CircFTO related to beef cattle intramuscular adipogenesis and application thereof

Abstract

The invention discloses circFTO related to beef cattle intramuscular fat generation and application of circFTO, and relates to the technical field of beef cattle intramuscular fat generation. The nucleotide sequence of the circFTO is as shown in SEQ ID NO: 1. The existence of circFTO is verified, the full length of the molecular sequence of the circular RNA is obtained through cloning, the circFTO ring structure is determined, and the molecule has resistance to exonuclease RNase R. The circFTO regulates beef cattle intramuscular fat generation to influence the beef quality, and is applied to an auxiliary genetic breeding process in the aspect of beef quality improvement. The circFTO disclosed by the invention can be used as a molecular marker related to beef quality, and has an important practical application value in genetic assisted breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of intramuscular fat production in beef cattle, and more specifically, to a circRNA related to intramuscular fat production in beef cattle and its application in evaluating beef quality. Background Art

[0002] The beef cattle industry, as an integral component of animal husbandry, plays a vital role in the national economy. In recent years, with the improvement of living standards and the upgrading of consumption patterns, consumer demand for beef products has continued to increase. Beef, a high-protein, low-cholesterol, premium meat, particularly high-end beef with rich marbled texture, is highly sought after. Marbling, a direct reflection of intramuscular fat (IMF) deposition, is a key economic indicator in beef cattle production and is closely associated with quality traits such as tenderness, flavor, and juiciness. However, due to long-term selective breeding for draft cattle, the proportion of IMF deposits in local Chinese cattle breeds is relatively low. Unlike subcutaneous and visceral fat, IMF is difficult to anatomically isolate, making its specific study and evaluation challenging. Therefore, the development of molecular markers associated with IMF tissue or cells is crucial for beef cattle breeding and improving beef quality.

[0003] Circular RNA (circRNA), typically composed of gene exons, is a type of endogenous noncoding RNA molecule that lacks a 5' cap and a 3' poly(A) tail and forms a covalently linked circular structure. CircRNAs are resistant to degradation by the RNA exonuclease RNase R, are highly stable, and are abundant in eukaryotic cells. They exhibit temporal, tissue, and disease specificity. Numerous circRNAs have been found in the intramuscular adipose tissue of species such as monkeys, cattle, sheep, and chickens, and are closely associated with adipocyte differentiation and lipid metabolism. This study proposes a circRNA associated with intramuscular adipogenesis in beef cattle and its identification method, providing new insights for breeding high-quality beef cattle breeds. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a circFTO related to intramuscular fat production in beef cattle.

[0005] Another object of the present invention is to provide applications of the circFTO.

[0006] The present invention used longissimus dorsi muscle tissue from 24-month-old adult Angus and Lufeng cattle as research materials. Comparison of muscle tissues from Angus and Lufeng cattle revealed significantly higher intramuscular fat content in Angus cattle than in Lufeng cattle. Therefore, combining next-generation sequencing and bioinformatics analysis, the present invention identified circRNAs that partially regulate intramuscular fat production in beef cattle, providing a theoretical basis for breeding high-quality beef cattle breeds (lines).

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention first discovered the existence of the circular transcript circFTO and found that it is closely related to intramuscular fat production in beef cattle. Therefore, the present invention claims the following:

[0009] A circular RNA related to intramuscular fat production in beef cattle is named circFTO, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0010] The application of the above circular RNA related to intramuscular fat production in beef cattle as a specific molecular marker for intramuscular adipocytes in beef cattle.

[0011] Furthermore, the aforementioned circular RNA or a product for detecting the expression of the circular RNA is used in analyzing the intramuscular fat content of beef cattle.

[0012] The analysis of the intramuscular fat content of beef cattle may specifically be used to determine the level of the intramuscular fat content.

[0013] Specifically, if the expression level of the circular RNA in the test sample is significantly higher than that in the control sample, the content of intramuscular fat in the test sample is lower than that in the control sample.

[0014] Furthermore, the above circular RNA or a product for detecting the expression of the circular RNA is used to identify or assist in identifying intramuscular adipose tissue or intramuscular adipocytes in beef cattle.

[0015] Furthermore, the use of the aforementioned circular RNA or a product for detecting the expression of the circular RNA in predicting or assisting in predicting beef quality;

[0016] Furthermore, the use of the aforementioned circular RNA or a product for detecting the expression of the circular RNA in the preparation of a kit for predicting or assisting in predicting beef quality;

[0017] Furthermore, the above-mentioned circular RNA or a product for detecting the expression of the circular RNA is used to increase the intramuscular fat content of beef cattle or improve the quality of beef cattle.

[0018] Furthermore, the aforementioned circular RNA or a product for detecting the expression of the circular RNA is used in breeding beef cattle breeds (lines) with different beef qualities.

[0019] The product for detecting the expression of the circular RNA includes a product for detecting the expression of the circular RNA using PCR technology.

[0020] Furthermore, the product for detecting the expression of the circular RNA using PCR technology includes PCR detection primers, including Divergent-circFTO-F and Divergent-circFTO-R, whose nucleotide sequences are shown in SEQ ID NOs: 2 to 3.

[0021] The product for detecting the expression of the circular RNA using PCR technology includes qPCR detection primers, including Convergent-circFTO-F and Convergent-circFTO-R, whose nucleotide sequences are shown in SEQ ID NOs: 4 to 5.

