Use of acetyl-coa carboxylase gene in regulating porcine lipid metabolism

Abstract

The present invention relates to the technical field of biology. Provided is the use of an acetyl-CoA carboxylase gene in regulating porcine lipid metabolism, disclosing for the first time the involvement of the ACACA gene in regulating porcine beige adipocyte differentiation, and clarifying that inhibiting the expression of the ACACA gene can directly promote adipocyte thermogenesis. The present invention provides a new solution for the treatment of obesity and obesity-related diseases and a new approach for the breeding of cold-resistant low-fat livestock and poultry breeds.

Description

Application of acetyl-CoA carboxylase gene in regulating porcine lipid metabolism

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 2024113993406, filed on October 9, 2024, entitled “Application of Acetyl-CoA Carboxylase Gene in Regulation of Porcine Lipid Metabolism”, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to the application of the acetyl-CoA carboxylase gene in regulating porcine lipid metabolism. Background Technology

[0004] Adipose tissue plays a crucial role in maintaining the body's energy balance, and is mainly divided into white adipose tissue, which stores energy, and brown adipose tissue, which has a thermogenic function. Beige adipose tissue, upon exposure to cold or activation by β-adrenaline, transforms from white adipose tissue into a form similar to brown adipose tissue, enhancing energy expenditure. Studies have shown that inducing the formation of beige adipose tissue can reduce fat deposition and combat obesity.

[0005] As an important source of meat, pig fat deposition directly affects meat quality, yield, and cold resistance. However, due to the lack of UCP1 and classic brown fat in pigs, research has focused on exploring the relationship between beige fat formation and fat deposition traits. Cold-resistant pig breeds, such as Tibetan pigs and Northeast Min pigs, possess stronger cold resistance through the formation of beige fat cells. Therefore, identifying key genes that promote beige fat formation in pigs is of great significance for improving their fat traits.

[0006] ACACA is the rate-limiting enzyme in fatty acid synthesis, primarily expressed in high-lipogenic tissues such as adipose tissue. Inhibiting ACACA can promote beige fat production by reducing fatty acid synthesis, thereby reducing fat deposition and enhancing thermogenesis. Therefore, the ACACA gene and its inhibitors have potential value in regulating pig fat metabolism and can serve as novel targets for feed additives in molecular breeding and production. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the acetyl-CoA carboxylase (ACACA) gene in regulating porcine lipid metabolism.

[0008] To achieve the objective of this invention, in a first aspect, this invention provides the application of the acetyl-CoA carboxylase gene in regulating porcine lipid metabolism, inhibiting the expression of the porcine acetyl-CoA carboxylase gene, and promoting fat burning.

[0009] Preferably, the nucleotide sequence of the coding region of the ACACA gene is shown in SEQ ID NO:1.

[0010] Preferably, the amino acid sequence of the protein (porcine acetyl-CoA carboxylase) encoded by the ACACA gene is shown in SEQ ID NO:2.

[0011] Secondly, this invention provides the application of the acetyl-CoA carboxylase gene in regulating the production of beige fat in pigs, inhibiting the expression of the acetyl-CoA carboxylase gene and promoting the production of beige fat.

[0012] Thirdly, the present invention provides any of the following applications of acetyl-CoA carboxylase gene inhibitors:

[0013] 1) Used in the preparation of products for the treatment and / or prevention of obesity;

[0014] 2) Used to promote the burning of white fat cells;

[0015] 3) Used in the preparation of products that promote weight loss;

[0016] 4) Used to inhibit the accumulation / gathering of white fat under the skin and / or in the abdomen;

[0017] 5) Used for breeding low-fat livestock and poultry breeds;

[0018] 6) Used for breeding cold-resistant livestock and poultry breeds.

[0019] The inhibitor can be selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, Cre-Loxp, TALEN, ZFN, or CRISPR / Cas9, etc.

[0020] Preferably, the inhibitor is siRNA, with the sequence shown in SEQ ID NO:3, whose base sequence is a nucleotide sequence that is reverse complementary to the 1741-1761 bases of the ACACA gene coding region in SEQ ID NO:1.

[0021] More preferably, the two U bases at the 3' end of the siRNA sequence can be replaced with T (i.e., GCUUCUGUCAGCUCAGAUATT), which does not affect the inhibitory effect, reduces costs, and enhances the ability to resist enzyme degradation.

