Antisense lncRNA CFL1-AS1 related to growth and development of cattle muscles and application of antisense lncRNA CFL1-AS1

Abstract

The invention discloses antisense l ncRNA CFL1-AS1 related to bovine muscle growth and development and application thereof, and belongs to the technical field of molecular biology, through high-throughput sequencing, differentially expressed l ncRNA TCONS00184439 is screened from adult bovine muscle tissue, RACE cloning is performed to obtain a full-length sequence 616bp, genomic sequences are compared, the positions of the genomic sequences and the antisense strand of a CFL1 gene are overlapped on a genome, and the antisense 1 ncRNA CFL1-AS1 related to bovine muscle growth and development is obtained. The antisense lncRNA is named as CFL1-AS1 according to the naming of the long-chain non-coding RNA; the CFL1-AS1 has specific low expression in adult cattle muscle tissues, the CFL1-AS1 is up-regulated to promote myoblast proliferation, myoblast differentiation is dynamically regulated and controlled, and the CFL1-AS1 participates in the muscle growth and development process; the CFL1-AS1 overcomes the defects of the antisense l ncRNA in muscle growth and development, and provides a new molecular marker and a new breeding idea for genetic improvement and efficient breeding of beef cattle.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a bovine muscle growth and development related antisense lncRNA CFL1-AS1 and application thereof. BACKGROUND

[0002] Beef is a kind of meat with high nutritional value, which has the characteristics of high protein and low fat, and is rich in essential amino acids, trace elements and mineral elements beneficial to health, and has delicious taste, and belongs to a high-quality protein source. Therefore, beef is favored by consumers in today's green and healthy diet. In recent years, the consumption of beef has been increasing, but the domestic production of beef is difficult to meet market demand. The import volume of beef has increased significantly, and the import volume of beef has increased continuously year by year. The proportion of imported beef in consumption has increased, and the dependence on foreign countries is high. With the improvement of residents' income level, the upgrading of consumption structure and the promotion of urbanization process will drive the growth of beef consumption demand. In the future, beef will still maintain a net import pattern, and beef supply will still be unable to meet the growth of consumption demand. This reveals that the beef market has great potential demand and industry space, which has attracted people's close attention to the quality and yield of beef.

[0003] Muscle is a core index for measuring animal growth speed, meat yield, meat quality and other important economic traits. As an important quantitative trait, the yield and quality of beef are closely related to the number and volume of muscle fibers, and the changes in the number and volume of muscle fibers are reflected in the proliferation and differentiation processes of muscle cells. In recent years, more and more studies have shown that lncRNAs widely exist in mammals, and as very important epigenetic regulatory factors, they are involved in various important regulatory processes such as genomic imprinting, chromosome silencing, chromatin modification, transcription activation and interference, and nuclear transport, and play a key role in the growth, development and differentiation processes of cells. However, in the field of agricultural animal research, there are few reports on antisense lncRNAs that have important functions in the growth and development of muscle. SUMMARY

[0004] The technical problems solved: In view of the technical defects that there are few reports on antisense lncRNA which has been confirmed to have important function for muscle growth and development in the prior art, an antisense lncRNA CFL1-AS1 related to bovine muscle growth and development and its application are disclosed. The lncRNA is significantly underexpressed in adult bovine muscle tissue, and when the lncRNA is overexpressed, it is found that the number of proliferating myoblasts is significantly increased and the expression of key genes CCND1 and PCNA is also significantly up-regulated. CFL1-AS1 negatively regulates the expression of myogenic determination factor MYOD in the late differentiation of myoblasts. It is proved that antisense lncRNA CFL1-AS1 promotes the proliferation of myoblasts, inhibits the differentiation of myoblasts, and participates in the muscle growth and development process. It is proved that the discovery of antisense lncRNA CFL1-AS1 helps to provide new molecular markers and breeding ideas for beef cattle genetic improvement and efficient breeding from the aspect of molecular breeding, and has important application prospect.

[0005] Technical scheme: An antisense lncRNA CFL1-AS1 related to bovine muscle growth and development, wherein the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development is differentially expressed in adult bovine muscle tissue, and the nucleotide sequence is shown as SEQ ID NO. 1.

[0006] Further, the preparation method of the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development uses adult bovine muscle tissue total RNA as a template to perform 3'RACE and 5'RACE amplification of CFL1-AS1, and obtains the complete CFL1-AS1 sequence.

