Long-chain non-coding lncALDH2 and application thereof in increasing intramuscular fat content of beef cattle

Abstract

The invention discloses a long-chain non-coding lncALDH2 and application of the long-chain non-coding lncALDH2 in increasing the intramuscular fat content of beef cattle. The sequence of lncRNA is shown as SEQ ID NO: 1. Experiments show that the newly found lncALDH2 provided by the invention is closely related to Qinchuan cattle precursor fat cell differentiation, and has a molecular marker value. The invention aims to provide the long non-coding RNA-lncALDH2 related to the adipocyte differentiation function, the lncALDH2 is used for regulating and controlling the differentiation behavior of Qinchuan cattle precursor adipocytes, and a cell model knocked down by the lncALDH2 is established and can be applied to molecular breeding of Qinchuan cattle and the like.

Description

Technical Field

[0001] The present invention relates to the technology of molecular marker-assisted breeding, and specifically relates to a long non-coding lncALDH2 and its application in increasing intramuscular fat content in beef cattle. Background Art

[0002] Qinchuan cattle is the top among the five major yellow cattle breeds in China and is a well-known local dual-purpose breed for draft and meat. The fat content in beef affects the tenderness, flavor, and juiciness of beef. In this context, increasing the fat content is crucial for improving the quality of beef.

[0003] Long non-coding RNA (lncRNA) is a class of non-coding RNA with a transcript length greater than 200 bp and a relatively long nucleotide chain. LncRNA has characteristics such as multiple types, multiple action modes, and large quantity, so the information it contains is very rich. Its molecular mechanisms involved in expression regulation are also diverse, mainly realizing the regulation of gene expression at three levels: epigenetics, transcriptional level, and post-transcriptional level. Existing research shows that lncRNA plays an important role in fat metabolism and can affect lipid synthesis in cells. Exploring new lncRNAs that play important roles in fat differentiation can not only reveal important mechanisms for regulating adipocyte differentiation but also provide new molecular breeding markers. Summary of the Invention

[0004] The present invention aims to provide a long non-coding RNA related to the function of adipocyte differentiation - lncALDH2, and use lncALDH2 to regulate the differentiation behavior of Qinchuan cattle preadipocytes, and establish a cell model with lncALDH2 knockdown, which can be used in applications such as molecular breeding of Qinchuan cattle. Specifically, it relates to a long non-coding lncALDH2 and its application in increasing intramuscular fat content in beef cattle.

[0005] To achieve the above object, the technical solutions adopted by the present invention include:

[0006] A long non-coding lncALDH2, wherein the sequence of the lncALDH2 is as shown in SEQ ID NO: 1.

[0007] A long non-coding lncALDH2, wherein the sequence of the lncALDH2 is as shown in SEQ ID NO: 2.

[0008] A cell model, wherein the cells of the cell model are bovine preadipocytes, and the long non-coding lncALDH2 of the present invention is knocked down in the bovine preadipocytes.

[0009] Optionally, the knockdown specifically includes:

[0010] Design corresponding siRNA and transfect cells using Lip3000 to achieve the purpose of knocking down the long non-coding lncALDH2.

[0011] Optionally, the siRNA sequences are: lncALDH2-sense GCAGCUCAGAAGAGUAUAATT; lncALDH2-antisense UUAUACUCUUCUGAGCUGCTT.

[0012] The long non-coding lncALDH2 described in the present invention is used for the application of preparing a model for regulating the fat content of cattle.

[0013] The long non-coding lncALDH2 described in the present invention is used for the application of cattle breeding with adjustable fat content.

[0014] The long non-coding lncALDH2 described in the present invention is used as a molecular marker for screening cattle breeds with high fat differentiation ability.

