LncRNA Related to the Proliferation of Porcine Skeletal Muscle Satellite Cells and Its Application

Abstract

The present invention provides an LncRNA related to the proliferation of porcine skeletal muscle satellite cells and its application. Based on the previous transcriptome sequencing data, the present invention found that the expression level of LncRNA Loc106505926 decreased extremely significantly with the increase of days of age, and PCR experiments proved the objective existence of Loc106505926; further, the role of Loc106505926 in the proliferation process of porcine skeletal muscle satellite cells was verified by real-time fluorescence quantitative PCR and EdU staining experiments, and it was found that after interfering with Loc106505926, the proliferation marker genes PCNA, Ki67, etc. decreased extremely significantly, and the number of positive cells decreased extremely significantly. The present invention proposes that Loc106505926 can be used as a biomarker to identify the proliferation of porcine skeletal muscle satellite cells, providing new ideas for the research on muscle growth and development.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetic technology, in particular to a LncRNA associated with the proliferation of porcine skeletal muscle satellite cells and an application thereof. Background Art

[0002] Skeletal muscle is the most abundant tissue in mammals. Besides being responsible for movement, it also generates heat, stores protein, and supports and protects soft tissues. As a vital organ for maintaining bodily function, skeletal muscle is closely linked to economic traits such as meat production and quality in livestock and poultry. Skeletal muscle development is complex and regulated by multiple factors, of which lncRNA is a key regulator.

[0003] LncRNAs, a key member of the noncoding RNA family, are widely present in diverse organisms, with functions implicated in various aspects of cellular life and individual development. LncRNAs are a class of ncRNAs greater than 200 nt in length, primarily transcribed by RNA polymerase II in eukaryotic organisms. Most lncRNAs exhibit structural features similar to mRNAs, containing promoters, exons, and introns. Their 5′ and 3′ ends are processed and modified to form a 5′ cap and a 3′ poly A tail. LncRNA sequences are relatively poorly conserved, with only approximately 12% of lncRNAs encoded in the human genome found in other organisms. Many lncRNAs share conserved secondary structures, specific splicing patterns, and subcellular localization. Furthermore, lncRNA expression exhibits spatiotemporal specificity. This conservation and specificity suggest that they possess functional properties.

[0004] Based on their location relative to protein-coding genes in the genome, lncRNAs can be categorized into five types: antisense lncRNAs, intergenic lncRNAs, divergent lncRNAs, enhancer lncRNAs, and intronic lncRNAs. Recent studies have demonstrated that lncRNAs regulate gene expression at multiple levels, including epigenetic, transcriptional, and post-transcriptional levels. At the epigenetic level, lncRNAs alter gene function through chromatin remodeling, DNA methylation, and histone modification. Transcriptional regulation involves lncRNAs modulating the transcription of adjacent genes, regulating transcription factors, and forming triple-helix complexes with DNA. At the post-transcriptional level, lncRNAs regulate genes by interacting with splicing factors to influence pre-mRNA splicing or by forming duplexes through base-specific complementary pairing with target mRNAs. Furthermore, lncRNAs can act as ceRNAs (ceRNAs) to competitively bind to miRNAs (miRNAs) to regulate gene expression. Furthermore, lncRNAs are involved in important regulatory processes, including X chromosome silencing, genomic imprinting, embryonic development, fat metabolism, skeletal muscle growth and development, and chromatin modification. Currently, research on the regulatory effects of LncRNA is mostly focused on model animals such as mice. The function and regulatory mechanism of LncRNA in pig skeletal muscle need further exploration. Summary of the Invention

[0005] The purpose of the present invention is to provide a LncRNA related to the proliferation of porcine skeletal muscle satellite cells and an application thereof.

[0006] To achieve the purpose of the present invention, in a first aspect, the present invention provides a LncRNA (Loc106505926) associated with the proliferation of porcine skeletal muscle satellite cells, and the cDNA sequence corresponding to the LncRNA is shown in SEQ ID NO: 1.

[0007] In a second aspect, the present invention provides use of the LncRNA as a marker for porcine skeletal muscle satellite cell proliferation in the preparation of a detection reagent or kit for identifying porcine skeletal muscle satellite cell proliferation.

[0008] In a third aspect, the present invention provides a pair of primers for detecting the LncRNA, comprising an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.

