A method for detecting the function of genes in metabolic processes

Abstract

The present invention provides a method for detecting the function of a gene in a metabolic process, which belongs to the field of functional gene technology. The present invention provides the application of gene editing combined with non-targeted metabolic flux detection in detecting the function of a gene in a metabolic process, and in particular provides a method for detecting the role of a gene in a metabolic process, by constructing a gene-edited cell line, thereby performing non-targeted metabolic flux detection of the target metabolism on the gene-edited cell line, thereby determining the role of the gene in the target metabolism, such as the main active pathway, the relative contribution of each pathway and the distribution change characteristics. The present invention can analyze the metabolic pathways related to differential metabolites, provide a theoretical reference for in-depth analysis of the lipid metabolism regulation mechanism in which the target gene participates, and help promote the application of the target gene in animal breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional genes, and in particular relates to a method for detecting the function of genes in metabolic processes. Background Art

[0002] The growth and development of skeletal muscle is crucial for meat quality and yield in livestock. The myostatin (Mstn) gene, a member of the transforming growth factor β family, typically negatively affects skeletal muscle growth. Compared with wild-type animals, knockout of Mstn significantly increased skeletal muscle development and reduced intramuscular fat content. Mstn has long been considered a key regulator of muscle mass. In multiple species, including sheep, dogs, and cattle, Mstn deficiency has been shown to increase the number and size of myofibers, leading to muscle hypertrophy.

[0003] Linoleic acid (LA) is an omega-6 fatty acid and a precursor of n-6 polyunsaturated fatty acids (PUFAs). LA is essential in human and animal nutrition, serving as an energy source. However, the role of the Mstn gene in LA metabolism is currently unclear, necessitating an alternative method to examine Mstn gene function. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting the function of genes in metabolic processes, which can quickly discover the role of genes in different metabolisms.

[0005] To solve the above technical problems, the present invention provides the application of gene editing combined with non-targeted metabolic flux detection in detecting gene function in the metabolic process.

[0006] In one embodiment of the present invention, the gene editing includes gene knockout.

[0007] A further object of the present invention is to provide a method for detecting the role of a gene in a metabolic process, comprising constructing a cell line in which the gene is knocked out, performing non-targeted metabolic flux detection on the cell line, and evaluating the main active pathways of the gene in cellular metabolic activities, the relative contributions of each pathway, and the distribution change characteristics.

[0008] In one embodiment of the present invention, the gene comprises a myostatin gene.

[0009] In one embodiment of the present invention, the basal cells of the cell line include skeletal muscle fibers or skeletal muscle fibroblasts.

[0010] In a specific embodiment of the present invention, when the skeletal muscle fibroblasts are used as the basic cells, gene knockout is achieved using sgRNAs with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0011] In a specific embodiment of the present invention, the gene knockout comprises using the pX458 vector as a backbone vector of the gene knockout vector.

[0012] In one embodiment of the present invention, the non-targeted metabolic flux detection includes detecting linoleic acid metabolism.

[0013] In one embodiment of the present invention, the detection of linoleic acid metabolism includes collecting primary and secondary mass spectrometry data using an Orbitrap Exploris 120 mass spectrometer.

[0014] In one embodiment of the present invention, the detection of linoleic acid metabolism includes detection of polar metabolites and non-polar metabolites;

[0015] Polar metabolites were separated by chromatography on a Vanquish ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEH Amide column. Phase A of the liquid chromatography system consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B consisted of acetonitrile.

[0016] For non-polar metabolites, the target compounds were chromatographed on a Vanquish ultra-high performance liquid chromatograph using a Phenomenex Kinetex C18 liquid chromatography column; the liquid chromatography phase A was an aqueous phase containing 0.01% acetic acid by volume, and the liquid chromatography phase B was a mixture of isopropanol and acetonitrile in a 1:1 volume ratio.

[0017] Beneficial effects: The present invention provides an application of gene editing combined with non-targeted metabolic flux detection in detecting gene functions in metabolic processes, and in particular provides a method for detecting the role of genes in metabolic processes. By constructing a gene-edited cell line, non-targeted metabolic flux detection of target metabolism is performed on the gene-edited cell line, thereby determining the role of the gene in the target metabolism, such as the main active pathways, the relative contributions of each pathway, and the distribution change characteristics.