[0022] The detection primer is used to detect the circular RNA.

[0023] A kit for detecting the circular RNA, comprising the PCR detection primer or qPCR detection primer.

[0024] Furthermore, when circular RNA was detected using PCR, the primers used included Divergent-circFTO-F and Divergent-circFTO-R, whose nucleotide sequences were shown in SEQ ID NOs: 2-3.

[0025] Furthermore, when circular RNA was detected using the qPCR method, the primers used included Convergent-circFTO-F and Convergent-circFTO-R, whose nucleotide sequences are shown in SEQ ID NOs: 4-5.

[0026] Furthermore, the kit further comprises internal reference primers, which include an upstream primer GAPDH-F and a downstream primer GAPDH-R of the GAPDH internal reference gene, and the nucleotide sequences thereof are shown in SEQ ID NOs: 6-7.

[0027] The above kit was used for the quantitative detection of circFTO in the longissimus dorsi muscle tissue of beef cattle.

[0028] The above kit is suitable for all types of fluorescent quantitative gene amplification instruments currently on the market. It has high sensitivity, rapid and accurate quantification, good stability, and good application prospects.

[0029] The present invention also provides a method for detecting the intramuscular fat content of beef cattle, comprising the following steps: detecting the expression level of circular RNA in beef cattle, and judging the intramuscular fat content of beef cattle according to the expression level of circular RNA.

[0030] The criteria for determining whether intramuscular fat is present or the level of intramuscular fat are as follows: if the expression level of circular RNA in beef cattle is significantly higher than that in the control sample, the content of intramuscular fat in the beef cattle is lower than that in the control sample.

[0031] The specific steps include:

[0032] (i) extracting total RNA from bovine muscle tissue as a test sample;

[0033] (ii) reverse transcribing the RNA obtained in step (i) to obtain cDNA;

[0034] (iii) amplifying and detecting the cDNA obtained in step (ii);

[0035] (iv) Through melting curve analysis, 2 -ΔΔCt Relative quantification was performed by the method.

[0036] Furthermore, the aforementioned circular RNA-related biological material is any one or more combinations of the following biological materials:

[0037] (1) an expression cassette containing the aforementioned circular RNA;

[0038] (2) a recombinant expression vector containing the aforementioned circular RNA;

[0039] (3) a recombinant expression vector containing the expression cassette described in (1);

[0040] (4) a recombinant cell containing the aforementioned circular RNA;

[0041] (5) A recombinant cell containing the expression cassette described in (1);

[0042] (6) A recombinant cell containing the recombinant expression vector described in (2) or (3);

[0043] Preferably, the starting vector of the recombinant expression vector described in (2) and (3) is a common eukaryotic expression vector of circular RNA or a lentivirus and adeno-associated virus packaging vector, further a pCD series vector, pK5ssAAV-ciR or pK25ssAAV-ciR, etc., wherein the pCD series vector includes pCD2.1-ciR, pCD-ciR, pCD5-ciR, pCD25-ciR, etc.; the present invention uses pCD2.1-ciR.

[0044] Preferably, the host cells of the recombinant cells in (4), (5) and (6) are beef cattle intramuscular preadipocytes, etc.

[0045] Furthermore, the aforementioned circular RNA-related biological material is any one or more combinations of the following biological materials:

[0046] (a) a sequence that inhibits or blocks the expression of the aforementioned circular RNA;

[0047] (b) A vector for inhibiting or blocking the expression of the circular RNA prepared using the sequence described in (a);

[0048] (c) Beef cattle intramuscular preadipocytes that inhibit or block the expression of the circular RNA, prepared using the sequence described in (a) or the vector described in (b).

[0049] Preferably, the sequence in (a) is antisense RNA, siRNA, shRNA or sgRNA of circular RNA.

[0050] Further preferably, the sequence is siRNA, named si-circFTO, and its nucleotide sequence is as follows:

[0051] Sense strand: 5′-GAUGGAGUGGUUGAUGAUCTT-3′ (SEQ ID NO: 40);

[0052] Antisense strand: 5′-GAUCAUCAACCACUCCAUCTT-3′ (SEQ ID NO: 41);

[0053] Application of the above circular RNA or related biological materials in regulating the intramuscular fat content of beef cattle.

[0054] Overexpression of circular RNA reduces the intramuscular fat content of beef cattle, while inhibition of circular RNA increases the intramuscular fat content of beef cattle.

[0055] The present invention has the following advantages and effects compared to the prior art:

[0056] This study verified the existence of circFTO, cloned the full-length molecular sequence of this circular RNA, and determined the circFTO loop structure. The molecule is resistant to the exonuclease RNase R. This circFTO regulates intramuscular fat production in beef cattle, affecting beef quality, and is being applied in assisted genetic breeding to improve beef quality. The circFTO disclosed in this invention can serve as a molecular marker associated with beef quality and has important practical applications in assisted genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the circularization site in the circFTO linker sequence and sequencing peak map.

[0058] Figure 2Quantitative changes of circFTO and linear FTO after RNase R treatment.

[0059] Figure 3 This is the agarose gel electrophoresis diagram of the double-primer method to verify the circular structure of circFTO.