[0009] In a fourth aspect, the present invention provides a kit for identifying the proliferation of porcine skeletal muscle satellite cells, wherein the kit comprises a reagent for detecting the expression level of the LncRNA.

[0010] Furthermore, the kit includes primers shown in SEQ ID NO: 2-3.

[0011] In a fifth aspect, the present invention provides an inhibitor of the LncRNA, wherein the inhibitor is an siRNA shown in SEQ ID NO: 8-9, SEQ ID NO: 10-11 or SEQ ID NO: 12-13, or a combination of the above three pairs of siRNA.

[0012] In a sixth aspect, the present invention provides a method for identifying the proliferation efficiency of pig skeletal muscle satellite cells, the method comprising: detecting the relative expression level of the LncRNA according to claim 1 in pig skeletal muscle satellite cells; compared with the control group, if the relative expression level of the LncRNA is low, it indicates that the proliferation efficiency of the pig skeletal muscle satellite cells is low.

[0013] Furthermore, the expression level of the LncRNA was detected using the primers shown in SEQ ID NO: 2-3.

[0014] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0015] Based on previous transcriptome sequencing data, the present invention discovered that the expression of the lncRNA Loc106505926 decreases significantly with age. PCR experiments confirmed the objective presence of Loc106505926. Real-time fluorescence quantitative PCR and EdU staining experiments further verified the role of Loc106505926 in the proliferation of porcine skeletal muscle satellite cells. Interference with Loc106505926 revealed a significant decrease in the expression of proliferation marker genes such as PCNA and Ki67, and a significant reduction in the number of positive cells. The present invention proposes that Loc106505926 can be used as a biomarker to identify the proliferation of porcine skeletal muscle satellite cells, providing new insights into muscle growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The PCR amplification results of Loc106505926 in a preferred embodiment of the present invention are shown in the right lane. The DNA marker is a DL2000 DNA molecular weight standard, which is 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp from top to bottom. The left lane is the amplified fragment of Loc106505926, which is 731 bp.

[0017] Figure 2 Figure 2 shows the expression changes of Loc106505926 in the longissimus dorsi muscle tissue of 1-day-old, 90-day-old and 180-day-old Jinfen white pigs in a preferred embodiment of the present invention. Different capital letters indicate extremely significant differences.

[0018] Figure 3This figure shows the knockdown efficiency of Loc106505926 in porcine skeletal muscle satellite cells in a preferred embodiment of the present invention. The NC group represents the control group, and the siLoc106505926-1, siLoc106505926-2, and siLoc106505926-3 groups represent knockdown groups treated with three pairs of Loc106505926 siRNAs. Values ​​are expressed as mean ± standard deviation. Significance between two groups was determined using an independent t-test. * indicates a significant difference, and ** indicates an extremely significant difference.

[0019] Figure 4 qRT-PCR was used to examine the expression of proliferation-related genes in the si group 48 hours after transfection in a preferred embodiment of the present invention. Values ​​are expressed as mean ± standard deviation. The significance between the two groups was determined using an independent t-test. * indicates a significant difference, and ** indicates a very significant difference.

[0020] Figure 5 This is a preferred embodiment of the present invention, in which EdU staining was used to examine the effect of Loc106505926 on the proliferation of porcine skeletal muscle satellite cells. Values ​​are expressed as mean ± standard deviation. The significance between the two groups was determined using an independent t-test. * indicates a significant difference, and ** indicates an extremely significant difference.

[0021] Figure 6 In the preferred embodiment of the present invention, the number of EdU-positive cells was statistically analyzed using Image J. The values ​​are expressed as "mean ± standard deviation". The significance between the two groups was analyzed using an independent t-test. * indicates a significant difference, and ** indicates an extremely significant difference. DETAILED DESCRIPTION

[0022] The present invention provides a porcine long noncoding RNA Loc106505926 and its application. High-throughput sequencing technology was used to analyze the differences in the whole transcriptome of the longissimus dorsi muscles of 1-day-old, 90-day-old, and 180-day-old pigs, and Loc106505926 was discovered. Subsequently, siRNA was designed and synthesized, and transfected into porcine skeletal muscle satellite cells using liposomes. This reduced the expression level of Loc106505926 and inhibited the proliferation of porcine skeletal muscle satellite cells.

