Application of RERGL gene in regulating bovine intramuscular fat deposition

Abstract

The present application relates to the field of bioengineering technology, and particularly relates to the application of RERGL gene in regulating intramuscular fat deposition of cattle. The present application discloses the function and application of Ras-related estrogen-regulated growth-inhibitory factor-like protein RERGL gene in regulating intramuscular fat deposition of cattle. Through cell experiments and animal model experiments, it is confirmed that overexpression of RERGL gene can significantly promote adipogenic differentiation of CD31 ‑ / CD45 ‑ / PDGFRα + fibroblast / adipocyte progenitor cells FAPs, increase lipid droplet accumulation, and can promote lipid deposition in muscle tissue and increase serum triglyceride content in mice in vivo; the mechanism is to activate the expression of key genes such as PPARγ, FASN, ADIPOQ, FABP4 and other adipogenic differentiation genes at the transcription and protein levels, so as to regulate fat metabolism and regional deposition. The present application proves that RERGL gene can promote intramuscular fat deposition of cattle, and provides a reference for cattle molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to... RERGL The function and application of genes in regulating intramuscular fat deposition in cattle. Background Technology

[0002] The beef cattle industry is an important component of my country's livestock system, occupying a key position in the agricultural economy and serving as a strategic necessity for ensuring national food security. With the continuous improvement of residents' consumption levels, the demand for beef is constantly growing, driving the continuous expansion of the industry. Market consumption demand is gradually shifting from "satisfying quantity" to "pursuing quality." Against this backdrop, how to improve beef quality has gradually become a key research focus in this field.

[0003] Adequate intramuscular fat (IMF) can significantly improve the tenderness, juiciness, flavor, and color of beef. Therefore, regulating fat metabolism and regional deposition has become a core scientific issue for improving beef carcass quality and producing high-quality beef. From a physiological perspective, fat deposition is essentially a result of nutrient allocation in the body. Appropriate reserves are necessary to maintain life activities, but excessive deposition leads to an imbalance in energy homeostasis, resulting in wasted feed resources and reduced beef quality. Based on this, developing intramuscular fat deposition regulation technology has become a current research focus for synergistically improving beef quality and beef cattle production efficiency.

[0004] Ras-related and Estrogen-Regulated Growth Inhibitor-like protein (RERGL) is a member of the Ras family, a small GTPase superfamily, and its encoding gene is located on human chromosome 12 (12p12.3). RERGL The protein is 205 amino acids long with a molecular weight of approximately 23.9 kDa. It can bind GDP / GTP and may possess intrinsic GTPase activity. Previous studies have shown that... RERGL The gene is widely expressed in the endometrium (RPKM 13.0) and adipose tissue (RPKM 12.7), with the highest expression level in the tibial artery (RPKM 48.29), suggesting that it may be involved in the regulation of adipose tissue metabolism.

[0005] CN202511051665.X discloses a technical solution that uses a high-energy, low-fiber diet in the early fattening stage, combined with a low-protein diet without soybean meal in the later fattening stage, to synergistically promote intramuscular fat deposition in yaks, thereby achieving high-efficiency production of yak marbled beef. CN202510926676.1 discloses that vitamin A can activate the expression of EBF2 through its active metabolite RA. EBF2 can target and inhibit the transcriptional process of CYP26B1 to maintain the activity of the retinol signaling pathway, thereby activating PPARγ and its downstream lipid metabolism-related genes, promoting fatty acid transport and lipid accumulation in bovine intramuscular fat cells, and thus promoting intramuscular fat deposition and improving beef quality. Currently, there is no application of the RERGL gene in regulating bovine intramuscular fat deposition. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to disclose RERGL This invention investigates the function of a gene in bovine intramuscular fat deposition, elucidates its regulatory role in adipogenic differentiation of bovine fat progenitor cells and lipid accumulation in muscle tissue, and applies it to beef quality improvement, breeding of high intramuscular fat cattle, and molecular marker breeding. This invention confirms... RERGL The gene can significantly promote the production and deposition of intramuscular fat in cattle, and related application methods and regulatory approaches are proposed.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides RERGL The application of the gene in the preparation of a formulation that promotes intramuscular fat deposition in bovine muscles, wherein the gene... RERGL The gene has the GenBank accession number NM_001286201.2, and its sequence is shown in SEQ ID NO:1, or a homologous gene of the same sequence with ≥95% homology that promotes intramuscular fat deposition in bovine muscle. Intramuscular fat deposition can be significantly promoted by increasing the expression level of the RERGL gene in target bovine tissues or cells.