[0007] Further, the 5'RACE amplification primer sequence is shown as SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 5.

[0008] Further, the 3'RACE amplification primer sequence is shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5.

[0009] Further, the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development is specifically underexpressed in adult bovine muscle tissue.

[0010] The application of an antisense lncRNA CFL1-AS1 related to bovine muscle growth and development in beef cattle genetic improvement or efficient breeding, wherein the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development is used as a molecular marker to regulate myoblast proliferation and differentiation or participate in the process of bovine muscle growth and development.

[0011] Further, the overexpression vector of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 is constructed by using a pcDNA3.1 plasmid, and the overexpression primer sequences of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 are shown as SEQ ID NO. 8 and SEQ ID NO. 9.

[0012] Further, the overexpression vector of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 is used to up-regulate the expression of CFL1-AS1.

[0013] Further, the overexpression vector of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 up-regulates the expression of CCND1 and PCNA and down-regulates the expression of MYOD.

[0014] Further, the real-time fluorescent quantitative PCR forward primer sequence of the proliferation key gene PCNA is shown as SEQ ID NO. 10, the real-time fluorescent quantitative PCR reverse primer sequence of the proliferation key gene PCNA is shown as SEQ ID NO. 11; the real-time fluorescent quantitative PCR forward primer sequence of the proliferation key gene CCND1 is shown as SEQ ID NO. 12, the real-time fluorescent quantitative PCR reverse primer sequence of the proliferation key gene CCND1 is shown as SEQ ID NO. 13; the real-time fluorescent quantitative PCR forward primer sequence of the myogenic determinant MYOD is shown as SEQ ID NO. 14, and the real-time fluorescent quantitative PCR reverse primer sequence of the myogenic determinant MYOD is shown as SEQ ID NO. 15.

[0015] Further, the bovine skeletal muscle tissue is subjected to primary culture, purification and subculture to obtain bovine primary muscle cells; the overexpression vector of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 is constructed by using a pcDNA3.1 plasmid, the overexpression pcDNA3.1-CFL1-AS1 vector is transfected into the bovine primary muscle cells, and the cells are cultured for another 36 hours, the proliferation of the cells is detected by an EdU experiment, the RNA of the cells is extracted, and the expression changes of CFL1-AS1 and muscle cell proliferation related marker factors are detected; after the overexpression pcDNA3.1-CFL1-AS1 vector is transfected into the bovine primary muscle cells, 2% pregnant horse serum is added to induce differentiation, and the cells are cultured for another 5 days, the RNA and protein of the cells are extracted, and the expression changes of muscle cell differentiation related marker factors are detected.

[0016] The application further discloses a reagent for detecting the expression level of the bovine muscle growth and development related antisense lncRNA CFL1-AS1 in different tissues, and the primer sequences for detection are shown as SEQ ID NO. 6 and SEQ ID NO. 7.

[0017] Further, the sample detected by the reagent is fetal bovine and adult bovine healthy muscle tissue.

[0018] Further, the sample detected by the reagent is adult bovine healthy muscle, heart, liver, spleen, lung, kidney, small intestine and fat.

[0019] Beneficial effects: 1. A new bovine muscle growth and development related antisense lncRNA is screened and identified in the fetal bovine and adult bovine muscle tissue, which is significantly down-regulated in the adult bovine muscle tissue, and the real-time fluorescent quantitative PCR experiment further verifies that the antisense lncRNA is specifically lowly expressed in the adult bovine muscle tissue; 2. The full-length sequence of the antisense lncRNA is cloned by RACE technology, and genome alignment analysis finds that the lncRNA and the CFL1 gene are located on bovine chromosome 29, and there is a large amount of overlap with the antisense strand sequence of CFL1, and the lncRNA is named CFL1-AS1 (CFL1 Antisense 1); 3. The overexpression vector pcDNA3.1-CFL1-AS1 of the antisense lncRNA CFL1-AS1 is further constructed, the bovine primary muscle cells are transfected, the proliferation of the cells is detected by the EdU experiment, and the expression changes of the proliferation key genes CCND1 and PCNA are detected by real-time fluorescent quantitative PCR, and it is found that the overexpression of the antisense lncRNA CFL1-AS1 significantly increases the number of proliferating myoblasts and significantly up-regulates the expression of the proliferation key genes CCND1 and PCNA; 4. After the bovine primary muscle cells are transfected with the overexpression vector pcDNA3.1-CFL1-AS1, the differentiation is induced for 5 days, and it is found that CFL1-AS1 negatively regulates the expression of the myogenic determinant MYOD in the process of myoblast differentiation by real-time fluorescent quantitative PCR and Western blotting experiment; 5. The bovine muscle growth and development related antisense lncRNA CFL1-AS1 promotes muscle cell proliferation, inhibits muscle cell differentiation, participates in the process of bovine muscle growth and development, helps to fill the deficiency of natural antisense lncRNA in the field of bovine muscle growth and development, provides a new molecular marker and selection idea for beef cattle genetic improvement and efficient breeding, and has important application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Fig. 1 is a diagram for screening and identifying the differentially expressed lncRNA CFL1-AS1 of adult bovine different tissues of the application, wherein A is a screening diagram, and B is an identification diagram;