[0023] The present invention also provides a LncRNA marker associated with the proliferation of pig skeletal muscle satellite cells. This method can be used to identify the proliferation of pig skeletal muscle satellite cells, providing new insights into the molecular mechanism of muscle growth and development.

[0024] The present invention adopts the following technical solutions:

[0025] The present invention provides a porcine long-chain non-coding RNA Loc106505926, and the cDNA sequence corresponding to the long-chain non-coding RNA Loc106505926 is shown in SEQ ID No: 1.

[0026] The present invention also provides a detection primer for Loc106505926, a LncRNA marker associated with the proliferation of pig skeletal muscle satellite cells. The detection primer is a primer for specifically amplifying Loc106505926.

[0027] The present invention also provides a product for identifying the proliferation of pig skeletal muscle satellite cells, wherein the product detects the expression level of Loc106505926 in a sample through bioinformatics analysis technology and nucleic acid amplification technology.

[0028] Furthermore, the nucleic acid amplification technology is selected from polymerase chain reaction, reverse transcription polymerase chain reaction, transcription-mediated amplification, ligase chain reaction or nucleic acid sequence-based amplification.

[0029] The present invention also provides a small interfering RNA of the long non-coding Loc106505926, wherein the small interfering RNA is the siRNA shown in SEQ ID NO: 8-9, SEQ ID NO: 10-11 or SEQ ID NO: 12-13, or a combination of the above three pairs of siRNAs.

[0030] The present invention also provides a method for identifying the proliferation efficiency of porcine skeletal muscle satellite cells, the method comprising:

[0031] The system expressing the Loc106505926 gene is treated with a candidate substance; the expression of the Loc106505926 gene in the system is detected; if the expression level of the Loc106505926 gene is low, it indicates that the proliferation efficiency of pig skeletal muscle satellite cells is low.

[0032] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0033] Example 1 Verification of the long non-coding RNA pig Loc106505926 gene

[0034] 1. Materials

[0035] Sample: The longissimus dorsi muscle tissue of 1-day-old Jinfen white pigs was collected, wrapped in tin foil, quickly frozen in liquid nitrogen, and stored at -80℃.

[0036] Reagents: Reagent was purchased from Life Technologies; PrimeScript RTreagent Kit with gDNA Eraser and DNA marker were purchased from TaKaRa; 2×Es Taq Master Mix (Dye) was purchased from Kangwei Century Biotechnology Co., Ltd.; 50×TAE buffer, nucleic acid dye, Triton X-100, and DAPI stain were purchased from Solebol; agarose was purchased from Shanghai Shenggong Bioengineering Co., Ltd.

[0037] 2. Methods

[0038] 2.1 Total RNA extraction

[0039] The longissimus dorsi muscle tissue of 1-day-old Jinfen white pig was taken out from the -80℃ refrigerator, 1 mL of Trizol reagent was added, and the tissue was thoroughly ground with a tissue homogenizer. After lysis, the tissue was homogenized at 4℃ and 12000 r·min. -1 Centrifuge for 10 minutes; transfer the supernatant to a new 1.5 mL centrifuge tube, add 200 μL of chloroform, shake vigorously for 15 seconds, and let it stand at room temperature for 3 minutes, then at 4°C and 12,000 rpm. -1 Centrifuge for 10 minutes; carefully transfer the supernatant to a new 1.5 mL centrifuge tube, discard the middle and lower organic phases, add an equal volume of isopropanol, shake gently to mix, and then stand on ice for 20 minutes at 4°C and 12,000 rpm. -1 Centrifuge for 10 minutes until RNA is precipitated at the bottom of the tube. Discard the supernatant. Add 1 mL of 75% ethanol to wash the precipitate. Invert the tube to suspend it in ethanol. Centrifuge for 10 minutes at 4°C and 12,000 rpm. -1 After centrifugation for 5 minutes, discard the supernatant and repeat three times; carefully remove excess ethanol with a pipette, invert the tube onto filter paper, and let it stand to evaporate the residual ethanol; after the ethanol evaporates completely, add an appropriate amount of RNase-free ddH2O, pipette to mix well, and let it stand at 4°C for 30 minutes; then use a nucleic acid protein analyzer to determine the purity and concentration of the total RNA, OD 260 / OD 280 Total RNA in the range of 1.9–2.1 was used for subsequent studies.