[0060] Figure 4 Oil red O staining was used to detect the number and size of lipid droplets in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0061] Figure 5 Figure 3 is the triglyceride content in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0062] Figure 6 Quantitative changes in adipocyte differentiation-related genes (PPARγ, C / EBPα) in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0063] Figure 7 Quantitative changes in fatty acid transport-related genes (FAT1, FAT4, FABP4, FABP5) in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0064] Figure 8 Quantitative changes in fatty acid synthesis-related genes (FASN, SCD, FADS1) in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0065] Figure 9 Quantitative changes in triglyceride synthesis-related genes (DGAT1, DGAT2, MGAT1) in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0066] Figure 10 Quantitative changes in lipid droplet maturation-related genes (PLIN1, PLIN2, and PLIN3) in beef cattle intramuscular preadipocytes after transfection with NC and OE-circFTO.

[0067] Figure 11 Oil red O staining was used to detect the number and size of lipid droplets in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0068] Figure 12 Figure 3 is the triglyceride content in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0069] Figure 13Quantitative changes in adipocyte differentiation-related genes (PPARγ, C / EBPα) in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0070] Figure 14 Quantitative changes in fatty acid transport-related genes (FAT1, FAT4, FABP4, FABP5) in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0071] Figure 15 Quantitative changes in fatty acid synthesis-related genes (FASN, SCD, FADS1) in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0072] Figure 16 Quantitative changes in triglyceride synthesis-related genes (DGAT1, DGAT2, MGAT1) in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO.

[0073] Figure 17 Quantitative changes in lipid droplet maturation-related genes (PLIN1, PLIN2, and PLIN3) in beef cattle intramuscular preadipocytes after transfection with NC and si-circFTO. DETAILED DESCRIPTION

[0074] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0075] Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer.

[0076] Example 1 Identification of Circularity of circFTO

[0077] This example provides a circular RNA associated with intramuscular adipocyte differentiation in beef cattle, including a circular RNA circFTO associated with intramuscular adipogenesis in beef cattle. The nucleotide sequence of the circular RNA circFTO is shown in SEQ ID NO: 1.

[0078] The primers used to detect the circular RNA circFTO include Divergent-circFTO-F and Divergent-circFTO-R. The nucleotide sequence of Divergent-circFTO-F is shown in SEQ ID NO: 2, the nucleotide sequence of Divergent-circFTO-R is shown in SEQ ID NO: 3, and the schematic diagram and nucleotide sequence of the linker sequence of the circular RNA circFTO are shown in Figure 1 shown.

[0079] 1. Experimental operation

[0080] A method for identifying circular RNA associated with intramuscular adipocyte differentiation in beef cattle comprises the following steps:

[0081] 1.1 Experimental Animals

[0082] The experiment used the longissimus dorsi muscle tissue of 24-month-old adult Angus cattle and Lufeng adult cattle as experimental materials, and 3 cows of each breed were used as research subjects.

[0083] 1.2. Sample collection and RNA extraction:

[0084] After slaughtering the experimental cattle, approximately 100 mg of longissimus dorsi muscle tissue was placed in a 1.5 mL homogenizer tube containing 1 mL of Trizol. High-speed homogenization was performed to completely disrupt the cells. After centrifugation, the sample was transferred to a new 1.5 mL centrifuge tube. After standing for 30 minutes, the sample was centrifuged at 12,000 rpm and 4°C for 5 minutes. The supernatant was removed, and 0.2 mL of chloroform was added per 1 mL of Trizol. The mixture was shaken for 20 seconds, allowed to stand at room temperature for 5 minutes, and centrifuged at 12,000 rpm for 20 minutes (speed reduction to zero). The supernatant was transferred to a new centrifuge tube, and 0.5 mL of pre-chilled isopropanol was added per 1 mL of Trizol. The sample was allowed to stand at -20°C overnight and centrifuged at 12,000 rpm and 4°C for 10 minutes. The supernatant was discarded, and the pellet was washed with 1 mL of 75% ethanol. The pellet was centrifuged at 7,500 rpm and 4°C for 5 minutes. The supernatant was discarded, and the RNA pellet was air-dried for approximately 10 minutes. An appropriate amount of DEPC-treated water was added to dissolve the RNA. The total RNA concentration was measured using a nucleic acid analyzer and the RNA concentration was recorded. 0.5 μL of total RNA was subjected to agarose gel electrophoresis to check the RNA quality. The extracted RNA was stored in a -80°C refrigerator until use.

[0085] 1.3. CircRNA Sequencing and Bioinformatics Analysis of Longissimus Dorsi Muscle Tissue from Different Beef Cattle Breeds

[0086] CircRNA sequencing was performed on the longissimus dorsi muscle tissue RNA obtained from the above-mentioned Angus adult cattle and Lufeng adult cattle. FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) software was used to perform an overall assessment of the sequencing data quality, including the quality value distribution of bases, the position distribution of quality values, GC content, and PCR duplication content.

[0087] circRNA identification and analysis were performed on circRNA-seq sequencing data using the internationally recognized algorithms CIRCexplorer2 (http: / / circexplorer2.readthedocs.io / en / latest / ), circRNA_finder (https: / / omictools.com / circrnafinder-tool), CIRI (https: / / sourceforge.net / projects / ciri / ), find_circ (https: / / github.com / orzechoj / circRNA_finder), and MapSplice (http: / / www.netlab.uky.edu / p / bioinfo / MapSplice2). Differential screening was performed using the internationally recognized algorithm edge (http: / / www.bioconductor.org / packages / release / bioc / html / edgeR.html). CircFTO, which was significantly differentially expressed, was finally selected through analysis. CircFTO was highly expressed in the longissimus dorsi muscle tissue of adult cattle in Lufeng. CircFTO is formed by the circularization of exons 5 to 6 of the bovine FTO gene, such as Figure 1 The nucleotide sequence is shown in SEQ ID NO: 1, and the linker region sequence of circFTO is shown in Figure 1 As shown;

[0088] 1.4 Reverse transcription

[0089] The RNA from the longissimus dorsi muscle tissue of beef cattle obtained in step 1.2 was used to obtain a cDNA template using a reverse transcription kit. The reverse transcription procedures are shown in Tables 1 and 2.