[0008] This invention also provides RERGL The application of a gene-encoded protein in the preparation of a formulation that promotes intramuscular fat deposition in bovine muscle, wherein the protein is encoded by the nucleotide sequence of GenBank number NM_001286201.2, the gene sequence of which is shown in SEQ ID NO:1, or by a protein mutant obtained by amino acid substitution, deletion, or insertion that retains the function of promoting intramuscular fat deposition. Preferably, the promotion of intramuscular fat deposition includes one or more of promoting the generation, maturation, and / or lipid accumulation of intramuscular adipocytes.

[0009] By using the aforementioned genes or proteins as direct intervention targets, various forms of formulations can be constructed, such as gene vector formulations and recombinant protein formulations, to increase the fat content in bovine muscle tissue.

[0010] The present invention further provides a recombinant expression vector for expression in mammalian cells. RERGL Genes, the ones mentioned RERGL The gene is as described above. By efficiently expressing the RERGL gene in bovine tissues or cells, direct intervention in intramuscular fat deposition can be achieved.

[0011] Based on this, the present invention provides the application of the recombinant expression vector in breeding beef cattle with high intramuscular fat content. By introducing the recombinant expression vector into relevant cells or tissues in the bovine body, ... RERGL The gene is overexpressed locally or systematically, thereby increasing intramuscular fat levels in beef cattle and improving meat quality. Preferably, the recombinant expression vector is a recombinant lentiviral vector of pLV3-CMV-RERGL(cattle)-3×FLAG-CopGFP-Puro, the vector plasmid sequence of which is shown in SEQ ID NO:5, and can achieve stable high expression of the RERGL gene in mammalian cells.

[0012] Preferably, the recombinant expression vector is applied to bovine fibroblast / adipocyte progenitor cells and / or bovine muscle tissue to enable... RERGL Overexpression within target cells significantly promotes intramuscular fat production and lipid accumulation; more preferably, the fibroblasts / adipocyte progenitors possess CD31. - / CD45 - / PDGFRα + Phenotype.

[0013] This invention also provides RERGL Application of genes as molecular markers in beef cattle quality selection. This involves detecting genes in individual beef cattle... RERGL Expression levels of genes and / or their encoded proteins, and / or detection RERGL Nucleotide polymorphisms in gene coding or regulatory regions, for screening RERGL Individuals with high expression or favorable genotypes can be used as breeding parents to obtain offspring with higher intramuscular fat content and better meat quality traits. This approach can be combined with traditional breeding indicators to provide new candidate markers for marker-assisted selection of beef cattle quality traits.

[0014] This invention further provides a method for regulating intramuscular fat deposition in bovine muscles, comprising: constructing a... RERGL The gene is an overexpression vector containing the inserted fragment, and is introduced into bovine fibroblast / adipocyte progenitor cells and / or bovine muscle tissue using viral transduction or other gene transfer methods, so that... RERGLThe overexpression vector efficiently expresses the drug in target cells, and through in vitro culture of transduced cells and / or feeding of treated beef cattle, it regulates intramuscular fat deposition in cattle, preferably promoting the generation, maturation, and lipid accumulation of intramuscular fat. The overexpression vector is preferably a recombinant lentiviral vector, more preferably the pLV3-CMV-RERGL(cattle)-3×FLAG-CopGFP-Puro vector.