[0018] An embodiment of the present invention completes the - / - The LA metabolic flux detection in C2C12 cells successfully revealed the Mstn - / -The metabolic changes of LA in C2C12 cells were analyzed. The metabolic ratio of LA to unsaturated fatty acids was reduced after Mstn gene knockout, and the metabolic pathways related to differential metabolites were analyzed, which provided a theoretical reference for in-depth analysis of the lipid metabolism regulation mechanism involved in the Mstn gene and helped to promote the application of the Mstn gene in animal breeding.

[0019] In one embodiment of the present invention, skeletal muscle fibers of Mstn gene knockout mice were isolated, and linoleic acid metabolic flux was detected in the isolated skeletal muscle fibers, revealing the metabolic changes of LA in the skeletal muscle of mice after Mstn gene knockout. After Mstn gene knockout, the metabolism of LA to DHA increased, providing a theoretical reference for in-depth analysis of the regulatory mechanism of skeletal muscle lipid metabolism involving the Mstn gene, and helping to promote the application of the Mstn gene in animal breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 3 is a morphological diagram of C2C12 cells. A: wild-type C2C12 cells (WT group); B: MSTN - / - C2C12 cells (KO group);

[0021] Figure 2 This is the principal component analysis diagram in Example 1;

[0022] Figure 3 This is a pie chart of the proportion of metabolite classification in Example 1;

[0023] Figure 4 This is the KEGG classification diagram of the labeled metabolites in Example 1;

[0024] Figure 5 This is a bar graph of the degree of metabolite labeling in Example 1, showing the proportion of each metabolite derived from the labeled substrate linoleic acid;

[0025] Figure 6 This is a diagram of muscle fiber separation morphology. A: Muscle tissue after digestion; B: Muscle fibers after blowing apart;

[0026] Figure 7 This is the scatter plot of the OPLS-DA model scores in Example 2;

[0027] Figure 8 This is a pie chart of the proportion of metabolite classification in Example 2;

[0028] Figure 9 This is a bar graph of metabolite labeling in Example 2, where A: linoleic acid; B: DHA. DETAILED DESCRIPTION

[0029] The present invention provides the application of gene editing combined with non-targeted metabolic flux detection in detecting gene function in metabolic processes.

[0030] In a specific embodiment of the present invention, the gene editing includes gene knockout, such as the CRISPR-Cas9-based method used in one embodiment to achieve gene knockout, and of course other methods can also be used.

[0031] The metabolic flux analysis (MFA) of the present invention uses stable isotope tracing technology to detect the isotope substitution form of downstream metabolites, thereby analyzing the flow and distribution of the compound in the metabolic pathway. By performing metabolic flux analysis on an organism, the activity level of a specific metabolic pathway of the organism can be obtained. 13 C 18 Metabolites can obtain LA in Mstn - / - The main active pathways in C2C12 cells, the relative contributions of each pathway and the distribution change characteristics will help to gain a deeper understanding of the function of the Mstn gene in affecting unsaturated fatty acid metabolism and provide a reference for the application of the Mstn gene in animal husbandry.

[0032] The present invention provides a method for detecting the role of a gene in a metabolic process, comprising constructing a cell line in which the gene is knocked out, performing non-targeted metabolic flux detection on the cell line, and evaluating the main active pathways of the gene in cellular metabolic activities, the relative contributions of each pathway, and the distribution change characteristics.

[0033] When constructing the cell line of the present invention, the basic cells can be directly knocked out by the CRISPR-Cas9 method, or they can be directly extracted from the gene knockout animal model. For example, in one embodiment of the present invention, pX458-sgRNA-Cas9 is used as the vector backbone to knock out the second exon of the Mstn gene in skeletal muscle fibroblasts (C2C12) cells, and then electrofection is performed using a nuclear transfection system, and positive cells are screened and identified to obtain MSTN. - / - Cell line. In the present invention, two sgRNAs are designed when knocking out the Mstn gene:

[0034] sgRNA1 (SEQ ID No. 1): 5'-TCCCCCAGGCCTCAATTACC-3';

[0035] sgRNA2 (SEQ ID No. 2): 5'-TTGGAGTAAGATACTTTGTC-3'.

