A method for identifying cold resistance of animals and its application

Abstract

The present invention relates to the field of biotechnology, and provides an identification method and application of animal cold resistance. Studies have found that compared with cold-intolerant pig breeds such as Bama pigs and Wuzhishan pigs, cold-tolerant pig breeds such as Tibetan pigs and Min pigs produce beige fat after acute cold stimulation, which was discovered through transcriptome data analysis and verified by Western blot and QPCR. PDK4 , pPDHA1 , CPT1A , p563HSL , pACACA , ACACA and BASIN Genes such as these are involved in the regulation of heat production in the body's fat cells and can be used to identify animals' ability to resist cold. They are of great significance for improving animals' resistance to cold, meat production performance and human health.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an identification method for animal cold resistance and application thereof. Background Art

[0002] Different pig breeds have obvious differences in their ability to adapt to cold. For example, the Northeast Min pigs and Tibetan pigs living in cold areas have strong cold resistance, while the Wuzhishan pigs and Bama pigs living in tropical areas are more sensitive to cold. Identifying the cold tolerance of livestock is crucial for breeding highly stress-resistant breeds and improving animal welfare. Animal adipose tissue plays a key role in the body's energy balance and temperature regulation. Adipose tissue is mainly divided into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is mainly used for energy storage, while BAT generates heat in cold environments through non-shivering thermogenesis (NST) to maintain body temperature. A large number of studies have shown that mammals can rely on non-shivering thermogenesis of uncoupling Protein 1 (UCP1) in BAT to maintain body temperature under cold conditions, but pigs have changed their genetic evolution. UCP1 The deletion of exons 3-5 of the gene results in the lack of functional BAT and UCP1 , which makes piglets sensitive to cold and increases the economic cost of pig farming. In recent years, studies have discovered a new type of fat cell between WAT and BAT, called "beige adipocytes" or "light brown adipocytes." Beige adipocytes can be transformed from WAT under cold stimulation or the action of specific hormones, and have similar heat production capacity as BAT. Cold-resistant pig breeds can produce beige fat in subcutaneous adipose tissue after acute cold stimulation, while cold-sensitive pig breeds cannot produce beige fat, which is an important reason for the difference in cold resistance among different pig breeds.

[0003] By comparing the molecular characteristics of beige fat formation in different pig breeds and identifying the related differential genes, a scientific basis will be provided for breeding cold-resistant breeds. At the same time, improving the ability of animals to adapt to cold can reduce the incidence of diseases, reduce breeding costs, improve animal welfare, and thus promote the sustainable development of animal husbandry. Summary of the invention

[0004] The purpose of the invention is to provide an identification method for animal cold resistance and application.

[0005] To achieve the purpose of the present invention, in a first aspect, the present invention provides marker genes for distinguishing cold-tolerant pig breeds from cold-intolerant pig breeds, wherein the marker genes consist of PDK4, pPDHA1, CPT1A, p563HSL, pACACA, ACACA and FASN.

[0006] In the present invention, the cold-resistant pig breeds include Northeast Min pigs and Tibetan pigs, and the cold-intolerant pig breeds include Wuzhishan pigs and Bama pigs.

[0007] In a second aspect, the present invention provides the application of the marker gene in the identification and breeding of cold-resistant pig breeds and non-cold-resistant pig breeds.

[0008] In a third aspect, the present invention provides the application of the marker gene in molecular marker-assisted breeding and variety improvement of cold-tolerant pig breeds and non-cold-tolerant pig breeds.

[0009] In a fourth aspect, the present invention provides a method for identifying cold-resistant pig breeds and cold-intolerant pig breeds, the method comprising: detecting the expression level of marker genes in the adipose tissue of the pig to be tested at the transcriptional level, the expression levels of marker genes PDK4, pPDHA1, CPT1A and p563HSL in cold-resistant pig breeds are significantly higher than those in cold-intolerant pig breeds, and the expression levels of marker genes pACACA, ACACA and FASN in cold-resistant pig breeds are significantly lower than those in cold-intolerant pig breeds.

[0010] In a fifth aspect, the present invention provides a detection reagent or a kit containing primers for detecting the marker gene.

[0011] In a sixth aspect, the present invention provides any of the following applications of the detection reagent or kit:

[0012] 1) Used for identification and breeding of cold-tolerant and non-cold-tolerant pig breeds;

[0013] 2) Used for molecular marker-assisted breeding and variety improvement of cold-tolerant and non-cold-tolerant pig breeds.