[0015] The advantages of the present invention are:

[0016] The long non-coding lncALDH2 of the present invention is a molecule that is first discovered and verified, and there is no existing patent or literature report, which has the originality of a basic patent. It can be used as a molecular marker to assist in screening cattle breeds with high fat differentiation ability and shorten the breeding cycle. Description of the Drawings

[0017] The drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the drawings:

[0018] Figure 1 It is the RT-qPCR reaction procedure in Example 2 of the present invention;

[0019] Figure 2 It is the relative expression level of lncALDH2 during the differentiation of Qinchuan cattle preadipocytes in Example 2 of the present invention;

[0020] Figure 3 It is the detection of the interference efficiency of lncALDH2 in Example 3 of the present invention;

[0021] Figure 4 It is the Oil Red O staining result and triglyceride result of adipocytes on the 6th day of induced differentiation after interfering with lncALDH2 in Example 4 of the present invention;

[0022] Figure 5Relative expression levels of mRNA of marker genes related to lipid metabolism and protein levels of marker genes on the 2nd and 6th days of induced differentiation after interfering with lncALDH2 in Example 5 of the present invention. Detailed implementation manners

[0023] The present invention will be specifically described below in conjunction with the accompanying drawings of the specification and the detailed implementation manners.

[0024] Existing studies have shown that lncRNAs play an important role in lipid metabolism and can affect cellular lipid synthesis. Exploring new lncRNAs that play important roles in adipocyte differentiation can not only reveal important mechanisms regulating adipocyte differentiation but also provide new molecular breeding markers. The newly discovered lncALDH2 provided by the present invention is experimentally shown to be closely related to the differentiation of Qinchuan cattle preadipocytes and has the value as a molecular marker. The present invention aims to provide a long non-coding RNA related to the function of adipocyte differentiation - lncALDH2, and use lncALDH2 to regulate the differentiation behavior of Qinchuan cattle preadipocytes, and establish a cell model with lncALDH2 knockdown. lncALDH2 can be used in applications such as molecular breeding of Qinchuan cattle.

[0025] Example 1: According to the sequence of lncALDH2 obtained by transcriptome sequencing, the full-length sequence of lncALDH2 was obtained by RACE

[0026] 1. Research object

[0027] Qinchuan cattle preadipocytes.

[0028] 2. Experimental method

[0029] RACE, the specific steps are as follows:

[0030] First, the isolation and culture of Qinchuan cattle preadipocytes, the specific steps are as follows

[0031] 1. Wash the collected perirenal adipose tissue of Qinchuan cattle 3 times with PBS containing 5% double antibiotics. Under sterile conditions, carefully remove the visible blood vessels and connective tissues in the fat mass with ophthalmic scissors and forceps, and then cut them into tissue blocks with a size of 1 mm 3 in size;

[0032] 2. Transfer the tissue blocks into a 50 ml centrifuge tube, add an equal volume of type I collagenase (0.1%) digestion solution, and digest in a constant temperature shaking water bath at 37°C for 60 - 90 min, with the shaking frequency of 90 rpm;

[0033] 3. Add an equal volume of complete medium to terminate digestion, and then pass through stainless steel cell sieves with pore sizes of 100 μM and 40 μM in sequence;

[0034] 4. The filtrate is collected into a 10-ml centrifuge tube and centrifuged at 1500 rpm for 10 min. The supernatant is discarded.

[0035] 5. Add 5 ml of red blood cell lysate, pipette gently to mix evenly, let stand at room temperature for 10 min, centrifuge at 1500 rpm for 10 min, and discard the supernatant.

[0036] 6. Add 5 ml of serum-free culture medium, pipette gently to mix evenly, centrifuge at 1500 rpm for 10 min, and discard the supernatant. Repeat this operation twice.

[0037] 7. Add an appropriate amount of complete medium, pipette gently to mix evenly to make a cell suspension, and count the cells.

[0038] 8. Inoculate the cells into a T75 cell culture flask at a density of 5×105, use complete medium, and culture in a 5% CO2, 37°C constant temperature incubator. Replace the complete medium every 2 days after inoculation. When the cell confluence reaches 80 - 90%, use trypsin to digest and cryopreserve the cells or passage them at a ratio of 1:3.

[0039] Then, the RNA of Qinchuan cattle preadipocytes was extracted. The total RNA of the cells was extracted using the Trizol method.

[0040] 1. Discard the culture medium, wash the culture dish once with PBS. Add 1 ml of Trizol to every 2.5x106 cells or to each well of a 6-well plate. Gently shake to evenly distribute the lysate on the cell surface, let stand at room temperature for 5 min, pipette the bottom of the dish to completely lyse the cells, and collect the liquid into a 1.5-ml centrifuge tube to obtain a homogenized cell lysate.