[0022] Preferably, the inhibitor is shRNA, and the DNA sequence encoding the shRNA is shown in SEQ ID NO:4, wherein the target sequence bases are identical to bases 3349-3369 of the ACACA gene coding region in SEQ ID NO:1.

[0023] Preferably, the inhibitor is 5-(tetradecyloxy)-2-furoic acid (TOFA).

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0025] (I) This invention reveals for the first time that the ACACA gene is involved in regulating the production of beige fat.

[0026] (II) This invention clarifies that inhibiting ACACA gene expression can directly promote in vivo fat burning.

[0027] (III) This invention provides a new solution for treating obesity and obesity-related diseases, avoiding the problems of insufficient effectiveness and questionable safety of weight loss drugs.

[0028] (iv) This invention provides a new approach for breeding cold-resistant and low-fat livestock and poultry breeds. Attached Figure Description

[0029] Figure 1 illustrates the regulation of thermogenic production in Tibetan pig adipocytes by ACACA gene expression in a preferred embodiment of the present invention. (A) Fluorescent staining images of cells treated with FSK and TOFA, BODIPY staining showing lipid droplets, and TMRE staining showing mitochondrial membrane potential of differentiated adipocytes, scale bar 10 μm; (B) Effects of FSK and TOFA treatment on mitochondrial respiration in differentiated Tibetan pig adipocytes, FSK and TOFA stimulation alone or in combination (n=5); (C) Immunohistochemical results of PRDM16, pHSL, and HSL (n=3); (DE) Protein and mRNA expression levels of beige adipocyte molecular markers in differentiated Tibetan pig adipocytes, FSK and TOFA stimulation alone or in combination (n=6); (F) ACACA overexpression and knockout. (G) Degradation of protein expression and Oil Red O staining in Tibetan pig adipocytes after shRNA transfection (n=5) by Seahorse; (H) Basal respiration and proton leakage calculated from the results of G (n=5); (I) Mitochondrial respiration of Tibetan pig adipocytes after ACACA overexpression by Seahorse (n=5); (J) Basal respiration and proton leakage calculated from the results of I (n=5); (K) Mitochondrial respiration of Tibetan pig adipocytes after siRNA transfection by Seahorse (n=5); (L) Basal respiration and proton leakage calculated from the results of K (n=5).

[0030] In the figure, *P < 0.05, **P < 0.01, and ***P < 0.001.

[0031] Figure 2 is a plasmid map of the ACACA overexpression vector in a preferred embodiment of the present invention. Detailed Implementation

[0032] Research has found that ACACA is a novel and important molecule regulating beige fat metabolism. Reducing its protein levels can promote thermogenesis in beige fat and burn white fat, thereby improving the body's cold resistance and reducing the content of white fat. Based on this, this invention focuses on the application of inhibiting ACACA protein or genes in the preparation of slimming and weight-loss products, improving the cold resistance of livestock and poultry, and breeding superior breeds that reduce the fat content of livestock and poultry.

[0033] The present invention adopts the following technical solution:

[0034] This invention provides the application of a reagent for inhibiting ACACA gene expression in the preparation of products having at least one of the following functions: 1)-6).

[0035] 1) Treatment and / or prevention of obesity;

[0036] 2) Promotes the burning of white fat cells;

[0037] 3) Promotes weight loss;

[0038] 4) Inhibits the accumulation / gathering of subcutaneous and / or abdominal white fat;

[0039] 5) Used for breeding low-fat livestock and poultry breeds;

[0040] 6) Used for breeding cold-resistant livestock and poultry breeds.

[0041] Preferably, the nucleotide sequence of the coding region of the ACACA gene is shown in SEQ ID NO:1.

[0042] Preferably, the amino acid sequence of the protein encoded by the ACACA gene is shown in SEQ ID NO:2.

[0043] Preferably, the siRNA of the ACACA gene has the sequence shown in SEQ ID NO:3, and its base sequence is a nucleotide sequence that is reverse complementary to the 1741-1761 bases of the ACACA gene coding region in SEQ ID NO:1.

[0044] More preferably, the two U bases at the 3' end of the siRNA sequence can be replaced with T (i.e., GCUUCUGUCAGCUCAGAUATT), which does not affect the inhibitory effect, reduces costs, and enhances the ability to resist enzyme degradation.

[0045] Preferably, the shRNA of the ACACA gene, the DNA sequence encoding the shRNA is shown in SEQ ID NO:4, wherein the target sequence bases are identical to bases 3349-3369 of the ACACA gene coding region in SEQ ID NO:1.