[0040] 2.2 Reverse transcription reaction

[0041] It is specifically divided into two steps. The first step is to remove genomic DNA from total RNA. Add the extracted RNA and the genomic removal reagent gDNA Eraser mixture to 10 μL and react at 42°C for 2 minutes. The second step is to reverse transcribe the total RNA into cDNA. Mix the liquid after the above reaction, buffer and reverse transcriptase, etc. to a final volume of 20 μL, react at 37°C for 15 minutes, 85°C for 5 seconds, and finally store in a -20°C refrigerator for use.

[0042] 2.3 Primer design

[0043] Based on the sequence of the Loc106505926 transcript (SEQ ID NO: 1), Loc106505926 amplification primers were designed using Oligo7 software as follows:

[0044] Upstream primer: 5′-AATGTGGCAGTAACTAGACC-3′ (SEQ ID NO: 2)

[0045] Downstream primer: 5′-GACTGTGGCAAATATATCTCC-3′ (SEQ ID NO: 3)

[0046] 2.4 PCR amplification

[0047] The reaction system was as follows: 5 μL of 2× Es Taq Master Mix, 0.5 μL each of 10 μmol / L upstream and downstream primers, 1 μL of cDNA, and ddH2O to 10 μL. The reaction procedure was: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension at 72°C for 60 s; followed by further extension at 72°C for 10 min; and storage at 4°C. After PCR amplification, fragment size analysis was performed by agarose gel electrophoresis.

[0048] 3. Results

[0049] The fragment obtained by agarose gel electrophoresis was 731 bp, which was consistent with the expected result ( Figure 1 Further sequencing confirmed that the amplified fragment was a partial sequence of Loc106505926, confirming the existence of this lncRNA.

[0050] Example 2: Temporal Expression Characteristics of Loc106505926

[0051] 1. Materials

[0052] Samples: The longissimus dorsi muscle tissues of 1-day-old, 90-day-old, and 180-day-old Jinfen white pigs were collected, wrapped in tin foil, and quickly frozen in liquid nitrogen and stored at -80°C.

[0053] Reagents: Reagent was purchased from Life Technologies; PrimeScript RTreagent Kit with gDNA Eraser and 2×SYBR Premix Ex TaqⅡ were purchased from TaKaRa.

[0054] 2. Methods

[0055] 2.1 Total RNA extraction and reverse transcription

[0056] The specific method is the same as that of Example 1.

[0057] 2.2 Primer design

[0058] Based on the sequence of the Loc106505926 transcript (SEQ ID NO: 1), the Loc106505926 quantitative primer pair was designed using Oligo7 software as follows:

[0059] Quantitative primer pair for Loc106505926:

[0060] Upstream primer: 5′-ACAAAGGGCTCTGGTTGAAGT-3′ (SEQ ID NO: 4)

[0061] Downstream primer: 5′-GTTGGCCATGTCCGAGTCTA-3′ (SEQ ID NO: 5)

[0062] 18S rRNA quantitative primer pair:

[0063] Upstream primer: 5′-CCCACGGAATCGAGAAAGAG-3′ (SEQ ID NO: 6)

[0064] Downstream primer: 5′-TTGACGGAAGGGCACCA-3′ (SEQ ID NO: 7)

[0065] 2.3 Fluorescence quantitative PCR

[0066] The total volume of fluorescence quantitative PCR reaction was 10 μL, including upstream and downstream primers (10 μmol·L -11 μL of the total volume (1 μL, 1:1 mix), 1 μL of cDNA, 5 μL of 2× SYBR Premix Ex TaqⅡ, and 10 μL of RNAase-free ddH2O. The reaction procedure followed the SYBR Premix Ex TaqⅡ instructions: pre-denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s; and melting curve generation at 95°C for 15 s, 65°C for 5 s, and 95°C for 50 s. Each sample was technically replicated three times, using 2 -△△CT The test results were analyzed by the method and expressed as mean ± standard error.

[0067] 3. Results

[0068] To analyze the temporal expression pattern of Loc106505926 during pig muscle growth and development, the present invention detected the expression of Loc106505926 in the longissimus dorsi muscle tissue of Jinfen white pigs at different ages by fluorescent quantitative PCR. The results showed that the expression of Loc106505926 was highest in the longissimus dorsi muscle tissue of Jinfen white pigs at 1 day old and lowest at 180 days old, indicating that its expression gradually decreased with the increase of Jinfen white pig age ( Figure 2 ), suggesting that Loc106505926 may be involved in regulating the growth and development of pig muscle.