[0021] Figure 2Gel electrophoresis map of RACE results of antisense lncRNA CFL1-AS1 of the application, wherein A is a 5'RACE gel electrophoresis map, B is a 3'RACE gel electrophoresis map, and C is a lncRNA CFL1-AS1 sequence full-length gel electrophoresis map;

[0022] Figure 3 Construction map of antisense lncRNA CFL1-AS1 overexpression vector of the application, wherein A is a full-length PCR product map of lncRNA CFL1-AS1, B is a CFL1-AS1 overexpression vector pcDNA3.1 ligation liquid PCR map, C is a CFL1-AS1 overexpression vector sequence alignment map, D is a CFL1-AS1 overexpression vector expression effect map in 293T cells, and E is a CFL1-AS1 overexpression vector mRNA expression level map;

[0023] Figure 4 Myoblast proliferation and mRNA expression level of key proliferation genes CCND1 and PCNA after overexpression of CFL1-AS1 of the application, wherein A is a mRNA expression level map, and B is a myoblast proliferation map;

[0024] Figure 5 mRNA and protein expression levels of myogenic determination factor MYOD after overexpression of CFL1-AS1 of the application and induction of myoblast differentiation for 5 days, wherein A is a myogenic determination factor MYOD mRNA expression level map, and B is a protein expression level map. DETAILED DESCRIPTION

[0025] The application will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only applicable to illustrate the application and are used to limit the scope of the application. If no special description is given in the examples, the experimental technical methods involved are common technical means used in cell biology or molecular biology, and are carried out according to conventional conditions.

[0026] Example 1: Extraction of RNA from different tissues of cattle and screening and identification of antisense lncRNA CFL1-AS1, which is specifically as follows:

[0027] I. Extraction of RNA from tissue samples:

[0028] (1) 150-200 mg of each of adult cattle healthy muscle, heart, liver, spleen, lung, kidney, small intestine and fat was taken and put into a mortar, and liquid nitrogen was added for grinding until powder was obtained.

[0029] (2) When the liquid nitrogen was basically volatilized, 1 mL of Trizol was added to collect the sample, and the Trizol was ground into powder and left to stand at room temperature.

[0030] (3) Until Trizol is completely dissolved with the sample into liquid, transfer into 1.5 mL centrifuge tube.

[0031] (4) Shake vigorously for 1 min, centrifuge at 4℃, 12000 r / min for 15 min.

[0032] (5) Transfer supernatant to a new 1.5 mL centrifuge tube, and add 0.2 mL of chloroform (one-fifth of the volume of Trizol).

[0033] (6) Shake vigorously for 1 min, stand at room temperature for 3 min, and centrifuge at 4℃, 12000 r / min for 15 min.

[0034] (7) Transfer supernatant to a new 1.5 mL centrifuge tube, and add 0.5 mL of pre-cooled isopropanol (half of the volume of Trizol).

[0035] (8) After mixing, stand at room temperature for 10 min, and centrifuge at 4℃, 12000 r / min for 10 min.

[0036] (9) Discard the supernatant, and add 1 mL of pre-cooled 75% alcohol (absolute ethanol: DEPC = 3:1) for washing.

[0037] (10) Centrifuge at 4℃, 12000 r / min for 10 min.

[0038] (11) Repeat step 9 and step 10 each once.

[0039] (12) Discard the supernatant, and dry.

[0040] (13) Add 20-50 μL of DEPC water or enzyme-free water to dilute the RNA.