[0046] Preferably, the full sequence of the ACACA overexpression vector is formed by SEQ ID NO:5 and 6 in tandem.

[0047] Preferably, the reagent for inhibiting ACACA gene expression is an antisense nucleic acid or a repressive transcription factor.

[0048] Preferably, the reagent for inhibiting ACACA gene expression includes Cre-Loxp, TALEN, ZFN, or CRISPR / Cas9.

[0049] Preferably, the reagent is capable of silencing or knocking out the ACACA gene.

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] Example 1: ACACA inhibitor TOFA enhances thermogenesis in porcine adipocytes

[0052] To investigate whether inhibition of ACACA regulates the thermogenesis of porcine adipocytes, stromal vascular fraction (SVF) cells were isolated from Tibetan pig cells and SVF cells were induced to differentiate into adipocytes. A cell permeability inhibitor, TOFA, was used to inhibit the activity of ACACA, and the thermoproduction of porcine adipocytes was analyzed.

[0053] 1. Isolation of porcine adipose matrix vascular components (SVF)

[0054] Subcutaneous adipose tissue (SAT) from 5-week-old male Tibetan and Bama pigs was surgically removed and minced into small pieces. Enzymatic digestion was performed using 2 mg / ml Type I collagenase in D-Hanks buffer at 37°C for 60 minutes, followed by filtration through a 70 μm filter to remove undigested tissue. The filtrate was centrifuged at 1500 rpm for 10 minutes to collect SVF cell clusters, red blood cells were removed, and the cells were centrifuged again. SVF cells were resuspended in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured in an incubator at 38.5°C and 5% CO2.

[0055] 2. Adipogenic differentiation of porcine SVF cells

[0056] SVF cells were seeded into culture plates until confluenced and cultured for 2 days (D0). The medium was then replaced with induction medium (containing 0.25 mM IBMX (3-isobutyl-1-methylxanthine), 1 μM dexamethasone, 5 μg / mL insulin, 17 μM pantothenic acid, 33 μM biotin, and 50 μM rosiglitazone) and cultured for 5 days. Afterward, the medium was replaced with maintenance medium (containing 5 μg / mL insulin, 17 μM pantothenic acid, and 33 μM biotin) and cultured until day 8.

[0057] 3. TOFA Inhibition of ACACA in Porcine Adipocytes Experiment

[0058] On day 8, the differentiated adipocytes were treated as follows: Control group: no drug intervention, only carrier solution was added; TOFA treatment group: treated with 10 μM TOFA for 24 hours; FSK (forskolin, which induces intracellular cAMP formation and stimulates lipolysis and mitochondrial respiration) treatment group: treated with 10 μM FSK for 24 hours; combined treatment group: treated with both 10 μM TOFA and 10 μM FSK for 24 hours.

[0059] 4. Cell fluorescence staining

[0060] Tibetan pig SVF cells were seeded in 35 mm glass dishes and cultured for induced differentiation. After differentiation and maturation, the cells were washed twice with preheated DPBS and then infused with DMEM complete medium containing 200 nM BODIPY493 / 503 and 20 nM tetramethylrhodamine ethyl ester (TMRE). The cells were stained in a 37°C cell culture incubator for 15-30 min and photographed using a laser confocal microscope. BODIPY493 / 503 stains lipid droplets green, while TMRE is a red dye used to stain mitochondrial membrane potential.

[0061] 5. The Seahorse XF energy metabolism detection system detects mitochondrial respiratory function in cells.

[0062] SVF cells from Tibetan pigs were seeded into XF24 cell culture plates, with 80-100 μL of cell suspension in each well. After sedimentation, the cells were incubated at 37°C in a 5% CO2 incubator and induced to differentiate. Differentiated cells were used for detection on day 8. The day before the experiment, 200 μL of sterile water was added to each well of the probe plate and hydrated overnight at 37°C in a CO2-free incubator. On the day of the experiment, the sterile water in the probe plate was discarded, and 200 μL of XF calibration solution (Agilent, 103575-100) was added, and the plates were equilibrated at 37°C in a CO2-free incubator for 45-60 minutes. Simultaneously, the detection solution was prepared (1 mL of 1M glucose, 1 mL of 100mM pyruvate, and 1 mL of 200mM glutamine were added to 100 mL of Seahorse XF DMEM medium), and a 10× drug solution of 1.5 μM oligomycin, 0.5 μM uncoupling agent (FCCP), and 0.5 μM rotenone / antimycin A (Rot / AA) was prepared. The culture medium was aspirated from the cell culture plates, and the plates were washed twice with 200 μL of detection solution. Finally, 160 μL of detection solution was added to each well, for a total volume of 180 μL. The plates were incubated at 37°C in a CO2-free incubator for 45–60 minutes before detection. Basal respiration, maximum respiratory rate, and proton leakage were measured using Wave software. Data analysis was performed using per-well DNA or protein content normalization.