[0041] 2. Add chloroform at 0.2 times the volume of Trizol to the above homogenized cell lysate, shake vigorously for 30 s, and incubate in the dark at room temperature for 3 min.

[0042] 3. Centrifuge at 12000 rpm at 4°C for 15 min. At this time, the homogenate is divided into three layers, and the RNA is in the upper aqueous phase.

[0043] 4. Carefully aspirate the supernatant and transfer it to a new 1.5-ml centrifuge tube. Add isopropanol at 0.5 times the volume of Trizol to the supernatant, invert up and down to mix thoroughly, and let stand at room temperature for 10 min.

[0044] 5. Centrifuge at 12000 rpm at 4°C for 10 min, carefully discard the supernatant, and a small amount of white RNA gel-like precipitate can be seen on the side wall and bottom of the centrifuge tube.

[0045] 6. Slowly add pre-cooled 75% ethanol (prepared with DEPC water) equal to the volume of Trizol, gently invert to wash the RNA precipitate, centrifuge at 12000 rpm at 4°C for 5 min, and carefully discard the supernatant.

[0046] 7. Repeat step (6) once;

[0047] 8. Open the centrifuge tube and let it stand in a laminar flow hood for 2 - 5 min to completely volatilize the ethanol.

[0048] 9. Add DEPC water preheated to 55 °C, dissolve the RNA pellet at room temperature, detect the purity and concentration of the RNA using a UV spectrophotometer, and then store it frozen at -80 °C in an ultra-low temperature freezer for later use.

[0049] Then perform RACE experiments using the total RNA, and use the SMARTer RACE 5’ / 3’ Kit to perform 5’RACE and 3’RACE experiments.

[0050] (1) Design 5’GSP specific primers, 3’GSP specific primers, and 3’NGSP specific primers. The primer design should meet the following requirements:

[0051] 1. The length is 23 - 28 nt to ensure specific annealing

[0052] 2. The GC content is between 50 - 70%

[0053] 3. Tm ≥ 65 °C; if Tm is greater than 70 °C, touchdown PCR can be used to obtain the best results. The calculation of Tm is based on the 3’ end of the gene-specific primer rather than the entire primer.

[0054] 4. It is not complementary to the 3’ end of the Universal Primer Mix.

[0055] 5. It is specifically complementary to the target gene

[0056] 6. There is a 15-base overlap with the 5’ end of the vector (add the sequence "GATTACGCCAAGCTT" at the 5’ end of the GSPs sequence), and the specific primer information is shown in Table 1;

[0057] (2) Synthesize the first strand of RACE-Ready cDNA. Prepare a 5.5 μl Buffer Mix reaction system. 4 μl of 5X First-Strand Buffer, 0.5 μl of DTT, 1 μl of dNTPs. Prepare 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA reaction systems respectively. 5’-RACE-Ready cDNA reaction system: 1 μl of RNA, 1 μl of 5’CDS Primer A, 9 μl of Sterile H 2O. 3'-RACE-Ready cDNA reaction system: 1 μl of RNA, 1 μl of 3' CDS Primer A, 10 μl of Sterile H 2 O. After mixing, incubate at 72 °C for 3 min and cool at 42 °C for 2 min. After the reaction, add 1 μl of SMARTer II A Oligonucleotide to the 5' RACE cDNA synthesis reaction solution. Prepare the Master Mix, 5.5 μl of Buffer Mix, 0.5 μl of RNase Inhibitor, 2 μl of SMARTScribe Reverse Transcriptase. Take 8 μl of the Master Mix and add it to the denatured RNA, for a total volume of 20 μl. After mixing evenly, incubate at 42 °C for 90 min, and then heat at 70 °C for 10 min. After the reaction, add 90 μl of Tricine-EDTA Buffer for dilution to obtain the first strand of RACE-Ready cDNA.