[0036] The present invention constructs the Mstn - / - After the cell line, the Mstn - / - Cell lines were identified to determine whether gene knockout occurred, and the Mstn - / -The identification primers are:

[0037] F (SEQ ID No. 3): 5'-GTGCTGAACTTGCAGTGAGAGG-3';

[0038] R (SEQ ID No. 4): 5'-AAAGATAAGCTAGGAGGCAAAG-3'.

[0039] In another embodiment of the present invention, skeletal muscle fibers were isolated from Mstn knockout mice as the cell line. The Mstn gene exon 2 knockout mice were constructed by Saiye Biotechnology Co., Ltd. using CRISPR / Cas technology and high-throughput electroporation of fertilized eggs to obtain Mstn knockout mice (male and female contract numbers: KOAIP220627XJ2 and KOAIP220729XJ1-B). The method for isolating mouse soleus muscle fibers described herein comprises the following steps: isolating soleus muscle from Mstn knockout mice, digesting the soleus muscle with collagenase until the muscle is swollen and hair-like fibers emerge, and then terminating the digestion; placing the terminated muscle in PBS buffer preheated at 37°C and repeatedly beating it into single muscle fibers, which are then aspirated using a capillary glass needle. The digestion temperature of the present invention is 37°C, the digestion time is 50-70 minutes, and the cells are shaken every 15 minutes during the digestion process.

[0040] The non-targeted metabolic flux detection of the present invention includes detecting linoleic acid metabolism, and the detection of linoleic acid metabolism includes using an Orbitrap Exploris 120 mass spectrometer to perform primary and secondary mass spectrometry data acquisition, and includes detection of polar metabolites and non-polar metabolites;

[0041] Polar metabolites were separated by chromatography on a Vanquish ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEH Amide column. Phase A of the liquid chromatography system consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B consisted of acetonitrile.

[0042] For non-polar metabolites, the target compounds were chromatographed on a Vanquish ultra-high performance liquid chromatograph using a Phenomenex Kinetex C18 liquid chromatography column; the liquid chromatography phase A was an aqueous phase containing 0.01% acetic acid by volume, and the liquid chromatography phase B was a mixture of isopropanol and acetonitrile in a 1:1 volume ratio.

[0043] To further illustrate the present invention, a method for detecting the function of a gene in a metabolic process provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] In the examples of the present invention, unless otherwise specified, the experimental methods used in the examples are conventional methods in the art, and the reagents and consumables used are all commercially available. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall be followed.

[0045] The mouse skeletal muscle fibroblasts (C2C12) (CTCC-001-0050) used in Example 1 were stored in the Shenzhen Agricultural Genomics Institute of the Chinese Academy of Agricultural Sciences.

[0046] Example 1 A method for detecting linoleic acid metabolic flux in Mstn gene knockout C2C12 cells I. Cultivation and passage of C2C12 cells

[0047] Complete medium: DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin for cell culture.

[0048] The C2C12 cells stored in liquid nitrogen were quickly thawed in a 37°C water bath. After thawing, the cells were resuspended in 1 mL of complete culture medium. Subsequently, the cell suspension was centrifuged at 800 rpm in a low-speed centrifuge for 5 minutes. After centrifugation, the supernatant was discarded. 1 mL of DMEM complete culture medium was taken and the cells were resuspended to ensure that the cells were evenly distributed. 4 mL of complete culture medium was added to the culture flask, and the cell suspension was evenly added to the culture flask using a pipette. Finally, the culture flask was placed in a 37°C, 5% CO2 constant temperature incubator for culture. After the cells attached to the wall of the culture flask, new culture medium was usually replaced after 24 hours.

[0049] When cells have grown to 60%-70% of the flask's volume, passage them. Wash the cells twice with PBS, remove the PBS, add 1 mL of 0.125% trypsin to digest the cells, and place them in a 37°C, 5% CO2 incubator for 1 minute. When the cells are observed to be round, add an equal volume of complete culture medium to terminate the cell digestion reaction. Remove the cells and centrifuge at 1000 rpm for 3 minutes. Remove the supernatant and disperse the cells with complete culture medium. Dispense the cell suspension into new culture flasks and culture in a cell culture incubator.

