Method for detecting effect of myostatin gene in alpha-linolenic acid metabolic flux

Abstract

The invention provides a method for detecting the effect of a myostatin gene in alpha-linolenic acid metabolic flux, and belongs to the technical field of gene detection. The invention provides a method for detecting the effect of a myostatin gene in alpha-linolenic acid metabolic flux, which combines gene editing and a non-targeted metabolic flux detection method to verify the effect of the Mstn gene in a target gene. Particularly, main active pathways of alpha-linolenic acid in gene editing cells and relative contribution and distribution change characteristics of the pathways are analyzed. The invention proves that the Mstn gene can regulate and control muscle growth by reducing metabolism of alpha-linolenic acid to prostaglandin E2; after the Mstn gene is knocked out, the synthesis and metabolic cycle rate of carnosine is slowed down.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular to a method for detecting the effect of myostatin gene in alpha-linolenic acid metabolic flow. 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] α-linolenic acid (ALA) is an 18-carbon polyunsaturated fatty acid and an essential polyunsaturated fatty acid for the human body. As an omega-3 essential fatty acid, ALA is a precursor for the synthesis of two important fatty acids, eicosapentaenoic acid and docosahexaenoic acid. These two fatty acids play a vital role in brain development, cardiovascular health, and inflammatory response. Adding α-linolenic acid to the diet of mice can significantly reduce the obesity rate of mice. However, the role of the Mstn gene in the α-linolenic acid metabolic flow is currently unclear, so a different 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 role of the myostatin gene in the α-linolenic acid metabolic flow, revealing the metabolic changes of ALA in gene-edited cells, and analyzing the metabolic pathways related to differential metabolites, providing a reference for genetic breeding of the Mstn gene.

[0005] The present invention provides a method for detecting the effect of myostatin gene in α-linolenic acid metabolic flow, comprising the following steps: knocking out the myostatin gene in target cells by gene editing method to obtain Mstn gene knockout cells;

[0006] A non-target metabolic flux detection method is used to detect the metabolic flux changes of α-linolenic acid in the Mstn gene knockout cells, and the metabolic flux changes include the main active pathways, the relative contributions of each pathway and the distribution change characteristics.

[0007] In a specific embodiment of the present invention, the target cells include mouse skeletal muscle fibroblasts.

[0008] In a specific embodiment of the present invention, the gene editing method includes the CRISPR-Cas9 method.

[0009] In a specific embodiment of the present invention, the sgRNA sequence used in the CRISPR-Cas9 method includes sgRNA1 with nucleotide sequences as shown in SEQ ID No.1 and sgRNA2 with nucleotide sequences as shown in SEQ ID No.2.

[0010] In a specific embodiment of the present invention, the pX458 vector is used as the vector backbone of CRISPR-Cas9.

[0011] In a specific embodiment of the present invention, the target cells include soleus muscle fibers isolated from MSTN gene knockout mice.

[0012] In a specific embodiment of the present invention, the separation method comprises the following steps: isolating the soleus muscle of the MSTN gene 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;

[0013] The digested muscle was placed in PBS buffer preheated at 37°C and repeatedly blown into single muscle fibers, and a capillary glass needle was used to aspirate the single muscle fiber.

[0014] In a specific embodiment of the present invention, the digestion temperature is 37° C., the time is 50 to 70 minutes, and the digestion is shaken every 15 minutes.

[0015] In one embodiment of the present invention, the detection includes collecting primary and secondary mass spectrometric data using an Orbitrap Exploris 120 mass spectrometer.

[0016] In a specific embodiment of the present invention, the detection includes detection of polar metabolites and non-polar metabolites;

[0017] For the detection of polar metabolites, the target compounds were separated by chromatography using a Vanquish ultra-high performance liquid chromatograph and a Waters ACQUITY UPLC 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.