[0069] Example 3 Effect of Loc106505926 on the Proliferation of Porcine Skeletal Muscle Satellite Cells

[0070] 1. Materials

[0071] Cells: Pig skeletal muscle satellite cells, isolated and cultured in the laboratory.

[0072] Reagents: Fetal bovine serum (FBS) and DME / F12 were purchased from Gibco; penicillin-streptomycin mixture, glycine, and 4% paraformaldehyde were purchased from Solebro; Triton X-100 was purchased from Sigma; 0.25% trypsin and PBS buffer were purchased from Hyclon; EdU proliferation detection kit and transfection reagent riboFECT™ CP were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0073] 2. Methods

[0074] The present invention targets the Loc106505926 sequence and synthesizes three pairs of siRNA sequences that inhibit its expression. The sequences are:

[0075] si-Loc1065059265-1:5′-GCCUUUCUUUGUCCUUUGUTT-3′(SEQ ID NO:8)

[0076] 5′-ACAAAGGACAAAGAAAGGCTT-3′(SEQ ID NO:9)

[0077] si-Loc1065059265-2: 5′-CCACCUUAUUGCAGAACAATT-3′ (SEQ ID NO: 10)

[0078] 5′-UUGUUCUGCAAUAAGGUGGTT-3′(SEQ ID NO:11)

[0079] si-Loc1065059265-3: 5′-GGCUCUGGUUGAAAGUGAAATT-3′ (SEQ ID NO: 12)

[0080] 5′-UUUCACUUCAACCAGAGCCTT-3′(SEQ ID NO:13)

[0081] The NC group sequence is:

[0082] 5′-UUCUCCGAACGUGUCACGUTT-3′(SEQ ID NO:14)

[0083] 5′-ACGUGACACGUUCGGAGAATT-3′(SEQ ID NO:15)

[0084] The Loc106505926 interference sequence si group and NC group used in the experiment were synthesized by Jima Co., Ltd.

[0085] 2.1 Detection of Loc106505926 siRNA interference efficiency

[0086] Porcine skeletal muscle satellite cells were seeded in 12-well plates and transfected when the cell density reached approximately 60%. 20 μmol of siRNA stock solution was diluted in 60 μL of 1× riboFECT™ CP Buffer and gently mixed. Then, 6 μL of riboFECT™ CP Reagent was added, gently pipetting to mix, and incubated at room temperature for 15 minutes. The mixture was added to 929 μL of complete culture medium, gently mixed, and then added to the cells. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours, after which the cells were harvested and assayed for knockdown efficiency using qRT-PCR.

[0087] 2.2 qRT-PCR detection of proliferation marker gene expression

[0088] Cells were seeded in 12-well plates and transfected when the cell density reached approximately 60%. The transfection method was the same as described in 2.1 of Example 3. 48 hours after transfection, cells from the different treatment groups were collected, total RNA was extracted and reverse transcribed into cDNA, and qRT-PCR was used to detect changes in key proliferation genes such as PCNA, ki67, CyclinD, CDK1, and CDK4. The specific operation method was the same as 2.3 of Example 2.

[0089] 2.3 EdU detection of cell proliferation

[0090] The cells were seeded in a 24-well plate and transfected when the cell density reached about 60%. The transfection method was the same as that described in 2.1 of Example 3. After 48 hours of transfection, the solution was replaced with 50 μmol·L -1 EdU culture medium was added and incubated in a 37°C constant temperature incubator with 5% CO2 for 2 h. After washing with PBS, 4% paraformaldehyde was added for fixation for 30 min, and 200 μL of 2 mg mL -1 The cells were incubated with glycine for 5 minutes at room temperature and washed three times with PBS. 300 μL of 0.5% Triton X-100 was added and incubated at room temperature for 30 minutes. 200 μL of Apollo staining solution was added and incubated at room temperature in the dark for 30 minutes. The cells were washed three times with 0.5% Triton X-100 for 10 minutes each. DAPI staining solution was added to stain the nuclei. The staining results were observed under a fluorescence microscope, and the number of EdU-positive cells was statistically analyzed using Image J.