[0090] Table 1 Genomic DNA removal reaction

[0091] Reagents volume 5×gDNAEraserBuffer 2μL gDNAEraser 1 μL Total RNA 1 μg <![CDATA[RNase-free ddH2O]]> Up to 10μL

[0092] The solution was mixed and incubated at 42°C for 2 min.

[0093] Table 2 RNA reverse transcription reaction

[0094] Reagents volume The above reaction mixture 10 μL PrimeScript RT Enzyme Mix I 1 μL <![CDATA[RNase-free ddH2O]]> 4μL RT Primer Mix 1 μL 5×PrimeScript Buffer 2(for Real Time) 4μL Total 20 μL

[0095] Mix well with a pipette and proceed to the second step: 37°C, 15 min; 85°C, 5 s; 4°C. The reverse-transcribed cDNA is stored in a -80°C refrigerator until use.

[0096] 1.5. PCR verification of the circular characteristics of circFTO

[0097] Using the full-length sequence of circFTO as a template, Primer 5 software was used to design primer sequences for RT-PCR identification of circular RNA circFTO. The sequences are as follows:

[0098] Divergent-circFTO-F: 5′-GTGGTGTCAGCCCATGAGTC-3′ (SEQ ID NO: 2);

[0099] Divergent-circFTO-R: 5′-CACTCGGTGGGTGGAACTAA-3′ (SEQ ID NO: 3);

[0100] PCR reaction was performed using the designed primers and the above cDNA as a template. The reaction system is shown in Table 3.

[0101] Table 3 PCR reaction system

[0102] Reagents volume 2×Taq Master Mix 10 μL Upstream primer 0.5μL Downstream primer 0.5μL cDNA template 2μL <![CDATA[ddH2O]]> 7μL Total 20 μL

[0103] The PCR program was as follows: 94°C pre-denaturation for 5 min; 39 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s; and 72°C for 10 min. The resulting products were detected by agarose gel electrophoresis, and the target bands were recovered by gel excision.

[0104] The recovered DNA was sent to Sangon Biotech for sequencing to identify the presence of circular RNA circFTO and its reverse circularization.

[0105] 2. Experimental results

[0106] Sequence alignment revealed that the junction position of circFTO exists. The junction position is formed by the 5' end of the 5'UTR and the 3' end of exon 5. Its peak diagram is single and there are no mixed peaks ( Figure 1 ).

[0107] Example 2 Analysis of RNase R Tolerance of circFTO Molecules

[0108] 1. Experimental operation

[0109] 1 μg of RNA from the longissimus dorsi muscle of bovine cattle (a mixed sample of RNA from the longissimus dorsi muscle of 24-month-old Angus and Lufeng cattle) was added with 2 U / μg of RNase R and incubated at 37°C for 30 minutes. As a control, another 1 μg of the above RNA was incubated with RNase-free water under the same conditions. After treatment with RNase R, the resulting RNA reaction solution was reverse transcribed. We used quantitative real-time PCR (qPCR) to detect the RNA expression levels of circRNA and its linear mRNA. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference for data analysis. CircRNA was detected using primers Divergent-circFTO-F (SEQ ID NO: 2) and Divergent-circFTO-R (SEQ ID NO: 3), linear mRNA was detected using primers Convergent-circFTO-F (SEQ ID NO: 3) and Convergent-circFTO-R (SEQ ID NO: 4), and the GAPDH internal reference gene was detected using primers GAPDH-F (SEQ ID NO: 5) and GAPDH-R (SEQ ID NO: 6) for qPCR quantitative detection.

[0110] 1.1 Primer design:

[0111] Linear mRNA FTO amplification:

[0112] Convergent-circFTO-F: 5′-CTCAATGCCACCCACCAACAC-3′ (SEQ ID NO: 4);

[0113] Convergent-circFTO-R: 5′-TCAAACTCGACCTCTGCCACTC-3′ (SEQ ID NO: 5).

[0114] GAPDH internal reference gene amplification:

[0115] GAPDH-F: 5′-CTGCCGCCTGGAGAAACCT-3′ (SEQ ID NO: 6);

[0116] GAPDH-R: 5'-GCTGTAGCCAAATTCATTGTCG-3' (SEQ ID NO: 7).

[0117] 1.2 RNase R treatment

[0118] Table 4 RNase R treatment system

[0119] Reagents volume RNA 1 μg 10×Reaction Buffer 2μL RNase R (20 U / μL) 2U / μg RNA RNase-Free Water Up to 10μL

[0120] 2 U / μg RNase R enzyme was added to 1 μg RNA, as shown in Table 4, and the reaction was carried out at 37° C. for 30 min. The resulting RNA reaction solution was reverse transcribed according to Tables 1 and 2.