[0063] 6. qPCR analysis

[0064] Total RNA was extracted from the adipose tissue of Bama piglets (TRIzol reagent, Thermo Fisher Scientific, 15596018) and purified according to the manufacturer's instructions. 1 μg of total RNA was reverse transcribed into cDNA (PrimeScript). TM The RT kit (Takara, RR047A) was used to perform quantitative PCR analysis on the QuantStudio 3 real-time PCR system (Life Technologies) using TB Green Premix Ex Taq (Takara, RR420A). The beige adipocyte marker genes PRDM16, EBF2, DIO2, UCP3, and CIDEA were analyzed. 18S rRNA (Gene ID: 100861538) was used as an internal reference gene for normalization. Primer sequences are shown in Table 1.

[0065] Table 1 Primer sequences for quantitative real-time PCR

[0066] 7. Immunoblot analysis

[0067] Cell lysates (100 μL per fraction) were extracted using the M-PER™ mammalian protein extraction reagent (Thermo Fisher Scientific, 78503) and supplemented with a protease inhibitor mixture (Roche, 04693159001). After centrifugation (12,000 × g, 4 °C, 20 min), the obtained proteins (20–50 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Merck-Millipore, IPVH00010). Detection was performed using primary antibodies (such as PRDM16 and HSL), and the immunoreaction bands were visualized using a FluorChem M fluorescence imaging system (Tanon, 5200) and ECL developing solution (Tanon, 180–501).

[0068] Results: TOFA is an allosteric inhibitor of ACACA that can inhibit fatty acid synthesis. Fluorescent staining revealed that FSK treatment promoted lipid droplet hydrolysis, significantly increasing the number of small lipid droplets. While many large lipid droplets remained in TOFA-treated cells, numerous small lipid droplets appeared. When FSK and TOFA were treated simultaneously, all large lipid droplets were hydrolyzed into small and micro lipid droplets (Figure 1A). We examined the effects of FSK and TOFA treatments on cellular OCR (Figure 1B), finding that both FSK and TOFA significantly increased proton leakage respiration and reduced ATP production, with the highest proton leakage observed in the FSK and TOFA simultaneous treatment group (Figure 1C). Furthermore, we found that both FSK and TOFA treatments increased the expression levels of thermogenic genes PRDM16, EBF2, PPARGC1A, CPT1A, and DIO2, with the highest expression levels of these thermogenic genes observed in the FSK and TOFA simultaneous treatment group (Figure 1, DE). These findings indicate that inhibiting ACACA activity with TOFA not only enhances thermogenesis in porcine adipocytes but also further enhances FSK-induced thermogenesis.

[0069] Example 2: Overexpression or inhibition of the ACACA gene regulates thermogenesis in Tibetan pig adipocytes

[0070] In addition to using the small molecule inhibitor TOFA of ACACA to study its regulatory effect on thermogenesis in adipocytes, we also used genetic methods to examine the regulatory effect of the ACACA gene on thermogenesis in porcine adipocytes, including overexpression or knockdown of the ACACA gene.

[0071] 1. RNA interference

[0072] (1) The mRNA sequence of the gene was found through the NCBI website. siRNA was designed using the online website siDirect version 2.0 (http: / / sidirect2.rnai.jp / design.cgi). The top 3 sequences were selected and synthesized by Shanghai Jima Biotechnology Co., Ltd. along with the negative control sequence.

[0073] (2) Seed SVF cells onto 12-well cell culture plates. When the cells reach 70-80% confluence, aspirate the culture medium and add 875 μL of DMEM complete culture medium to each well. Place the plates in a cell culture incubator.

[0074] (3) Prepare interference reagents according to the number of cells to be treated in the cell culture plate. Solution A: Add 62.5 μL OPTI and 2.5 μL siRNA or negative control to each well. Solution B: Add 62.5 μL OPTI-MEM to each well. TM Serum-free culture medium and 2.5 μL RNAIMAX transfection reagent, solution A and solution B are mixed separately and incubated at room temperature for 5 min;

[0075] (4) Add solution B to solution A and mix thoroughly. Incubate at room temperature for 15 minutes.