[0091] According to the mRNA sequences of porcine proliferation-related marker genes PCNA (NM_001291925.1), ki67 (NM_001101827.1), CyclinD, CDK1, and CDK4 published in the NCBI database, primers were designed using Oligo7 software as follows:

[0092] PCNA upstream primer: 5′-AACCTGCAGAGCATGGACTC-3′ (SEQ ID NO: 16)

[0093] Downstream primer: 5′-CTAGTGCCAAGGTGTCTGCA-3′ (SEQ ID NO: 17)

[0094] Upstream primer of ki67: 5′-CTTCGACAAAAGCTGCGGAT-3′ (SEQ ID NO: 18)

[0095] Downstream primer: 5′-CCGTAAAGCCTGGTGCTTTCT-3′ (SEQ ID NO: 19)

[0096] Upstream primer for Cyclin D: 5′-GTGCTGGGCAAGTTGAAGTG-3′ (SEQ ID NO: 20)

[0097] Downstream primer: 5′-TTGAGCAGCACGACCTCAAT-3′ (SEQ ID NO: 21)

[0098] Upstream primer for CDK1: 5′-CAGGAAGCCTAGCATCCCAC-3′ (SEQ ID NO: 22)

[0099] Downstream primer: 5′-TTGCCAGAAATTCGCTTGGC-3′ (SEQ ID NO: 23)

[0100] Upstream primer for CDK4: 5′-TGTGTCTCTACCCCGAGGAG-3′ (SEQ ID NO: 24)

[0101] Downstream primer: 5′-GGCAGAGATCGACTTCTCGG-3′ (SEQ ID NO: 25)

[0102] 3. Results

[0103] 3.1 Interference efficiency of siRNA against Loc106505926

[0104] In order to study the function of Loc106505926, the present invention inhibited the expression of Loc106505926 through siRNA-mediated interference experiments. The results of fluorescence quantitative PCR showed that compared with the control group, the first pair (si-Loc1065059265-1) and the second pair (si-Loc1065059265-2) of synthesized siRNAs could not significantly reduce the expression of Loc106505926. The third pair (si-Loc1065059265-3) of synthesized siRNAs could significantly reduce the expression of Loc106505926 in pig skeletal muscle satellite cells, indicating that the designed and synthesized third pair of siRNAs (si-Loc1065059265-3) can effectively inhibit the expression of Loc106505926. Figure 3 ).

[0105] 3.2 Effects of Loc106505926 Interference on the Proliferation of Porcine Skeletal Muscle Satellite Cells

[0106] The present invention uses qRT-PCR and EdU staining tests to detect the effect of interfering with Loc106505926 on the proliferation of porcine skeletal muscle satellite cells. The results show that both qRT-PCR and EdU staining tests show that interfering with Loc106505926 can significantly reduce the proliferation rate of porcine skeletal muscle satellite cells ( Figures 4 to 6 ), suggesting that Loc106505926 can participate in regulating the growth and development of pig muscle.