[0121] 1.3 qPCR reaction

[0122] The qPCR reaction system is shown in Table 5.

[0123] Table 5 qPCR reaction system

[0124] Reagents volume 2×SYBR qPCR Master Mix 5μL Upstream primer 0.5μL Downstream primer 0.5μL cDNA template 2μL <![CDATA[ddH2O]]> 2μL Total 10 μL

[0125] The qPCR program settings were as follows: pre-denaturation at 95°C for 2 min; 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 40 s, with fluorescence signal collection at the end of extension; and finally, 1 min at 95°C, annealing at 60°C for 30 s, and the corresponding melting curve was drawn.

[0126] 3. Experimental results

[0127] The relative expression of genes in each group of samples was analyzed by t-test. The data were presented as mean ± standard error (Mean ± SEM). P < 0.05 was considered significant.

[0128] Since circRNA has no free 5' and 3' ends and is resistant to nuclease exonucleases, the total RNA was treated with RNase R and quantitative analysis showed that the amount of circFTO did not change significantly before and after treatment, while the change in the linear control group reached an extremely significant level ( Figure 2 ), successfully verifying the ring structure of cirFTO.

[0129] Example 3: Verification of circFTO circularization by double primer method

[0130] 1. Experimental operation

[0131] Genomic DNA (gDNA) was extracted from the longissimus dorsi muscle of beef cattle using the Ezup column-based animal genomic DNA extraction kit from Shanghai Bioengineering Co., Ltd. PCR amplification was then performed using the cDNA and gDNA as templates with the divergent primers Divergent-circFTO-F (SEQ ID NO: 2) and Divergent-circFTO-R (SEQ ID NO: 3), respectively. As controls, PCR amplification was performed using the convergent primers Convergent-circFTO-F (SEQ ID NO: 4) and Convergent-circFTO-R (SEQ ID NO: 5), respectively. The results were analyzed by agarose gel electrophoresis.

[0132] 2. Experimental results

[0133] The convergent primers for circRNA successfully amplified the corresponding bands with cDNA and gDNA as templates, while the divergent primers for circRNA successfully amplified the corresponding bands only in cDNA ( Figure 3 ), verifying the circularization of circFTO.

[0134] Example 4 Fluorescence quantitative PCR detection of the effect of overexpression of circFTO on the expression of adipogenesis-related genes in beef cattle intramuscular preadipocytes

[0135] 1. Experimental operation

[0136] 1.1 Isolation, transfection, and processing of beef cattle intramuscular preadipocytes

[0137] The longissimus dorsi muscle tissue of newborn calves was taken, and the connective tissue and blood vessels visible to the naked eye were removed. After washing three times with PBS buffer containing 2‰ double antibodies (penicillin and streptomycin), the longissimus dorsi muscle tissue was cut into pieces of about 1-2 mm. 3 For the tissue blocks, add 30mL type I collagenase digestion solution (add 1g type I collagenase to 500mL serum-free DMEM / F12 medium, filter with a 22μm filter and set aside), digest in a 37℃ water bath for 1.5-2.0h, shaking continuously during the period. The digestion solution is neutralized with an equal volume of medium containing 10% FBS, passed through a 70μm and 40μm cell sieves, and the filtrate is collected. Centrifuge at 1500×g for 10min, discard the supernatant, add serum-free DMEM / F12 medium, centrifuge at 1500×g for 10min, and repeat twice. Resuspend the cells with DMEM / F12 medium containing 10% FBS, count the cells, and calculate the percentage of 4.0×10 4 pieces / cm 2Live cell density was inoculated and cultured in a 37°C, 5% CO2 incubator for 1.5-2.0 hours. Beef cattle intramuscular preadipocytes were isolated using the differential attachment principle, non-adherent cells were removed, and the cells were washed three times with sterile PBS. After switching to conventional culture medium, the cells were cultured in a 37°C, 5% CO2 incubator until the cells reached 80%-90% density. The cells were harvested by trypsinization and digested with freezing solution (90% FBS + 10% DMSO). After pipetting, the cells were transferred to cryovials and graded frozen in liquid nitrogen for storage.

[0138] Beef cattle intramuscular preadipocytes were inoculated into 12-well plates (density 5×10 4 Cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum until the contact inhibition stage and then cultured for 2 days to ensure that the cells reached the contact inhibition state. Type I differentiation induction medium (3-isobutyl-1-methylxanthine: 500 μM, dexamethasone: 1 μM, insulin: 10 μg / mL) was added to induce differentiation. After 2 days of culture, cells were transfected using Lipofectamine 2000 reagent and divided into the following groups: experimental group, transfected with circFTO overexpression plasmid (OE-circFTO, concentration 2 μg / well); negative control group, transfected with empty vector plasmid (NC, concentration 2 μg / well). After transfection, the cells were switched to type II differentiation induction medium (insulin: 10 μg / mL) and cultured for another 48 hours. Cells were then harvested for subsequent analysis.

[0139] The construction of the circFTO overexpression plasmid includes the following steps:

[0140] First, the full-length sequence primers of circFTO were designed as follows:

[0141] Full-circFTO-F: 5'-GGGGTACCTGAAATATGCTATCTTACAGATGATCTCAATGCCACCCAC-3' (SEQ ID NO: 8);

[0142] Full-circFTO-R: 5'-CGGGATCCTCAAGAAAAAATATATTCACCAACCACTCCATCTTTCTCCAC-3' (SEQ ID NO: 9);

[0143] The full-length sequence of circFTO was cloned using cDNA obtained by reverse transcription of RNA from the longissimus dorsi muscle tissue of beef cattle as a template.