[0041] (14) Detect the quality of RNA and save: detect the concentration and OD value of the extracted RNA by ultraviolet spectrophotometer, and detect the integrity of the RNA by agarose gel electrophoresis. Save the RNA sample at -80℃ or proceed to the next step.

[0042] II. Screening and identification of antisense lncRNA CFL1-AS1 related to bovine muscle growth and development:

[0043] According to the high-throughput sequencing results of the subject group in the present application, 28 lncRNAs (including lncRNA CFL1-AS1) with differential expression and highly conserved muscle differentiation related miRNA binding sites were screened for real-time fluorescence quantitative PCR test. β-actin was used as an internal reference gene, and the specific information of the quantitative primer is shown in Table 1 below:

[0044] Table 1

[0045]

[0046]

[0047] Remove the RNA from the -80℃ freezer, thaw it on ice, and prepare the following reverse transcription system:

[0048] The reverse transcription system consisted of 20.0 μL of total RNA (1.0 μg), 4×gDNA wiper Mix (4.0 μL), and Nuclease-free H2O added to a final volume of 16.0 μL. The reaction conditions were 42 °C for 2 min. Subsequently, 4.0 μL of 5×No RT Control Mix was added, and the reaction conditions were 50 °C for 15 min and 85 °C for 2 min.

[0049] Components and volume of the qRT-PCR system:

[0050] Total reaction volume 20.0 μL: 10.0 μL 2×AceQ Universal SYBR qPCR Master Mix; 0.4 μL each of forward and reverse primers; 2.0 μL cDNA; and 7.2 μL enzyme-free water. Reaction conditions: pre-deformation at 95℃ for 5 min, followed by 40 cycles of (95℃ for 10 s, 60℃ for 30 s).

[0051] PCR reactions were performed using SYBR Green as a fluorescent label on a Light Cycler real-time quantitative PCR instrument. The target band was identified by melting curve analysis and electrophoresis. β-actin was used as an internal control, and 2^ -△△Ct The relative expression levels of the target gene were calculated. P-values ​​were determined using one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001. All experimental data are expressed as mean ± SEM of three replicates and plotted using GraphPad Prism 7.0 software (San Diego, CA, USA).

[0052] Quantitative results such as Figure 1 As shown, TCONS_00184439 (lncRNA CFL1-AS1) was specifically and significantly lowly expressed in adult bovine muscle tissue (P<0.001). This suggests that the antisense lncRNA CFL1-AS1 may be involved in the muscle growth and development process in cattle.

[0053] Example 2: Amplification of the full-length lncRNA CFL1-AS1 sequence using RACE technology, as detailed below:

[0054] Using adult bovine muscle tissue RNA (6210A) as a template, a kit (Takara Bio USA, Inc. RACE 5’ / 3’Kit User Manual) to perform rapid amplification of the 3' and 5' ends of the cDNA of the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development.

[0055] I. Reverse transcription:

[0056] 1. RNA denaturation and annealing:

[0057] (1) The reagents required for the experiment were thawed on ice, and after complete thawing, the reaction was prepared according to Table 2 below:

[0058] Table 2

[0059] 3' RAC amplification 5' RAC amplification Component Name Volume (μL) Volume (μL) RNA 1 1 3' RACE RT Primer (12 μM) 1.0 - 5' RACE RT Primer (12 μM) - 1.0 Nuclease free water 9.5 8.5 Total Volume 11.5 10.5

[0060] (2) Mix the solution, centrifuge briefly, and then place it in a PCR instrument for reaction. The reaction program is 72°C for 3 min, then reduce to 4°C for incubation for 5 min. After annealing is completed, centrifuge briefly and immediately place on ice.

[0061] 2. Synthesis of cDNA:

[0062] (1) Prepare the reverse transcription reaction solution in Table 3 below on ice, mix gently, and centrifuge briefly.

[0063] Table 3

[0064] Component Name Volume (μL) 5X RACE RT Buffer 4.0 dNTP Mix (10 mM each) 2.0 RNase Inhibitor (40 U / μL) 0.5 Evo M-MLV RTase for RACE (100 U / μL) 2.0 Total Volume 8.5

[0065] (2) As shown in Table 4 below, take 8.5 μl of the reverse transcription reaction solution prepared above and add it to the reagents after the first step of annealing is completed.

[0066] Table 4

[0067]

[0068]

[0069] (3) Gently mix the solution, centrifuge briefly to collect the solution at the bottom of the tube, and perform the reaction in a PCR instrument with the program set. The reaction conditions are 42°C for 90 min, 70°C for 15 min, and 4°C for holding.