[0014] In the seventh aspect, the present invention provides a method for identifying the cold resistance of animals. After the animals are subjected to acute cold stimulation, beige fat cells are produced in the animal's adipose tissue, and the expression of genes involved in the de novo synthesis of fatty acids, such as pACACA, ACACA and FASN in the animal's adipose tissue is downregulated, while the expression of genes involved in the fatty acid oxidation process, such as PDK4, pPDHA1, CPT1A and p563HSL, is upregulated, then the animal is judged to have the ability to resist cold.

[0015] Furthermore, the conditions for acute cold stimulation are: 4-6°C, 4-6 hours.

[0016] After acute cold stimulation, animals (such as Northeast Min pigs and Tibetan pigs) produce beige fat cells, that is, the original fat is formed with the characteristics of multiple chambers and small fat droplets, while the fat cells of the cold-intolerant pig breeds Bama pigs and Wuzhishan pigs are still in the form of single large fat droplets.

[0017] Furthermore, the animal includes a pig.

[0018] In an eighth aspect, the present invention provides application of the method in animal breeding.

[0019] Breeding objectives include improving animals' resistance to cold and meat production.

[0020] In a ninth aspect, the present invention provides a method for establishing a beige cell thermogenic model (beige fat in vitro thermogenic model), comprising: treating beige adipocytes (such as mature beige adipocytes of Tibetan pigs) with 5 μM FSK (forskolin, FSK; adenylate cyclase activator) for 24 hours, i.e., the condition for beige adipocyte thermogenicity.

[0021] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, lipid droplets became smaller.

[0022] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, mitochondria were depolarized and their number increased.

[0023] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, the cellular proton leak oxygen consumption was significantly increased.

[0024] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, the expression of PDK4 and CPT1A genes involved in fatty acid oxidation was upregulated.

[0025] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, the expression levels of ACACA, ACLY, FASN, and SCD genes involved in the de novo synthesis of fatty acids decreased.

[0026] Furthermore, after beige adipocytes were treated with 5 μM FSK for 24 h, the thermogenic genes PPARGC1A and CIDEA, TAG hydrolyzing genes ATGL and HSL, and lipid droplet protein gene PLIN4 were upregulated.

[0027] In a tenth aspect, the present invention provides an application of a beige adipocyte thermogenic model in the study of beige fat function.

[0028] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] The present invention provides a method for identifying the cold resistance of animals. It is found that compared with the cold-intolerant pig breeds Bama pig and Wuzhishan pig, the cold-tolerant pig breeds Tibetan pig and Min pig will produce beige fat after acute cold stimulation. This is found through transcriptome data analysis and verified by Western blot. PDK4 , pPDHA1 , CPT1A , p563HSL , PcA , ACACA and FASNGenes such as these are involved in the regulation of heat production in the body's fat cells and can be used to identify animals' ability to resist cold. They are of great significance for improving animals' resistance to cold, meat production performance and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 In the preferred embodiment of the present invention, the DNL gene of cold-resistant pig breeds is down-regulated after acute cold stimulation. (A) Acute cold stimulation induces the formation of beige adipocytes in cold-tolerant pig breeds; (B) Distribution of beige fat DEGs in Tibetan pigs and Min pigs; (C) Distribution of white fat DEGs in Bama pigs and Wuzhishan pigs; (D) GO enrichment pathway bubble diagram of beige fat DEGs and white fat DEGs; (E) GSEA spectrum shows that genes in the fatty acid biosynthesis pathway are downregulated after cold stimulation in Tibetan pigs; (F) Expression change pattern of fatty acid metabolism genes in four pig breeds after acute cold stimulation; (G) mRNA expression levels of fatty acid oxidation (FAO) and de novo lipogenesis (DNL) related genes in Tibetan pig IWAT; (H) Expression levels of FAO and DNL related proteins in Tibetan pig IWAT; (I) mRNA expression levels of FAO and DNL related genes in Bama pig IWAT; (J) Expression levels of FAO and DNL related proteins in Bama pig IWAT.

[0031] Figure 2 The optimal time and concentration for FSK treatment of cells in a preferred embodiment of the present invention are determined. (A) Schematic diagram of FSK treatment of cells at different times; (B) Schematic diagram of FSK treatment of cells at different concentrations; (C) Pictures of fat cells treated with FSK at different times, with a scale of 50 μM; (D) Expression levels of genes related to beige fat and hydrolysis; (E) Pictures of cells treated with FSK at different concentrations after hydrolysis, with a scale of 50 μM; (F) Expression levels of genes related to fatty acid oxidation and hydrolysis.