[0018] The detection of non-polar metabolites includes the use of a Vanquish ultra-high performance liquid chromatograph and a Phenomenex Kinetex C18 liquid chromatography column to perform chromatographic separation of the target compounds. The liquid chromatography phase A is an aqueous phase containing 0.01% acetic acid by volume, and the phase B is a mixture of isopropanol and acetonitrile in a volume ratio of 1:1.

[0019] Beneficial effects: The present invention provides a method for detecting the role of the myostatin gene in the α-linolenic acid metabolic flux, which combines gene editing and non-targeted metabolic flux detection methods to verify the role of the MSTN gene in the target gene, especially to analyze the main active pathways of ALA in gene-edited cells, the relative contributions of each pathway and the distribution change characteristics.

[0020] In one embodiment of the present invention, after verification with C2C12 cells, it was found that a total of 160 labeled metabolites were identified, and the two largest categories of substances were lipids and lipid molecules and organic acids and their derivatives. The identified differential metabolites belong to 15 metabolic pathways, including fatty acid and unsaturated fatty acid biosynthesis, arachidonic acid metabolism, etc. In the arachidonic acid metabolic pathway, the prostaglandin E2 labeling degree was significantly reduced, indicating that the metabolic ratio of ALA to prostaglandin E2 was reduced after the Mstn gene was knocked out, that is, the Mstn gene may regulate muscle growth by reducing the metabolism of ALA to prostaglandin E2.

[0021] In one embodiment of the present invention, after verification with skeletal muscle fibers isolated from Mstn gene knockout mice, non-targeted metabolic flux detection identified a total of 30 labeled metabolites belonging to lipids and lipid molecules and organic acids and their derivatives, respectively. There was no significant difference in the labeling degree of α-linolenic acid and carnosine. According to the ratio of each homologous body of carnosine, the ratio of m+0 carnosine increased and the ratio of m+1 and m+2 carnosine decreased in the KO group compared with the WT group (P<0.05), indicating that the synthesis and metabolic cycle rate of carnosine slowed down after Mstn gene knockout. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 shows the morphology of C2C12 cells. A: wild-type C2C12 cells (WT group); B: MSTN - / - C2C12 cells (KO group);

[0023] Figure 2 For wild-type C2C12 cells and Mstn - / - Principal component analysis of C2C12 cells;

[0024] Figure 3 This is a pie chart showing the classification percentage of the labeled metabolites in Example 1;

[0025] Figure 4 The KEGG classification diagram of the labeled metabolites in Example 1;

[0026] Figure 5 This is a bar graph of metabolite labeling, and the data in the figure are the proportions of each metabolite from the labeled substrate linoleic acid;

[0027] Figure 6 This is the result of muscle fiber separation. A: muscle tissue after digestion; B: muscle fibers after blowing apart;

[0028] Figure 7 is a scatter plot of the muscle fiber OPLS-DA model scores in Example 2;

[0029] Figure 8 This is a pie chart of the metabolite classification ratio in Example 2;

[0030] Fig. 9 This is a bar graph of metabolite labeling in Example 2;

[0031] Fig.10 It is the ratio of each homologous body of carnosine in Example 2. DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting the effect of myostatin gene in α-linolenic acid metabolic flow, comprising the following steps: knocking out the myostatin gene in target cells by gene editing method to obtain Mstn gene knockout cells;

[0033] A non-target metabolic flux detection method is used to detect the metabolic flux changes of α-linolenic acid in the Mstn gene knockout cells, and the metabolic flux changes include the main active pathways, the relative contributions of each pathway and the distribution change characteristics.

[0034] In a specific embodiment of the present invention, mouse skeletal muscle fibroblasts (C2C12, CTCC-001-0050, stored in the Shenzhen Agricultural Genomics Institute of the Chinese Academy of Agricultural Sciences) are used as target cells, and gene editing is completed using the CRISPR-Cas9 method. When performing the gene editing, the pX458 vector is used as the vector skeleton of CRISPR-Cas9, and the sgRNA is inserted into the vector skeleton to construct a pX458-sgRNA-Cas9 recombinant vector to knock out the second exon of the Mstn gene. The sgRNA sequence used in the CRISPR-Cas9 method of the present invention includes sgRNA1 shown in SEQ ID No.1 and sgRNA2 shown in SEQ ID No.2, whose nucleotide sequences are shown in SEQ ID No.1.