[0058] (3) Perform 5' RACE and 3' RACE experiments using the first strand of RACE-Ready cDNA. Prepare the PCR Master Mix, 15.5 μl of PCR-Grade H 2 O, 25 μl of 2X SeqAmp Buffer, 1 μl of SeqAmp DNA Polymerase, for a total of 41.5 μl. For the 5' RACE reaction, add 2.5 μl of 5' RACE-Ready cDNA, 5 μl of 10X UPM, and 1 μl of 5' GSP in the following order; for the 3' RACE reaction, add 2.5 μl of 3' RACE-Ready cDNA, 5 μl of 10X UPM, and 1 μl of 3' GSP in the following order. The total volume of the system is 50 μl. Perform touchdown PCR on the system: 94 °C for 30 sec, 72 °C for 3 min, for a total of 5 cycles; 94 °C for 30 sec, 70 °C for 30 sec, 72 °C for 3 min, for a total of 5 cycles; 94 °C for 30 sec, 68 °C for 30 sec, 72 °C for 3 min, for a total of 25 cycles. Perform agarose gel electrophoresis on the PCR products to determine the amplified specific bands, and select the specific bands for PCR product cloning. If no specific bands are obtained, nested PCR can be performed to obtain specific fragments. Use the touchdown PCR products diluted with Tricine-EDTA Buffer as the template for PCR. Prepare the PCR Master Mix, 15.5 μl of PCR-Grade H 2O, 25 μL of 2X SeqAmp Buffer, 1 μL of SeqAmp DNA Polymerase, for a total of 41.5 μL. The reaction was added with 2.5 μL of PCR template, 5 μL of 10X UPS, and 1 μL of 5’NGSP in the following order respectively. The total system was 50 μL. The system was subjected to nested PCR reaction: 94 °C for 30 sec, 68 °C for 30 sec, 72 °C for 3 min, for a total of 25 cycles. The PCR products were subjected to agarose gel electrophoresis to determine the specific bands amplified, and the specific bands were selected for PCR product cloning.

[0059] (4) Gel extraction and purification. The gel extraction and purification of DNA fragments were carried out according to the gel extraction kit (Beijing Omega, D2500-01). The steps of gel extraction and purification were as follows:

[0060] 1. Prepare an agarose EB gel and electrophoretically separate the DNA fragments;

[0061] 2. After electrophoresis for 30 min, cut out the desired DNA fragment from the gel, and cut off as much excess gel as possible, and try to cut the target band into small pieces;

[0062] 3. Weigh an empty centrifuge tube, cut the gel with the target fragment into a 1.5 mL centrifuge tube, add Binding Buffer with a volume 2-3 times that of the gel weight after weighing, place it in a water bath at 55 °C and incubate until the gel completely melts, and mix it every 2-3 min;

[0063] 4. Discard the waste liquid after centrifugation. Then place the adsorption column in the ultra-clean bench and turn on the fan to level 8 for 5-8 min to dry the residual absolute ethanol in the adsorption column;

[0064] 5. Place the adsorption column into a clean 1.5 mL centrifuge tube, add an appropriate amount of elution buffer in the middle of the adsorption column, let it stand at room temperature for several minutes or place it in a 4 °C refrigerator for 1-2 h and then centrifuge. The solution in the centrifuge tube is the purified DNA product.

[0065] (5) T-vector ligation and transformation experiment of Escherichia coli. Pipette 1 μL of the cloning vector pMD19-T vector (simple) (50 ng, 0.03 pmol) and 3 μL of the gel-extracted and purified DNA (0.1-0.3 pmol) into a 1.5 mL centrifuge tube, add ddH2O to 5 μL, mix well, centrifuge briefly, and then add 5 μL of Solution I. Incubate overnight at 4 °C or for 2 h at 16 °C. The subsequent steps are as follows:

[0066] 1. Take out 50 μL of DH5α Escherichia coli competent cells from an -80 °C refrigerator, place them on ice to thaw, take 5.5 μL of the ligated product and 50 μL of competent cells, gently pipette and mix well, and place on ice for 30 min.

[0067] 2. Heat shock at 42 °C in a water bath for 45 s, then quickly cool on ice for 2 - 3 min.

[0068] 3. Then add 800 - 1000 μL of LB liquid medium without ampicillin or the SOD medium provided in the kit (Beijing Tsingke, 9057) to the centrifuge tube, seal with a sealing film, and culture the Escherichia coli at 37 °C with shaking for 60 min (constant temperature shaking incubator, rotation setting range is 160 - 225 rpm).

[0069] 4. After centrifuging the above bacterial solution at 4000 rmp for 1 min, discard 900 μL of the culture supernatant, repeatedly pipette the remaining 100 μL of the culture medium until the bacterial cells are fully suspended, then spread the bacterial solution on an LB solid medium containing ampicillin (100 μg / mL), and spread evenly until there is no flowing bacterial solution on the surface of the medium. Then, place the plate upside down in a constant temperature incubator overnight.