[0015] In the above method, the regulatory effect on intramuscular fat deposition can be evaluated by optionally detecting the expression levels of genes such as PPARγ, FASN, ADIPOQ, and FABP4 (key adipogenic factors) and / or detecting the expression levels of proteins such as FASN, PPARγ, ADIPOQ, and FABP4, and / or by assessing lipid accumulation through methods such as lipid droplet staining. This evaluation step helps optimize vector construction, administration methods, and application conditions, but it is not a necessary condition for the realization of the technical solution of this invention.

[0016] The advantages of this invention compared to the prior art are as follows: 1. This invention clarifies that RERGL The gene is a key regulator of intramuscular fat deposition in cattle, filling the gap in "intramuscular fat regulation targets" in bovine molecular breeding. It can replace the inefficient traditional phenotype-dependent breeding model and provide a precise molecular breeding direction for improving the quality of beef cattle.

[0017] 2. Validated by both bovine FAPs cell experiments and mouse model experiments. RERGL Gene overexpression can stably activate adipogenic differentiation-related pathways, ensuring reliable regulation of intramuscular fat deposition during beef cattle breeding and avoiding bias in results from a single experimental scenario.

[0018] 3. The related technical solutions proposed in this invention have the characteristics of standardization: RERGL The core technical processes, including overexpression vector construction, bovine FAPs cell sorting, and lentivirus transfection, are clearly defined and require no complex specialized equipment. This facilitates rapid learning and application by various breeding institutions, effectively lowering the technical threshold for bovine molecular breeding and accelerating the development of high intramuscular fat beef cattle breeds. Furthermore, it allows for... RERGL Genes are used as an indicator for evaluating breeding effectiveness. By detecting the gene expression levels and intramuscular fat content of selected offspring, the effectiveness of breeding programs can be quickly verified, reducing the trial-and-error costs during the breeding cycle. Attached Figure Description

[0019] Figure 1 Map of RERGL (cattle) overexpression plasmid vectors Figure 2 Flowchart for bovine FAPs cell extraction Figure 3BODIPY fluorescence staining image of bovine FAPs cells overexpressing the RERGL gene. Figure 4 Oil Red O staining for overexpression of the RERGL gene in bovine FAPs cells. Figure 5 Western blot assay for RERGL gene overexpression in bovine FAPs cells Figure 6 BODIPY fluorescence staining image of RERGL gene overexpression in mice. Figure 7 Oil Red O staining image of mouse muscle overexpressing the RERGL gene. Figure 8 Western blot assay for RERGL gene overexpression in mice Figure 9 qRT-PCR detection of RERGL gene overexpression in mice Figure 10 Detection of serum triglyceride levels in mice with RERGL gene overexpression Detailed Implementation The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0020] Example 1: Construction of overexpression plasmids and preparation of lentivirus solution (a) Construction of overexpression plasmids 1. Gene amplification: The target gene RERGL was synthesized, and its nucleotide sequence is shown in SEQ ID NO:1.

[0021] atgaatgatgtgaagcttactgttttgggaggtgaaggaacagggaaatctgcccttatagtaaggtttcttaccaagcgcttcattggagaatatgcttctaattttgaatctatctataacaaacatttgtgtttggaagggaagcaattg aatctagaaatatatgacccttgttctcagccacagaaagcaaaattttccctcacaagtgagctgcattgggcagatgggtttgttattgtgtatgacatcagtgacaggtcttcctttgcatttgcaaaagcattaatctacagaattcgg gagccacagacaagtcattgtaaaagacctgtggagtcagcagtgcttttggtgggtaacaagcaagatctctgtcatgtgcgagaggttggctgggaagaagggcacaaactggcattggataaccggtgccaattctgtgaactgtctgca gcagagcaatctctggaggtggaaatgatgtttatcagaattatcagggacatcctgacaaacttcaaactcaaagagaagagaagatacagtggatctaaatccatggccaagctgatcaataatgtatttggaaagagaaggaaatctgtt The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 is shown in SEQ ID NO:2.