[0032] Figure 3In the preferred embodiment of the present invention, FSK enhances thermogenesis of beige adipocytes in Tibetan pigs and inhibits the expression of DNL gene. (A) Fluorescence staining image of beige adipocytes, green (BODIPY493 / 503): lipid droplets; red (TMRE): mitochondrial membrane potential; blue (HOECHST): cell nucleus; scale 10μM; (B) Electron microscope image of beige adipocytes, LD: lipid droplets, red arrows: mitochondria, scale 1μM; (C) Statistical frequency graph of lipid droplet size in beige adipocytes; (D) Flow cytometer analysis of cell mitochondrial membrane potential, abscissa: fluorescence signal of TMRE dye, ordinate: cell number; (E) qPCR detection of COX2 gene expression level (mitochondrial copy number); (F) Seahorse determination of OCR of beige adipocytes after FSK treatment; (G) Proton leak and ATP synthesis obtained by calculation. (HI) qPCR detection of the expression levels of DNL (H) and FAO (I) genes in beige adipocytes of Tibetan pigs; (J) Western blot detection of the expression levels of FAO and DNL proteins in beige adipocytes of Tibetan pigs.

[0033] DETAILED DESCRIPTION

[0034] The present invention provides a method for identifying the cold resistance of animals and a method for constructing a cell heat production model and its application. Animal fat is used as a research object to identify the cold resistance of animals, establish a heat-producing cell line model, and apply it to the analysis of the heat production mechanism of animal fat.

[0035] In order to further study the thermogenic mechanism of adipocytes, stromal vascular fraction (SVF) cells were isolated from inguinal white adipose tissue (iWAT) of Tibetan pigs, and a culture system for directional differentiation of SVF cells into adipocytes was established. Treatment of adipocytes with 5μM forskolin (FSK; adenylate cyclase activator) for 24h successfully activated the thermogenic program of cells, which was manifested as ① smaller lipid droplets of adipocytes, ② depolarization of mitochondria and increased copy number, ③ increased cellular oxygen consumption rates (OCR), significantly increased proton leak, reduced ATP synthesis, and ④ increased production of fatty acid during fatty acid oxidation (FAO). PDK4 (NCBI Gene ID: 100286778), CPT1A(NCBIGene ID: 399527) gene expression is upregulated, PDK4 protein expression is increased, that is, FAO is upregulated, ⑤ fatty acid de novo synthesis (denovo lipogenesis, dNL) process ACACA (NCBI Gene ID: 397324)、 ACLY (NCBI Gene ID:100125957), FASN (NCBI Gene ID: 397561)、 SCD The expression of genes (NCBI Gene ID: 396670) and ACACA, pACACA, and FASN proteins decreased, i.e., DNL was downregulated. ⑥ Thermogenic genes PPARGC1A (NCBI Gene ID:397013) and CIDEA (NCBI Gene ID: 100127171), triglyceride hydrolysis gene ATGL (NCBI ID:100049704) and HSL (NCBI ID: 397583) 、 Lipoprotein gene PLIN4 Increase.

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0037] Example 1 Identification of cold-resistant pig breeds

[0038] First, an acute cold stimulation test was conducted on cold-tolerant Tibetan pigs and Min pigs, and cold-intolerant Bama pigs and Wuzhishan pigs: 8 healthy male Tibetan pigs, Bama pigs, Min pigs and Wuzhishan pigs of similar weight at 1 month old were selected and randomly divided into a normal temperature control group and an acute cold stimulation group, with 4 biological replicates in each group, with free access to food and water. The ambient temperature of the normal temperature control group was controlled at 25±2℃, and the acute cold stimulation test was 4-6℃ for 4h. The adipose tissue was collected from iWAT, which was quickly frozen in liquid nitrogen after collection and stored in a -80℃ refrigerator for use.

[0039] The results showed that the iWAT of cold-tolerant pigs could undergo browning of white fat and produce beige fat cells after acute cold stimulation. Specifically, the fat formation of Tibetan pigs and Min pigs was characterized by multi-chamber and small fat droplets, while the fat cells of Bama pigs and Wuzhishan pigs, which were not cold-tolerant, were still in the form of single large fat droplets ( Figure 1 A).