[0035] sgRNA1: 5'-TCCCCCAGGCCTCAATTACC-3';

[0036] sgRNA2: 5'-TTGGAGTAAGATACTTTGTC-3'.

[0037] In this embodiment of the present invention, the Lonza nuclear transfection system is used for electrotransfection to obtain Mstn - / - The present invention obtains the Mstn - / - After the C2C12 cells are generated, identification is required. The primers used for identification include the following:

[0038] F(SEQ ID No.3):5'-GTGCTGAACTTGCAGTGAGAGG-3';

[0039] R (SEQ ID No. 4): 5'-AAAGATAAGCTAGGAGGCAAAG-3'.

[0040] In another specific embodiment of the present invention, the soleus muscle fibers isolated from MSTN gene knockout mice are used as target cells, wherein the second exon knockout mice of the Mstn gene are commissioned to be constructed by Saiye Biotechnology Co., Ltd., and the CRISPR / Cas technology is used to obtain the Mstn gene knockout mice by applying high-throughput electroporation of fertilized eggs (contract numbers of 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 gene knockout mouse, digesting the soleus muscle with collagenase until the muscle is swollen and accompanied by the hair-like muscle fibers, and then terminating the digestion; placing the terminated digested 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 the 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.

[0041] In the present invention, non-targeted metabolomics and fully labeled 13 C 18 Analysis of ALA in Mstn by ALA Tracer Method - / - The main active pathways in C2C12 cells and mouse soleus muscle fibers, the relative contribution of each pathway and the distribution change characteristics. In a specific embodiment of the present invention, the detection includes using an Orbitrap Exploris 120 mass spectrometer to collect primary and secondary mass spectrometry data; the detection includes detection of polar metabolites and non-polar metabolites;

[0042] For the detection of polar metabolites, the target compounds were separated by chromatography using a Vanquish ultra-high performance liquid chromatograph and a Waters ACQUITY UPLC 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.

[0043] The detection of non-polar metabolites includes the use of a Vanquish ultra-high performance liquid chromatograph and a Phenomenex Kinetex C18 liquid chromatography column to perform chromatographic separation of the target compounds. The liquid chromatography phase A is an aqueous phase containing 0.01% acetic acid by volume, and the phase B is a mixture of isopropanol and acetonitrile in a volume ratio of 1:1.

[0044] In order to further illustrate the present invention, a method for detecting the role of myostatin gene in α-linolenic acid metabolic flow 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.

[0045] 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.

[0046] 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.

[0047] Example 1 A method for detecting α-linolenic acid metabolic flux in Mstn gene knockout C2C12 cells

[0048] 1. C2C12 cell culture and passaging

[0049] Complete medium: Add 10% FBS and 1% penicillin-streptomycin for cell culture to DMEM medium.

[0050] 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.

[0051] Use an inverted fluorescence microscope to observe the cell growth status. When the cells grow to 60% to 70% of the culture flask, perform cell passage. Wash the cells twice with PBS, remove PBS, add 1mL of 0.125% trypsin to digest the cells, and place them in a 37°C, 5% CO2 constant temperature 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. Take out the cells, centrifuge at 1000rpm for 3 minutes, remove the supernatant, and then blow the cells with complete culture medium. According to the specific needs of the experiment, distribute the cell suspension into new culture flasks and place them in a cell culture incubator for culture.

[0052] 2. MSTN homozygous knockout (Mstn - / - Construction of C2C12 cell line

[0053] pX458-sgRNA-Cas9 was selected as the vector backbone to knock out the second exon of the Mstn gene.

