Application of ESRRA protein and coding gene thereof in regulation and control of porcine skeletal muscle satellite cell differentiation

Abstract

The invention belongs to the technical field of molecular biotechnology and cell engineering engineering, discloses application of ESRRA protein or a coding gene thereof in regulation and control of porcine skeletal muscle satellite cell differentiation, and provides application of the ESRRA protein, the coding gene thereof or a biological material carrying the coding gene thereof in regulation and control of porcine skeletal muscle satellite cell differentiation. The encoding gene of the ESRRA protein is as shown in SEQ ID NO. 1. The ESRRA positively regulates myoblast differentiation and myotube formation, and the gene can be applied to regulation of pig skeletal muscle satellite cell differentiation, regulation of pig muscle development and pig variety breeding. The differentiation of the porcine skeletal muscle satellite cells can be promoted by increasing the expression quantity of the ESRRA gene, and the differentiation of the porcine skeletal muscle satellite cells can be inhibited by reducing the expression quantity of the ESRRA gene. The protein and the application thereof lay a foundation and provide a direction for further research on the regulation effect of the ESRRA gene in the aspects of pig muscle development, variety breeding and meat quality traits and the application of the ESRRA gene in the future.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology and cell engineering technology, and in particular to application of ESRRA protein and its encoding gene in regulating the differentiation of pig skeletal muscle satellite cells. Background Art

[0002] With the improvement of people's living standards and the strengthening of health awareness, the market demand for high-quality pork is also growing. Improving pork production and production efficiency has become an important issue that the pig farming industry needs to address urgently.

[0003] Skeletal muscle, as the primary component of pork, directly determines pork production through its growth rate and degree of development. Skeletal muscle development is complex, tightly regulated, and highly spatiotemporally specific. During the fetal period, mammals undergo two major waves of myofiber formation—primary and secondary myofibers—which largely determine the number of muscle fibers. After birth, myofibers further differentiate into slow-twitch and fast-twitch fibers, and increases in skeletal muscle mass primarily depend on the volume increase of existing myofibers. Previous studies have shown that the development and differentiation of skeletal muscle are regulated by multiple myogenic genes and transcription factors. Therefore, in-depth analysis of the key transcriptional regulators involved in myoblast differentiation has important theoretical and practical value for uncovering the core molecular mechanisms influencing porcine skeletal muscle growth and development, thereby contributing to genetic improvement and molecular breeding practices in porcine breeds. However, the number of publicly available regulatory genes involved in porcine skeletal muscle satellite cell differentiation is limited, and currently insufficient to meet the needs of current research and development and production practices.

[0004] Therefore, the existing technology needs to be further improved. Summary of the Invention

[0005] To address the above problems, the present invention provides the use of ESRRA protein or its encoding gene in regulating the differentiation of porcine skeletal muscle satellite cells. The ESRRA is an important transcriptional regulatory factor in the myogenesis process and plays a key role in regulating the differentiation of porcine skeletal muscle satellite cells.

[0006] By analyzing transcriptome data from the porcine longissimus dorsi muscle from embryonic days 33 to 105, combined with single-cell transcriptome data from porcine skeletal muscle, this study discovered for the first time that ESRRA is specifically highly expressed in late myoblast development. Furthermore, an in vitro myoblast differentiation model was constructed and RNA interference experiments were conducted. The results showed that knocking out ESRRA significantly inhibited myoblast differentiation and myotube formation. These results indicate that ESRRA plays a key role in regulating myoblast differentiation and is an important transcriptional regulator in the myogenesis process.

[0007] Based on the above research, this application provides the following technical solutions: In a first aspect, the present application provides the use of ESRRA protein, its encoding gene or a biomaterial carrying its encoding gene in regulating the differentiation of porcine skeletal muscle satellite cells. The encoding gene of the ESRRA protein is shown in SEQ ID NO. 1.

[0008] Experiments have shown that the expression level of the ESRRA protein encoded by the gene as shown in SEQ ID NO.1 shows a consistent upward trend with the key myogenic transcription factor MYOG, and significantly increases with the advancement of the differentiation process. This result suggests that ESRRA plays an important functional role in myoblast differentiation.