[0070] II. First round of rapid amplification of the cDNA ends:

[0071] (1) PCR reaction preparation: Prepare the PCR reaction solution on ice according to Table 5 below, and mix gently using a pipette.

[0072] Table 5

[0073] 5' cDNA end rapid amplification configuration system: Component Final concentration Amount (μL) 5' RACE cDNA - 2.5 SEQ ID NO. 4 (10X) 1X 5 SEQ ID NO. 2 (10 μM) 0.2 μM 1 5X L-Exp Taq PCR Buffer(Mg 2+ plus) 1X 10 L-Exp Taq HS DNA Polymerase (5 U / μl) 2.5U 0.5 dNTP Mix (10 mM each) 0.4 mM 2 Nuclease free water - 29 3' cDNA end rapid amplification configuration system: Component Final concentration Amount (μL) 3' RACE cDNA - 2.5 SEQ ID NO. 4 (10X) 1X 5 SEQ ID NO. 3 (10 μM) 0.2 μM 1 5X L-Exp Taq PCR Buffer(Mg 2+ plus) 1X 10 L-Exp Taq HS DNA Polymerase (5 U / μl) 2.5U 0.5 dNTP Mix (10 mM each) 0.4 mM 2 Nuclease free water - 29 Total Volume - 50

[0074] (3) The PCR reaction program adopts the touchdown PCR method, as shown in Table 6 below:

[0075] Table 6

[0076]

[0077]

[0078] Three, two rounds of cDNA nested PCR (Nested PCR) amplification, primer sequence is:

[0079] (1) Take 5 μl of the 3' end and 5' end cDNA end rapid amplification PCR product of the first round of amplification, respectively, add 245 μl of Tricine-EDTA Buffer, shake and mix.

[0080] (2) According to Table 7 below, prepare the Nested PCR reaction solution on ice, gently mix and then place in the PCR instrument for reaction.

[0081] Table 7

[0082] 5' cDNA end rapid amplification Component Final concentration Amount (μL) Step (1) diluted PCR product - 5 SEQ ID NO. 5 (10X) 0.2 μM 1 SEQ ID NO. 2 (10 μM) 0.2 μM 0.5 5X L-Exp Taq PCR Buffer(Mg 2+ plus)]]> 1X 10 L-Exp Taq HS DNA Polymerase (5 U / μl) 2.5U 0.5 dNTP Mix (10 mM each) 0.4 mM 2 Nuclease free water - 31 Total Volume - 50 3' cDNA end rapid amplification Component Final concentration Amount (μL) Step (1) diluted PCR product - 5 SEQ ID NO. 5 (10X) 1X 1 SEQ ID NO. 3 (10 μM) 0.2 μM 0.5 5X L-Exp Taq PCR Buffer (Mg2+ plus) 1X 10 L-Exp Taq HS DNA Polymerase (5 U / μl) 2.5U 0.5 dNTP Mix (10 mM each) 0.4 mM 2 Nuclease free water - 31 Total Volume - 50

[0083] (4) The PCR reaction program adopts the touchdown PCR method, as shown in Table 8 below:

[0084] Table 8

[0085]

[0086] By 5' RACE technology, the lncRNA CFL1-AS1 5' end sequence full length is 470 bp (such as Figure 2 Figure A); by 3' RACE technology, the lncRNA CFL1-AS1 3' end sequence full length is 433 bp (such as Figure 2 Figure B); according to the predicted sequence splicing, the lncRNA CFL1-AS1 full length sequence is 616 bp (such as Figure 2 Figure C, SEQ ID NO. 1). Genomic sequence comparison found that this lncRNA and the antisense strand composed of four exons of the actin-binding protein gene 1 (Cofilin1, CFL1) on the bovine chromosome 29 The location overlaps on the genome, so according to the naming principle of long-chain non-coding RNA, this newly identified antisense lncRNA is named CFL1-AS1 (Antisense CFL1).

[0087] Example 3: Construction of antisense lncRNA CFL1-AS1 overexpression vector, as follows:

[0088] I. Primer design and synthesis: Referring to the pcDNA3.1-CFP plasmid map, the CFL1-AS1 target fragment was inserted between the HindIII and KpnI enzyme sites. The Primer Premier 5 software was used to design full-length primers CFL1-AS1-HindIII-F (SEQ ID NO. 8) and CFL1-AS1-KpnI-R (SEQ ID NO. 9) with enzyme sites.