[0050] 2. MSTN homozygous knockout (Mstn - / - Construction of C2C12 cell line

[0051] pX458-sgRNA-Cas9 was selected as the vector backbone to knock out the second exon of the MSTN gene.

[0052] The sgRNA sequences are:

[0053] sgRNA1 (SEQ ID No. 1): 5'-TCCCCCAGGCCTCAATTACC-3';

[0054] sgRNA2 (SEQ ID No. 2): 5'-TTGGAGTAAGATACTTTGTC-3'.

[0055] The cells were counted and 5×10 5 Cells were electroporated using the Lonza nucleofection system.

[0056] After 48 hours of cell culture, positive cells were screened using a BD flow cytometer and plated onto 10 cm culture dishes (100 cells per dish). Monoclonal cells were picked using a cloning ring, half of which were transferred to a 48-well plate for further culture, and the other half were used for genotyping.

[0057] 25 μL PCR reaction system: 12.5 μL Taq DNA polymerase, 1 μL upstream and downstream primers, 1 μL DNA, and the balance RNase-free water;

[0058] PCR reaction program: pre-denaturation at 95°C for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 62°C for 15 s, and extension at 72°C for 30 s; and further extension at 72°C for 5 min.

[0059] 2% agarose gel electrophoresis was used to preliminarily determine whether the gene was knocked out based on the size of the amplified product. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing and sequence analysis was performed using SnapGene software. Qualified cells were selected for expansion culture to obtain Mstn. - / - C2C12 cells ( Figure 1 ).

[0060] Mstn - / - The identification primers are:

[0061] F (SEQ ID No. 3): 5'-GTGCTGAACTTGCAGTGAGAGG-3';

[0062] R (SEQ ID No. 4): 5'-AAAGATAAGCTAGGAGGCAAAG-3'.

[0063] 3. Non-target metabolic flux detection of linoleic acid C labeled samples

[0064] Drug preparation: Prepare 50mM stock solution, (1) dissolve linoleic acid and linoleic acid in 0.1M NaOH solution respectively 13 C 18 The final concentration is 50mM and the mixture is heated in a 70℃ water bath. (2) Prepare 10% bovine serum albumin (BSA) and filter it through 0.22mm filter before use. Mix (1) and (2) in proportion to achieve a final concentration of 5mM metabolites. Incubate at 55℃ for 10 minutes after mixing. Filter again before use.

[0065] When the cell count reaches approximately 70-80%, drug treatment is prepared. Cell culture medium is prepared to a final concentration of 0.1 mM and added to the cells for 24 hours.

[0066] The cell suspension was collected by trypsin digestion, centrifuged at 4°C, 1000 rpm for 5 min, the supernatant was removed, the cells were gently washed with PBS, centrifuged again, the supernatant was completely removed, and the cells were quick-frozen in liquid nitrogen for 1 min.

[0067] Add 25 μL of water to the collected cell pellet, freeze in liquid nitrogen for 1 minute, remove, thaw, and vortex mix for 30 seconds. Repeat this step three times. Sonicate in an ice-water bath for 10 minutes. Take 50 μL of the homogenate for protein quantification. Add 800 μL of methanol:acetonitrile (1:1 v / v) to the remaining 200 μL sample, transfer to a 2 mL centrifuge tube, vortex mix for 30 seconds, and incubate at -40°C for 1 hour. Centrifuge the sample at 12,000 rpm for 15 minutes at 4°C. Take an equal amount of 800 μL of the supernatant from all samples, dry them, reconstitute them in a 1:1 acetonitrile:water mixture (containing an isotopically labeled internal standard mixture), and vortex mix for 30 seconds. Sonicate the sample in an ice-water bath for 10 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the supernatant to a vial for analysis.

[0068] Polar metabolites were separated by chromatography on a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEHAmide column (2.1 mm × 100 mm, 1.7 μm). The HPLC phase A consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and the HPLC phase B consisted of acetonitrile. The sample tray temperature was 4°C, and the injection volume was 2 μL.

[0069] Nonpolar metabolites were separated by chromatography on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. Phase A consisted of aqueous phase containing 0.01% acetic acid, and phase B consisted of isopropanol:acetonitrile (1:1, v / v). The sample tray temperature was 4°C, and the injection volume was 2 μL.