[0009] The protein expression levels of MYOG (a key regulatory factor for early myoblast differentiation) and MYHC (a terminal differentiation marker for myotube formation) in myoblasts after ESRRA knockdown were significantly reduced.

[0010] In addition, knockdown of ESRRA significantly reduced the differentiation index and fusion index of C2C12 cells, indicating that the expression of ESRRA plays an important positive regulatory role in the differentiation process of myoblasts and myotube fusion.

[0011] In a second aspect, the present application provides the use of an ESRRA protein, its encoding gene, or a biomaterial carrying its encoding gene in regulating porcine muscle development. The gene encoding the ESRRA protein is shown in SEQ ID NO. 1. By regulating the expression level of the ESRRA protein, the differentiation of porcine skeletal muscle satellite cells and myotube formation can be promoted or reduced, thereby regulating porcine muscle development.

[0012] In a third aspect, the present application provides the use of ESRRA protein, its encoding gene or biological materials carrying its encoding gene in pig breed breeding. The encoding gene of the ESRRA protein is shown in SEQ ID NO. 1.

[0013] Optionally, in the above application, the regulation is positive regulation.

[0014] Optionally, the application method is: 1) Increase the expression of the ESRRA gene to promote the differentiation of porcine skeletal muscle satellite cells and myotube formation; or 2) inhibiting the differentiation and myotube formation of porcine skeletal muscle satellite cells by reducing the expression of the ESRRA gene.

[0015] Optionally, in the above application, the application method is: the biological material is an expression cassette vector, a vector, a host cell or a recombinant bacterium.

[0016] Optionally, in the above application, the method of increasing the expression level of the ESRRA gene is selected from one or a combination of the following methods: 1) by introducing an expression vector containing the ESRRA gene; 2) by increasing the copy number of the ESRRA gene on the cell chromosome; 3) by operably linking a strong promoter to the gene; 4) By introducing enhancers.

[0017] Optionally, in the above application, the method of reducing the expression level of the ESRRA gene is selected from at least one of siRNA, shRNA, dsRNA, miRNA, CRISPR, TALEN and ZFN (zinc finger nuclease technology) gene editing technologies.

[0018] Optionally, the nucleotide sequence encoding the siRNA includes: siRNA-sense as shown in SEQ ID NO. 2, and siRNA-antisense as shown in SEQ ID NO. 3.

[0019] In a fourth aspect, the present application further provides a method for regulating pig muscle development, which regulates the expression of the ESRRA gene in the pig through a transgenic method. The gene encoding the ESRRA protein is shown in SEQ ID NO. 1.

[0020] The present invention has the following beneficial effects: 1. The present invention discovered for the first time that the ESRRA gene is specifically and highly expressed in the late developmental stage of myoblasts and positively regulates the differentiation of myoblasts.

[0021] 2. The present invention demonstrates that knocking out ESRRA significantly inhibits myoblast differentiation and myotube formation, clarifying that the porcine ESRRA gene plays an important regulatory role in the process of myoblast differentiation and myotube formation. This not only provides new ideas for studying the expression regulatory mechanism of muscle development, but also lays the foundation for further research on the regulatory role of the ESRRA gene in porcine muscle development, breed selection and meat quality traits in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a line graph of gene expression trends from embryonic days 33 to 105 in Example 1; Figure 2 Figure 2 is a cell type-specific gene expression map in the single-cell transcriptome of the longissimus dorsi muscle of pigs; A is a global clustering of the longissimus dorsi muscle cell population using the UMAP map; B is a graph showing the expression characteristics of specific marker genes in different cell types; C is a cell type indicator map; Figure 3 UMAP images of gene expression in porcine skeletal muscle satellite cell subpopulations; A shows the expression pattern of marker genes using UMAP images; B is a cell type indicator image; Figure 4 This is the pseudo-temporal developmental trajectory of pig skeletal muscle satellite cells; Figure 5 This is a heat map of transcription factor activity in porcine skeletal muscle satellite cells; Figure 6 The dynamic change trend of ESRRA transcription expression at different developmental stages; Figure 7 Figure 3 shows the effect of ESRAA on the myogenic differentiation of porcine skeletal muscle satellite cells; A and B show the changes in protein expression of ESRRA and myogenic marker MYOG on days 0, 2, 4, and 6 of myogenic differentiation during porcine skeletal muscle satellite cells; C and D show the changes in protein expression of ESRRA and myogenic-related factors on day 4 of myogenic differentiation after interference with ESRRA. Figure 8 Figure 3 is the effect of Esrra on the myogenic differentiation of C2C12 cells; A and B are the changes in the protein expression of Esrra and myogenic-related factors during the myogenic differentiation of C2C12 cells, i.e., on days 0, 2, 4, and 6 of induction; C and D are the changes in the protein expression of Esrra and myogenic-related factors on the fourth day of induction of myogenic differentiation after interference with Esrra; E is the cell immunofluorescence result on the fourth day of induction of myogenic differentiation after interference with Esrra; F is the analysis of the myotube differentiation index of Si-Esrra and Si-NC based on Figure A, and G is the quantitative analysis of the fusion index of Si-Esrra and Si-NC based on Figure A. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in this field. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0024] Example 1 Identification of ESRRA gene regulation of porcine skeletal muscle satellite cell differentiation based on multi-omics data 1. Establishment of a transcriptional map of myotube formation (1) Experimental methods: In this example, the transcriptome data of the longissimus dorsi muscle at embryonic days 33, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, and 105 of the pig were selected to construct a dynamic transcriptional map during myotube formation.

