Method for detecting function of gene in metabolic process

Abstract

The invention provides a method for detecting functions of genes in a metabolic process, and belongs to the technical field of functional genes. The invention provides application of gene editing in combination with non-targeted metabolic flux detection in detection of gene functions in a metabolic process, and particularly provides a method for detecting functions of genes in the metabolic process, and non-targeted metabolic flux detection of target metabolism is performed on a gene editing cell line by constructing the gene editing cell line, so that the gene editing cell line can be used for detecting the metabolic function of the gene in the metabolic process. Therefore, the functions of the gene in target metabolism, such as main activity pathways and relative contribution and distribution change characteristics of each pathway, are determined. According to the invention, metabolic pathways related to differential metabolites can be analyzed, a theoretical reference is provided for deep analysis of a lipid metabolism regulation mechanism in which the target gene participates, and the application of the target gene in animal breeding is promoted.

Description

Technical Field

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

[0002] The growth and development of skeletal muscle is essential for the quality and yield of meat in livestock. The myostatin (Mstn) gene is a member of the transforming growth factor β family that usually has a negative impact on the growth of skeletal muscle. Compared with wild-type animals, knocking out Mstn significantly increased skeletal muscle development and reduced intramuscular fat content. Mstn has long been considered a key regulator of muscle quality. In multiple species, including sheep, dogs, and cattle, Mstn deficiency has shown an increase in 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 acid (PUFA). LA is an essential fatty acid in human and animal nutrition and can be used as an energy source. However, the role of the Mstn gene in the LA metabolic flow is currently unclear, so another method is needed to detect the function of the Mstn gene. 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] In order 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 a specific 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 cell metabolic activities, the relative contributions of each pathway, and the distribution change characteristics.

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

[0009] In a specific 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 such as those shown in SEQ ID No. 1 and SEQ ID No. 2.

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

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

[0013] In a specific embodiment of the present invention, the detection of linoleic acid metabolism includes using an OrbitrapExploris 120 mass spectrometer to collect primary and secondary mass spectrometry data.

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

[0015] For polar metabolites, the target compounds were separated by chromatography using a Vanquish ultra-high performance liquid chromatograph and a Waters ACQUITYUPLC BEH Amide liquid chromatography column; the liquid chromatography phase A was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the phase B was acetonitrile;

[0016] For non-polar metabolites, a Vanquish ultra-high performance liquid chromatograph was used to perform chromatographic separation of the target compounds 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 volume ratio of 1:1.

[0017] Beneficial effects: The present invention provides an application of gene editing combined with non-targeted metabolic flux detection in detecting gene function in the metabolic process, and in particular provides a method for detecting the role of genes in the metabolic process. By constructing a gene-edited cell line, non-targeted metabolic flux detection of target metabolism is performed on the gene-edited cell line to determine 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 Mstn-based - / - The LA metabolic flux detection in C2C12 cells successfully revealed the - / -The metabolic changes of LA in C2C12 cells were analyzed, and the metabolic ratio of LA to unsaturated fatty acids was reduced after Mstn gene knockout. 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 on the isolated skeletal muscle fibers, revealing the metabolic changes of LA in the skeletal muscle of mice after Mstn gene knockout. The metabolism of LA to DHA increased after Mstn gene knockout, providing a theoretical reference for in-depth analysis of the skeletal muscle lipid metabolism regulation mechanism 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 It is the principal component analysis diagram in Example 1;

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

[0023] Figure 4 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, in which the proportion of each metabolite derived from the labeled substrate linoleic acid;

[0025] Figure 6 This is a morphological diagram of muscle fiber separation. A: digested muscle tissue; B: blown-away muscle fibers;

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

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

[0028] Fig. 9 This is a bar graph of metabolite labeling in Example 2, wherein 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 the metabolic process.

[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 may 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 cell 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 skeleton to knock out the second exon of the Mstn gene in skeletal muscle fibroblasts (C2C12) cells, and then electro-transfection is performed using a nuclear transfection system, positive cells are screened and identified, and MSTN is obtained. - / - 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 primers for identification 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 are isolated from Mstn knockout mice as the cell line, wherein the Mstn gene exon 2 knockout mice are commissioned to be constructed by Saiye Biotechnology Co., Ltd., and CRISPR / Cas technology is used to obtain Mstn knockout mice by applying high-throughput electroporation of fertilized eggs (contract numbers for male and female mice: KOAIP220627XJ2, KOAIP220729XJ1-B). The mouse soleus muscle fiber isolation method of the present invention comprises the following steps: isolating the soleus muscle of the Mstn knockout mouse, digesting the soleus muscle with collagenase until the muscle is swollen and accompanied by hair-like muscle fibers, and then terminating the digestion; placing the terminated muscle in a PBS buffer solution preheated at 37°C and repeatedly blowing it into a single muscle fiber, and using a capillary glass needle to aspirate a single muscle fiber. The digestion temperature of the present invention is 37°C, the time is 50-70min, and shaking once every 15min 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 collect primary and secondary mass spectrometry data, and includes detection of polar metabolites and non-polar metabolites;

[0041] For polar metabolites, the target compounds were separated by chromatography using a Vanquish ultra-high performance liquid chromatograph and a Waters ACQUITYUPLC BEH Amide liquid chromatography column; the liquid chromatography phase A was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the phase B was acetonitrile;

[0042] For non-polar metabolites, a Vanquish ultra-high performance liquid chromatograph was used to perform chromatographic separation of the target compounds 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 volume ratio of 1:1.