[0070] 5. In a laminar flow hood, randomly select monoclonal colonies in the culture dish with a sterilized white pipette tip, place the pipette tip with the attached colonies in a centrifuge tube, let the attached colonies soak in the LB liquid medium (corresponding to the resistance), seal the centrifuge tube with a sealing film, and shake and culture in a 37 °C constant temperature incubator for about 4 - 5 h (until the medium becomes turbid), then send it to the company for first-generation sequencing.

[0071] Finally, according to the sequences obtained from the experimental results of 5’RACE and 3’RACE, splice to obtain the full-length sequence of lncALDH2. The obtained sequence is compared with the NCBI gene database to determine the sequence information and chromosomal location.

[0072] Table 1 Primer information for RACE experiments

[0073]

[0074]

[0075] 3. Experimental conclusions

[0076] SEQ ID NO: 1 is the sequence of lncALDH2 obtained by transcriptome sequencing. The sequence length is 831 bp, and the full-length sequence of lncALDH2 is obtained through RACE experiments. SEQ ID NO: 2 is the full-length sequence of lncALDH2, and the sequence length is 2117 bp. The sequence of lncALDH2 is located on bovine chromosome 17.

[0077] Example 2: According to the sequence information of lncALDH2, specific primers were designed, and the changes of lncALDH2 during the differentiation of Qinchuan cattle preadipocytes were analyzed by qRT-PCR.

[0078] 1. Research object

[0079] Qinchuan cattle preadipocytes.

[0080] 2. Experimental method

[0081] (1) Inductive differentiation of Qinchuan cattle preadipocytes.

[0082] When the cell density was about 80%, induction reagent 1 was added. The formula of induction reagent 1 was 45 ml of F12 medium, 5 ml of fetal bovine serum (FBS), 0.5 ml of penicillin-streptomycin double antibody, 0.5 ml of insulin, 5 μl of Dex, 150 μl of IBMX, and 3.9 μl of rosiglitazone. The medium was changed on the second day of inductive differentiation, and induction reagent 2 was added. The formula of induction reagent 2 was 45 ml of F12 medium, 5 ml of fetal bovine serum (FBS), 0.5 ml of penicillin-streptomycin double antibody, and 0.5 ml of insulin. Among them, insulin was 0.01 g of recombinant bovine insulin, dissolved in 10 mL of 0.02 mol / L HCl solution (17.2 μL of 36 - 38% HCl, add water to 10 mL), prepared into a 1 mg / mL stock solution, left at room temperature for 24 h, filtered and aliquoted, and stored at -20 °C for later use. Dex was 25 mg of dexamethasone dissolved in 6.37 ml of absolute ethanol. IBMX was 100 mg of IBMX dissolved in 2.7 ml of DMSO. Rosiglitazone was 10 mg of rosiglitazone dissolved in 1 ml of DMSO.

[0083] (2) Extract the total RNA of cells. As above.

[0084] (3) Reverse transcription of total RNA to synthesize cDNA

[0085] The genomic DNA removal reverse transcription kit (PrimeScriptTM RT reagent Kit with gDNA Eraser) from Takara Biotechnology (Dalian) Co., Ltd. was used. According to the instructions, reaction solution I was prepared to remove genomic DNA. The reaction system was as shown in Table 2, and the reaction program was as shown in Table 3; according to the reaction system shown in Table 4 and the reaction program shown in Table 5, the total RNA was reverse transcribed into cDNA and stored at -20 °C for later use.

[0086] Table 2 Reaction system for removing genomic DNA

[0087]

[0088]

[0089] Table 3 Genomic DNA Removal Reaction Program

[0090]

[0091] Table 4 Reverse Transcription Reaction System

[0092]

[0093] Table 5 Reverse Transcription Reaction Program

[0094]

[0095] (4) Perform qRT-PCR experiments. The primer information for qRT-PCR is shown in Table 6, and β-actin is selected as the internal reference.