[0025] Specifically, based on the characteristics of porcine embryonic muscle development, the primary myotube formation stage was defined as embryonic days 33 to 65, and the secondary myotube formation stage was defined as embryonic days 70 to 105. Subsequently, a fuzzy C-means clustering algorithm was used to perform cluster analysis on gene expression patterns at different stages.

[0026] (2) Experimental results and analysis The experimental results are as follows Figure 1 As shown in the figure, seven distinct temporal expression clusters were identified during the primary myotube formation stage: Clusters 4 and 5 comprise genes with continuously increasing expression levels, Clusters 2, 3, and 7 comprise genes with gradually decreasing expression levels, and Clusters 1 and 6 exhibit a bimodal expression trend. Eight expression clusters were identified during the secondary myotube formation stage, with Clusters 1 and 3 containing genes with continuously upregulated expression levels, Cluster 4 containing genes with continuously downregulated expression levels, and the remaining clusters showing no significant temporal expression trends.

[0027] 2. Identification of cell types in porcine longissimus dorsi muscle (1) Experimental methods This example performed unsupervised cluster analysis on the single-cell transcriptome data of the longissimus dorsi muscle of pig based on Seurat software.

[0028] To further analyze the heterogeneity of myocytes, the UMAP dimensionality reduction algorithm was used to cluster them again, and the spatial expression distribution of MYOD1, S100A2 and ACTA1 in myocytes was analyzed.

[0029] To verify the accuracy of the above subpopulation annotations, pseudo-temporal developmental trajectory analysis was further carried out.

[0030] (2) Experimental results and analysis like Figure 2 As shown in the results, a total of 6 cell populations were identified, namely fibroadipogenic cells (FAPs, characteristic gene: DCN), immune cells (CORO1A), endothelial cells (VWF), muscle cells (PAX7, MYOD1, ACTA1, MYL9), red blood cells (GATA2) and glial cells (PLP1).

[0031] The results of the spatial expression distribution experiment in muscle cells showed that MYOD1 is a typical marker gene of myoblasts, S100A2 is highly expressed in the late stage of myoblast differentiation, and ACTA1 is a marker of mature muscle cells. Combining the expression patterns of the above marker genes, muscle cells are further annotated into early myoblasts, late myoblasts and mature myocytes, such as Figure 3 shown.

[0032] The results of pseudo-temporal developmental trajectory analysis revealed that early myoblasts, late myoblasts and mature myocytes exhibited a continuous and dynamic developmental trajectory, further supporting the biological rationality of the classification of myoblast subpopulations (e.g. Figure 4 shown).

[0033] 3. Analysis of the transcription factor regulatory network in porcine longissimus dorsi myoblasts (1) Experimental methods To deeply analyze the key regulatory mechanisms in the process of myoblast differentiation, this example uses the RSSZ method to accurately identify cell-specific transcription factors in early myoblasts, late myoblasts, and mature myocytes, and divides them into seven functional modules based on transcription factor activity.