[0043] In order 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 can be purchased commercially. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are 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: Add 10% FBS and 1% penicillin-streptomycin for cell culture to DMEM medium.

[0048] Place the C2C12 cells stored in liquid nitrogen in a 37°C water bath for rapid thawing. After thawing, resuspend the cells with 1 mL of complete culture medium. Subsequently, centrifuge the cell suspension at 800 rpm for 5 minutes in a low-speed centrifuge. After centrifugation, discard the supernatant. Take 1 mL of DMEM complete culture medium and resuspend the cells to ensure that the cells are evenly distributed. Add 4 mL of complete culture medium to the culture flask, and use a pipette to evenly add the cell suspension to the culture flask. Finally, place the culture flask in a 37°C, 5% CO2 constant temperature incubator for culture. After the cells attach to the wall of the culture flask, replace the new culture medium usually after 24 hours.

[0049] When the cells grow to 60%-70% of the culture flask, perform cell passage. Wash the cells twice with PBS, remove PBS, add 1mL 0.125% trypsin to digest the cells, place in a 37°C, 5% CO2 constant temperature incubator, and digest 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. Take out the cells, centrifuge at 1000rpm for 3 minutes, remove the supernatant, and then blow the cells with complete culture medium, distribute the cell suspension into new culture flasks, and place them in a cell culture incubator for culture.

[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 the cells were plated in 10 cm culture dishes (100 cells per dish). Monoclonal cells were picked using a cloning ring, half of the cells 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 TaqDNA polymerase, 1 μL upstream and downstream primers, 1 μL DNA, and the rest RNase free water;

[0058] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 30 s, 30 cycles; and extension at 72°C for another 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 comparison analysis was performed using SnapGene software. Qualified cells were selected for expansion culture to obtain Mstn - / - C2C12 cells ( Figure 1 ).

[0060] Mstn - / - The primers for identification 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), filter through 0.22mm and set aside. Mix (1) and (2) in proportion, and the final concentration of the metabolite is 5mM. After mixing, incubate at 55℃ for 10min. Filter again and set aside.

[0065] When the cell number reaches about 70-80%, prepare for drug treatment. Prepare cell culture medium according to the final concentration of 0.1mM, add it to the cells and culture for 24h.

[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 it in liquid nitrogen for 1 min, take it out and thaw it, vortex mix it for 30 s, and repeat this step 3 times. Ultrasonicate it in an ice water bath for 10 min, take 50 μL of homogenate for protein quantification, add 800 μL of methanol: acetonitrile = 1:1 (V / V) to the remaining 200 μL sample, transfer it to a 2mL centrifuge tube, vortex mix it for 30 s, and let it stand at -40℃ for 1h. Centrifuge the sample at 4℃, 12000rpm for 15min, take an equal amount of 800 μL of supernatant from all samples, dry it, add acetonitrile: water = 1:1 (containing isotope labeled internal standard mixture) in proportion, and vortex mix it for 30 s. Ultrasonicate the sample in an ice water bath for 10 min, and centrifuge it at 4℃, 12000rpm for 15 min. Take the supernatant and place it in a sample injection bottle for detection on the machine.

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

[0069] For non-polar metabolites, the target compounds were chromatographed using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The liquid chromatography phase A was an aqueous phase containing 0.01% acetic acid, and the phase B was 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 can collect primary and secondary mass spectrometry data under the control of the control software (Xcalibur). The detailed parameters are as follows: Sheath gas flow rate: 50Arb, Aux gas flow rate: 15Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8kV (positive) or -3.4kV (negative).

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

[0072] Wild-type C2C12 cells were WT group, Mstn - / - The C2C12 cells were KO group. The principal component analysis results are shown in Figures 2 to 4 As shown in Figure 2, the WT group and the KO group had good repeatability and obvious separation between the two groups. The variance contribution of the first principal component (PC1) was 40.2% ( Figure 2 A total of 177 marker metabolites were identified, with the two largest categories being lipids and lipid 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 group and the WT group, 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 decreased 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 sole muscle tissue in mice

[0076] Saiye Biotechnology Co., Ltd. was commissioned to construct mice with the second exon knockout of the Mstn gene. CRISPR / Cas technology was used to obtain Mstn knockout mice by applying high-throughput electroporation of fertilized eggs. Three wild-type mice (Mus-WT group) and three Mstn 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 preheated at 37°C containing 3-5 ml of 0.2% type I collagenase, 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 (A).