[0022] MNDVKLTVLGGEGTGKSALIVRFLTKRFIGEYASNFESIYNKHLCLEGKQLNLEIYDPCSQPQKAKFSLTSELHWADGFVIVYDISDRSSFAFAKALIYRIREPQTSHCKRPVESAVLLVGNKQDLCHVREVGWEEGHKLALDNRCQFCELSAAEQSLEVEMMFIRIIRDILTNFKLKEKRRYSGSKSMAKLINNVFGKRRKSV was amplified using I-5™ 2×High-Fidelity Master Mix polymerase. The specific reaction system is as follows: The primer sequences are as follows: Primer F1: ATGAATGATGTGAAGCTTACTGTTTTG, SEQ ID NO:3; Primer R1: AACAGATTTCCTTCTCTTTCCAAATAC, SEQ ID NO:4.

[0023] Amplification conditions: The above products were subjected to electrophoresis, and the target band was recovered. 2. Vector enzyme digestion: The above products were subjected to electrophoresis, and the target band was recovered. 3. Gene and vector ligation: Connect according to the following reaction system: All of the above products were transformed into competent Escherichia coli DH5α, plated on plates with the corresponding antibiotics, and incubated overnight at 37°C; among them, RERGL The gene's GenBank ID is NM_001286201.2; after screening for positive clones, plasmids were extracted and sequenced. The vector map for the bovine (cattle) source is pLV3-CMV-RERGL(cattle)-3×FLAG-CopGFP-Puro, used to verify element integrity, such as... Figure 1 As shown.

[0024] Sequencing results of the pLV3-CMV-RERGL (cattle)-3×FLAG-CopGFP-Puro plasmid are shown in SEQ ID NO:5: GAATTC GCCACC ATGAATGATGTGAAGCTTACTGTTTTGGGAGGTGAAGGAACAGGGAAATCTGCCCTTATAGTA AGGTTTCTTACCAAGCGCTTCATTGGAGAATATGCTTCTAATTTTGAATCTATCTATAACAAACATTTGTGTTTGGA AGGGAAGCAATTGAATCTAGAAATATATGACCCTTGTTCTCAGCCACAGAAAGCAAAATTTTCCCTCACAAGTGAGC TGCATTGGGCAGATGGGTTTGTTATTGTGTATGACATCAGTGACAGGTCTTCCTTTGCATTTGCAAAAGCATTAATC TACAGAATTCGGGAGCCACAGACAAGTCATTGTAAAAGACCTGTGGAGTCAGCAGTGCTTTTGGTGGGTAACAAGCA AGATCTCTGTCATGTGCGAGAGGTTGGCTGGGAAGAAGGGCACAAACTGGCATTGGATAACCGGTGCCAATTCTGTG AACTGTCTGCAGCAGAGCAATCTCTGGAGGTGGAAATGATGTTTATCAGAATTATCAGGGACATCCTGACAAACTTC AAACTCAAAGAGAAGAGAAGATACAGTGGATCTAAATCCATGGCCAAGCTGATCAATAATGTATTTGGAAAGAGAAG GAAATCTGTT GGATCC The italicized and bolded sections represent the EroR I and BamH I restriction sites, while the double-underlined and bolded sections represent the RERGL gene.

[0025] (II) Preparation of Lentiviral Fluid 1. Plasmid co-transfection of packaging cells The RERGL overexpression plasmid pLV3-CMV-RERGL-3×FLAG-CopGFP-Puro, which was verified by sequencing, was mixed with a lentiviral helper plasmid at an optimized ratio (target plasmid (pLV3-CMV-RERGL-3×FLAG-CopGFP-Puro): packaging plasmid (e.g., psPAX2): envelope plasmid (e.g., pMD2.G) = 3:2:1). 293T packaging cells were co-transfected using Lipofectamine 3000 Reagent (catalog number: L3000015). After transfection, the cells were incubated at 37°C in a 5% CO2 incubator for 48–72 h to allow the lentiviral particles to complete packaging within the cells and be released into the culture medium.