[0034] (2) Experimental results and analysis The results show Figure 5 As shown in the figure, modules 1 and 2 showed higher activity in late myoblasts, the activity of modules 5 and 7 was significantly enhanced in early myoblasts, module 6 was mainly active in the myoblast stage, module 3 showed higher activity in both late myoblasts and mature myocytes, and module 4 was specifically active in the mature myoblast stage.

[0035] It is worth noting that the activity levels of multiple transcription factors in module 1 are significantly higher in late myoblasts than in other developmental stages, suggesting that the transcription factors in this module may play a key regulatory role in driving the differentiation of myoblasts into mature muscle cells.

[0036] 4. Identification of key functional genes regulating myoblast differentiation (1) Experimental methods Given that both the primary and secondary myotube formation stages are accompanied by the active differentiation of myoblasts, and that genes associated with myoblast differentiation generally show a trend of continuous upregulation in these two stages, this example integrates genes whose expression levels continuously increase during the primary and secondary myotube formation stages, as well as transcription factors with specific high activity in late myoblasts, and further combines gene expression levels for screening.

[0037] (2) Experimental results and analysis According to the above method, the key transcription factor regulating myoblast differentiation was finally identified as ESRRA. Figure 6 As shown, ESRRA showed a continuously upregulated expression pattern in both the primary and secondary myotube formation stages, suggesting that it may play a core regulatory role in the myoblast differentiation process and is an important functional transcription factor in this process. The sequence of this gene is shown in SEQ ID NO. 1 in the sequence listing.

[0038] Example 2 Application of ESRRA gene knockdown in regulating pig skeletal muscle satellite cell differentiation 1. Design siRNA In this example, the mRNA sequence of the porcine ESRRA gene was used as a template to design the interference target of the ESRRA gene, and the interference sequence was synthesized by Shanghai Gene Gene Co., Ltd. The primer sequences of the siRNA are as follows: P-siRNA-sense: 5'- GUGGGCGACAGAAGUACAATT-3' (SEQ ID NO.2) P-siRNA-antisense: 5'-UUGUACUUCUGUCGCCCACTT-3' (SEQ ID NO.3) 2. Purification and culture of porcine skeletal muscle satellite cells Prepare an appropriate amount of sterile PBS buffer containing 3% penicillin-streptomycin. Aseptically isolate the longissimus dorsi muscle tissue from the pig and immediately rinse it three times in this buffer. Transfer the muscle to a sterile, dry Petri dish. Use ophthalmic scissors and forceps to remove visible nerves, blood vessels, and connective tissue from the tissue surface and mince it to approximately 0.5–1 mm³. Place an appropriate amount of the tissue into a 50 mL centrifuge tube. Add enough 0.1% type II collagenase to completely cover the tissue. Mix thoroughly, seal the tube, and digest in a 37°C incubator for 2 hours, gently shaking the tube every 10–15 minutes to promote thorough digestion. After digestion, add an equal volume of complete culture medium supplemented with 10% fetal bovine serum (FBS) to terminate the reaction. Filter the digestion solution through 100-, 200-, and 400-mesh disposable sterile cell strainers, sequentially. Collect the filtrate into a 50 mL centrifuge tube and centrifuge at 1500 rpm for 10 minutes. After discarding the supernatant, resuspend the pellet in 10 mL of serum-free DMEM high-glucose medium, repeat centrifugation (1500 rpm, 10 minutes), and discard the supernatant. Finally, resuspend the resulting cell pellet in complete medium. The resulting cells were pipetted and mixed to prepare a single-cell suspension, and the cells were counted at 1×10 6 Cells were seeded at a density of 10 cells / mL in a T25 cell culture flask. An appropriate amount of complete culture medium was added, labeled P0, and cultured in a 37°C, 5% CO2, humidified incubator. After 2 hours of culture, the supernatant from flask P0 (containing non-adherent cells) was transferred to a new flask labeled P01. Twelve hours later, the supernatant was transferred again to another flask labeled P02. The resulting P01 and P02 cells are purified porcine skeletal muscle satellite cells. When the cells reached approximately 90% confluency, the medium was changed to DMEM high-glucose medium supplemented with 2% horse serum for myogenic differentiation. The medium was changed daily to maintain the induction environment.