[0070] The Orbitrap Exploris 120 mass spectrometer was capable of acquiring both primary and secondary mass spectrometric data under the control of Xcalibur software. Detailed parameters were as follows: sheath gas flow rate: 50 Arb, aux gas flow rate: 15 Arb, capillary temperature: 320°C, full MS resolution: 60,000, MS / MS resolution: 15,000, collision energy: SNCE 20 / 30 / 40, spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0071] 4. Mstn - / - Inhibit the metabolism of linoleic acid to unsaturated fatty acids

[0072] Wild-type C2C12 cells were designated as the WT group, and Mstn - / - The C2C12 cells were KO group. The results of principal component analysis are shown in Figures 2 to 4 As shown in Figure 2, the WT and KO groups had good repeatability and were clearly separated from each other. The first principal component (PC1) contributed 40.2% of the variance ( Figure 2 A total of 177 marker metabolites were identified, with the two largest categories being lipids and lipid-like molecules (60%) and organic compounds (14%) ( Figure 3 The differential metabolites belonged to 15 metabolic pathways, including fatty acid and unsaturated fatty acid biosynthesis, linoleic acid metabolism, and arachidonic acid metabolism ( Figure 4 ).

[0073] In the unsaturated fatty acid synthesis pathway, there was no significant difference in the linoleic acid labeling between the KO and WT groups, while the labeling of unsaturated fatty acids such as palmitoleic acid, stearic acid, erucic acid, and neuraminic acid decreased, indicating that the metabolism of linoleic acid to unsaturated fatty acids was reduced after Mstn gene knockout ( Figure 5 ).

[0074] Example 2: Method for detecting linoleic acid metabolic flux in skeletal muscle fibers isolated from MSTN gene knockout mice

[0075] 1. Digestion of flounder muscle tissue in mice

[0076] Saiye Biotechnology Co., Ltd. was commissioned to construct mice with knockout of the second exon of the Mstn gene. CRISPR / Cas technology was used to obtain Mstn gene knockout mice by applying high-throughput electroporation of fertilized eggs. Three wild-type mice (Mus-WT group) and three Mstn gene knockout mice (Mus-KO group) were taken respectively, and killed by cervical dislocation. The whole soleus muscle was removed; rinsed once with 95% alcohol and three times with PBS, and the muscle was immediately transferred to a test tube containing 3-5 ml of 0.2% type I collagenase preheated at 37°C, placed in a water bath, and digested for 50-70 minutes, shaking every 15 minutes. Under a microscope, when the muscle is swollen and accompanied by the emergence of hair-like muscle fibers, the digestion is terminated ( Figure 6 Middle A).

[0077] 2. Muscle Fiber Separation

[0078] Remove the muscle from the test tube, rinse it once in a culture dish containing 37°C preheated PBS, and then transfer it to another culture dish containing 37°C preheated PBS. Use a 1000μL pipette to repeatedly pipette. Use a 1mm inner diameter capillary glass tube to stretch and make a glass needle to aspirate intact undamaged muscle fibers. Each time a muscle fiber is aspirated, rinse it in three culture dishes containing PBS in turn, for a total of three rinses ( Figure 6 Middle B).

[0079] 3. Non-targeted metabolic flux detection of linoleic acid C-labeled muscle fibers

[0080] Drug preparation: Prepare 50mM stock solution, (1) dissolve linoleic acid and linoleic acid in 0.1M NaOH solution respectively 13 C 18 The final concentration is 50mM and heat treatment is performed in a 70℃ water bath. (2) Prepare 10% bovine serum albumin (BSA) and filter it through 0.22mm filter before use. Mix (1) and (2) in proportion to achieve a final metabolite concentration of 5mM. Incubate at 55℃ for 10 minutes after mixing. Filter again and set aside. Isolate approximately 200-300 muscle fibers and prepare for drug treatment. Prepare cell culture medium at a final concentration of 0.1mM and add it to the cells after preparation. Culture for 24 hours.