[0026] 2. Virus fluid collection and purification Cell supernatants were collected at 24 h, 48 h, and 72 h post-transfection. Cell debris was removed by centrifugation, and the virus solution was filtered through a 0.4 μm filter membrane. The virus solution was then concentrated by ultracentrifugation or column purification to obtain high-titer RERGL lentivirus solution.

[0027] 3. Virus titer determination Viral genomic RNA was detected by quantitative real-time PCR or infectious particles were detected by limiting dilution method. Lentiviral titers were determined to ensure that the quality of the viral solution met the experimental requirements.

[0028] Example 2, Cell Model Experiment (a) CD31 - / CD45 - / PDGFRα + Fibroblast / adipocyte progenitor (FAP) extract 1. Sample pretreatment: Take fresh longissimus dorsi muscle from cattle, remove surface fat and connective tissue, cut about 1 g of muscle tissue, add washing solution, and centrifuge at 1000 g for 5 min.

[0029] 2. Enzymatic digestion: Discard the supernatant, add 2-3 times the volume of collagenase, thoroughly mince the muscle tissue, and digest it in a shaker at 120 rpm and 37°C for 1 hour.

[0030] 3. Digestion termination: Transfer the digestion solution to a 50 mL centrifuge tube and add 10 times the volume of pre-cooled washing solution to terminate the digestion.

[0031] 4. Filtration and centrifugation: Filter through 100 μm and 70 μm filter membranes in sequence, collect the filtrate and centrifuge at 500 g for 15 min.

[0032] 5. Cell washing: Discard the supernatant, add 8 mL of washing buffer to resuspend the cells, transfer to a 10 mL centrifuge tube, centrifuge at 500 g for 5 min at room temperature, and repeat this step twice.

[0033] 6. Initial culture: Discard the supernatant, add pre-warmed complete culture medium to resuspend the cells, seed them in culture dishes, and transfer them to an incubator for 4-6 hours of culture.

[0034] 7. Change the medium for culture: Discard the supernatant, rinse once with washing solution, replace with new complete medium and continue culture for 8 hours, then change the medium again for later use.

[0035] 8. Flow cytometry (after 48 h of plate culture) ① Discard the supernatant, rinse once with 2 ml of washing buffer, discard the washing buffer, digest with 2 ml of trypsin for 2 min, add 2 ml of complete culture medium and resuspend the cells by pipetting, centrifuge at 500 g for 5 min at 4℃. ② Discard the supernatant, add 1 ml of washing solution and count the contents, then centrifuge at 500 g for 5 min; ③ Discard the supernatant and mix the cells with CD31 + CD45 + PDGFRα + The fluorescent antibody was incubated at 4°C in the dark for 30 min (mixed once every 5 min), and an unstained control group and a single-stained control group were set up.

[0036] Note: Add 1 µL of antibody to every 100 µL of washing buffer for incubation of 1 million cells; the control group is incubated with 100,000 cells.

[0037] 5 million cells were sorted (using 5 ml tubes): Whole-stain group: 5 µL CD31 + 5 µL CD45 + 5 µL PDGFRα + 485 µL of washing solution containing 5 million cells; CD31 single-stain control group: 1 µL CD31 + 99 µL of washing solution containing 100,000 cells; CD45 monostaining control group: 1 µL CD45 + 99 µL of washing solution containing 100,000 cells; PDGFRα single staining control group: 1 µL PDGFRα + 99 µL of washing solution containing 100,000 cells; Unstained control group: 500 µL of loading solution containing 100,000 cells (stored at 4°C).

[0038] ④ Add 1 mL of washing buffer to the staining tube, centrifuge at 500 g for 5 min at 4℃, discard the supernatant, and repeat twice.