[0089] II. PCR reaction: Amplify CFL1-AS1 using bovine muscle tissue cDNA as a template, and centrifuge the mixture shown in Table 9.

[0090] Table 9

[0091] Component Volume (μL) SEQ ID NO. 8 0.5 SEQ ID NO. 9 0.5 Taq Polymerase 5 cDNA template (muscle tissue) 2 ddH2O 2 Total 10

[0092] The reaction program was performed on a PCR instrument: 95°C, pre-denaturation for 5 min; 95°C, denaturation for 30 s, 67°C, annealing for 30 s, 72°C, extension for 1 min 30 s, 38 cycles; 72°C, final extension for 10 min; 16°C, storage for 10 min.

[0093] III. PCR product purification and recovery: After 25 min of 2% agarose gel electrophoresis, the target band was recovered, and the PCR product was recovered according to the DNA purification kit (TaKaRa) instructions.

[0094] IV. Enzymatic digestion and ligation: The purified PCR product and pcDNA3.1-GFP plasmid were digested with HindIII and KpnI at 37°C for 30 min, and the reaction system is shown in Table 10.

[0095] Table 10

[0096]

[0097] The digested pcDNA3.1-GFP plasmid and CFL1-AS1 were mixed at a ratio of 1:3 using T4 ligase at 16°C for 1 h, and the ligation system is shown in Table 11, then 4°C overnight.

[0098] Table 11

[0099] Component Volume (μL) 10×T4 DNA Ligase Buffer 2 pcDNA3.1 plasmid 4 CFL1-AS1 12.5 T4 DNA Ligase 1.5 Total 20

[0100] V. Vector transformation: The pcDNA3.1-CFL1-AS1 ligation product was transformed into DH5a using the heat shock method. After plating, the plate was incubated at 37°C for 14h. Single colonies were picked and identified by PCR amplification using CFL1-AS1 primers and sequencing. The bacterial liquid was preserved and the plasmid was extracted for cell transfection.

[0101] The designed pcDNA3.1-CFL1 primers were used to amplify the target fragment (as shown in Figure 3 A), and the plasmid containing the recombinant vector pcDNA3.1-CFL1-AS1 was extracted (as shown in Figure 3 B) and sent for sequencing. The sequence obtained using the universal primer T7 was compared with the CFL1-AS1 sequence in NCBI Blast. The sequencing results matched the CFL1-AS1 target sequence successfully, with a match of 100% (as shown in Figure 3 C). The pcDNA3.1-CFL1-AS1 expression vector was successfully constructed. The CFL1-AS1 overexpression vector pcDNA3.1-CFL1-AS1 and the control vector pcDNA3.1 were transfected into 293T cells. It was found that the expression of green fluorescent protein GFP was higher in the control group (pcDNA3.1) and the experimental group (pcDNA3.1-CFL1-AS1) (as shown in Figure 3 D), indicating that the transfection efficiency of the pcDNA3.1-CFL1-AS1 expression vector was high. RT-qPCR results showed that the expression of CFL1-AS1 at the mRNA level was significantly increased in the pcDNA3.1-CFL1-AS1 transfection group (p<0.001) (as shown in Figure 3 E). The constructed pcDNA3.1-CFL1-AS1 expression vector can be used for subsequent cell experiments.

[0102] Example 4: Antisense lncRNA CFL1-AS1 regulates the proliferation and differentiation of bovine primary muscle cells, as follows:

[0103] I. Culture of bovine primary muscle cells:

[0104] (1) The leg muscles of a 90-day-old calf were taken to a flat dish, and the leg was sterilized with ethanol. In a clean bench, the muscle was taken to a flat dish, washed with Hank's solution for 3 times, and the fat and connective tissue was removed. The muscle specimen was cut into about 0.1 cm 3 pieces;

[0105] (2) The cut muscle was moved to a centrifuge tube, washed with Hank's solution for 3 times, and incubated for 1 min. The upper liquid and floating tissue were discarded.

[0106] (3) Add 0.25% trypsin to the centrifuge tube, and digest at 37°C for 20 min, shake or blow the centrifuge tube every 5 min, and then add growth medium to terminate the digestion;

[0107] (4) After repeated blowing, sieve with 100, 200, and 400 meshes in turn, collect the filtrate, and centrifuge at 1000 r / min for 10 min;

[0108] (5) Discard the supernatant, resuspend the cells with growth medium, add the suspension to a culture bottle not coated with polylysine (PPL), remove the fibroblasts by differential adhesion, culture at 37°C for 1 h, transfer to a coated bottle, culture with growth medium, replace the medium after 4 days, and then replace the medium once a day.