[0081] The cell suspension was collected and centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was removed, the cells were gently washed with PBS, centrifuged again, the supernatant completely removed, and the pellet was snap-frozen in liquid nitrogen for 1 min. 25 μL of water was added to the collected cell pellet, and the pellet was frozen in liquid nitrogen for 1 min. After removal, the pellet was thawed and vortexed for 30 s. This step was repeated three times. The pellet was sonicated in an ice-water bath for 10 min. A 50 μL homogenate was collected for protein quantification. To the remaining 200 μL sample, 800 μL of methanol:acetonitrile (1:1 v / v) was added. The pellet was transferred to a 2 mL centrifuge tube, vortexed for 30 s, and allowed to stand at -40°C for 1 h. The samples were centrifuged at 12000 rpm for 15 min at 4°C. An equal amount of 800 μL of supernatant from all samples was dried and reconstituted in a 1:1 acetonitrile:water mixture (containing an isotopically labeled internal standard mixture) and vortexed for 30 s. Place the sample in an ice-water bath for 10 minutes, ultrasonicate it, and centrifuge it at 12,000 rpm for 15 minutes at 4°C. Take the supernatant and place it in a sample injection bottle for analysis.

[0082] Polar metabolites were separated by chromatography on a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. Phase A consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B consisted of acetonitrile. The sample tray temperature was 4°C, and the injection volume was 2 μL.

[0083] Nonpolar metabolites were separated by chromatography on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph. Phase A consisted of aqueous phase containing 0.01% acetic acid, and phase B consisted of isopropanol:acetonitrile (1:1, v / v). The sample tray temperature was 4°C, and the injection volume was 2 μL.

[0084] The Orbitrap Exploris 120 mass spectrometer was capable of acquiring both primary and secondary mass spectrometric data under the control of Xcalibur software. Detailed parameters were as follows: sheath gas flow rate: 50 Arb, aux gas flow rate: 15 Arb, capillary temperature: 320°C, full MS resolution: 60,000, MS / MS resolution: 15,000, collision energy: SNCE 20 / 30 / 40, spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0085] (3) Data Analysis

[0086] Labeled metabolites were extracted from the non-targeted metabolic flux data, and R (ggplot2) and SIMCA software were used to filter the data, perform OPLS-DA score analysis, classify labeled metabolites, and analyze metabolic flux. The results showed that after Mstn gene knockout, the metabolic ratio of LA to DHA in mouse skeletal muscle fibers increased, which was beneficial to muscle growth and development.

[0087] OPLS-DA analysis was performed using SIMCA software, and the results showed that there was good separation between the Mus-WT group and the Mus-KO group ( Figure 7 ). A total of 26 marker metabolites were identified by non-targeted metabolic flux detection, which belonged to lipids and lipid molecules, organic acids and their derivatives, and organic heterocyclic compounds ( Figure 8 ).

[0088] In the lipid metabolism pathway, there was no significant difference in linoleic acid labeling between the Mus-KO group and the Mus-WT group, while the DHA labeling increased, indicating that Mstn gene knockout promoted the metabolism of linoleic acid to DHA ( Figure 9 ).

[0089] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the role of myostatin gene in metabolic processes, characterized in that: The method comprises constructing a cell line in which the gene is knocked out, performing non-targeted metabolic flux detection on the cell line, and evaluating the main active pathways of the gene in cellular metabolic activities, the relative contributions of each pathway, and the distribution change characteristics; the non-targeted metabolic flux detection includes detecting linoleic acid metabolism; The basal cells of the cell line include skeletal muscle fibers or skeletal muscle fibroblasts; When the skeletal muscle fibroblasts are used as the basic cells, gene knockout is achieved using sgRNA with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; The detection of linoleic acid metabolism includes primary and secondary mass spectrometry data acquisition using an Orbitrap Exploris 120 mass spectrometer; The detection of linoleic acid metabolism includes detection of polar metabolites and non-polar metabolites; Polar metabolites were separated by chromatography on a Vanquish ultra-high performance liquid chromatograph using a Waters ACQUITY UPLCBEH Amide column. Phase A of the liquid chromatography system consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B consisted of acetonitrile. For non-polar metabolites, the target compounds were chromatographed on a Vanquish ultra-high performance liquid chromatograph using a Phenomenex Kinetex C18 liquid chromatography column; phase A of the liquid chromatography was an aqueous phase containing 0.01% acetic acid by volume, and phase B was a mixture of isopropanol and acetonitrile in a 1:1 volume ratio.

2. The method according to claim 1, characterized in that The gene knockout comprises using the pX458 vector as a backbone vector of the gene knockout vector.

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