[0039] ⑤ For the fully stained group, add 2 mL of loading solution and transfer the cells to a flow cytometry sorting tube. For the single-stained control group, add 500 µL of loading solution and transfer the cells to a flow cytometry sorting tube. Incubate at 4°C and transport to the machine.

[0040] ⑥ Use the unstained control group for gate setting and set the basic fluorescence; use the monostained control group for gate setting and set the compensation fluorescence.

[0041] ⑦ Sorting FAPs cells into CD31 - / CD45 - / PDGFRα + .

[0042] ⑧ At the end of sorting, transfer the FAPs cells to a 15 mL tube for storage.

[0043] (ii) CD31 - / CD45 - / PDGFRα + FAPs cell adipogenesis culture Experiments were conducted using pre-adipogenic FAPs cells (60% confluence) in three groups: saline group (CON), empty vector plasmid group (NC), and key gene overexpression group (OE-RERGL). Subsequently, adipogenic differentiation culture was induced and cultured according to the adipogenic differentiation culture protocol, and the results were detected.

[0044] The methods for inducing differentiation culture are as follows: 1. Adipogenic Differentiation Medium (ADM) Formulation 2. Preparation method of stock solution (freeze at -20℃, shelf life 6 months) 3. FAPs cell adipogenesis culture steps (1) Cell seeding: Resuspend the sorted FAPs cells in complete culture medium, adjust the cell density and seed them into culture wells / dishes, and incubate them in an incubator until the cell confluence reaches 80%-90% (about 2-3 days).

[0045] (2) Pre-induction treatment (days 1-3): Discard the supernatant and rinse once with washing solution; add freshly prepared ADM inducer I solution and incubate at 37℃ in a 5% CO2 incubator.

[0046] (3) Late induction treatment (days 4-8): After day 3, discard the early induction culture medium and rinse once with washing buffer; add freshly prepared ADM inducer II solution; change the late induction culture medium every 2 days and continue culturing until day 8. The adipogenesis process of FAPs cells is as follows... Figure 2 As shown.

[0047] (III) Detection of adipogenic differentiation capacity of cells The assay for adipogenic differentiation capacity of cells mainly includes: (1) BODIPY and Oil Red O staining methods were used to detect cell differentiation status.

[0048] (2) Western Blot was used to detect the expression levels of differentiation-related proteins such as PPARγ, FASN, ADIPOQ, and FABP4.

[0049] The specific experimental design and results are as follows: 1. FAPs cell culture and adipogenesis induction FAPs cells were isolated from the longissimus dorsi muscle of bovines and cultured in normal medium for 2–3 days until they were nearly confluent. Then, they were replaced with adipogenic induction medium (ADM inducer I solution was used for the first 3 days, and ADM inducer II solution was used for the next 5 days) to induce adipogenic differentiation.

[0050] 2. Experimental group processing Differentiated FAPs cells were divided into three groups: control group (CON, with added physiological saline), empty vector transfection group (NC, transfected with empty vector plasmid), and... RERGL Overexpression group (OE-RERGL, transfected with RERGL overexpression plasmid pLV3-CMV-RERGL(cattle)-3×FLAG-CopGFP-Puro).

[0051] 3. BODIPY staining analysis BODIPY staining results showed that the fluorescence intensity, number, and distribution density of lipid droplets in the OE-RERGL group were significantly higher than those in the CON and NC groups. Figure 3 ),show RERGL Overexpression significantly promoted the formation and accumulation of intracellular lipid droplets.

[0052] 4. Oil Red O staining analysis Oil Red O staining further revealed large clusters of lipid droplets in the OE-RERGL group, while only a few scattered lipid droplets were observed in the CON and NC groups. Figure 4 ),prove RERGL Overexpression can significantly enhance the adipogenic differentiation ability and lipid accumulation level of bovine FAPs cells.