[0039] 3. Cell transfection (1) One day before transfection, the cultured porcine skeletal muscle satellite cells were inoculated into a 6 cm dish at a density of 5 × 10 5 , placed in a 37°C, 5% CO2 cell culture incubator, and the cells grew to a density of 60%-80% for transfection.

[0040] (2) Aspirate the culture medium from the 6 cm dish and add 2 ml of DMEM high glucose medium without double antibody and containing serum to each dish. Then add the transfection mixture. The transfection mixture for each group is as follows: Prepare the GP-transfect-Mate-medium mixture. For one dish, add 500 μL of high-glucose DMEM (serum-free and antibiotic-free) to a 1.5 ml sterile centrifuge tube, add 10 μL of transfection reagent, mix gently with a pipette, and let stand at room temperature for 5 minutes.

[0041] Group A: Control group: The transfection mixture included: 500 μL DMEM high glucose medium and 15 μL NC, which was gently mixed with a pipette and allowed to stand for 5 minutes.

[0042] Group B: Experimental group: The transfection mixture included: 500 μL DMEM high glucose medium, 15 μL Si-ESRRA, gently mixed with a pipette, and let it stand for 5 minutes.

[0043] Add the GP-transfect-Mate-medium mixture dropwise to the RNA oligo-medium mixture, mix gently with a pipette, let it stand at room temperature for 20 minutes, and then transfect immediately.

[0044] (3) Add the above liquids dropwise to the cell culture dish and shake the dish to evenly disperse the transfection mixture.

[0045] (4) 24 hours after transfection, replace the differentiation medium and continue culturing.

[0046] (5) On the fourth day of differentiation, cells were collected for analysis.

[0047] 4. Detection of myoblast differentiation marker proteins Cells were harvested by centrifugation and lysed using RIPA buffer. Extracted proteins were then separated by 10% SDS-PAGE gel electrophoresis using a pre-run at 80 V for 20 minutes followed by 120 V for 2.5 hours. Proteins were transferred to a PVDF membrane using a Bio-Rad Trans-Blot system. The membrane was blocked on ice with 5% BSA in PBST buffer for 4 hours and then incubated with primary and secondary antibodies overnight and 2 hours, respectively. Protein bands were detected using the BeyoECL Star chemiluminescence kit, and Western blots were quantified using AlphaView SA software using the default user guide parameters.

[0048] During the differentiation of porcine skeletal muscle satellite cells, i.e., on days 0, 2, 4, and 6 after replacing the induction medium containing 2% horse serum (HS), cells were lysed to obtain proteins for Western blot. Figure 7 The Western blot experimental results of A showed that the expression level of ESRRA showed a consistent upward trend with the key myogenic transcription factor MYOG, and increased significantly with the advancement of differentiation. This result suggests that ESRRA plays an important functional role in myoblast differentiation.

[0049] Further ESRRA knockdown experiments revealed that after transfection of Si-ESRRA or Si-NC into porcine skeletal muscle satellite cells and induction of myogenic differentiation for four days, the cells were lysed to obtain proteins for Western blot analysis. The experimental results were as follows: Figure 7 As shown in Figure C, the protein expression levels of MYOG and MYHC in myoblasts after ESRRA knockdown were significantly reduced. The downregulation of MYHC, a terminal differentiation marker for myotube formation, reflects the blockage of myoblast differentiation. The simultaneous decrease in MYOG, a key regulator of early myoblast differentiation, further suggests that ESRRA may exert dual regulatory effects by mediating both the early and terminal stages of the differentiation cascade, thereby interfering with the differentiation process of myoblasts into mature myotubes.