[0040] Example 2 Screening of key genes for the formation of beige fat

[0041] In order to explore the mechanism of beige fat formation and heat production in cold-resistant pig breeds under acute cold stimulation, we collected 8 samples from the normal temperature control group and cold stimulation group of 4 pig breeds for transcriptome sequencing. The transcriptome data were divided into two groups, cold-resistant and cold-intolerant, for analysis. All differentially expressed genes of the two breeds with stronger white fat browning ability, Tibetan pig and Min pig, were defined as "beigedifferentially expressed genes (BeigeDEGs)", and all differentially expressed genes of the two breeds with lower white fat browning ability, Bama pig and Wuzhishan pig, were defined as "whitedifferentially expressed genes (WhiteDEGs)". Full transcriptome analysis of iWAT of four pig breeds before and after acute cold stimulation (FC>1.5, P <0.05; n=4)

[0042] The results showed that the two cold-tolerant pig breeds, Tibetan pigs and Min pigs, had 2030 up-regulated BeigeDEGs and 2281 down-regulated BeigeDEGs ( Figure 1 B); Bama pig and Wuzhishan pig, two cold-intolerant pig breeds, had 1681 up-regulated WhiteDEGs and 2366 down-regulated WhiteDEGs ( Figure 1 C). Then, GO enrichment analysis was performed on all the common genes, and a bubble diagram of the significantly enriched pathways was drawn. It can be seen from the figure that the up-regulated genes of BeigeDEGs were mainly enriched in pathways such as angiogenesis and inflammatory response; the down-regulated genes were mainly enriched in fatty acid biosynthetic process, cholesterol biosynthesis, SREBF activated gene expression, and lipid biosynthetic process ( Figure 1 D).

[0043] To further analyze the distribution of DEGs before and after acute cold stimulation in the four pig breeds, we performed gene set enrichment analysis (GSEA) on the transcriptome data of iWAT. The fatty acid biosynthetic process was significantly downregulated after acute cold stimulation in cold-tolerant pig breeds (FDR=0.118, NES=-1.52) ( Figure 1 E).

[0044] The results of iWAT transcriptome data analysis showed that the fatty acid biosynthesis pathway was significantly downregulated in cold-tolerant pigs, indicating that the formation of beige fat is related to the activation of heat production and the de novo synthesis of fatty acids. In order to screen genes that regulate thermogenesis in adipose tissue of cold-tolerant pigs, we drew a heat map of the expression of genes in the fatty acid metabolism pathway in four pig breeds. From the figure, we can see that DNL-related genes SLC25A1, ACLY, ACACA, FASN and SCD were significantly downregulated in cold-tolerant pigs, while fatty acid oxidation (FAO)-related genes CPT1A and PDK4 were significantly upregulated ( Figure 1 F).

[0045] We selected cold-tolerant Tibetan pigs and cold-intolerant Bama pigs as representatives and verified the mRNA and protein expression levels of related genes. From the fluorescence quantitative results, it can be seen that after acute cold stimulation, the mRNA expression of fatty acid de novo synthesis (DNL)-related genes SLC25A1, ACLY, ACACA, FASN and SCD in the iWAT tissue of Tibetan pigs was downregulated, while the mRNA expression of fatty acid oxidation (FAO)-related genes PDK4 and CPT1α was upregulated ( Figure 1 G). In addition, Western blot results showed that the expression of PDK4, pPDHA1, CPT1A and p563HSL proteins in iWAT tissue of Tibetan pigs was upregulated, and the expression of pACACA, ACACA and FASN proteins was downregulated after acute cold stimulation ( Figure 1 H), indicating that acute cold stimulation promoted the FAO process in the adipose tissue of Tibetan pigs and inhibited the DNL process. However, before and after acute cold stimulation, the expression levels of DNL and FAO related gene mRNA and protein in the adipose tissue of Bama pigs did not change significantly ( Figure 1 , I and J).

[0046] It shows that during the acute cold stimulation of the cold-resistant Tibetan pig, genes / proteins such as PDK4, pPDHA1, CPT1A, p563HSL, pACACA, ACACA and FASN are involved in the regulation of adipocyte heat production in the body.

[0047] Example 3 Beige fat differentiation and heat generation model in vitro

[0048] Under acute cold stimulation conditions, Tibetan pigs and Min pigs produce beige fat cells in their subcutaneous adipose tissue to generate heat to help them resist the cold. In order to study the thermogenic mechanism of beige fat, SVF cells were isolated from IWAT tissue, and a culture system for the directed differentiation of SVF cells into beige adipocytes was established. An animal thermogenic cell model was established based on beige fat, laying the foundation for better studying the function of beige fat.