[0096] Table 6 Primer Information for qRT-PCR

[0097]

[0098] Use the TB Green Premix Ex Taq II (TliRNaseH Plus) kit from Beijing Baosheng Biotechnology Co., Ltd. to prepare the reaction system shown in Table M-1 according to the operation manual; perform RT-qPCR reactions using the ABI 7500 real-time fluorescence quantitative system. The reaction program is shown in Figure M-1, and the experimental results are analyzed using the 2 -ΔΔCt method, and the formula is as follows:

[0099] ΔΔCt = [Ct target gene (待测样品) - Ct internal reference gene (待测样品) - [Ct target gene (校正样品) - Ct internal reference gene (校正样品) ;

[0100] In the formula, the Ct value represents the number of cycles when the fluorescence reaches the threshold.

[0101] (1) RT-qPCR reaction system, see Table 7;

[0102] Table 7 RT-qPCR Reaction System

[0103]

[0104] (2) RT-qPCR reaction program, see Figure 1 ;

[0105] 3. Experimental Conclusions

[0106] Figure 2It is the relative expression level of lncALDH2 during the differentiation of Qinchuan cattle preadipocytes. The results showed that the expression level of lncALDH2 changed during the differentiation of Qinchuan cattle preadipocytes, and the highest expression level was observed on the second day.

[0107] Example 3: Detection of the interference efficiency of lncALDH2

[0108] 1. Research object

[0109] Qinchuan cattle preadipocytes.

[0110] 2. Experimental method

[0111] (1) Cell transfection

[0112] 1. When the cell density reaches about 70%, replace the medium with DMEM without double antibiotics.

[0113] 2. Prepare solution A by mixing 3.15 μL of lipo3000 and 125 μL of serum-free medium (opti) per well in a 6-well plate, and prepare solution B by mixing 5 μL of siRNA and 125 μL of opti; the siRNA sequence information is shown in Table 8.

[0114] 3. Mix solutions A and B, centrifuge, and let stand for 10 to 15 minutes.

[0115] 4. Add 250 μL of the mixed solution to each well of the 6-well plate.

[0116] (2) Extract total cellular RNA. Extract total RNA 48 hours after transfection, reverse transcribe, and perform RT-qPCR to analyze the interference efficiency of lncALDH2.

[0117] Table 8 siRNA sequence information

[0118]

[0119] 3. Experimental conclusion

[0120] Combined Figure 3 , in the figure, NC represents the untreated control group, and SI represents the treatment group with lncALDH2 interference. After interference, the expression level of lncALDH2 decreased by about 70%, showing a significant difference from the NC group, and further research can be carried out.

[0121] Example 4: Analyze the effect of interfering with the expression of lncALDH2 on the differentiation of Qinchuan cattle preadipocytes using Oil Red O staining experiment and triglyceride experiment

[0122] 1. Research object

[0123] Qinchuan cattle preadipocytes.

[0124] 2. Experimental Methods

[0125] (1) Oil Red O Staining

[0126] Ⅰ Preparation of Staining Solution:

[0127] 1. Stock solution of Oil Red O: Dissolve 0.25 g of Oil Red O in 50 ml of isopropanol, wrap it with tin foil (light protection), shake overnight at 37 °C, filter with filter paper and store at room temperature. Filter again before use.

[0128] 2. Working solution of Oil Red O: Prepare it by mixing the stock solution of Oil Red O and double-distilled water at a ratio of 3:2 when in use. Filter before use.

[0129] Ⅱ Steps of Oil Red O Staining:

[0130] 1. Take the cells to be stained, discard the culture medium, and wash them 3 times with PBS;

[0131] 2. Fix with 4% paraformaldehyde for 20 - 30 min, in the dark;

[0132] 3. Wash 3 times with PBS;

[0133] 4. Stain with the working solution of Oil Red O, just cover the cells, in the dark for 30 min;

[0134] 5. Wash 3 times with PBS;

[0135] 6. Add a small amount of PBS to prevent the lipid droplets from melting;

[0136] 7. Take pictures under an inverted microscope.