[0076] (5) Slowly add the above mixture to the cell culture plate at 125 μL per well, gently mix the culture medium and interfering reagent in the cell culture plate, and place it in a cell culture incubator.

[0077] (6) On day 0 and day 3 of cell differentiation induction, the cells were subjected to interference treatment in accordance with the above steps, and the DMEM complete medium was replaced with a medium containing differentiation induction reagent.

[0078] 2. Construct shRNA and overexpression plasmid

[0079] (1) shRNA design: Using the CDS region of the porcine ACACA gene (NCBI number NM_001114269.1) as a template, shRNA was designed using an online website (https: / / portals.broadinstitute.org / gpp / public). The top 3 shRNA sequences were selected, and the primers were annealed and ligated to the enzyme-digested plasmid (PB-pACACA-shRNA). The ligase system is shown in Table 2.

[0080] Construction of overexpression plasmid: The CDS region of the ACACA gene was amplified using Tibetan pig cDNA as a template, and the ACACA gene was ligated into the enzyme-digested plasmid (PB513B-1-CAG) to construct the overexpression plasmid (the full sequence of the overexpression plasmid is shown in SEQ ID NO:5 and 6).

[0081] Table 2 Ligase System

[0082] (2) Add the above-connected plasmid (Figure 2) to 50 μL of Escherichia coli DH5α competent cells, gently tap the bottom of the centrifuge tube to mix, and place on ice for 30 min;

[0083] (3) Heat shock at 42℃ for 30s, quickly insert into ice, stand for 2min, add to 500μL LB medium, and shake at 200rpm for 1h in a 37℃ incubator;

[0084] (4) Centrifuge at 3000 rpm for 2 min, discard the supernatant, leave about 100 μL, and resuspend the E. coli precipitate at the bottom;

[0085] (5) Spreading: Spread the above 100 μL bacterial solution evenly onto a plate containing ampicillin resistance, place it in a 37℃ incubator upright for 1 hour, and then invert it overnight for incubation.

[0086] (6) Picking bacteria: Pick 10-15 colonies of each plasmid in 5 mL of LB liquid medium containing ampicillin, shake at 37℃ and 200 rpm for 5-6 h, and when the bacterial solution becomes turbid, take 500 μL of the bacterial solution for sequencing.

[0087] (7) Plasmids with correct sequencing results are inoculated into 200 mL of LB liquid medium and cultured for 12-16 h. Plasmids are extracted according to the instructions of the plasmid extraction kit and sequenced. Plasmids with correct sequences can be used for cell transfection.

[0088] 3. Cell transfection and screening

[0089] (1) Tibetan pig SVF cells were cultured in 100 mm culture dishes and electroporation was started when they reached about 80-90% of the cell line.

[0090] (2) Add 1 mL of culture medium to a 12-well plate and place it in a cell culture incubator to balance CO2.

[0091] (3) Wash the cells with DPBS, digest the cells with 0.25% trypsin, and centrifuge at 80g for 10min;

[0092] (4) Prepare the electroporation buffer during centrifugation. Use 100 μL of electroporation buffer for each plasmid, and take 10 μL to dilute the plasmid. Calculate the amount of electroporation based on the molar mass ratio of the plasmid.

[0093] (5) After centrifuging the cells, aspirate the supernatant, gently disperse the precipitate by hand, gently resuspend the cell precipitate with 90 μL of electroporation buffer, mix it with the plasmid, and then transfer it all into the electroporation cup.

[0094] (6) Gently shake the electric transfer cup on the table a few times, put it into the electric transfer instrument, and start the electric transfer;

[0095] (7) Remove the CO2-equilibrated 12-well plate from the incubator, use a Pasteur pipette to draw 500 μL of culture medium and mix it with the cells in the electroporation cuvette, then add 5 × 10⁻⁶ cells per well. 5 Hole / hole plate;

[0096] (8) After electroporation for 24 hours, the culture medium was replaced with complete culture medium, and the transfection efficiency was observed under an inverted fluorescence microscope.

[0097] (9) After 48 hours, switch to a complete culture medium containing 2 mg / mL puromycin. Change the medium every 2 days until all cells are successfully electroporated. Then proceed with subsequent experiments or freeze the cells.

[0098] 4. Red O staining and quantification

[0099] (1) The culture medium was removed from the cells that were induced to differentiate and mature. The cells were washed twice with preheated DPBS and fixed with 4% paraformaldehyde for 30-60 min.