[0109] (6) Microscopic identification: the freshly isolated primary cells are small and spherical, and have strong refractive properties. After 12 h, the cells start to adhere, and after 72 h, the adhesion is complete, the cells gradually extend into spindle-shaped, and with the extension of the culture time, the cells proliferate, migrate, and gradually arrange in a regular direction. When the cells are fused to more than 80%, muscle tubes begin to form.

[0110] II. Transfection of antisense lncRNA CFL1-AS1 overexpression vector into bovine primary muscle cells: well-grown bovine primary muscle cells were inoculated into a 6-well plate, and when the cell confluence reached 80%, CFL1-AS1 overexpression vector was transfected into bovine primary muscle cells using ExFect Transfection Reagent, and cultured at 37°C in a 5% CO2 incubator for 24-36 h, and then the cell RNA or protein was collected.

[0111] III. Induction of myoblast differentiation: bovine primary muscle cells were cultured at low density with proliferation medium (complete medium). When the cell number reached the required amount, the cells were passaged into a 6-well cell culture plate, and continued to be cultured until the cell density was about 80%-90%, the proliferation medium was replaced with differentiation medium, and C2C12 cell differentiation was induced, and the cells were cultured at 37°C in a 5% CO2 cell incubator for 5 d, and the differentiation medium was replaced once a day.

[0112] Preparation of proliferation medium: DMEM high-sugar medium: fetal bovine serum = 9:1.

[0113] Preparation of differentiation medium: DMEM high-sugar medium: horse serum = 98:2.

[0114] IV. EdU staining to detect cell proliferation: BeyoClick TMEdU-555 cell proliferation detection kit and inverted fluorescence microscope to detect the effect of antisense lncRNA CFL1-AS1 on the proliferation of bovine primary muscle cells. After treating bovine primary muscle cells in a 6-well plate, discard half of the culture medium, add 2x EdU working solution with the same volume as the culture medium, continue to incubate myoblasts for 2h, then fix the myoblasts with 4% paraformaldehyde, and permeabilize the cells. After washing the cells, add Click reaction solution and incubate at room temperature for 30min. After washing the cells 3 times, use Hoechst 33342 for nuclear staining, incubate at room temperature for 10min, and then wash 3 times. Finally, perform fluorescence staining imaging analysis under an inverted fluorescence microscope.

[0115] V. qRT-PCR detects the expression of genes:

[0116] (1) Extract total RNA from cells by TRIzol method, synthesize cDNA using reverse transcription kit (R323), first remove genomic DNA, and prepare the following table 12 reaction solution in an enzyme-free centrifuge tube:

[0117] Table 12

[0118] RNase-free ddH2O to 16 μL 4×gDNA wiper Mix 4 μL Template RNA Total RNA: 1 μg

[0119] (2) Mix gently with vortex shaker, centrifuge with centrifuge, run 42℃, 2min on PCR instrument. Add 4μL of 5x HiScript III qRT SuperMix, mix, and perform reverse transcription reaction in PCR instrument at 37℃ for 2min and 85℃ for 5s. The obtained cDNA is diluted 5 times with RNase-free ddH2O, and the concentration is determined on a UV spectrophotometer. Further dilute to a uniform concentration, and the product can be used for qPCR reaction, or stored at -20℃ for standby.

[0120] (3) Use AceQ universal SYBR qPCR Master Mix kit to detect the relative expression level of genes in cells or tissues, and perform relative quantitative analysis by 2 -ΔΔ(CT) method.

[0121] Perform operation in the dark on ice, and the RT-qPCR reaction system is as follows table 13:

[0122] Table 13

[0123]

[0124] The reaction program is as follows table 14:

[0125] Table 14

[0126]

[0127] (4) Statistical method: each test was repeated three times, and the statistical analysis was performed using Graphpad Prism 8.0 statistical software, the difference between the two was analyzed by t test, and it was considered that when P < 0.05, it had statistical significance.