[0144] Next, the full-length circFTO sequence and the pCD2.1-ciR plasmid were double-digested with KpnI and BamHI, ligated with T4 ligase at 16°C for 12-16 hours, and transformed into competent E. coli DH5α cells. Single colonies were selected and cultured at 37°C for 5 hours. After verification by PCR, the culture was subjected to Sanger sequencing. The culture medium containing the correct sequence was cultured at 37°C for 12-16 hours, and the endotoxin-free plasmid was extracted for use, resulting in the circFTO overexpression plasmid, designated OE-circFTO.

[0145] 1.2 Oil Red O staining

[0146] Slowly add 4% paraformaldehyde solution to a 12-well plate and fix at room temperature for 30 minutes. After fixation, wash the cells three times with DEPC water and stain with Oil Red O for 1 hour. The Oil Red O stain was then discarded and the cells were washed twice with DEPC water. Finally, the treated cells were placed under an inverted microscope to observe the lipid droplet staining effect and photographed.

[0147] 1.3. Determination of triglyceride content

[0148] The specific operation steps refer to the Prilai high-fat sample triglyceride content enzymatic assay kit (E1024-105), and the triglyceride content is calculated according to the formula in the instructions.

[0149] 1.4 RNA Extraction

[0150] Add 500 μL of Trizol to a 12-well plate, scrape the cells to completely disrupt them, centrifuge, and transfer to a new 1.5 mL microcentrifuge tube. Transfer the Trizol to a 1.5 mL nuclease-free microcentrifuge tube and incubate at room temperature for 5 minutes. Centrifuge at 4°C, 12,000 rpm, and 4°C for 5 minutes. Add 0.2 mL of chloroform per 1 mL of Trizol. Cap the tube tightly and shake the sample vigorously for 15 seconds. Let it sit at room temperature for 3 minutes. Centrifuge the sample at 12,000 rpm for 10 minutes at 4°C. The sample will separate into three layers. Carefully aspirate the upper aqueous phase and transfer it to a new microcentrifuge tube. Add an equal volume of isopropanol (approximately 0.2 mL) to the aqueous phase, mix, and incubate at room temperature for 10 minutes to precipitate RNA. Centrifuge the sample at 12,000 rpm for 10 minutes. The precipitated RNA will form a white or transparent fibrous material at the bottom of the tube. Carefully remove the supernatant and rinse the RNA pellet with 75% ethanol. Gently pipette the RNA pellet and centrifuge at 7500 rpm for 5 minutes at 4°C. Remove the supernatant and add 75% ethanol again. Repeat the washing process once. After removing the ethanol, air-dry the RNA pellet at room temperature for approximately 5-10 minutes. Dissolve the RNA pellet in an appropriate amount of RNase-free water.

[0151] 1.5. qPCR reaction

[0152] The reverse transcription experimental method described in step 1.4 of Example 1 was used to reverse transcribe the cell RNA into cDNA. The experimental method described in step 1.3 of Example 2 was used to detect the mRNA expression levels of adipogenesis-related genes by qPCR. The related genes detected included adipocyte differentiation (PPARγ, C / EBPα), fatty acid transport (FAT1, FAT4, FABP4, FABP5), fatty acid synthesis (FASN, SCD, FADS1), triglyceride synthesis (DGAT1, DGAT2, MGAT1) and lipid droplet maturation (PLIN1, PLIN2, PLIN3). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal reference for data analysis. The GAPDH internal reference gene was detected using primers GAPDH-F (SEQ ID NO: 6) and GAPDH-R (SEQ ID NO: 7). Three replicates were designed for each sample during the reaction. 2 -△△Ct The gene expression in each sample was measured.

[0153] 2. Primer design

[0154] Primer design for adipogenesis-related genes:

[0155] PPARγ-F: 5'-CGCTGATGCACTGCCTATGA-3' (SEQ ID NO: 10);

[0156] PPARγ-R: 5'-AGAGGTCCACAGAGCTGATTCC-3' (SEQ ID NO: 11);

[0157] C / EBPα-F: 5'-GCGGGAACGCAACAACATC-3' (SEQ ID NO: 12);

[0158] C / EBPα-R: 5'-GTCACTGGTCAACTCCAGCAC-3' (SEQ ID NO: 13);

[0159] FAT1-F: 5'-CATTACACGCTGATCGTCAAGG-3' (SEQ ID NO: 14);

[0160] FAT1-R: 5'-AAAGAGACGCTGTAGGAGGTG-3' (SEQ ID NO: 15);

[0161] FAT4-F: 5'-CAGTGGTGATCCAGGTACGG-3' (SEQ ID NO: 16);

[0162] FAT4-R:5′-TCATGCGCTGTCACGGAAATA-3′(SEQ ID NO:17);

[0163] FABP4-F:5'-ATCAGCGTAAATGGGGATTTGG-3'(SEQ ID NO:18);

[0164] FABP4-R:5′-GTCTGCGGTGATTTCATCGAA-3′(SEQ ID NO:19);

[0165] FABP5-F:5'-TGAAAGAGCTAGGAGTAGGACTG-3'(SEQ ID NO:20);

[0166] FABP5-R:5′-CTCTCGGTTTTGACCGTGATG-3′(SEQ ID NO:21);