[0077] 2. Muscle Fiber Separation

[0078] The muscle was removed from the test tube and rinsed once in a culture dish containing 37°C preheated PBS, then transferred to another culture dish containing 37°C preheated PBS and repeatedly pipetted with a 1000μL pipette. A 1mm inner diameter capillary glass tube was stretched to make a glass needle to absorb intact, undamaged muscle fibers. Each time the muscle fibers were absorbed, they were rinsed in 3 culture dishes containing PBS in turn, for a total of 3 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 the cells are heated in a 70℃ water bath. (2) Prepare 10% bovine serum albumin (BSA), filter through 0.22mm and set aside. Mix (1) and (2) in proportion, and the final concentration of the metabolite is 5mM. Incubate at 55℃ for 10min after mixing. Filter again and set aside. Separate about 200-300 muscle fibers and prepare for drug treatment. Prepare the cell culture medium at a final concentration of 0.1mM, add it to the cells, and culture for 24h.

[0081] Collect the cell suspension, centrifuge at 4°C, 1000rpm, for 5min, remove the supernatant, wash the cells gently with PBS, centrifuge again, completely remove the supernatant, and freeze in liquid nitrogen for 1min. Add 25μl of water to the collected cell pellet, freeze in liquid nitrogen for 1min, take out and thaw, vortex mix for 30s, and repeat this step 3 times. Ultrasonicate in an ice water bath for 10min, take 50μl of homogenate for protein quantification, add 800μL of methanol: acetonitrile = 1:1 (V / V) to the remaining 200μL sample, transfer to a 2mL centrifuge tube, vortex mix for 30s, and stand at -40°C for 1h. Centrifuge the sample at 4°C, 12000rpm for 15min, take an equal amount of 800μL supernatant from all samples and dry them, add acetonitrile: water = 1:1 in proportion to re-dissolve (containing isotope-labeled internal standard mixture), and vortex mix for 30s. Place the sample in an ice-water bath for 10 minutes and centrifuge at 12,000 rpm for 15 minutes at 4°C. Take the supernatant and place it in a sample injection bottle for testing.

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

[0083] For non-polar metabolites, the target compounds were chromatographed using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph on a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The liquid chromatography phase A was an aqueous phase containing 0.01% acetic acid, and the phase B was 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 can collect primary and secondary mass spectrometry data under the control of the control software (Xcalibur). The detailed parameters are as follows: Sheath gas flowrate: 50Arb, Aux gas flowrate: 15Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8kV (positive) or -3.4kV (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 the development of muscle growth.

[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 lipid 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, but the DHA labeling increased, indicating that Mstn gene knockout promoted the metabolism of linoleic acid to DHA ( Fig. 9 ).

[0089] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. Application of gene editing combined with non-targeted metabolic flux detection in detecting gene function in metabolic processes.

2. The use according to claim 1, characterized in that: The gene editing includes gene knockout.

3. A method for detecting the role of a gene in a metabolic process, characterized in that: The method includes 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 cell metabolic activities, the relative contributions of various pathways, and the distribution change characteristics.

4. The method according to claim 3, characterized in that: The genes include the myostatin gene.

5. The method according to claim 3, characterized in that: The basic cells of the cell line include skeletal muscle fibers or skeletal muscle fibroblasts.

6. The method according to claim 5, characterized in that: When the skeletal muscle fibroblasts are used as basic cells, gene knockout is achieved using sgRNAs with nucleotide sequences such as those shown in SEQ ID No.1 and SEQ ID No.

2.

7. The method according to claim 6, characterized in that: The gene knockout comprises using the pX458 vector as a backbone vector of the gene knockout vector.

8. The method according to claim 3, characterized in that: The non-targeted metabolic flux detection includes detecting linoleic acid metabolism.

9. The method according to claim 8, characterized in that: The detection of linoleic acid metabolism includes using an OrbitrapExploris 120 mass spectrometer to collect primary and secondary mass spectrometry data.

10. The method according to claim 8, characterized in that: The detection of linoleic acid metabolism includes detection of polar metabolites and non-polar metabolites; For polar metabolites, the target compounds were separated by chromatography using a Vanquish ultra-high performance liquid chromatograph and a Waters ACQUITY UPLCBEH Amide liquid chromatography column; the liquid chromatography phase A was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the phase B was acetonitrile; For non-polar metabolites, a Vanquish ultra-high performance liquid chromatograph was used to perform chromatographic separation of the target compounds 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 phase B was a mixture of isopropanol and acetonitrile in a volume ratio of 1:1.

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