[0050] Example 3 ESRRA gene mediates mouse myoblast differentiation 1. Design siRNA In this example, the mRNA sequence of the mouse Esrra gene was used as a template to design an interference target for the Esrra gene, and Shanghai Gene Gene Co., Ltd. was commissioned to synthesize the corresponding siRNA interference sequence. The primer sequences are as follows: M-siRNA-sense: 5'- CCUCCAAUGAGUGUGAGAUTT-3' (SEQ ID NO.4) M-siRNA-antisense: 5'- AUCUCACACUCAUUGGAGGTT-3' (SEQ ID NO.5) 2. C2C12 cell culture and passaging The cells used in this experiment were C2C12 (CL-0044) cells, provided by Wuhan Punosai Life Science Co., Ltd. Aspirate the original culture medium, add 1 ml of 1× PBS, and gently shake the 6 cm culture dish to rinse the cells three times. Discard the 1× PBS. Add 1 ml of 0.25% trypsin solution (containing EDTA) and gently shake evenly. Place the dish in an incubator for digestion. When cells in the center of the cell clump become noticeably rounded and interspaced under a microscope, add 1 ml of DMEM high-glucose medium containing 10% fetal bovine serum to terminate digestion. Pipette the cells to minimize the size of a single-cell suspension. Collect the cell suspension and centrifuge at 1200 rpm for 3 minutes. Aspirate and discard the supernatant. Add fresh complete medium, inoculate the cells into a new dish according to the desired ratio, top up the medium, and incubate at 37°C.

[0051] 3. Myogenic differentiation of mouse C2C12 myoblasts When the cell density reached 80%-90%, the differentiation medium was replaced with DMEM high glucose medium containing 2% horse serum and 1% penicillin-streptomycin to induce differentiation. The differentiation medium was replaced every 48 hours.

[0052] 4. Cell transfection (1) One day before transfection, C2C12 cells within ten generations were selected and inoculated in a 24-well plate at a density of 5×10 4 , transfection was performed when the cells covered about 50% of the bottom of the dish; (2) Aspirate the culture medium from the 24-well plate and add 400 μL of DMEM high-glucose medium without double-antibody and containing serum to each well. Then add the transfection mixture. The transfection mixtures for each group are as follows: Prepare the GP-transfect-Mate-medium mixture. Take one well as an example: add 50 μL of high-glucose DMEM (serum-containing, antibiotic-free) to a 1.5 ml sterile centrifuge tube, and add 2 μL of transfection reagent. Mix gently with a pipette and let it stand at room temperature for 5 minutes.

[0053] Group A: Control group: The transfection mixture included: 50 μL DMEM high glucose medium and 2 μL NC, which was gently mixed with a pipette and allowed to stand for 5 minutes.

[0054] Group B: Experimental group: The transfection mixture included: 50 μL DMEM high glucose medium, 2 μL Si-Esrra, gently mixed with a pipette, and let it stand for 5 minutes.

[0055] Add the GP-transfect-Mate-medium mixture dropwise to the RNA oligo-medium mixture, mix gently with a pipette, let it stand at room temperature for 20 minutes, and then transfect immediately.

[0056] (3) Add the above liquid to the cell culture dish dropwise and shake the 24-well plate to evenly disperse the transfection mixture.

[0057] (4) After 24 hours of transfection, the differentiation medium was replaced and cultured.

[0058] (5) On the fourth day of differentiation, cells were collected for analysis.

[0059] 5. Cell Immunofluorescence Detection (1) Detection method C2C12 cells were passaged and plated in 24-well plates. When cell confluency reached 80%–90%, differentiation was induced by replacing the culture medium with 2% horse serum. On day 4 of differentiation (D4), cells were washed three times with 1× PBS, fixed with 4% paraformaldehyde for 20 minutes, washed three times with 1× PBS, treated with 5% Triton X-100 for 15 minutes to increase permeability, and washed with 1× PBS for 10 minutes. Subsequently, cells were blocked with 5% BSA blocking solution for 1 hour and washed again with 1× PBS for 10 minutes. Primary antibodies were diluted 1:100 in 5% BSA and incubated overnight at 4°C. The following day, fluorescently labeled secondary antibodies were incubated at 37°C for 1 hour in the dark. Nuclei were stained with DAPI for 10 minutes and washed three times with 1× PBS (10 minutes each). Cells were then observed and photographed using a fluorescence microscope. C2C12 cells were transfected with si-Esrra and si-NC, respectively. After 4 days of differentiation, the cells were fixed and MyHC immunofluorescence staining was performed to evaluate myotube formation.