[0128] Six, Western blot detection of MYOD gene expression changes: discard the culture medium of myoblasts in 6-well plates and wash twice with PBS, add 100 μL RIPA lysis buffer and lyse on ice for 10 min, scrape the cells with a scraper and collect the cells in a centrifuge tube, and determine the protein concentration with a BCA kit. According to the protein loading buffer instructions, the protein sample is boiled and denatured, then subjected to SDS-PAGE precast gel electrophoresis for about 45-60 min, the gel is transferred to PVDF membrane and electrophoresed for about 90 min, BSA or skim milk is used to block the transfer membrane for 1 h, after washing, the primary antibody is incubated overnight, the primary antibody is recovered, after washing, the secondary antibody is incubated for 2 h, the secondary antibody is recovered and washed, finally, ECL luminescent liquid is used to image in the protein gel imager, and then ImageJ software is used for protein gray scale analysis. The antibody information used is as follows Table 15.

[0129] Table 15

[0130] Antibody Name Use Source Company GAPDH primary antibody Rabbit MultiSciences MYOD primary antibody Rabbit Beyotime

[0131] Results: Overexpression of CFL1-AS1 up-regulates the mRNA expression levels of proliferation key genes CCND1 and PCNA (P < 0.05) (as shown in Figure 4 A), and increases the number of EdU positive cells (as shown in Figure 4 B); after the differentiation of bovine primary muscle cells (5d), antisense lncRNA CFL1-AS1 negatively regulates the mRNA level (as shown in Figure 5 A) and protein level expression (as shown in Figure 5 B) of myogenic determination factor MYOD. These results show that CFL1-AS1 can promote bovine primary muscle cell proliferation and inhibit bovine primary muscle cell differentiation to a certain extent.

[0132] The above-described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. The protection scope of the present application is subject to the scope claimed in the claims, and all other embodiments obtained by those of ordinary skill in the art without creative labor based on the embodiments in the present application belong to the protection scope of the present application.

Claims

1. A bovine muscle growth and development-related antisense lncRNA CFL1-AS1, characterized in that, The antisense lncRNA CFL1-AS1, which is associated with bovine muscle growth and development, is differentially expressed in adult bovine muscle tissue, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The antisense lncRNA CFL1-AS1 related to bovine muscle growth and development according to claim 1, characterized in that, The method for preparing the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development involves using Total RNA from adult bovine muscle tissue as a template to amplify CFL1-AS1 via 3'RACE and 5'RACE to obtain the complete CFL1-AS1 sequence.

3. The antisense lncRNA CFL1-AS1 related to bovine muscle growth and development according to claim 2, characterized in that, The 5'RACE amplification primer sequences are shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.

5.

4. The antisense lncRNA CFL1-AS1 related to bovine muscle growth and development according to claim 2, characterized in that, The 3'RACE amplification primer sequences are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

5. The antisense lncRNA CFL1-AS1 related to bovine muscle growth and development according to claim 1, characterized in that, The antisense lncRNA CFL1-AS1, which is associated with bovine muscle growth and development, is specifically lowly expressed in adult bovine muscle tissue.

6. The application of the antisense lncRNA CFL1-AS1 related to bovine muscle growth and development as described in claim 1 in the genetic improvement or high-efficiency breeding of beef cattle, characterized in that, The antisense lncRNA CFL1-AS1, which is associated with bovine muscle growth and development, serves as a molecular marker to regulate myoblast proliferation and differentiation or participate in the process of bovine muscle growth and development.

7. The application according to claim 6, characterized in that: An overexpression vector for the antisense lncRNA CFL1-AS1, which is related to bovine muscle growth and development, was constructed using the pcDNA3.1 plasmid. The primer sequences for the overexpression of the antisense lncRNA CFL1-AS1, which is related to bovine muscle growth and development, are shown in SEQ ID NO.8 and SEQ ID NO.

9.

8. The application according to claim 7, characterized in that: The overexpression vector of the antisense lncRNA CFL1-AS1, which is associated with bovine muscle growth and development, is used to upregulate the expression of CFL1-AS1.

9. The application according to claim 7, characterized in that: The overexpression vector of the bovine muscle growth and development-related antisense lncRNA CFL1-AS1 upregulated the expression of CCND1 and PCNA, and downregulated the expression of MYOD.

10. A reagent for detecting the expression level of the antisense lncRNA CFL1-AS1 associated with bovine muscle growth and development as described in claim 1 in different tissues, characterized in that: The primer sequences used for detection are shown in SEQ ID NO.6 and SEQ ID NO.7.

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