[0167] FASN-F:5′-GGCTCTATGGATTACCCAAGC-3′(SEQ ID NO:22);

[0168] FASN-R:5′-CCAGTGTTCGTTCCTCGGA-3′(SEQ ID NO:23);

[0169] SCD-F:5′-TTCTTGCGATACACTCTGGTGC-3′(SEQ ID NO:24);

[0170] SCD-R:5'-CGGGATTGAATGTTCTTGTCGT-3'(SEQ ID NO:25);

[0171] FADS1-F:5′-AGCACATGCCATACAACCATC-3′(SEQ ID NO:26);

[0172] FADS1-R:5′-TTTCCGCTGAACCACAAAATAGA-3′(SEQ ID NO:27);

[0173] DGAT1-F:5'-CTGATCCTGAGTAATGCAAGGTT-3'(SEQ ID NO:28);

[0174] DGAT1-R:5'-TGGATGCAATAATCACGCATGG-3'(SEQ ID NO:29);

[0175] DGAT2-F: 5'-GCGCTACTTCCGAGACTACTT-3' (SEQ ID NO: 30);

[0176] DGAT2-R: 5'-GGGCCTTATGCCAGGAAACT-3' (SEQ ID NO: 31);

[0177] MGAT1-F: 5'-GCGCTTGCTTGGATAAGTTGTT-3' (SEQ ID NO: 32);

[0178] MGAT1-R: 5'-CACGGCAATGTTACTCAGGTC-3' (SEQ ID NO: 33);

[0179] PLIN1-F: 5'-CTGTGTGCAATGCCTATGAGA-3' (SEQ ID NO: 34);

[0180] PLIN1-R: 5'-CTGGAGGGTATTGAAGAGCCG-3' (SEQ ID NO: 35);

[0181] PLIN2-F: 5'-GACCTTGTTGTCCTCCGCTTAT-3' (SEQ ID NO: 36);

[0182] PLIN2-R: 5'-CAACCGCAATTTGTGGCTC-3' (SEQ ID NO: 37);

[0183] PLIN3-F: 5'-GACGGCCAGTGAGTATGC-3' (SEQ ID NO: 38);

[0184] PLIN3-R: 5'-TGTTTCCTTCGCACTAGACAC-3' (SEQ ID NO: 39).

[0185] 3. Experimental results

[0186] like Figure 4 and Figure 5 After transfection and overexpression of circFTO, the size and number of lipid droplets in cells were significantly reduced, and the triglyceride content was reduced. qPCR results showed that after transfection and overexpression of circFTO, the relative expression levels of PPARγ and C / EBPα mRNA were significantly reduced, and the fat differentiation process was reduced ( Figure 6 ); significantly reduced the relative mRNA expression level of FABP4, and had a downward trend on the levels of FAT1, FAT4 and FABP5, reducing the fatty acid transport process in cells ( Figure 7); significantly reduced the relative expression levels of FASN, SCD and FADS1, thereby inhibiting fatty acid synthesis ( Figure 8 ); significantly reduced the relative expression levels of DGAT1, DGAT2, and MGAT1 to inhibit triglyceride synthesis ( Figure 9 ); At the same time, the relative expression levels of PLIN2 and PLIN3 were significantly reduced, so OE-circFTO can significantly reduce the development and maturation of lipid droplets ( Figure 10 ).

[0187] Example 5 Fluorescence quantitative PCR detection of the effect of circFTO on the expression of lipogenesis-related genes in beef cattle intramuscular preadipocytes

[0188] 1. Experimental operation

[0189] Beef cattle intramuscular preadipocytes were treated and transfected using Lipofectamine 2000 as described in step 1.1 of Example 4 and divided into the following groups: experimental group, transfected with siRNA interfering with circFTO (si-circFTO, concentration 2 μg / well); negative control group, transfected with negative control (NC, concentration 2 μg / well). si-circFTO and negative control were obtained from Guangzhou Jisai Co., Ltd. The number of lipid droplets and triglyceride content in the cells were determined according to the methods described in steps 1.2 and 1.3 of Example 4. Cellular RNA was extracted and reverse transcribed into cDNA according to the method described in step 1.4 of Example 4 and step 1.4 of Example 1. The mRNA expression levels of lipogenesis-related genes were detected by qPCR using the experimental method described in step 1.3 of Example 2. The primers described in step 2 of Example 4 were used to detect related genes including adipocyte differentiation (PPARγ, C / EBPα), fatty acid transport (FAT1, FAT4, FABP4, FABP5), fatty acid synthesis (FASN, SCD, FADS1), triglyceride synthesis (DGAT1, DGAT2, MGAT1) and lipid droplet maturation (PLIN1, PLIN2, PLIN3). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference for data analysis. The GAPDH internal reference gene was detected using primers GAPDH-F (SEQ ID NO: 6) and GAPDH-R (SEQ ID NO: 7). Three replicates were designed for each sample during the reaction. 2 -△△Ct The gene expression in each sample was measured.

[0190] Among them, si-circFTO:

[0191] Sense strand: 5′-GAUGGAGUGGUUGAUGAUCTT-3′ (SEQ ID NO: 40);

[0192] Antisense strand: 5′-GAUCAUCAACCACUCCAUCTT-3′ (SEQ ID NO: 41);

[0193] NC, negative siRNA:

[0194] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 42)

[0195] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 43)

[0196] si-cicFTO and NC are chemically synthesized by Genema and can be directly introduced into cells using Lipofectamine 2000 transfection reagent to specifically block the expression of circFTO, resulting in a gene function loss phenotype.