[0053] 5. Western blot detection Western blot analysis showed that, compared with the NC group, the OE-RERGL group exhibited significantly increased FASN protein expression (approximately 3.2-fold), upregulated PPARγ protein expression (approximately 2.3-fold), increased ADIPOQ protein expression (approximately 2.2-fold), and increased FABP4 protein expression (approximately 3.5-fold), with all differences being statistically significant. P＜0.05 () Figure 5 ), confirmed at the protein level RERGL It promotes adipogenic differentiation. (Note: ns indicates that the difference is not statistically significant.) P＞0.05 * indicates a statistically significant difference ( P＜0.05 )) Example 3: Mouse model experiment 1. Animal materials and experimental grouping Thirty-six healthy 4-week-old C57BL / 6 mice with identical growing environments were purchased and randomly divided into three groups (n=12): wild-type mice (CON), negative control group (NC), and gene-overexpressing mice (OE-RERGL). After grouping, the left gastrocnemius muscle of each group of mice underwent targeted intervention: the CON group was injected with saline, the NC group was injected with empty lentivirus, and the OE-RERGL group was injected with RERGL overexpressing lentivirus. All mice were housed in the same environment until 10 weeks of age.

[0054] The RERGL overexpressing lentivirus injected into the OE-RERGL group was prepared using the same method as the lentivirus in Example 1.

[0055] 2. Tissue differentiation capacity testing The specific tests for tissue differentiation capacity include: (1) BODIPY and Oil Red O staining were used to observe the morphology, size and distribution of intramuscular adipocytes.

[0056] (2) Mouse serum triglyceride (TG) detection kit to detect serum TG content.

[0057] (3) qRT-PCR and Western Blot were used to detect the expression of adipocyte differentiation genes and proteins such as PPARγ, FASN, ADIPOQ, and FABP4.

[0058] The specific experimental design and results are as follows: 1. Mouse grouping and sample collection Thirty-six C57BL / 6 mice were grouped and treated as described above. After being raised in the same environment until they were 10 weeks old, blood and muscle tissue samples were collected for later use.

[0059] 2. BODIPY staining analysis Muscle tissue samples were stained with BODIPY stain to observe lipid droplet content. Results showed that the intensity and number of lipid droplet fluorescence signals in the OE-RERGL group were significantly higher than those in the CON and NC groups. Figure 6 This indicates that lipid accumulation was more pronounced in the cells of this group.

[0060] 3. Oil Red O staining analysis Muscle tissue samples were stained with Oil Red O. The results showed that the area and staining intensity of lipid droplets in the OE-RERGL group were significantly higher than those in the CON and NC groups. Figure 7 The results suggest that RERGL overexpression promotes lipid deposition in mouse muscle tissue.