[0100] (2) Prepare Oil Red O working solution by mixing Oil Red O stock solution with distilled water at a ratio of 3:2, filter with a 0.45μm filter, and let stand at room temperature in the dark for 5-10 minutes before use;

[0101] (3) After the cells are fixed, 4% paraformaldehyde is removed, and the cells are washed once with distilled water and once with 60% isopropanol.

[0102] (4) Add 2-3 mL (for a six-well plate) of Oil Red O working solution to each well and stain at room temperature in the dark for 15-30 min;

[0103] (5) Discard Oil Red O working solution, wash away excess Oil Red O working solution with 60% isopropanol, and wash cells with distilled water. Add 1 mL of distilled water to each well and take pictures with an inverted microscope.

[0104] (6) Oil Red O quantification: Remove distilled water, add 1 mL of 100% isopropanol to each well, place on a shaker, shake at room temperature for 10 min, pipette 200 μL from each well into a 96-well plate, and measure the absorbance at 510 nm using an ELISA reader.

[0105] Results: By detecting the expression level of ACACA protein in differentiated and mature adipocytes, it was found that ACACA protein expression was significantly increased in the overexpression (OE) group and significantly decreased in the shRNA knockdown group (Figure 1F). Oil Red O staining results showed that ACACA gene overexpression increased lipid accumulation, while gene knockdown significantly inhibited lipid accumulation (Figure 1F). To investigate the effects of ACACA gene knockdown and overexpression on thermogenesis in porcine adipocytes, mitochondrial respiration was measured. The results showed that stable knockdown of ACACA expression using shRNA significantly enhanced proton leakage respiration, resulting in a decrease in ATP production and oxygen consumption, indicating enhanced thermogenesis (Figure 1, GH). Conversely, overexpression of ACACA significantly inhibited proton leakage respiration, indicating suppressed thermogenesis (Figure 1, IJ). Further knockdown of ACACA expression using siRNA during porcine adipocyte differentiation also showed a significant increase in proton leakage respiration and a decrease in ATP production respiration (Figure 1, KL), confirming that inhibiting ACACA can promote thermogenesis in adipocytes.

[0106] Although the present invention has been described in detail above with general descriptions 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 fall within the scope of protection claimed by the present invention. Industrial applicability

[0107] This invention provides the application of the acetyl-CoA carboxylase gene in regulating porcine lipid metabolism. This invention reveals for the first time that the ACACA gene participates in regulating the differentiation of beige adipocytes in pigs, and clarifies that inhibiting ACACA gene expression can directly promote thermogenesis in adipocytes. This invention provides a new solution for treating obesity and obesity-related diseases, and offers a new approach for breeding cold-resistant, low-fat livestock and poultry breeds, possessing significant economic value and application prospects.

Claims

1. The application of the acetyl-CoA carboxylase gene in regulating porcine lipid metabolism, characterized in that, Inhibits the expression of porcine acetyl-CoA carboxylase gene and promotes fat burning; The amino acid sequence of porcine acetyl-CoA carboxylase is shown in SEQ ID NO:

2.

2. The application of the acetyl-CoA carboxylase gene in regulating beige fat production in pigs, characterized in that, Inhibit the expression of porcine acetyl-CoA carboxylase gene and promote the production of beige fat; The amino acid sequence of porcine acetyl-CoA carboxylase is shown in SEQ ID NO:

2.

3. Any of the following applications of acetyl-CoA carboxylase gene inhibitors: 1) Used in the preparation of products for the treatment and / or prevention of obesity; 2) Used to promote fat cell burning; 3) Used in the preparation of products that promote weight loss; 4) Used to inhibit the accumulation / gathering of white fat under the skin and / or in the abdomen; 5) Used for breeding low-fat livestock and poultry breeds; 6) Used for breeding cold-resistant livestock and poultry breeds; wherein, The amino acid sequence of porcine acetyl-CoA carboxylase is shown in SEQ ID NO:2; The inhibitors are selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, Cre-Loxp, TALEN, ZFN, or CRISPR / Cas9.

4. Use according to claim 3, characterized in that, The inhibitor is siRNA, with the sequence shown in SEQ ID NO:

3.

5. The application according to claim 3, characterized in that, The inhibitor is shRNA, and the DNA sequence encoding the shRNA is shown in SEQ ID NO:

4.

6. The application according to claim 3, characterized in that, The inhibitor is 5-(tetradecyloxy)-2-furoic acid.

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