTFDC1 by qRT-PCR. OETwo days after MuSCs (i.e. MuSCs overexpressing PRTFDC1) were induced to differentiate, the relative mRNA expression levels of PRTFDC1, PAX7, MYOD, MYOG and MYH1 were measured. 18S rRNA was used as an internal reference gene, and cells infected with PLVX-IRES-ZsGreen1-Scramble lentivirus were used as negative control (NC). There were 6 biological replicates in each group. Figure 3 Figure B shows the Western blot analysis of PRTFDC1. OE The expression changes of PRTFDC1 and MYOG proteins after 2 days of MuSCs induced differentiation were studied, with β-TUBULIN as an internal control. Figure 3 Figure C shows the MYHC immunofluorescence staining assessment of PRTFDC1. OE Myotube formation of MuSCs at 1d and 2d of induced differentiation, Scale bar=200μm; Figure 3 Figure D shows the statistical analysis of the differentiation index, calculating the proportion of nuclei in myotubes containing ≥2 nuclei out of the total nuclei. Data were collected after 2 days of induced differentiation, with 5 biological replicates per group. P <0.05,** P <0.01,*** P <0.001.

[0035] Among them, myotube formation was observed by MYHC immunofluorescence staining. Two days after induction of differentiation, the PRTFDC1 overexpression group showed a significant increase in the number of myotubes, with longer tubes and a higher degree of fusion (results are shown in Figure 1). Figure 3 (As shown in Figure C). Further statistical analysis showed that its differentiation index (number of myotube nuclei with ≥2 nuclei / total number of nuclei) was significantly increased (26.5% vs. 40.1%). P <0.05, the result is as follows Figure 3 As shown in Figure D). RT-qPCR results showed that the mRNA level of PRTFDC1 was significantly increased in the overexpression group (as shown in Figure D). P <0.01), while the transcriptional levels of muscle differentiation-related genes MYOD, MYOG, and MYH1 were significantly upregulated (results are shown in Figure 1). Figure 3 (As shown in Figure A). Western blot results further confirmed the above trend, showing that PRTFDC1 overexpression significantly promoted MYOG protein expression (results are shown in Figure A). Figure 3 (As shown in Figure B). Detection Example 3

[0036] For Example 2 (corresponding) Figure 4 PRTFDC1-siRNA and Comparative Example 2 (corresponding to) Figure 4 Cells induced to differentiate in NC (non-nuclear) cells for 4 days were examined, and the results are as follows: Figure 4 As shown. Among them, Figure 4 Figure A shows the relative mRNA expression levels of PRTFDC1, PAX7, MYOD, MYOG, and MYH1 after 4 days of PRTFDC1-siRNA-induced differentiation by RT-qPCR. 18S rRNA was used as the internal reference gene, and non-target siRNA was used as the negative control (NC). Each group had 4 biological replicates. Figure 4 Figure B in the middle shows the Western blot analysis of the expression changes of PRTFDC1 and MYOG proteins 4 days after PRTFDC1-siRNA MuSCs induced differentiation, with GAPDH as an internal control. Figure 4 Figure C shows the myotube formation of PRTFDC1-siRNA MuSCs at 1d and 4d after induction of differentiation using MYHC immunofluorescence staining, with a scale bar of 200μm. Figure 4 Figure D shows the differentiation index of cells induced to differentiate on day 4, with 5 biological replicates per group. P <0.05,** P <0.01.

[0037] The above results show that myotube formation was inhibited and the differentiation index was significantly reduced in the PRTFDC1 knockdown group (74.0% vs. 61.3%). P <0.05, such as Figure 4 As shown in Figures C and D). RT-qPCR and Western blot results showed that the expression level of PRTFDC1 was significantly reduced in the knockdown group (as shown in Figures C and D). P <0.05), while the expression level of MYOG also decreased significantly (e.g., Figure 4 (As shown in Figures A and B). In summary, PRTFDC1 overexpression promotes myotube formation, while knockdown inhibits differentiation, indicating that PRTFDC1 plays a positive regulatory role in the differentiation of MuSCs in Sujiang pigs. Detection Example 4

[0038] The expression levels of cGAS, STING, and their downstream signaling molecules TBK1 and IRF3 in PRTFDC1-overexpressing MuSCs were detected by Western blot. The results are as follows: Figure 5 As shown. Among them, Figure 5 Figure A shows the expression and phosphorylation levels of key proteins in the cGAS-STING pathway, cGAS, STING, TBK1, and IRF3, detected by Western blot after overexpression of PRTFDC1 in MuSCs, with GAPDH as an internal control. Figure 5 Figure B shows the gray-scale quantitative statistical analysis of protein bands, with three biological replicates per group.P <0.05,** P <0.01.

[0039] pass Figure 5 It can be seen that PRTFDC1 overexpression significantly enhanced the activation levels of STING and its downstream molecules TBK1 and IRF3 (e.g., Figure 5 (As shown in Figures A and B). These results suggest that the regulatory role of PRTFDC1 is related to the cGAS–STING signaling pathway.

[0040] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0041] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0042] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. Use of the PRTFDC1 gene for promoting differentiation of porcine skeletal muscle satellite cells for purposes other than disease treatment.

2. Use according to claim 1, characterized in that, The pig is selected from the group consisting of a Suyang pig.

3. Use of the PRTFDC1 gene for accelerating myotube formation and skeletal muscle growth and development for purposes other than disease treatment.

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