[0061] 4. Western Blot Detection Western blot analysis showed that, compared with the NC group, the OE-RERGL group had a significantly increased FASN protein expression level of approximately 3.1 times; PPARγ protein expression upregulated by approximately 2.4 times; ADIPOQ protein expression increased by approximately 3.4 times; and FABP4 protein expression increased by approximately 2.5 times. All differences were statistically significant. P＜0.05 () Figure 8 This confirms that it effectively enhances the body's ability to deposit fat. (Note: ns indicates that the difference is not statistically significant.) P＞0.05 * indicates a statistically significant difference ( P＜0.05 )) 5. Real-time quantitative PCR detection RNA was extracted from muscle tissue samples and reverse transcribed before qRT-PCR. The results showed that, compared with the NC group, the OE-RERGL group exhibited significantly increased C / EBPα gene mRNA expression levels by approximately 1.82-fold (mean values ​​1.8970, 1.8766, 1.7048, with an average upregulation of approximately 82%); PPARγ gene mRNA expression was upregulated by approximately 2.39-fold (mean values ​​2.2101, 2.5602, 2.4011, with an average upregulation of approximately 139%); FABP4 gene mRNA expression increased by approximately 4.32-fold (mean values ​​4.3087, 4.0277, 4.6254, with an average upregulation of approximately 332%); and ADIPOQ gene mRNA expression increased by approximately 5.78-fold (mean values ​​5.5040, 6.5107, 5.3291, with an average upregulation of approximately 478%), with all indicators showing highly significant differences (P < 0.01). Figure 9 The promoting effect of RERGL on lipid deposition was confirmed at the transcriptional level. (Note: ns indicates no statistically significant difference (P>0.05), ** indicates a highly statistically significant difference (P<0.01)) 6. Detection of triglycerides (TG) in mice After blood was allowed to stand, it was centrifuged at 3000 g for 10 min at 4℃, and the supernatant was collected and processed according to the ELISA kit. Results showed that compared with the NC group, the TG content in the OE-RERGL group was significantly increased. Statistical analysis showed that the average TG content in the NC group was approximately 1.05 mmol / gprot, and the average content in the OE-RERGL group was approximately 1.13 mmol / gprot, an upregulation of approximately 7.6%, and the difference was highly significant (P < 0.01); while there was no significant difference between the CON group and the NC group (P > 0.05). Figure 10 As a core indicator of lipid metabolism, the significant increase in TG levels in the bloodstream indicates that RERGL overexpression can promote lipid synthesis and transport, providing sufficient lipid precursors for intramuscular fat deposition, thus confirming the promoting effect of RERGL on increasing muscle fat content. (Note: ns indicates no statistically significant difference (P>0.05), ** indicates a highly statistically significant difference (P<0.01)) Based on the aforementioned cell and animal experiments, it was confirmed that RERGL significantly promotes adipogenic differentiation at both the transcriptional and protein levels. In bovine FAP cells, RERGL overexpression significantly promoted lipid droplet accumulation and significantly upregulated the expression of key adipogenic factors such as FASN, PPARγ, ADIPOQ, and FABP4. In mouse models, RERGL overexpression also enhanced intramuscular fat deposition, increased adipogenic gene expression, and serum triglyceride levels, suggesting that it may systemically regulate lipid metabolism. This study validates the pro-adipogenic effect of RERGL at multiple levels, providing a theoretical basis for exploring its potential applications in meat quality improvement.

[0062] Although the present invention has been described in detail with general description and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The application of the RERGL gene in promoting intramuscular fat deposition in bovine muscle for non-therapeutic purposes, characterized in that, The sequence of the RERGL gene is shown in SEQ ID NO:

1.

2. The application of the RERGL gene in molecular breeding to promote intramuscular fat deposition in mammals, characterized in that... The sequence of the RERGL gene is shown in SEQ ID NO:1, and the mammal is a mouse or a cow.

3. The application as described in claim 2, characterized in that, The amino acid sequence of the protein encoded by the RERGL gene is shown in SEQ ID NO:

2.

4. An application of a recombinant expression vector in the cultivation of mammals with high intramuscular fat content for non-therapeutic purposes, characterized in that, The recombinant expression vector is pLV3-CMV-RERGL (cattle)-3×FLAG-CopGFP-Puro, and its vector plasmid sequence is shown in SEQ ID NO:5; the mammal is a mouse or a cow.

5. The application as described in claim 4, characterized in that, The mammal is the cow.

6. A method for promoting intramuscular fat deposition in mammals for non-therapeutic purposes, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector; the recombinant expression vector contains the RERGL gene as shown in any one of claims 1-3; (2) The recombinant expression vector was introduced into mammalian muscle tissue using lentiviral transfection; (3) Culture transfected cells and / or feed treated mammals to regulate intramuscular fat deposition in mammals; The mammal in question is either a cow or a mouse.

7. The method as described in claim 6, characterized in that, The recombinant expression vector is pLV3-CMV-RERGL(cattle)-3×FLAG-CopGFP-Puro, and its vector plasmid sequence is shown in SEQ ID NO:

5.

8. The method as described in claim 6 or 7, characterized in that, The mammal in question is a cow.

Images