[0060] (2) Test results and analysis The results showed that downregulation of ESRRA expression significantly reduced the number of MyHC-positive cells. ＋ The fusion index is defined as the percentage of the total number of nuclei in the differentiated myotubes to the total number of nuclei (for statistical convenience, myotubes are defined as containing MYHC cells with 3 or more nuclei). ＋ cell).

[0061] Figure 8 F. Figure 8 The quantitative results of G showed that knockdown of ESRRA significantly reduced the differentiation index and fusion index of C2C12 cells. This result indicates that the expression of ESRRA has an important positive regulatory effect on the differentiation process of myoblasts and myotube fusion.

[0062] 6. Protein detection of C2C12 cell myogenic differentiation ability (1) Detection method After C2C12 cell samples were collected on days 0, 2, 4, and 6 of myogenic differentiation induction, the protein expression levels of MYOG and Esrra were detected by Western blot. Figure 8 The detection results of B showed that the protein level of Esrra showed a gradual upward trend during the differentiation process of C2C12 cells, that is, on days 0, 2, 4, and 6 of induced differentiation. Its expression pattern was consistent with the classic marker proteins MYOG and MYHC in the process of myogenic differentiation, suggesting that Esrra may play an important role in the differentiation of myoblasts.

[0063] After four days of differentiation induction, cell samples were collected from C2C12 cells transfected with Si-Esrra or Si-NC, and the protein expression levels of MYOG, MYHC and Esrra were detected by Western blot. Figure 8 C and Figure 8 As shown in Figure D, the results show that after interfering with Esrra, the differentiation marker proteins MYOG and MYHC decreased significantly, which further verified the positive regulatory function of Esrra in myoblast differentiation. In addition, this expression pattern is highly consistent with the change trend observed in Example 2 during the differentiation of porcine skeletal muscle satellite cells.

[0064] The above results indicate that the function of ESRRA in regulating myogenic differentiation may be conserved in different species.

[0065] It is to be understood that, for those skilled in the art, equivalent replacements or changes can be made according to the technical solutions and the inventive concept of the present invention, and all such changes or replacements should fall within the scope of protection of the claims appended hereto. Without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented in a wider range with equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application includes any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied within the scope of the following claims.

Claims

1. Application of ESRRA protein, its encoding gene or biomaterial carrying its encoding gene in regulating the differentiation of porcine skeletal muscle satellite cells, characterized in that: The gene encoding the ESRRA protein is shown in SEQ ID NO.

1.

2. Application of ESRRA protein, its encoding gene or biomaterial carrying its encoding gene in regulating pig muscle development, characterized in that: The gene encoding the ESRRA protein is shown in SEQ ID NO.

1.

3. Application of ESRRA protein, its encoding gene or biological material carrying its encoding gene in pig breed selection, characterized in that: The gene encoding the ESRRA protein is shown in SEQ ID NO.

1.

4. The use according to any one of claims 1 to 3, characterized in that The regulation is positive regulation.

5. The use according to claim 4, characterized in that The application method is: 1) Increase the expression of the ESRRA gene to promote the differentiation of porcine skeletal muscle satellite cells and myotube formation; or 2) inhibiting the differentiation and myotube formation of porcine skeletal muscle satellite cells by reducing the expression of the ESRRA gene.

6. The use according to claim 4, characterized in that The application method is as follows: the biological material is an expression cassette vector, a vector, a host cell or a recombinant bacterium.

7. The use according to claim 4, characterized in that The method for increasing the expression level of the ESRRA gene is selected from one or a combination of the following methods: 1) by introducing an expression vector containing the ESRRA gene; 2) by increasing the copy number of the ESRRA gene on the cell chromosome; 3) by operably linking a strong promoter to the gene; 4) By introducing enhancers.

8. The use according to claim 4, characterized in that The method of reducing the expression level of the ESRRA gene is selected from at least one of siRNA, shRNA, dsRNA, miRNA, CRISPR, TALEN and ZFN.

9. The use according to claim 8, characterized in that The nucleotide sequence encoding the siRNA includes: P-siRNA-sense as shown in SEQ ID NO. 2, and P-siRNA-antisense as shown in SEQ ID NO.

3.

10. A method for regulating pig muscle development, characterized in that: The expression of the ESRRA gene in pigs is regulated by a transgenic method. The gene encoding the ESRRA protein is shown in SEQ ID NO. 1.

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