[0197] 2. Experimental results

[0198] Oil red O staining results Figure 11 , we found that si-circFTO can significantly increase the size and number of lipid droplets. And the triglyceride test results are as follows Figure 12 , si-circFTO significantly increased the triglyceride content in cells. qPCR results showed that si-circFTO significantly increased the relative expression level of C / EBPα mRNA and promoted the adipogenic differentiation process ( Figure 13 ); significantly increased the relative expression levels of FAT1, FABP4 and FABP5 mRNA, and promoted the fatty acid transport process in cells ( Figure 14 ); significantly promoted the relative expression levels of FASN and FADS1, thereby increasing fatty acid synthesis ( Figure 15 ); significantly increased the relative expression levels of DGAT1, DGAT2, and MGAT1 to enhance triglyceride synthesis ( Figure 16 ); At the same time, the relative expression levels of PLIN1, PLIN2, and PLIN3 were significantly increased, indicating that si-circFTO can significantly promote the development and maturation of lipid droplets ( Figure 17 ).

[0199] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A circular RNA associated with intramuscular fat production in beef cattle, characterized in that: The circular RNA is circFTO, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. Use of the circular RNA related to intramuscular fat production in beef cattle according to claim 1 as a specific molecular marker for intramuscular adipocytes in beef cattle.

3. The use of the circular RNA or a product for detecting the expression of the circular RNA according to claim 1, characterized in that: The application is any one of the following applications: 1) Use of circular RNA or a product for detecting the expression of said circular RNA in analyzing the intramuscular fat content of beef cattle; 2) Use of circular RNA or a product for detecting the expression of said circular RNA in identifying or assisting in identifying intramuscular adipose tissue or intramuscular adipocytes in beef cattle; 3) Use of circular RNA or products for detecting the expression of such circular RNA in predicting or assisting in predicting beef quality; 4) Use of circular RNA or products for detecting the expression of said circular RNA in the preparation of a kit for predicting or assisting in predicting beef quality; 5) Use of circular RNA or products for detecting the expression of said circular RNA in increasing the intramuscular fat content of beef cattle or improving the quality of beef cattle; 6) Use of circular RNA or products for detecting the expression of said circular RNA in breeding beef cattle breeds with different beef qualities.

4. A product for detecting the expression of the circular RNA according to claim 1, characterized in that: The product includes a detection primer or a kit containing a detection primer; The detection primers include PCR detection primers or qPCR detection primers; The PCR detection primers include Divergent-circFTO-F and Divergent-circFTO-R, whose nucleotide sequences are shown in SEQ ID NOs: 2-3; The qPCR detection primers include Convergent-circFTO-F and Convergent-circFTO-R, and their nucleotide sequences are shown in SEQ ID NOs: 4-5.

5. Use of the product according to claim 4 in the quantitative detection of circFTO in the longissimus dorsi muscle tissue of beef cattle.

6. A method for detecting the intramuscular fat content of beef cattle, characterized in that: The method comprises the following steps: detecting the expression level of the circular RNA according to claim 1 in beef cattle, and judging the intramuscular fat content of the beef cattle according to the expression level of the circular RNA.

7. The method according to claim 6, characterized in that The specific steps include: (i) extracting total RNA from bovine muscle tissue as a test sample; (ii) reverse transcribing the RNA obtained in step (i) to obtain cDNA; (iii) amplifying and detecting the cDNA obtained in step (ii); (iv) Through melting curve analysis, 2 -ΔΔCt Relative quantification was performed.

8. The circular RNA-related biological material according to claim 1, characterized in that The biomaterial is any one or more combinations of the following biomaterials: (1) An expression cassette containing the circular RNA according to claim 1; (2) A recombinant expression vector containing the circular RNA according to claim 1; (3) a recombinant expression vector containing the expression cassette described in (1); (4) A recombinant cell containing the circular RNA according to claim 1; (5) A recombinant cell containing the expression cassette described in (1); (6) A recombinant cell containing the recombinant expression vector described in (2) or (3); And / or, the biomaterial is any one or more combinations of the following biomaterials: (a) a sequence that inhibits or blocks the expression of the circular RNA according to claim 1; (b) A vector for inhibiting or blocking the expression of the circular RNA prepared using the sequence described in (a); (c) Beef cattle intramuscular preadipocytes that inhibit or block the expression of the circular RNA, prepared using the sequence described in (a) or the vector described in (b).

9. The biomaterial according to claim 8, characterized in that: The sequence in (a) is antisense RNA, siRNA, shRNA or sgRNA of circular RNA; Furthermore, the sequence described in (a) is siRNA, named si-circFTO, and its nucleotide sequence is as follows: Sense strand: 5′-GAUGGAGUGGUUGAUGAUCTT-3′; Antisense strand: 5′-GAUCAUCAACCACUCCAUCTT-3′.

10. Use of the circular RNA according to claim 1 or the circular RNA-related biomaterial according to any one of claims 8 to 9 in regulating the intramuscular fat content of beef cattle, characterized in that: Overexpression of circular RNA reduces the intramuscular fat content of beef cattle, while inhibition of circular RNA increases the intramuscular fat content of beef cattle.

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