[0107] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Shanxi Agricultural University Shanxi Provincial Animal Husbandry Technology Extension Service Center <120> LncRNA related to porcine skeletal muscle satellite cell proliferation and its application <130> KHP221114634.4 <160> 25 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1706 <212> DNA <213> Pig (Sus scrofa) <400> 1 gtggttcagg tcactgctat ggcatgggtt tgtccctggc ctgggaattt gcacatgctg 60 tgggtgtggc caaaaataaa tagtgaaatc atcaaaaaaa gcacaaacat gaaaaaatgt 120 ggcagtaact agaccatgaa aaggccactt gtttacagca agagctgaga cacaaaggca 180 aagggtcacc ttgttctacc tcagcaagtc ggcaaatttg caaataatga aaacatgaat 240 aatggagatg aagtgtttat ctcctctagc catatgcatt agttacagat accctaaata 300 ctacctccta aaaccaggga cattccccca caattccatg atcaaattaa ggaagtttga 360 tattgatgca gtactctttt ctaacacaat tttcattttc aattttgttc ttgtttcaat 420 catgtcgttt cttgccatga ttattttcat cagggatcag gcactgcatt gaattgtcat 480 gtctctttat tcacctttaa tttggaacag cctttctttg tcctttgtaa catcaacatt 540 tttcaagagt acaagccagt ggatttgtag agtgccacgt ccatctagtt tatctgatat 600 tttctcatta taattatcag gttttcattt tggcaggagt tttatctcca ttttgcagat 660 gacaaaagtt gatgcccaga gagggtaagt aattactggg cagaatttga gcctagaagt 720 ctggctcaga gcttaggcct cccaaaatc tcaggctaat tctggctttg cttaagaaat 780 tggaaggtcc agcaaggagc attactgggc ccaccttat gcagaacaat aatgaacagg 840 agctgagttg cagctactct tccccaggag atatatttgc cacagtccct gcccttccct 900 attgtcttgt tcctgtctaa cttcatttat gttacttgcc tggccccagt gggccatgga 960 cttggagaac cctgtactct ccccaaacct gatgtagaaa ggggtttttt caaaacaaag 1020 ggctctggtt gaagtgaaag ttgattgggg gaaagtgggc ttaagcgctt gtctttctgt 1080 gtagactcgg acatggccaa cagcagtttt catgggctga gtgggggagt gggccttctt 1140 ttatcctatt gcagcttctt tcctccatcc tgcgtcacat gcaccaggac ccacccctcc 1200 cctgtcccca gatgaccctc taactgtgac agagtaagat ggggcgggtg cccctgtgtg 1260 aaagtcactg ctcctcagag aagagctgag catcttgcat ccttccctga aagacaccca 1320 cctctctgct tctgcagagt actgggatcc agctggaata atctcagggc tgtgactctg 1380 tagtctttta ttttctttcc taaacttgcc ttaactcaag tatcatcaga ttggcttata 1440 aaatgtggat atccaagccc ttctgcagat tcctattcaa gttggtctgg gagattgcac 1500 attccacaag gacacagact ggtctatttt gttcattgct ctatttccag cgtggagaa 1560 cagtgcctgg catgcagcag ggaacttgtg caaggtgata aagatggtct ccaggagttc 1620 ccgccctggc acagtgagtt aagaacgtga gtgcagcatc tcaggtcact gcagagatgc 1680 aggcgcagtg gatccagcat tgccac 1706 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 aatgtggcag taactagacc 20 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 gactgtggca aatatatctc c 21 <210> 4 <211> twenty one <212> DNA <213> Artificial Sequence <400> 4 acaaagggct ctggttgaag t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gttggccatg tccgagtcta 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 cccacggaat cgagaaagag 20 <210> 7 <211> 17 <212> DNA <213> Artificial Sequence <400> 7 ttgacggaag ggcacca 17 <210> 8 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 8 gccuuucuuu guccuuugut t 21 <210> 9 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 9 acaaaggaca aagaaaggct t 21 <210> 10 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 10 ccaccuuauu gcagaacaat t 21 <210> 11 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 11 uuguucugca auaagguggt t 21 <210> 12 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 12 ggcucugguu gaagugaaat t 21 <210> 13 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 13 uuucacuuca accagagcct t 21 <210> 14 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 14 uucuccgaac gugucacgut t 21 <210> 15 <211> twenty one <212> DNA / RNA <213> Artificial Sequence <400> 15 acgugacacg uucggagaat t 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 aacctgcaga gcatggactc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 ctagtgccaa ggtgtctgca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 cttcgacaaa agctgcggat 20 <210> 19 <211> twenty one <212> DNA <213> Artificial Sequence <400> 19 ccgtaaagcc tggtgctttc t 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 gtgctgggca agttgaagtg 20 <210> twenty one <211> 20 <212> DNA <213> Artificial Sequence <400> twenty one ttgagcagca cgacctcaat 20 <210> twenty two <211> 20 <212> DNA <213> Artificial Sequence <400> twenty two caggaagcct agcatcccac 20 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <400> twenty three ttgccagaaa ttcgcttggc 20 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <400> twenty four tgtgtctcta ccccgaggag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 ggcagagatc gacttctcgg 20

Claims

1. Use of a reagent for detecting the expression level of LncRNA in the preparation of a detection reagent or kit for identifying the proliferation of porcine skeletal muscle satellite cells; The cDNA sequence corresponding to the LncRNA is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that The reagent for detecting the expression level of LncRNA includes an upstream primer shown in SEQ ID NO: 2 and a downstream primer shown in SEQ ID NO: 3.

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