[0054] The sgRNA sequences are:

[0055] sgRNA1 (SEQ ID No. 1): 5'-TCCCCCAGGCCTCAATTACC-3';

[0056] sgRNA2 (SEQ ID No. 2): 5'-TTGGAGTAAGATACTTTGTC-3'.

[0057] The cells were counted and 5×10 5 Cells were electroporated using the Lonza nucleofection system.

[0058] 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.

[0059] 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;

[0060] 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.

[0061] 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 ).

[0062] Mstn - / - The primers for identification are:

[0063] F (SEQ ID No. 3): 5'-GTGCTGAACTTGCAGTGAGAGG-3';

[0064] R (SEQ ID No. 4): 5'-AAAGATAAGCTAGGAGGCAAAG-3'.

[0065] 3. Non-target metabolic flux detection of α-linolenic acid C-labeled samples

[0066] Drug preparation: Prepare 50 mM stock solution, (1) dissolve α-linolenic acid and α-linolenic acid in 0.1 M 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 4. Mstn gene affects α-linolenic acid metabolism

[0074] Wild-type C2C12 cells were WT group, Mstn - / - The C2C12 cells were the KO group. The principal component analysis results showed that the WT group and the KO group had good repeatability and good separation between the two groups. The variance contribution of the second principal component (PC2) was 23.6% ( Figure 2 A total of 160 marker metabolites were identified, with the two largest categories being lipids and lipid molecules (59.52%) and organic acids and their derivatives (14.29%) ( Figure 3 The identified differential metabolites belonged to 15 metabolic pathways, including fatty acid and unsaturated fatty acid biosynthesis, arachidonic acid metabolism, etc. ( Figure 4 ).

[0075] In the arachidonic acid metabolic pathway, there was no significant difference in arachidonic acid, prostaglandin G2, leukotriene B4 and thromboxane A2 between the KO group and the WT group, and the prostaglandin E2 labeling was significantly reduced, indicating that the metabolic ratio of α-linolenic acid to prostaglandin E2 was reduced after Mstn gene knockout ( Figure 5 ). Prostaglandin E2 is a major metabolite of arachidonic acid produced by cyclooxygenase, which can stimulate the transcription of IL-6 and MuRF-1 in skeletal muscle to regulate muscle mass. The Mstn gene may regulate muscle growth by reducing the metabolism of α-linolenic acid to prostaglandin E2.

[0076] Example 2 A method for detecting the metabolic flux of α-linolenic acid isolated from skeletal muscle fibers of Mstn gene knockout mice

[0077] 1. Digestion of sole muscle tissue in mice

[0078] Saiye Biotechnology Co., Ltd. was commissioned to construct mice with the second exon knockout of the Mstn gene. Three wild-type mice (Mus-WT group) and three Mstn knockout mice (Mus-KO group) were taken, killed by cervical dislocation, and 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, digestion was terminated when the muscle was swollen and accompanied by hair-like muscle fibers.

[0079] 2. Muscle Fiber Separation

[0080] The muscle was removed from the test tube, rinsed once in a culture dish containing 37°C preheated PBS, and then transferred to another culture dish containing 37°C preheated PBS ( Figure 6A), repeatedly blow with a 1000μL pipette. Use a 1mm inner diameter capillary glass tube to stretch and make a glass needle to absorb intact and undamaged muscle fibers. Each time the muscle fibers are absorbed, they must be rinsed in 3 culture dishes filled with PBS in turn, for a total of 3 rinses ( Figure 6 (middle B).

[0081] 3. Non-targeted metabolic flux detection of α-linolenic acid C labeled muscle fibers

[0082] Separate 200 to 300 muscle fibers and use the prepared linoleic acid and linoleic acid 13 C 18 The muscle fibers were cultured with the stock solution for 24 h.