[0137] (2) Determination of Triglyceride (TG) Content

[0138] 1. Lysis of Adipocytes:

[0139] Discard the cell culture medium, wash 3 times with PBS to remove glycerol; Digest with 0.25% trypsin at 37 °C for 3 - 5 min, collect the cells, centrifuge at 2000 rpm for 5 min, discard the supernatant; Add 200 μl of lysis buffer to each 2.5x106 cells or per well of a 6-well plate to treat the cell pellet, pipette and mix well, and let it stand at room temperature for 10 min;

[0140] 2. Treatment of Lysis Buffer:

[0141] Centrifuge briefly for 10 s, transfer an appropriate amount of the supernatant lysis buffer to a 1.5 ml centrifuge tube, heat in a water bath at 70 °C for 10 min, and flocculent precipitates may appear when the cell amount is large; Centrifuge at 2000 rpm for 5 min at room temperature, and take the supernatant for determination;

[0142] 3. Determination of Triglyceride Content:

[0143] Prepare the working solution by mixing reagent R1 and R2 at a ratio of 4:1. Mix well and use immediately or store at 4°C for <1 day. Observe whether it changes color before use. If it changes color, discard it;

[0144] Dilute the 4 mM glycerol standard with distilled water in a serial dilution to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 μmol / L. Usually, take 4 - 6 of these tubes to construct a standard curve. Note that the 0 concentration tube is set as the control reaction tube;

[0145] Using a 96 - well plate, add samples according to the ratio of 10 μl of distilled water / standard / sample to be tested + 190 μl of working solution. React at 37°C for 10 min. After the reaction reaches equilibrium, the color is stable within 60 min;

[0146] Use an enzyme - linked immunosorbent assay (ELISA) reader to measure the OD value of each tube at a wavelength of 550 nm. Plot the standard curve and calculate the concentration of triglyceride in the sample. Finally, correct the content of triglyceride according to the dilution factor and the number of cells.

[0147] 3. Experimental conclusions

[0148] Combined with Figure 4 , in the figure, NC represents the untreated control group, and SI represents the treatment group with interference of lncALDH2. The upper photo is the result after Oil Red O staining, and the lower bar graph is the triglyceride result; according to the Oil Red O staining result and the triglyceride result, it shows that after interfering with the expression of lncALDH2, the lipid accumulation of Qinchuan cattle preadipocytes can be significantly promoted.

[0149] Example 5: Changes in marker genes related to lipid metabolism on the 2nd and 6th days after interference in quantitative and protein analysis

[0150] 1. Research object

[0151] Qinchuan cattle preadipocytes.

[0152] 2. Experimental method

[0153] Collect total RNA on the 2nd and 6th days after induction of differentiation with interference respectively, and perform RT - qPCR to analyze the changes in the mRNA level and protein level of marker genes.

[0154] Primer information of marker genes

[0155]

[0156] 3. Experimental conclusions

[0157] Combined with Figure 5, in the figure, NC represents the untreated control group, and SI represents the treatment group with lncALDH2 interference. The upper figure is the quantitative result, and the lower figure is the protein result; the experimental results show that on the 2nd day after interference, the mRNA levels of PPARγ and C / EBPα did not change significantly, but the protein level of PPARγ increased significantly. On the 6th day after interference, the mRNA levels of FABP4 and DGAT2 increased significantly, and at the same time, the protein level of FABP4 also increased significantly.

[0158] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A long non-coding lncALDH2, characterized in that: The sequence of lncALDH2 is shown in SEQ ID NO:

1.

2. A long non-coding lncALDH2, characterized in that: The sequence of lncALDH2 is shown in SEQ ID NO:

2.

3. A cell model, characterized in that The cells of the cell model are bovine preadipocytes, and the long non-coding lncALDH2 according to claim 1 or 2 is knocked down in the bovine preadipocytes.

4. The cell model according to claim 3, characterized in that The knockdown specifically includes: The corresponding siRNA was designed and the cells were transfected with lip3000 to achieve the purpose of knocking down the long noncoding lncALDH2.

5. The cell model according to claim 3 or 4, characterized in that The siRNA sequences are: lncALDH2-senseGCAGCUCAGAAGAGUAUAATT; lncALDH2-antisenseUUAUACUCUUCUGAGCUGCTT.

6. Use of the long non-coding lncALDH2 according to claim 1 or 2 for preparing a model for regulating bovine fat content.

7. Use of the long non-coding lncALDH2 according to claim 1 or 2 in breeding cattle with adjustable fat content.

8. Use of the long non-coding lncALDH2 according to claim 1 or 2 as a molecular marker for screening cattle breeds with high fat differentiation ability.

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