[0049] The steps for isolating and culturing SVF cells are as follows: (1) IWAT tissue of a one-month-old male cold-resistant Tibetan pig was selected and washed with 75% alcohol and DPBS containing 3% penicillin / streptomycin (PS). An appropriate amount of adipose tissue was minced in a clean bench and 2 times the volume of 2 mg / ml DPBS was added. (1) Add an equal volume of DMEM / F12 complete medium to the digested tissue to terminate digestion, filter the tissue suspension with a 70 μM cell filter, and centrifuge at 1500 r / min for 10 min at room temperature; (2) Aspirate the supernatant, add 3-5 ml of red blood cell lysis buffer to resuspend the cell pellet, let it stand at 4°C for 10 min, and centrifuge at 1500 r / min for 10 min; (3) Aspirate the supernatant, add 1-2 ml of dMEM / F12 complete medium to resuspend the cell pellet, inoculate it in a 100 mm cell culture dish, add up to 10 ml of medium, shake well, and culture in a 38.5°C cell culture incubator containing 5% CO2, changing the medium every 2 days; (4) When the cells grow to 80-90% confluency, they are passaged, plated or frozen to obtain SVF cells, which contain primary adipocytes.

[0050] The method for inducing beige fat differentiation is as follows: SVF cells are seeded on a culture plate until confluence and cultured for another 2 days (D0). The medium is replaced with an induction medium (components include 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. The medium is then replaced with a maintenance medium (components include: 5 μg / mL insulin, 17 μM pantothenic acid, 33 μM biotin) and cultured until day 8.

[0051] FSK (Forskolin) is an adenylate cyclase activator that can induce the formation of intracellular cAMP and stimulate fat hydrolysis. It is widely used in humans and mice, but no research has been reported in pig cells. Therefore, we need to explore the conditions for FSK to treat pig beige adipocytes. Figure 2 A's experimental design induced SVF cells to differentiate into white fat. On the 8th day of differentiation, 10 μM FSK was used to treat for 0, 6, 24 and 72 hours respectively. The state of the cells and the changes in lipid droplet size were observed under a microscope at any time. From the pictures of fat cells, it can be seen that many small lipid droplets appeared after 24 hours of FSK treatment, and the number of large lipid droplets decreased significantly. After 72 hours of FSK treatment, the large lipid droplets were basically hydrolyzed into small lipid droplets ( Figure 2 C). FSK treatment of cells for 24 hours and adaptive heat production ( PPARGC1A)、Fatty acid oxidation / energy metabolism( PDK4 ), hydrolysis gene HSL The expression levels of Figure 2 D). In order to observe the morphological changes of cell lipid droplets and count the size of lipid droplets in subsequent experiments, the time of FSK treatment of cells was determined to be 24h.

[0052] Table 1 Primers required for fluorescence quantitative PCR (SEQ ID NO: 1-26)

[0053]

[0054] After determining that the FSK treatment time is 24h, it is necessary to determine the optimal FSK concentration for treating cells. Figure 2 Experimental design B, induced SVF cells to differentiate into white fat. On the 8th day of differentiation, the cells were treated with 0, 0.1, 1, 5 and 10 μM FSK for 24 hours. After the treatment, the changes in lipid droplet size were observed under a microscope. From the pictures of adipocytes, it can be seen that a large number of small lipid droplets appeared in the 1 μM FSK treatment group, and a small number of large lipid droplets appeared in the 5 μM and 10 μM FSK treatment groups, and the fat hydrolysis effect was the best ( Figure 2 E). Thermogenesis-related genes ( PPARGC1A , PDK4 , CIDEA ), hydrolysis gene HSL The expression level was significantly increased in the 0.1μM and 10μM FSK treatment groups, and reached a higher level in the 5μM and 10μM FSK treatment groups (primer information is shown in Table 1, Figure 2 F) In order to better observe the changes in lipid droplet morphology, the optimal concentration of FSK treatment is 5 μM.

[0055] In cold-tolerant pigs, beige adipocytes are responsible for heat production after acute cold stimulation. Therefore, after screening the optimal concentration and time for FSK treatment, we established an in vitro heat production model of beige adipocytes using Tibetan pig cells. First, SVF cells were differentiated into mature beige adipocytes and then treated with 5 μM FSK or DMSO (control, the solvent for FSK is DMSO) for 24 hours. From the fluorescence staining ( Figure 3 A) and electron microscopy ( Figure 3 As can be seen in the picture B), the lipid droplets of beige adipocytes in the FSK-treated group became smaller, and the position and morphology of mitochondria changed. We counted the size of lipid droplets and found that the proportion of small lipid droplets in adipocytes in the FSK-treated group increased significantly: less than 2 μM in the FSK-treated group 2 The lipid droplet frequency of the control group was about 4 times that of the control group ( Figure 3 C).