[0083] Collect the cell suspension, centrifuge at 4°C, 1000rpm, for 5min, remove the supernatant, wash the cells gently with PBS, and freeze in liquid nitrogen for 1min. Add 25μL of water to the collected cell pellet, freeze it in liquid nitrogen for 1min, take it out and thaw it, vortex mix it 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 it to a 2mL centrifuge tube, vortex mix it for 30s, and let it stand at -40°C for 1h. Centrifuge the sample at 4°C, 12000rpm for 15min, take an equal amount of 800μL of 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 it for 30s. Place the sample in an ice water bath for 10min of ultrasonication, and centrifuge it at 4°C, 12000rpm for 15min. The supernatant was placed in a sample injection bottle for mass spectrometry detection.

[0084] 4. Effect of MSTN gene on α-linolenic acid metabolism

[0085] OPLS-DA analysis was performed using SIMCA software, and the results are shown in Figure 7 As shown in Figure 3, there was good separation between the Mus-WT group and the Mus-KO group. A total of 30 marker metabolites were identified by non-targeted metabolic flux detection, which belonged to lipid and lipid molecules and organic acids and their derivatives ( Figure 8 ).

[0086] There was no significant difference in the labeling of α-linolenic acid and carnosine between the Mus-KO group and the Mus-WT group ( Fig. 9 ). Carnosine plays an important role in exercise performance and skeletal muscle homeostasis. According to the ratio of each homologue of carnosine, the ratio of m+0 carnosine increased in the Mus-KO group compared with the Mus-WT group, while the ratio of m+1 and m+2 carnosine decreased (P<0.05), indicating that the synthesis and metabolic cycle rate of carnosine slowed down after Mstn gene knockout ( Fig.10 ).

[0087] 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. A method for detecting the effect of myostatin gene in α-linolenic acid metabolic flux, characterized in that: The following steps are involved: The myostatin gene in the target cells was knocked out using gene editing methods to obtain Mstn gene knockout cells; A non-target metabolic flux detection method is used to detect the metabolic flux changes of α-linolenic acid in the Mstn gene knockout cells, and the metabolic flux changes include the main active pathways, the relative contributions of each pathway and the distribution change characteristics.

2. The method according to claim 1, characterized in that: The target cells include mouse skeletal muscle fibroblasts.

3. The method according to claim 1, characterized in that: The gene editing method includes the CRISPR-Cas9 method.

4. The method according to claim 3, characterized in that: The sgRNA sequence used in the CRISPR-Cas9 method includes sgRNA1 with nucleotide sequences as shown in SEQ ID No.1 and sgRNA2 with nucleotide sequences as shown in SEQ ID No.

2.

5. The method according to claim 3, characterized in that: Including using pX458 vector as the vector backbone of CRISPR-Cas9.

6. The method according to claim 1, characterized in that: The target cells include soleus muscle fibers isolated from Mstn knockout mice.

7. The method according to claim 6, characterized in that: The separation method comprises the following steps: isolating the soleus muscle of the Mstn gene 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; The digested muscle was placed in PBS buffer preheated at 37°C and repeatedly blown into single muscle fibers, and a capillary glass needle was used to aspirate the single muscle fiber.

8. The method according to claim 7, characterized in that: The digestion temperature was 37° C., the time was 50 to 70 min, and the mixture was shaken every 15 min during the digestion process.

9. The method according to claim 1, characterized in that: The detection includes primary and secondary mass spectrometry data acquisition using an Orbitrap Exploris 120 mass spectrometer.

10. The method according to claim 1 or 9, characterized in that: The detection includes detection of polar metabolites and non-polar metabolites; For the detection of 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. The detection of non-polar metabolites includes the use of a Vanquish ultra-high performance liquid chromatograph and a Phenomenex Kinetex C18 liquid chromatography column to perform chromatographic separation of the target compounds. The liquid chromatography phase A is an aqueous phase containing 0.01% acetic acid by volume, and the phase B is a mixture of isopropanol and acetonitrile in a volume ratio of 1:1.

Images