[0056] Tetramethylrhodamine, ethyl ester (TMRE) is a highly sensitive cationic dye used to assess mitochondrial membrane potential. A decrease in fluorescence signal indicates mitochondrial depolarization. To evaluate whether FSK treatment would enhance cellular metabolic capacity, we used BODIPY493 / 503, TMRE, and HOECHST to stain the lipid droplets, mitochondrial membrane potential, and nuclei of beige adipocytes, respectively, and performed flow cytometry analysis on the fluorescently stained cells. The results showed that the red fluorescence signal was weakened in cells containing green fluorescence, indicating that the mitochondrial membrane potential of beige adipocytes was depolarized. TMRE depolarization represents increased heat production ( Figure 3 D).

[0057] The COX2 gene was quantitatively analyzed (primer information is shown in Table 1) to determine the mitochondrial copy number, and the results showed that the mitochondrial copy number of beige adipocytes was significantly increased after FSK treatment ( Figure 3 E). The Seahorse XF Cell Mitochondrial Stress Kit was used to measure the OCR of cells. The calculation results show that after FSK treatment, the proton leakage of beige adipocytes was significantly increased and ATP synthesis was reduced ( Figure 3 , F and G). The above results showed that FSK treatment of pig beige adipocytes can activate the cell thermogenic program, indicating that the in vitro thermogenic model of pig beige adipocytes was successfully established.

[0058] In order to explore the expression of DNL genes while beige adipocytes produce heat, we detected the expression of DNL and FAO genes and proteins in beige adipocytes differentiated from mature Tibetan pig SVF cells before and after FSK treatment. The results of qPCR showed that DNL-related genes in FSK-treated cells ACACA , FASN The expression of genes such as Figure 3 H), but FAO-related genes PDK4 and CPT1A The expression level of Figure 3 I). Western blot results showed that the expression levels of PDK4 and pPDHA1 proteins in the FSK-treated group increased, while the expression levels of pACACA and FASN proteins decreased significantly ( Figure 3 J), indicating that FSK promoted thermogenesis in beige adipocytes of Tibetan pigs and significantly inhibited the expression of DNL gene, while up-regulating the expression of FAO gene.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A marker gene for distinguishing cold-tolerant pig breeds from cold-intolerant pig breeds, characterized in that: The marker genes consist of PDK4, pPDHA1, CPT1A, p563HSL, pACACA, ACACA and FASN.

2. Use of the marker gene according to claim 1 in the identification and breeding of cold-tolerant and non-cold-tolerant pig breeds.

3. Application of the marker gene according to claim 1 in molecular marker-assisted breeding and variety improvement of cold-tolerant and non-cold-tolerant pig breeds.

4. A method for identifying cold-tolerant pig breeds and cold-intolerant pig breeds, characterized in that: The method comprises: detecting the expression level of the marker gene in the adipose tissue of the tested pig at the transcription or protein level, wherein the expression levels of the marker genes PDK4, pPDHA1, CPT1A and p563HSL in the cold-tolerant pig breeds are significantly higher than those in the cold-intolerant pig breeds, and the expression levels of the marker genes pACACA, ACACA and FASN in the cold-tolerant pig breeds are significantly lower than those in the cold-intolerant pig breeds.

5. A detection reagent or kit comprising the primers for detecting the marker gene according to claim 4.

6. Any of the following uses of the detection reagent or kit according to claim 5: 1) Used for identification and breeding of cold-tolerant and non-cold-tolerant pig breeds; 2) Used for molecular marker-assisted breeding and variety improvement of cold-tolerant and non-cold-tolerant pig breeds.

7. A method for identifying the cold resistance of animals, characterized in that: After the animal is subjected to acute cold stimulation, beige adipocytes are produced in the animal's adipose tissue, and the expression of pACACA, ACACA and FASN genes in the animal's adipose tissue is downregulated, while the expression of PDK4, pPDHA1, CPT1A and p563HSL genes is upregulated, and the animal is judged to have cold resistance; The animal is a pig.

8. The method according to claim 7, characterized in that The conditions for acute cold stimulation are: 4-6℃, 4-6 hours.

9. Application of the method according to claim 7 or 8 in animal breeding; Breeding objectives include improving animals’ resistance to cold weather and meat production; The animal is a pig.

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